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. 2010 Nov;83(1 Suppl):4.

8/2/2010

PMCID: PMC3399395
Biol Reprod. 2010 Nov;83(1 Suppl):4.

Genomic Imprinting in Invasive Trophoblast in Mammalian Interspecies Hybrids.

Douglas F Antczak, Xu Wang, Donald C Miller, Andrew G Clark 1

Genomic imprinting is a special form of epigenetic modification of the genome in which gene expression is determined by parent-of-origin. Imprinted genes are important in normal fetal and placental development, and their dysregulation has been implicated in several important abnormalities, including intrauterine growth retardation. Although the phenomenon of genomic imprinting has been recognized for a quarter century, only about 160 imprinted genes total have been identified in humans and mice, the species most widely studied, despite strenuous efforts of many research groups around the world to identify more. Computational bioinformatic approaches aimed at predicting the imprinting status of genes have generated long lists of candidates, but few of those genes have yet been shown to be imprinted. We hypothesized that it should be possible to identify imprinted genes more easily in tissue samples from interspecies hybrids, in which there are more genetic differences between the maternal and paternal genome than in intraspecies matings. We chose to study gene expression in invasive trophoblast tissues from the reciprocal equine hybrids, the mule and hinny. There is evidence for differences in placental anatomy and physiology between mules and hinnies that is consistent with an imprinting origin. We used massively parallel Illumina short-read sequencing (RNA-seq) to examine parent-of-origin gene expression in the transcriptomes of chorionic girdle trophoblast samples from conceptuses of mule and hinny. Genes with parent-of-origin bias in their expression patterns were identified based on Single Nucleotide Polymorphism (SNP) differences in orthologous genes in the two parent species. Fourteen of these genes are known to be imprinted from studies of other species, and seven are novel. Using pyrosequencing, the biased parent-of-origin expression patterns for these genes were confirmed in mule and hinny placental tissue. The expression of a subset of these genes was examined in lymphocytes from mules and hinnies, and in this adult tissue the imprinted genes were expressed bi-allelically, as has been shown for some imprinted genes in humans. The novel and known imprinted genes of mule and hinny thus appear to be regulated like imprinted genes identified in other species. A substantial core of the imprinted genes identified in humans show similar imprinting patterns in mice, pigs, and cattle. Thus, it seems reasonable that some fraction of the new imprinted genes identified in interspecies equid hybrids would also be imprinted in humans and other mammals.

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Endogenous Retroviruses: From Infectious Elements to Essential Genes.

Thomas E Spencer 1, Massimo Palmarini 2

Endogenous retroviruses (ERVs) are present in the genome of all vertebrates and are remnants of ancient exogenous retroviral infections of the host germline transmitted vertically from generation to generation. Syncytins are envelope (env) genes of retroviral origin that have been co-opted by the host to mediate placental development. Two syncytin genes have been identified in primates (HERV-W (ERVWE1) termed syncytin-1 and HERV-FRD termed syncytin-2), as well as distinct, nonorthologous genes in rodents (syncytin-A and -B) and rabbits (syncytin-Ory1). The syncytins can induce cell-cell fusion, and placental development in those mammals requires the fusion of trophoblast cells into a highly specialized, multinucleated syncytiotrophoblast layer, which is essential for absorption of nutrients from the mother. All of the syncytins are expressed in the syncytiotrophoblasts, and null mutation of the syncytin-A gene in mice results in disruption of the syncytiotrophoblast-containing labyrinth placenta. The null placentae exhibit decreased vascularization and poor transport, resulting in fetal growth retardation and ultimately embryo loss between 11.5 and 13.5 days of gestation. In both humans and mice, one of the two syncytins (human syncytin-2 and mouse syncytin-B) is also immunosuppressive, suggesting they may be also involved in immunotolerance of the semiallogeneic fetus. The sheep genome contains 27 JSRV-related endogenous betaretroviruses (enJSRVs) related to the pathogenic Jaagsiekte sheep retrovirus (JSRV) that have been integrating in the host genome for the last 5-7 million years. The exogenous JSRV is a causative agent of a transmissible lung cancer in sheep, and enJSRVs are able to protect their host against JSRV infection by blocking different steps of the viral replication cycle via receptor interference and late replication steps. Indeed, endogenization and positive selection of ERVs acting as restriction factors is a mechanism used by the host to fight exogenous retroviral infections. In sheep, the enJSRVs are most abundantly expressed in the uterine luminal and glandular epithelia as well as in the conceptus (embryo and associated extraembryonic membranes) trophectoderm. Sixteen of the 27 enJSRV loci contain an env gene with an intact open reading frame, and in utero loss-of-function experiments found the enJSRVs envelope (env) to be essential for conceptus elongation and trophoblast growth and differentiation. Collectively, available evidence supports the ideas that genes captured from ancestral retroviruses have been pivotal in the acquisition of new, important functions in mammalian evolution and were positively selected for biological roles in genome plasticity, protection of the host against infection of related pathogenic and exogenous retroviruses, and placental morphogenesis. This work supported by NIH Grant HD052745 and by a Strategic Research Developmental Grant by the Scottish Funding Council.

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Functional Genomics and Epigenetics in Pig Cloning Research.

X Cindy Tian 3, Joonghoon Park 3, Richard Bruno 3, Richard French 2, Randall S Prather 3

Abstract not available.

Biol Reprod. 2010 Nov;83(1 Suppl):4.

The Race Between Ovarian Cancer and Immunity: How Hormones and Inhibitory Molecules Set the Pace.

Kunle O Odunsi 1

Hormones and inhibitory molecules play an important role in determining the fate of anti-tumor effector cells. We have addressed this issue by studying NY-ESO-1, a "cancer-testis" antigen frequently expressed in epithelial ovarian cancer (EOC) and among the most immunogenic tumor antigens defined to date. In an effort to understand in-vivo tolerance mechanisms, we assessed the phenotype and function of NY-ESO-1 specific CD8+ T cells derived from peripheral blood (PBL), tumor-infiltrating lymphocytes (TILs) and ascites (TALs) of EOC patients with NY-ESO-1 expressing tumors, with or without humoral immunity to NY-ESO-1. Whereas NY-ESO-1-specific CD8+ T cells were readily detectable ex vivo with tetramers in TILs and TALs of seropositive patients, they were only detectable in PBL following in vitro stimulation. Compared with PBL, tumor derived NY-ESO-1-specific CD8+ T cells demonstrated impaired effector function, preferential usage of dominant TCR, and enriched co-expression of inhibitory molecules LAG-3 and PD-1. Expression of LAG-3 and PD-1 on CD8+ T cells was upregulated by IL-10, IL-6 (cytokines found in tumor ascites), and tumor-derived APCs. Functionally, CD8+LAG-3+PD-1+ T cells were more impaired in IFN-γ/TNF-α production compared with LAG-3+PD-1- or LAG-3-PD-1- subsets. Dual blockade of LAG-3 and PD-1 during T cell priming efficiently augmented proliferation and cytokine production by NY-ESO-1-specific CD8+ T cells, indicating that anti-tumor function of NY-ESO-1 specific CD8+ T cells could potentially be improved by therapeutic targeting of these inhibitory receptors.

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Endometrial Cancer: A Hormonally Responsive Malignancy.

Kimberly Leslie 1

Endometrial carcinogenesis is related to overexposure to estrogen that is not modulated by the differentiating effects of progesterone. Our work has focused on the downstream genomic and proteomic effects of progesterone through PRA and PRB that inhibit endometrial cancer proliferation, induce apoptosis and differentiation, and prevent metastatic spread. We have identified a group of effectors, including cell adhesion molecules, cytokines, growth factors, DNA remodeling proteins, and other transcription factors that are controlled by, and in turn control, progesterone's actions in the endometrium. These progesterone-controlled pathways, including inhibition of angiogenesis and PI3K/AKT/mTOR, are now being exploited as new therapeutic avenues in endometrial cancer clinical trials and will be discussed.

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Aromatase and Breast Cancer: From Bench to Bedside.

Serdar Bulun 1

Aromatase, encoded by a single gene, is the key enzyme for estrogen biosynthesis; its inhibitors effectively eliminate all estrogen production in the human body. A number of partially tissue-specific promoters regulate aromatase expression in a variety of human tissues including the ovary, adipose tissue, skin and breast cancer. Increased aromatase expression in breast tumor tissue occurs via selective activation of a distinct promoter region in adipose fibroblasts surrounding malignant epithelial cells. This breast cancer-associated promoter region is regulated by a PGE2-cAMP-dependent signaling pathway in breast adipose fibroblasts. Moreover, this promoter region is induced by C/EBPβ and members of the Jun transcription factors and suppressed by BRCA1. Pathology and physiology associated with aromatase provide novel and exciting opportunities for the treatment of endocrine-responsive breast cancer. Although currently available aromatase inhibitors are superior to antiestrogens as adjuvant agents to prolong disease-free survival in postmenopausal breast cancer, they are associated with osteoporosis and inadvertent ovarian stimulation in premenopausal women. A key challenge is the refinement of aromatase inhibitor treatment of breast cancer, so that this class of drugs would not cause osteoporosis and may be used in premenopausal women without stimulating ovarian cyst formation. Development of new breast tumor tissue-specific aromatase inhibitors via selective targeting of promoters of the aromatase gene may minimize these side effects without sacrificing clinical efficacy.

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Cell Adhesion Junctions in the Testis: Are They a Target for Contraception?

C Yan Cheng 1, Dolores D Mruk 1, Bruno Silvestrini 2

During spermatogenesis, there are extensive restructuring of cell junctions at the Sertoli-Sertoli and Sertoli-germ cell interface during (i) spermatogonial renewal via mitosis, (ii) germ cell cycle progression and meiosis, and (iii) spermiogenesis and spermiation. However, developing germ cells remain attached to the Sertoli cell in the seminiferous epithelium which is mediated by desmosome, gap junction and apical ectoplasmic specialization (apical ES, a testis-specific atypical adherens junction type), whereas adjacent Sertoli cells near the basement membrane are held together by tight junction (TJ), basal ES, desmosome, and gap junction that constitute the blood-testis barrier (BTB) in the mammalian testes. Studies in recent years have shown that these junctions are the prime target of male contraceptive development, in particular the apical ES. For instance, studies by using a micropipette pressure transducing system (MPTS) have shown that the adhesive force conferred by apical ES between Sertoli cells and spermatids (step 8-19 spermatids) is at least twice as strong as desmosome and gap junctions between Sertoli cells and pre-step 8 spermatids or spermatocytes, yet apical ES is significantly more vulnerable to the disruption induced by adjudin [1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydraizde] and other related compounds. Apical ES is uniquely found in the testis which is typified by the presence of hexagonally packed actin-based filaments sandwiched between cisternae of endoplasmic reticulum and the apposing plasma membranes of the Sertoli cell and the spermatid, and this unique ultrastructural feature is limited to the Sertoli cell side at the Sertoli-spermatid interface. Once apical appears in step 8 spermatids during spermiogenesis, this is the only anchorage device that attaches developing spermatids to the Sertoli cell in the epithelium till spermiation. However, apical ES is not found in any other organs in the mammalian body. Interestingly, basal ES at the Sertoli-Sertoli cell interface at the BTB, however, is coexisting with TJ, desmosome and gap junction and the BTB determines which drugs and how much drugs can have access to the apical compartment of the epithelium via drug transporters. In short, cell adhesion junctions in the seminiferous epithelium are the prime target for contraceptive development if a compound can exert its effects primarily at this ES in the testis. In this presentation, we will briefly review the unique features of apical ES and the latest findings regarding the mechanism of action of a candidate compound on this adhesion junction in the testis. The work in the authors' laboratory was supported by NIH grants, NICHD U54 HD029990 Project 5 and R01 HD056034 to CYC.

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Claudin 5, Germ Cells, and the Blood-Testis Barrier.

Rex A Hess, Carla MK Morrow 1

Claudins, in addition to occludin and junctional adhesion molecules (JAMs), are integral membrane proteins that contribute to blood-testis-barrier (BTB) function between Sertoli cells. Of the claudin proteins, claudins 3 and 11 have received the most attention; however, recent studies have found claudin 5 (CLDN5) expression in both Sertoli and germ cells, in contrast to prior reports of Cldn5 expression only in vascular endothelium. The transcription factor ets variant 5 (ETV5) is essential for self-renewal of spermatogonial stem cells and was found to regulate Cldn5. The BTB was compromised in Etv5-/- testes and Cldn5 expression was reduced significantly, while occludin and JAMs were not. Therefore, it was hypothesized that CLDN5 was important for BTB function in the seminiferous epithelium. Cldn5 mRNA shows a significant increase in testis on d20 and immunostaining was increased in Stage VIII of the wild type adult testis, coincident with development and importance of BTB formation. Surprisingly, CLDN5 was also demonstrated in spermatogonia and preleptotene spermatocytes and its expression in the seminiferous epithelium was depended upon both the presence of germ cells and ETV5. However, CLDN5 expression in testicular vascular endothelium and rete testis epithelium was ETV5 independent. CLDN5 was absent in both aspermic Etv5 and W/Wv mice testes. However, in W/Wv tubules showing limited spermatogenesis, CLDN5 was present at the BTB. In conclusion, germ cells are necessary for Sertoli cell CLDN5 expression and lack of CLDN5 at the BTB is associated barrier leakiness.

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Adhesion Properties of Spermatogonial Stem Cells.

A MM van Pelt 1, M PA van Bragt 1, Gianfranco Carlomagno 1, C M Korver 1, S Repping 1, Dirk G De Rooij 2

Spermatogonial stem cells (SSCs), at the beginning of spermatogenesis, can either selfrenew to form two new stem cells or differentiate to form pairs of spermatogonia that stay connected by intercellular bridges and eventually develop into spermatozoa. Spermatogenesis is maintained throughout life, which implies an efficient balancing between self-renewal and differentiation of the SSCs. In many stem cell systems, the balance between self-renewal and differentiation is regulated by the niche. Recently, it was shown that SSCs preferentially localize in areas of the tubule close to the interstitial tissue, suggesting that the niche is located in this area. Furthermore, SSCs migrate out of the niche during differentiation as differentiating spermatogonia have a random distribution in the seminiferous tubules. A recent study sheds light on the important role of adhesion molecules in driving the proper localization of SSCs in the niche and shows that the adhesion molecule ITGB1 is important for the homing of SSCs to the basal membrane after transplantation. In addition, we now found that during BMP4-induced differentiation of the spermatogonial stem cell line GC-6spg, pathways involved in adherens junctions, focal junctions, gap junctions, cell adhesion molecules (CAMs) and regulation of actin cytoskeleton are highly affected. Among the genes belonging to several of the most affected adhesion pathways was cdh1 (known as E-cadherin), an adhesion molecule known to be expressed by a subpopulation of spermatogonia including SSCs. Taken together, adhesion molecules, known to be linked to the stem cell niche, are involved in spermatogonial stem cell maintenance and differentiation.

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Adrenomedullin Signaling at the Maternal-Fetal Interface.

Kathleen M Caron 1

Adrenomedullin (AM) is a peptide vasodilator that is elevated nearly 5-fold in normal human pregnancies but often blunted in pregnancies that are complicated by fetal growth restriction, gestational diabetes or preeclampisa. Using gene targeted mouse models, we showed that haploinsufficiency for maternal AM leads to poor uterine receptivity, reduced pinopode formation and sub-fertility. Pregnant AM+/- females exhibit abnormal implantation, ectopic placentation and fetal growth restriction that is largely independent of fetal genotype. These data indicate that a modest genetic reduction in maternal levels of AM is sufficient to cause pregnancy complications. Although AM-/- mice die at mid-gestation with lymphatic vascular defects, we have recently discovered that fetal AM is required for branching morphogenesis of the placental labyrinth layer and remodeling of maternal spiral arteries. Morphometric analysis, immunostaining and 3D placental casting shows that AM-/- fetal vessels are larger and under-branched compared to those of their wildtype littermates. Furthermore, the maternal spiral arteries which feed the AM-/- placenta fail to remodel compared to the spiral arteries of neighboring AM+/+ placentas. Therefore, AM serves as a trophoblast-derived factor that is critical for fetal and maternal placental vascularization. These preeclampsia associated pathologies in AM-/- placentas suggests that the genetic dosage of AM provided by the fetus may impact on the probability or severity of maternal preeclampsia. Collectively, our studies demonstrate that the precise genetic dosage of both maternal and fetal AM is required for the establishment and maintenance of a healthy pregnancy and normal fetal growth.

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Maternal-Fetal Immune Dialog in Nonhuman Primates.

Thaddeus G Golos, Svetlana V Dambaeva, Gennadiy I Bondarenko, Maureen Durning 1

The cells at the human maternal-fetal interface that engage in the placental-immune dialog have been studied extensively, where HLA-G-expressing trophoblasts and putative targets (natural killer (NK) cells, macrophages, T cells) are readily available for in vitro studies. However, the significance of MHC-leukocyte interactions for pregnancy success is exceedingly difficult to interrogate with human in vivo studies. We have developed two approaches for studying the trophoblast-leukocyte dialog in vivo in the rhesus monkey, where the primary trophoblast MHC class I molecule, Mamu-AG, is considered homologous to HLA-G. Recent passive immunization studies with anti-Mamu-AG antibodies have demonstrated disruption of placental villous growth, decidual differentiation, and spiral arteriole remodelling by endovascular trophoblasts, in vivo evidence of a physiological role in early pregnancy. Taking a cue from success with this approach, we have adapted immunodepletion of peripheral blood leukocytes with rhesus monkeys as an approach to modulate the endometrial and decidual populations. Successful paradigms for depleting peripheral blood NK cells have been established, and although studies are in the early stages, changes in endometrial/decidual leukocyte populations, vascularization, and glandular and stromal differentiation with immunodepletion support the hypothesis that leukocytes play an important role in implantation and the establishment of a successful pregnancy. In addition, initial results suggest that nonhuman primate implantation is sensitive to immune manipulation and could provide a model for early pregnancy loss.

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Dendritic Cells in Implantation: New Players During the Angiogenesis Process.

Sandra M Blois 1

Implantation of mammalian embryos into their mother's uterus ensures optimal nourishment and protection throughout development. Successful pregnancy relies on the establishment of an adequate blood supply to support the metabolic demands of the growing embryo/fetus. Pregnancy is indeed one of the unique situations in which angiogenesis (the development of new blood vessels from pre-existing ones) takes place under physiological conditions. A delicate balance between stimulatory and inhibitory signals regulates angiogenic responses in the decidua, where trophoblasts and NK cells appear to play a crucial role through their proven ability to produce growth factors and cytokines that modulate endothelial cell responsiveness. Dendritic cells (DC) are also considered an important regulatory component during pregnancy, mainly due to their role in the establishment of maternal immunologic tolerance. DC are known to increase their numbers at the peri-implantation period in mice, where this accumulation is accompanied by a transitory decrease in the relative number of DC producing the Th2 cytokine interleukin 10 (IL-10). However, the recent finding that DC subsets can promote angiogenesis in a variety of physiopathological settings suggests that the classical functions ascribed to DC during pregnancy need to be revised, as these cells may not only promote immune tolerance but also influence other processes such as decidualization and placentation and the vascular changes associated to them. Thus, by linking immunoregulatory and pro-angiogenic functions, DC may represent a pivotal component of the uterine signaling network involved in the establishment and maintenance of pregnancy.

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Mitochondrial DNA Mutations in Reproductive Medicine.

Weiwei Fan 1, Douglas C Wallace 2

We have created the first mouse model of directional segregation of a heteroplasmic mtDNA frameshift mutation during oogenesis. The same mice also developed maternally inherited mitochondrial cardiomyopathy caused by a homoplasmic mtDNA missense mutation. A mouse cell line was first isolated containing a homoplasmic COI T6589C missense mutation and a homoplasmic ND6 13885insC frameshift mutation. The mutant mtDNA was next introduced into the female mouse embryonic stem (mES) cells. One of the resulting mES cybrids contained both homoplasmic mutations but 4% of its mtDNAs acquired a T deletion adjacent to the ND6 13885insC (ND6 13885insCdelT), which restored the normal ND6 coding sequence. After transmitted into the mouse germline, the COI T6589C mutation remained homoplasmic but the ND6 frameshift mutation dropped to 47% in the founder mouse and to 14% to 6% to 0% in the successive generations. The resulting homoplasmic COI T6589C + ND6 13885insCdelT mutant mice had a 37%-48% complex IV deficiency in brain, heart, liver, and skeletal muscle and increased heart mitochondrial proliferation, disordered mitochondrial distribution, and cristae-lysis. Echo cardiograms revealed that these animals developed a hypertrophic cardiomyopathy associated with a 26% increase in left ventricular wall thickness, a 30% decrease in left ventricular diastolic internal dimension, a 39% decrease in circumferential strain, and a 74% decrease in radial strain. This stable maternally inherited mouse model now opens new avenues for studying the pathophysiology and therapeutic of mitochondrial diseases.

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Germline Transmission of Mitochondrial DNA in the Mouse.

Timothy Wai 1, Asangla Ao 2, Xiaoyun Zhang 2, Daniel Dufort 2, Daniel Cyr 3, Eric A Shoubridge 2

In mammals, mitochondria and mitochondrial DNA (mtDNA) are transmitted through the female germline. Mature oocytes contain nearly 200,000 copies of mtDNA, organized at 1-2 copies per organelle. Despite the high genome copy number, mtDNA sequence variants are observed to segregate rapidly between generations, and this has led to the concept of a genetic bottleneck for the transmission of mtDNA. Here, we demonstrate that a subgroup of replicating genomes in the early post-natal ovary is responsible for the rapid segregation of mtDNA and show that the rate of segregation of mtDNA can be accelerated when mtDNA copy number is further reduced in heteroplasmic germline-specific knockout mouse models, yet very extreme reductions in germ cell mtDNA content seem to cause female-specific infertility. Low copy number embryos can be fertilized and proceed through preimplantation development yet fail to develop normally after implantation. Tracking the developmental outcome of embryos containing a range of mtDNAs points to a developmental threshold of about 50,000 copies of mtDNA in the oocyte Thus, we advance the hypothesis that the large number of mitochondria and mtDNAs present in the oocyte represent a genetic mechanism to ensure their distribution to the gametes and somatic cells of the next generation. If true, mtDNA copy number may be the most important determinant of oocyte quality not because of the effects on oocyte metabolism, but because too few would result in a maldistribution in the embryo.

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Nonhuman Primate Model for Mitochondrial Gene Therapies.

Shoukhrat M Mitalipov 1

Mitochondria are found in all eukaryotic cells and are essential for basic cellular function due to their principal role in the production of energy. Mitochondria contain their own genome (mt)DNA which, unlike the nuclear genome, is almost exclusively transmitted maternally from the egg. Mutations in mtDNA contribute to a diverse range of still incurable human diseases and disorders. To establish preclinical models for new therapeutic approaches, we recently demonstrated that the mitochondrial genome can be efficiently replaced in mature metaphase II arrested rhesus oocytes by chromosome transfer. Newly reconstructed oocytes consist of nuclear genetic material from one female and cytoplasmic components including mitochondria and mtDNA from another female. This approach yields developmentally competent oocytes suitable for fertilization and producing embryonic stem cells or healthy offspring. Potential clinical applications of this novel reproductive technology include mitochondrial gene replacement therapy to prevent transmission of mtDNA mutations and treatment of infertility caused by cytoplasmic defects in oocytes.

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Developmental Potential and Plasticity of Stem Cells in the Placenta.

James (Jay) C Cross 1

Abstract not available.

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Transcriptomics of Human Preplacental Trophoblast Development.

Paul Robson, Wishva Herath, Lili Sun, Wenlong Luo, Mikael Huss 1

Human embryonic stem cells (hESCs) readily give rise to placental cell types derived from the developing embryo. This provides experimental access to the earliest extraembryonic cell types of the developing human embryo that would otherwise be inaccessible. This is an invaluable research tool particularly considering the biological novelties derived in the primate extraembryonic tissues which remain in accessible in other animal models. Here we first define a robust protocol to differentiation hESCs uniformly along the trophoblast lineage by inhibition of FGF signaling and induction by BMP4. This treatment results in the immediate and dramatic up-regulation (within 4 hrs) of a suite of trophoblast transcription factors (eg. CDX2, GATA3, GATA2, MSX2, and TFAPA2) and the down-regulation (within 8 hrs) of key pluripotency factors. BeadArray gene expression profiling through a time course of differentiation reveals similarities to in vivo expression dynamics with, for instance, the up-regulation of CGA preceding that of CGB. In addition, we provide evidence for the hard wiring of the hypoxic response within the trophoblast developmental genetic regulatory network, in anticipation of the low oxygen environment within the first trimester placenta. Finally, to fully explore the preplacental transcriptome we have employed RNA-seq to generate >60 million 50 base, strand-specific reads from each of 5 time points of differentiation using the ABI SOLiD3+ NextGen sequencing system. This data identifies mesenchymal to epithelial cell splicing transitions, monoallelic expression presumably resulting from imprinting, and many novel transcribed regions. In sum, our data provide a resource enabling insight into the molecular control and characterization of the human preplacental trophoblast.

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

TWIST Is a Key Molecular Regulator of Cadherin-Dependent Terminal Differentiation of Human Trophoblastic Cells In Vitro.

York Hunt Ng, Hua Zhu, Spiro Getsios, Colin D MacCalman 1

The formation of the multinucleated syncytial trophoblast of the human placenta by the terminal differentiation and fusion of mononucleate villous cytotrophoblasts and the invasion of extravillous cytotrophoblasts (EVTs) into the underlying maternal tissues and vasculature are key steps in human placentation. The molecular mechanisms underlying the development of an invasive phenotype in EVTs include many of those first identified as having a role in cancer cell metastasis. Previous studies from our laboratory have demonstrated that a cellular event involved in syncytial trophoblast formation is the continuous and progressive decrease in expression of the cell-adhesion molecule E-CADHERIN (E-CAD). In view of these observations, and the integral role of the basic helix-loop-helix transcription factor TWIST in the onset and progression of cancer in a wide variety of tissues, we have examined the expression, regulation and function of TWIST in human trophoblastic cells in vitro. Our initial studies using Western blotting and immunofluorescent staining revealed that TWIST and E-CAD expression in cultured BeWo cells are differentially regulated by the second messenger cyclic AMP. Similarly, interleukin-1beta and transforming growth factor-beta1, two cytokines which promote and restrain human trophoblastic cell invasion respectively, were found to differentially regulate TWIST expression in primary cultures of EVTs in time-dependent manner. Gain- and loss-of-function studies were respectively performed using either a mammalian expression vector containing a cDNA encoding Twist or siRNA specific for this transcription factor. Our results show that Twist promotes the formation of the syncytial trophoblast through down-regulation of E-CAD, and promotes the invasive ability of EVTs through up-regulating N-CADHERIN (N-CAD) expression. Collectively, our findings demonstrate that TWIST is a key regulator of cadherin-mediated terminal differentiation of human trophoblastic cells. This research was funded by a grant from the Canadian Institutes of Health Research (CDM). YHN is the recipient of a studentship from the Child and Family Research Institute.

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Arginine Stimulates Migration of Ovine Trophectoderm Cells Through the MTOR-RPS6-RPS6K Signaling Cascade and Synthesis of Nitric Oxide, Polyamines, and Interferon Tau.

Fuller W Bazer, Jinyoung Kim, Robert C Burghardt, Guoyao Wu, Greg A Johnson, Thomas E Spencer 1

L-Arginine (Arg) in the uterine lumen of ewes increases significantly between Days 10 and 15 of pregnancy, and it is an essential substrate for synthesis of nitric oxide (NO), by nitric oxide synthase (NOS), and of polyamines, by ornithine decarboxylase (ODC), via arginase required for growth and development of conceptuses (embryo and its extra-embryonic membranes). Arg activates the FK506 binding protein 12-rapamycin associated protein 1 (MTOR) and the ribosomal protein S6 kinase (RPS6K) cell signaling pathways to affect cell proliferation and mRNA translation. Studies were conducted using an established ovine trophectoderm cell line (oTr cells) cultured in customized DMEM-F12 medium containing physiological levels of each amino acid and glucose. The cells were starved for 6 h in each respective nutrient-free customized medium (e.g., Arg-free medium for Arg treatment) and then cultured in either 0.2 mM Arg, 0.2 mM leucine (Leu), 0.5 mM glutamine (Gln), or 4 mM glucose for the duration of the treatment period. After culture, the cells were lysed and assayed for protein and the lysate stored at -80°C until analyzed for mRNAs and proteins of interest. This study examined effects of Arg on: 1) activation of the MTOR signal transduction pathway; 2) cell migration; 3) metabolism of Arg to NO to affect cell migration; and 4) production of IFNT. In addition, effects of L-NAME (an inhibitor of NOS), Nor-NOHA (an inhibitor of arginase), and SNAP and DETA (NO donors) on cell migration were examined. Pertinent results were that: 1) Arg increased abundance of phosphorylated MTOR, RPS6K and 4EBP1, key components of the MTOR cell signaling pathway; 2) Arg enhanced migration of oTr cells principally through NO as both SNAP and DETA stimulated migration by 1.5- and 1.8-fold (P<0.01); and 3) Arg increased abundance of IFNT. These results indicate that Arg, acting via the MTOR cell signaling pathway, increases generation of NO via NOS, production of polyamines via ODC, and production of IFNT. Thus, Arg affects cell proliferation, cell migration and mRNA translation which are key mechanisms whereby dietary supplementation with Arg may improve establishment and maintenance of pregnancy as well as growth and development of conceptuses in animals as well as humans. This research was supported by USDA CSREES National Research Initiative Grant 2006-35203-17283.

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Effects of Maternal Plane of Nutrition, Placental Tissue Type, and Stage of Gestation on 3B-Hydroxysteroid Dehydrogenase, 17A-Hydroxylase, and Aromatase Activity of Sheep Placenta.

Lawrence P Reynolds 1, Joel S Caton 1, Kimberly A Vonnahme 1, Dale A Redmer 1, C Jo Corbin 2, Alan J Conley 2

We and others previously have shown that when ewes are undernourished (LOW intake) or overnourished (HIGH intake) during pregnancy, they exhibit a 15-40% reduction in placental weight and blood flow, leading to reduced lamb birthweight compared with moderately-fed (MOD intake) ewes. Further, nutrient intake during pregnancy also affects maternal circulating estrogen concentrations, which are either similar or elevated in LOW and are reduced in HIGH compared with MOD intake ewes. We hypothesized that maternal dietary intake level would affect the ability of the placental to synthesize estrogen. We also wanted to examine whether placental tissue type or stage of gestation affects placental estrogen synthesis. We therefore examined the effects of: (1) maternal dietary intake level, (2) placental tissue type (i.e., whole placentome [PLAC], maternal caruncle [CAR], or fetal cotyledon [COT]), and (3) stage of gestation on placental activity of the 3 key enzymes in the pathway from progestins to estrogens; namely, 3B-hydroxysteroid dehydrogenase (3BHSD), 17A-hydroxylase (P450C17), and aromatase using microsomal preparations, radioactive substrates (H3-DHEA, 14C-P4, and H3-androstenedione for 3BHSD, P450C17, and aromatase, respectively), and thin-layer chromatography, with methods similar to those we have previously described. All enzyme activities are reported as pmol of product per mg of microsomal protein per min. In experiment 1, samples of PLAC, CAR, and COT were obtained from MOD intake ewes (n=4) on day 135 of gestation. Based on the results of exp. 1, for experiment 2 only samples of COT were obtained from LOW (n=7), MOD (n=6), and HIGH intake ewes (n=7) at term (approx. day 145-150). In exp. 1, at day 135 of gestation, activity of 3BHSD and aromatase were less (P<0.01) for CAR than COT or PLAC, which were similar (52 and 0.45 vs. 592 and 4.7 or 541 and 4.4, respectively; overall SEM 81 and 0.7 for 3BHSD and aromatase); activity of P450C17, in contrast, was undetectable. For exp. 2, at day 131 (approx. 15-20 days prepartum) maternal systemic estrogen concentrations (pg/ml) were similar for LOW and MOD and reduced (P<0.05) in HIGH ewes (30.9 and 29.6 vs. 12.1; overall SEM 3.2). In exp. 2, at term, placental and total COT weights were similar among LOW, MOD, and HIGH ewes. However, activity of 3BHSD and aromatase in COT at term were less (P<0.05) for MOD compared with LOW or HIGH ewes (238 and 0.2 vs. 686 and 2.5 or 366 and 1.6, respectively; overall SEM 86 and 0.7 for 3BHSD and aromatase). Similarly, P450C17 activity was less (P<0.05) for MOD compared with LOW or HIGH ewes (5.4 vs. 14.9 or 13.3; overall SEM 3.1). Thus, activity of placental steroidogenic enzymes at term was affected by maternal intake, with 3BHSD, P450C17, and aromatase being less in moderately fed compared with undernourished or overnourished ewes. In addition, fetal COT seems to be the major sight of estrogen synthesis in the sheep placenta. Lastly, the most dramatic gestational change was for P450C17, which was undetectable at day 135 but elevated dramatically at term. Supported by USDA-NRI grants Grant No. 2003-35206-13621 and 2005-35206-15281 and by NIH grant HD045784.

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Placental Transferrin Receptor Expression with Uterine Space Restriction in Ovine Pregnancy.

Mary Yue Sun, Jason Habeck, Jill Koch, Katie Meyer, Ronald Magness, Pamela Kling 1

Iron transport via the placenta to the fetus is impaired in growth-restricted fetuses, resulting in lower tissue levels of iron. We utilized a sheep uterine space restriction model to study the effects of insufficient uterine space on placental and fetal development. In humans, cell surface transferrin receptor (TfR) is involved in placental iron transport, but it is not known if TfR is similarly involved in sheep pregnancy. Therefore, we hypothesized that placental TfR expression increases in uterine space restriction. To promote uterine space restriction, nonpregnant ewes underwent a surgical severing of the intercorneal vascular connections and then ligating a single uterine horn 2-3 months prior to conception. Singletons, twins, and triplets were studied at 120 or 130 days of gestation (term=145d). Non-space-restricted (NS, Controls) were defined as ligated and nonligated singletons and nonligated twins. Uterine space-restricted (USR) sheep were defined as ligated twins, nonligated triplets, ligated triplets. Therefore NSR had an average of 53.6±2.0, whereas the USR groups averaged 26.9±1.6 placentomes per fetus. Placentomes were either frozen or fixed in formalin, paraffin-blocked, and tissues stained in Gomori Trichrome (collagen I) and Perl's Prussian Blue (hemosiderin iron). Immunohistochemistry slides were stained and Western blots were probed with anti CD-71 TfR. Microscopic placentome structures were visualized. In NSR, trichrome staining was seen mostly in the supporting structures of the blood vessels, but extravascular staining, indicating significant scarring, was observed to a great extent in the USR group. Prussian Blue stain was minimal, except for small areas in the hemophagic zone. On Western blot analysis, ovine TfR mobility was consistent with human TfR. With the immunohistochemistry, TfR was found on the maternal and fetal chorionic epithelia, endometrial glands, vascular endothelium/smooth muscle, and Hofbauer cells. Immunostaining was more intense in the maternal compared to fetal epithelia in NSR, but the staining appeared more evenly distributed in the maternal and fetal compartments in USR. Endothelial, vascular smooth muscle and vascular stroma TfR staining was more evident in USR than NSR. Greater collagen and TfR staining was observed in placentomes from the USR fetuses, compared to NSR. These data support the hypothesis that TfR is involved in the regulation of ovine fetal iron transport. More research is necessary to further investigate the role of TfR in USR. NIH HL49210, HD38843, HL87144

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Adiponectin Regulation of the Immune Modulators, CD24 and Siglec10, in the Human Placenta.

Emily A McDonald, Michael Wayne Wolfe 1

A successful pregnancy is contingent on maternal tolerance of the immunologically foreign fetus. Prevalent diseases such as preeclampsia are thought to be due to an inappropriate immune response at the maternal-fetal interface. The interaction of two immune molecules, CD24 and Siglec10, has recently been described as negatively regulating the inflammatory response induced by intracellular components such as high mobility box 1 (HMGB1), heat-shock protein (Hsp)70, and Hsp90, collectively referred to as danger-associated molecular patterns (DAMPs). We have recently reported that adiponectin, produced by maternal adipose, has negative effects on hormone production by the human placenta. Here we expand the role of adiponectin at the maternal-fetal interface to include that of immune modulation. Immunohistochemistry of sections of term villi revealed abundant expression of CD24 in cytotrophoblasts and syncytiotrophoblasts, while Siglec10 was only expressed by syncytiotrophoblasts. Interestingly, a divergent pattern of expression was observed for CD24 and Siglec10. Primary trophoblasts treated with EGF (an inducer of syncytialization) initially (d4) show increased CD24 and decreased Siglec10 mRNA expression, however the expression patterns reverse as the cells remain in culture (d7). Treatment of cytotrophoblast cells from healthy term placenta with globular adiponectin (2μg/ml) however during the course of spontaneous syncytialization in vitro resulted in a significant increase in both Siglec10 and CD24 mRNA and protein levels, as determined by microarray and western blot analysis. These data suggest adiponectin is capable of overriding the normal cellular control mechanisms regulating CD24/Siglec10 expression. In addition, exposure of term trophoblasts to theCD24-Siglec10 ligand HMGB1, resulted in increased expression of the protein tyrosine phosphatases shp-1 and shp-2. These proteins function within the Siglec10 signaling cascade and suppress responses to inflammation. To examine the role of Siglec10/CD24 in the inflammatory response to DAMPs we utilized a chimeric Siglec10 that forms complexes with CD24/HMGB1 but does not allow intracellular signaling. Treatment of term trophoblasts with HMGB1 in vitro caused an increase in IL-6 mRNA expression by these cells. Addition of the Siglec10 chimeric protein attenuated the ability of HMGB1 to stimulate IL-6 expression in term trophoblasts. Our findings are novel in two respects: 1) this represents the first time the CD24-Siglec10 pathway has been implicated in a trophoblast response to intracellular debris, and 2) these data describe a role for adiponectin in enhancing the CD24-Siglec10 pathway in syncytiotrophoblasts. Thus, CD24-Siglec10 may promote tolerance during pregnancy through reduction of inflammatory responses to cellular debris released upon cell death/lysis. Furthermore, adiponectin may prime the trophoblast cell to respond more efficiently to the presence of these DAMPs by increasing the expression of both CD24 and Siglec10 even in the absence of inflammatory signals.

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Prostaglandin F2-alpha Stimulates Cyclooxygenase-2 Expression and Prostaglandin F2-alpha Synthesis Through NF-kappaB Activation via Reactive Oxygen Species in the Corpus Luteum of Pseudopregnant Rats.

Ken Taniguchi, Fumie Kizuka, Isao Tamura, Norihiro Sugino 1

Prostaglandin F2-alpha (PGF2alpha) is the most important factor that controls regression of the corpus luteum (CL) in various species. The CL has been found to produce PGF2alpha in various species, supporting an important role for intraluteal PGF2alpha in the regulation of luteal function. PGF2alpha is synthesized via cyclooxygenase-2 (COX-2), which is the rate-limiting enzyme in prostaglandin biosynthesis in the CL. Interestingly, PGF2alpha has been reported to induce COX-2 expression in the CL, suggesting the presence of a positive feedback pathway in the CL undergoing functional luteolysis. However, the mechanism by which PGF2alpha induces COX-2 is not fully clarified. The present study was undertaken to investigate whether PGF2alpha increases PGF2alpha synthesis by stimulating COX-2 expression in the CL of pseudopregnant rats. We also investigated the molecular mechanism by which PGF2alpha stimulates COX-2 expression. A luteolytic dose of PGF2alpha (3 mg/kg body) was administered subcutaneously on day 7 of pseudopregnancy. Control rats received phosphate buffer. Each group of rats was autopsied 2 h, 6 h, and 24 h later. Serum progesterone concentrations, expression of COX-2 and COX-1 mRNA and PGF2alpha concentrations in the CL were measured. PGF2alpha significantly increased COX-2 mRNA expression at 2 h and PGF2α concentrations at 24 h, while it had no significant effects on COX-1 mRNA expression. PGFalpha significantly decreased serum progesterone levels compared with the control at any of the times studied. To investigate whether the increase of COX-2 mRNA expression 2 h after PGF2alpha administration is responsible for the increase in PGF2alpha concentrations in the CL at 24 h, a selective COX-2 inhibitor, NS-398 (10 mg/kg body), was administered simultaneously with PGF2alpha. NS-398 completely abolished the increase in PGF2α concentrations in the CL induced by PGF2alpha administration, indicating that PGF2α increased PGF2alpha synthesis by stimulating COX-2 expression. We examined transcriptional factors responsible for the increase in COX-2 mRNA expression induced by PGF2alpha. To examine the involvement of NF-kappaB, NF-kappaB protein expression and NF-kappaB binding activities in the nucleus were analyzed in the CL 30 min after PGF2alpha administration by western blotting and electrophoretic mobility shift assay (EMSA), respectively. PGF2alpha increased NF-kappaB protein expression in the nucleus, and EMSA revealed that PGF2alpha increased binding activities of NF-kappaB to the NF-kappaB consensus sequence of the COX-2 gene promoter. We further examined whether PGF2alpha activates NF-kappaB through the production of reactive oxygen species (ROS). Both superoxide dismutase (SOD), a specific scavenger of superoxide radicals, and catalase, a scavenger of hydrogen peroxide, were administered from tail vain just before PGF2alpha administration. The NF-kappaB protein expression in the nucleus was measured 30 min and COX-2 mRNA levels in the CL were measured 2 h after PGFalpha administration. The increases in NF-kappaB protein levels and COX-2 mRNA expression induced by PGF2alpha administration were significantly inhibited by administration of SOD and catalase, indicating that PGF2alpha activated NF-kappaB through ROS production. In conclusion, the present study showed that PGF2α stimulates COX-2 expression and PGF2alpha synthesis through NF-kappaB activation via ROS in the CL of pseudopregnant rats, suggesting an important role of the PGF2alpha-induced positive feedback pathway in the CL regression of pseudopregnant rats.

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Deciphering the Luteal Transcriptome: Insights into Mechanisms Regulating Bovine Corpus Luteum Regression.

Rina Meidan 1, Mohan Mondal 2, Beau Schilling 2, Heli Buchnik 1, Eyal Klipper 1, Yulia Zalman 1, Joseph K Folger 2, Juan Pedro Stiebel 2, James J Ireland 2, George W Smith 2

In spite of substantial investigation, the mechanisms controlling corpus luteum (CL) life span and the stage-specific luteolytic response to PGF2alpha remain unresolved. The objective of this study was to identify PGF2alpha-induced changes in gene expression profiles in bovine CL after PGF2alpha administration that occur specifically on d 4 (PGF2alpha refractory CL) or on d 11 (PGF2alpha responsive CL) of the estrous cycle and are associated with the luteolytic response to PGF2alpha. CL were collected from cross bred beef heifers (n = 30) at 0, 4 and 24 h following administration of 25 mg PGF2alpha on d 4 and d 11 of the estrous cycle (n = 5 animals per treatment) and isolated RNA labeled and hybridized to Affymetrix GeneChip Bovine Genome Arrays. At 4 h post PGF2alpha injection, a total of 18 (d 4) and 130 (d 11) genes with decreased expression and 204 (d 4) and 532 (d 11) genes with increased expression were revealed. At 24 h post PGF2alpha injection, a total of 178 (d 4) and 579 (d 11) genes with decreased expression and 71 (d4) and 842 (d 11) genes with increased expression were identified. Bioinformatics analyses revealed categories of genes and gene networks/canonical pathways prominently regulated in response to PGF2alpha injection on d11 but not d 4 of the estrous cycle and potential transcriptional mechanisms responsible for differential regulation. Of particular interest were results indicating a potential association of activity of specific transcription factor families (e.g. ETS) with PGF2α-induced gene expression at 4 h post PGF2α administration in d 11 (PGF2α-responsive), but not in d 4 (PGF2alpha-nonresponsive) CL. Upregulation of protein abundance for the predicted ETS-responsive genes NRG1, ATF3, and PTX3 was noted 4 h after PGF2alpha administration specifically in d 11, but not d 4 CL. However, no differences in basal or PGF2alpha-induced expression of mRNA for six ETS family transcription factors in d 4 versus d11 CL were observed. Further, FGF2, a proangiogenic factor, was markedly elevated by PGF2alpha in d 4 CL, at this stage there was also a higher basal expression of cell cycle regulatory molecules (such as CDC25C, CCNA2, CCNB1 and CCNE2) than on d 11. Thrombospondins (THBS1 and THBS2), known to inhibit angiogenesis, and SELE, an adhesion molecule involved in leukocyte recruitment, were induced by PGF2alpha only in the mature, PGF2alpha-responsive CL (d 11). We also noted an inverse relationship between expression of FGF2 and PTX3, which binds and inhibits FGF2 action. As the previous experiment used whole CL tissue consisting of large luteal cells, small luteal cells, and endothelial cells (EC) we next wanted to examine if genes differentially expressed in d 4 vs d 11 CL were confined to a specific cell type. To determine cell-specific expression, RNA was isolated from EC and steroidogenic cells, enriched from the CL using BS-1-coated magnetic beads. While mRNAs for FGF2, PTX3, THBS1, and THBS2 were present in both the steroidogenic and EC compartments of the CL, NRG1 expression was more abundant in the steroidogenic luteal cells. In contrast, expression of mRNA for the adhesion molecules SELE and SELP was highly enriched in luteal EC. Results provide novel insight into PGF2α-induced changes in bovine CL and indicate a stage-specific and cell-specific profile of gene expression that may be functionally associated with the luteolytic response. Supported by BARD (IS 3987-07) to GWS and RM

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Luteinization of Bovine Granulosa Cells for 8 Days Increased Major Histocompatibility Complex (MHC) II Molecules, T-Cell Co-stimulatory Ligands, and Prostaglandin F2 Alpha-induced Activation of T Lymphocytes During Cocultures.

Wenxiang Luo, Milo C Wiltbank 1

As the corpus luteum (CL) develops it acquires luteolytic capacity (bovine CL after Day 7 regress after prostaglandin F2 alpha (PGF) treatment) by mechanism(s) that remain incompletely defined. The process of luteolysis involves in vivo interactions between multiple cell types that may be key to the development of luteolytic capacity. One possible key interaction is between large luteal cells (LLC) and T lymphocytes (TC). Interferon gamma (IFNG) secreted by TC has dramatic effects on luteal cells; however, it is unclear how the TC is activated to produce IFNG in CL. In this study, we hypothesized that LLC act as antigen-presenting cells (APC) to activate TC. We designed 3 experiments to test this hypothesis. Real-time PCR was used to measure mRNA concentrations and Ficoll-Hypaque method was used in isolating Peripheral Blood Mononuclear Cells (PBMC) from bovine blood. Bovine granulosa cells were luteinized for 2, 4, 6, and 8 d (LuGC) to mimic changes in LLC during acquisition of luteolytic capacity. Experiment 1: The LuGC on Day 4 or 8 were cocultured with or without PGF, Staphylococcal enterotoxin B (SEB, to enhance MHCII and TC receptor binding), SEB+PGF, or Concanavalin A (ConA as positive control for TC activation). Expression of IFNG did not occur in response to treatments in cultures of LuGC or PBMC alone (except with ConA in PBMC). However, cocultures of Day 8 LuGC with PBMC had a significant induction of IFNG after treatment with SEB+PGF (569%) or ConA (1187%) but not after other treatments. In contrast, cocultures of Day 4 LuGC did not express IFNG after SEB+PGF but only after ConA. An IFNG-responsive gene, IDO1, was also dramatically induced in Day 8 LuGC+PBMC cocultures treated with SEB+PGF (791%) or ConA (1458%). Thus, LuGC can activate TC but only after luteinization and PGF+SEB treatment. Experiment 2: Changes in MHCI and II were investigated during luteinization of GC. MHCI (BoLA) was not changed during luteinization or in response to PGF (P>0.1). However, MHCII BoLA-DRB3 and BoLA-DQB mRNA increased during luteinization and were greatest in Day 8 LuGC (Day 8>Day 2 by 4-fold for DRB3 and 17-fold for DQB). Further, PGF induced BoLA-DRB3 (191%) and BoLA-DQB (130%) in Day 8 but not Day 2, 4, or 6 LuGC. Thus, activation of TC by LLC may be due to changes in MHCII during luteinization and this may underlie stage-specific activation of TC by LuGC. Experiment 3: Optimal TC responses are facilitated by co-stimulatory ligands coming from APC. The LuGC expressed all 5 of the costimulatory ligands that were measured TNFSF4, TNFSF9, ICOSLG, CD80, and TNFSF14; however, only TNFSF9 increased during luteinization (14-fold) and after PGF (Day 6-326%; Day 8-237%). In addition, genes for degradation of antigen proteins were expressed in LuGC including immuno-proteasome genes PSMB9, PSMB8, and PSMB10, and lysosomal pathway genes CTSS, IFI30, and UBA7; although, there was no effect of luteinization time or PGF on their expression. In conclusion, the coculture system used in these studies elucidated interactions between LuGC and TC that required differentiation of the LuGC for about 8 d to produce an APC-like phenotype (MHCII and TNFSF9 expression). These cocultures demonstrated PGF-induced LuGC-dependent activation of TC (IFNG expression) and subsequent TC regulation of luteal cells (IDO1 induction by IFNG). These intercellular interactions are likely to be critical in luteolysis. Supported by NIH R01 HD050616.

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Microarray Analysis of the Transcriptome in the Primate Corpus Luteum During Chorionic Gonadotropin Administration Simulating Early Pregnancy.

Cecily V Bishop 1, Shannon Satterwhite 1, Liangzhi Xu 2, Jon D Hennebold 1, Richard L Stouffer 1

The primate corpus luteum (CL) becomes desensitized to tonic, pulsatile luteinizing hormone (LH) action in the late luteal phase of the non-gravid cycle. However, the CL responds to the embryo-derived, LH-like molecule chorionic gonadotropin (CG) with extended luteal function. To explore CG-regulated gene expression in the CL, adult female rhesus macaques (n=20) were randomly assigned to a model of simulated early pregnancy (SEP). Human CG (hCG; Novarel) was administered 2x daily in escalating doses of 15 - 2880 IU beginning on Day 9 of the luteal phase and CL were collected on Days 10, 12, 15, and 18 (n=4/group). Additional CL (n=4) were collected on Day 10 of the luteal phase without CG treatment to serve as the "non-gravid" controls. Total RNA was isolated from individual CL, fluorescently labeled and hybridized to Affymetrix GeneChip Rhesus Macaque Genome Arrays at the OHSU Gene Microarray Shared Resource core. Array data were normalized by the robust multichip average (RMA) algorithm, and GeneSifter software (www.genesifter.net) was used to identify changes in transcript expression between Day 10 control and SEP groups. The level of 1192 transcripts changed expression >2-fold (one-way ANOVA, FDR correction; P<0.05) during SEP when compared to Day 10 controls. Pair-wise analysis revealed significant acute affects, with 292 transcripts increasing and 127 transcripts decreasing in expression following one day of hCG treatment. Hierarchical clustering of treatment groups revealed that the majority of changes occurred between Days 10 and 12 of SEP. To compare transcript levels between CG-rescued and regressing CL, previously banked rhesus GeneChip array data (NCBI GEO Series GSE10367) from CL collected during the mid- to late and very late luteal phase were analyzed with time-matched intervals in SEP. Comparing RMA-normalized transcripts from the natural cycle (Group 1, CL days 10, 12, 14-16, and 18-19) with those from luteal rescue (Group 2, hCG-treated samples) revealed 7677 transcripts changing in expression pattern >2-fold (one-way ANOVA, FDR correction; P<0.05) between the two groups. Clustering of samples revealed that the SEP samples possessed the most related transcript expression profiles. Regressed CL (days 18-19, around menses) were the most unlike all other CL. KEGG analysis indicated the most affected pathway was Steroid Biosynthesis; an acute stimulatory response to hCG during SEP was observed for HSD3beta2 and CYP19 mRNA, whereas a chronic stimulatory response occurred for StAR, CYP11A1, CYP17A1, and LHCGR mRNA as measured by real-time PCR. Acute stimulation by CG was noted for PGR, and GCRα mRNA, while acute inhibition by CG was measured for VEGF, CRHBP and 11βHSD2 mRNA. Chronic stimulation was detected for RLN1, CAT, and PGRMC1 mRNA; while message for 11βHSD1 was chronically inhibited during SEP. Transcript levels for the prostaglandin F2α receptor PTGFR, which are up-regulated during luteolysis, were mostly down-regulated by SEP treatment. The most significantly absent pathway (negative z-score) following SEP treatment includes a group of genes whose products promote cell-death. By further comparing the genome-wide changes in luteal gene expression during rescue in SEP, with those in CL during luteolysis in the natural menstrual cycle, it is possible to identify key regulatory pathways promoting fertility. R01 HD20869, RR00163, T32-HD07133 (CVB), China Scholarship Council (CSC; LX).

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Liver X Receptor Modulation of Gene Expression Leading to Pro-Luteolytic Effects in Primate Luteal Cells.

Randy L Bogan, Jon D Hennebold 1

The expression of genes necessary for reverse cholesterol transport (RCT) and cholesterol metabolism is controlled by the liver x receptors (LXR) alpha and beta, members of the nuclear hormone receptor superfamily. Previously, we reported an acute induction of RCT component expression occurs (mRNA and protein) coinciding with the fall in progesterone (P4) synthesis at the end of the luteal phase (i.e., functional regression) in the rhesus macaque corpus luteum (CL). Additionally, we identified three endogenous LXR ligands (22R-hydroxycholesterol and 27-hydroxycholesterol, or 22ROH and 27OH, respectively; and desmosterol) that are present in the CL during the luteal phase. Therefore, LXR regulation of gene expression may be a key determinant of functional regression in the primate CL. To test this hypothesis, we used dispersed primate luteal cell cultures obtained from mid-luteal stage CL (days 7-8 post LH-surge, peak P4 production) to determine whether synthetic and endogenous LXR ligands: 1) increase expression of known LXR target genes, 2) inhibit expression of cholesterol uptake and steroidogenic genes, and 3) increase cholesterol efflux. The well characterized synthetic LXR agonist T0901317 (T09, 1 µM) as well as the endogenous ligands 22ROH, 27OH, and desmosterol (1 or 5 µM, physiologic range) were incubated with luteal cells for 24 hr. To facilitate uptake of sterols, 0.5% (w:v) beta-cyclodextrin (beta-CDX) was added to the culture media. T09 caused significant (p<0.05) increases compared to vehicle in mRNA levels of known LXR target genes including the cholesterol efflux proteins ATP binding cassette subfamily A1 (ABCA1), and G1 (ABCG1), as well as LXRalpha itself (n = 8). T09 also caused a significant decrease in 3beta-hydroxysteroid dehydrogenase (HSD3B2) mRNA compared to vehicle (p<0.05), but had no effect on low and high density lipoprotein receptors, or steroidogenic acute regulatory protein expression. Surprisingly, beta-CDX itself caused a significant (p<0.05) decrease in ABCA1, ABCG1, and LXRalpha expression in vehicle-treated cells. As beta-CDX is known to deplete cells of sterols, this indicates that there is a high degree of basal LXR gene expression controlled by endogenous sterols. LXR target gene responsiveness to the endogenous ligands varied between luteal preparations. When replicates were divided based on initial LXRalpha mRNA levels (as an indicator of endogenous LXR activity), the 4 cultures with the lowest basal LXRalpha expression had significant (p< 0.05) increases in ABCA1 and/or ABCG1 mRNA levels in response to endogenous ligand (5 µM) treatment. The cultures with the highest LXRalpha levels did not respond to endogenous ligands with statistically significant increases in LXR target gene expression, indicating that luteal cells with higher LXR levels may be refractory to further stimulation. Cholesterol efflux assays were used as a functional endpoint of LXR activation. T09 and the 5 µM doses of 22ROH, 27OH and desmosterol significantly (p<0.05, n = 5) increased cholesterol efflux compared to controls. Collectively, these data indicate that: 1) activation of LXR-mediated RCT may inhibit luteal steroidogenesis by increasing cholesterol efflux and decreasing HSD3B2 expression ultimately causing functional regression, and 2) there is a high degree of basal LXR activity in the primate CL that may need to be repressed during the luteal phase to maintain P4 synthesis. This research was supported by NICHD R01-HD42000 and NCRR RR00163.

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Factor(s) Secreted from Bovine Luteal Cells Alter Function of Bovine T Lymphocytes.

Koji Toyokawa, JL Pate 1

The overall goal of this research is to understand the function of the bovine corpus luteum (CL), ultimately leading to improved reproductive efficiency in the dairy industry. The specific research focus is to understand how T lymphocytes (T cells) in the bovine CL are regulated during the estrous cycle. We hypothesized that T cell function could be modified not only by cell-cell contact but also via secreted factor(s) from the CL. There are two major protein secretory pathways, termed conventional and unconventional secretory pathways. Most secreted proteins transit through the ER-Golgi system (conventional secretion), whereas some secreted proteins bypass the traditional secretory pathway (unconventional), and the latter secretory pathways are not inhibited by brefeldin A (BFA). In order to test our hypothesis, we utilized BFA to block secretion of conventionally secreted proteins, which also results in upregulation of proteins secreted by unconventional pathways. In this study, conditioned media from luteal cells alone (LC) and from luteal cells treated with BFA (LC+BFA) were utilized to understand autocrine and/or paracrine interactions between T cells and luteal cells. The primary objectives of this study were to determine T cell responses and cytokine production stimulated by factor(s) secreted from luteal cells. First, the efficacy of BFA in altering protein secretion by luteal cells was examined. Secretion of IL-4, one of the conventionally secreted cytokines, was significantly inhibited in LC+BFA compared to conditioned medium from LC (50% reduction, P<0.01). Secondly, we determined if conditioned medium from luteal cells would mediate T cell responses, and further examined if BFA-restricted secretion would alter function of T cells. Conditioned media from LC and LC+BFA significantly stimulated T cell proliferation compared to media alone with or without BFA (P<0.01). In addition, LC+BFA-conditioned medium stimulated even higher T cell proliferation than LC-conditioned medium alone (30% and 20%, respectively, P<0.01). However, addition of PGF2α to the culture medium had no additive or synergistic effect on T cell responses. Finally, we investigated if factor(s) secreted from LC- and LC+BFA-conditioned media altered cytokine expression in T cells. Gene expression of IL2, IL2R, IFNγ and TGFβ was examined in T cells cultured in LC- or LC+BFA-conditioned medium. Unexpectedly, expression of IL2 was significantly downregulated in T cells cultured in LC- and LC+BFA-conditioned media compared to medium alone (P<0.01), whereas expression of IL2R and IFNγ in T cells was not changed. However, expression of TGFβ (anti-inflammatory cytokine) in T cells was also downregulated in LC- and LC+BFA-conditioned media (P=0.05 and 0.08, respectively). In conclusion, this study provides evidence that autocrine and/or paracrine factor(s) are involved in activation of T cells by luteal cells. Furthermore, it is speculated that different factor(s) may be secreted from luteal cells via unconventional secretions, and factor(s) secreted from luteal cells stimulated by BFA may have more potent stimulatory effects on T cell proliferation. However, the exact molecular mechanisms and identification of luteal-derived factors to modulate T cell functions remain unknown. This project was supported by National Research Initiative Competitive Grant no. 2004-35203-14789 from the USDA Cooperative State Research, Education, and Extension Service to JLP.

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Olfactomedin 1 (Olfm 1) in Fallopian Tube May Modulate Tubal Ectopic Pregnancy in Humans: Evidence from Immunohistochemistry and an In Vitro Coculture Model.

Suranga P Kodithuwakku, William SB Yueng, Pak Chung Ho, Kai Fai Lee 1

Olfactomedins are secretary glycoprotein constituted in the extracellular matrix (ECM) of various cell types. Recent studies suggested that Olfm-1 is down-regulated during the window of implantation (WOI) in the human endometrium and up-regulated in pathological condition like endometriosis and recurrent spontaneous abortions. Ectopic pregnancy is a gynaecological emergency and fertility threatening phenomenon which occurs in 1-2% of normal pregnancies and shows an increasing trend. Yet, tubal ectopic pregnancy accounts for more than 98% of the cases and the distal part of the fallopian tube accounts for 85-95% of all tubal pregnancies. Our unpublished data demonstrated that the expressions of Olfms are steroid hormone-dependent and Olfm-1 affects spheroid attachment onto endometrial epithelial cells in vitro. In the present study, we investigated the spatiotemporal expression of Olfm-1 protein and the transcript of different isoforms (Olfm-1, -2, -3, and -4) in normal Fallopian tubes and tubes with ectopic pregnancies from human samples. Furthermore, an in-vitro-trophoblastic-spheroids and Fallopian-tube-epithelial-cells co-culture model was developed to investigate the possible role of Olfm-1 in tubal ectopic pregnancies using a human Fallopian tube epithelium cell line (OE-E6/E7) and recombinant Olfm-1 protein. Olfm-1 mRNA in the ampullary region showed a significantly lower level (p<0.05) at luteal phase (n=12) than the follicular phase (n=15); whereas there was no significant difference in the infundibullary or isthmic regions between the two phases.The Olfm-1 protein was strongly expressed in the epithelium of the three regions in the human Fallopian tube. The expression of Olfm-1 protein in the infundibulary region is significantly higher (p<0.05) than in the isthmic and ampullary regions in both the follicular (H-SCORE=3.6±0.3 vs 2.5±0.3 vs 2.3±0.7, respectively, n=8) and luteal (H-SCORE=3.5±0.2 vs 2.4±0.5 vs 2.3±0.6, respectively, n=10) phases of the cycle. Interestingly, the ampullary tubal ectopic sections (n=10) showed a significantly lower level of Olfm-1 expression in the epithelium (H-SCORE=1.3±0.2 when compared to normal ampullae (p<0.05). A trophoblastic-spheroids (JAr) and human-Fallopian-tube-epithelial-cell (OE-E6/E7) co-culture system was established. Treatment of OE-E6/E7 with recombinant human Olfm-1 for 24 hrs dose-dependently (0.01-1μg/ml) reduced JAr spheroids attachment to the OE-E6/E7 monolayers when compared to the untreated controls. Furthermore, activation of the Wnt-signalling pathway using Wnt3a or LiCl was associated with reduced Olfm-1 expression in OE-E6/E7 and increased spheroids attachment to OE-E6/E7 cells, suggesting a possible interaction of Olfm-1 with the Wnt-signalling pathway in the tubal ectopic pregnancy. In sum, Olfm-1 in the human Fallopian tube may function to reduce tubal ectopic embryo attachment. Activation of the Wnt-signaling pathway and down-regulation of Olfm-1 expression favour the spheroid/embryo attachment onto human Fallopian tube epithelium. [This project is supported in part by an RCG grant HKU7514/05M to PCH]

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Potential Role of Wnt in Neonatal Uterine Development.

Kanako Hayashi 1, Shin Yoshioka 1, Edmund B Rucker 2, Thomas E Spencer 3, Paul S Cooke 4, Francesco J DeMayo 6, John P Lydon 5, James A MacLean 1

WNT genes encode secreted glycoproteins that control cell fate, mortality, proliferation, differentiation, and tissue growth, and subsets (Wnt4, Wnt5a, and Wnt7a) are involved in Mullerian duct patterning and differentiation during embryonic development of the female reproductive tract. The present study investigated potential role of Wnts during postnatal mouse uterine development, because disruption of endometrial adenogenesis and mesenchymal specification and differentiation can cause permanent fertility problems in the adult. The expression of 19 Wnt genes and 10 Fzd receptors was determined by RT-PCR. The spatial expression patterns of the most highly expressed genes: Wn4, Wnt5a, Wnt7a, Wnt7b, Wnt11, Wnt16, Fzd6 and Fzd10 were further examined by in situ hybridization in the neonatal mouse uterus. Wnt4, Wnt5a and Wnt16 were localized in the endometrial stroma, whereas Wnt7a, Wnt7b and Wnt11 were in the uterine epithelia of neonatal mice. Exposure of mice to estrogen or progesterone during critical development periods inhibits endometrial adenogenesis. Diethylstilbestrol (DES) or progesterone-induced disruption of endometrial gland development was associated with reduction or ablation of Wn4, Wnt5a, Wnt7a, Wnt11, Wnt16 and Fzd10 mRNA during endometrial morphogenesis. Next, we have characterized postnatal uterine morphogenesis by conditionally ablating the expression of Wnt11 in the uterine epithelium of mouse after birth using progesterone receptor (PR) Cre knockin mouse. To increase the efficiency in the production of homozygous null alleles and to decrease the incidence of mosaic deletion, PRcre/+ Wnt11f/- mice were crossed with PRcre/+Wnt11+/- mice to PR+/+Wnt11f/f mice. Wnt11d/d mice did not show disrupted endometrial gland development, and adult Wnt11d/d mice had normal estrus cycles and were fertile. However, we observed disorganized luminal epithelium on postnatal (P) day 7 in Wnt11d/d mice, and levels of Wnt4, Wnt5a, Wnt7a, Wnt7b and Wnt16 in neonatal uteri were decreased in Wnt11d/d mice compared to those in Wnt11f/f mice. Interestingly, ablation of Wnt11 appears to increase the numbers of endometrial glands on P21 and P28. Wnt7a was stimulated on P14 and P21 in uteri of Wnt11f/f mice, whereas Wnt5a and Wnt16 were suppressed by loss of Wnt11. These results implicate that temporal and spatial expression of Wnts are critical factors for endometrial adenogenesis via autocrine and paracrine effects on neonatal uterine development. Further, Wnt11 actions may involve changes in the expression of other members of the Wnt system to regulate uterine morphogenesis. Supported by NIH HD058222.

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Steroidal Regulation of Uterine Macrophage Migration Inhibitory Factor Expression Is Mediated via miRNA-451.

Warren B Nothnick, Caitlin Healy 1

Macrophage migration inhibitory factor (MIF) is a multifunctional cytokine which regulates cell proliferation, angiogenesis and immune cell trafficking. Within the uterus, MIF is expressed primarily by uterine epithelial cells in a cycle stage-dependent fashion. Despite characterization of the pattern of uterine MIF expression in a variety of species, virtually no information exists on MIF regulation. As such, the objective of this study was to examine steroidal regulation of Mif in the mouse uterus. Two-month-old, ovariectomized female mice (N=6/treatment group/time point) were treated with estrogen (E2; 1 μg/kg BW) or E2 + progesterone (P4; 2 mg/kg BW) then sacrificed at 0, 4, 8, and 24h post steroid treatment. Uterine Mif was examined by qRT-PCR, Western blot analysis, and immunohistochemical localization. E2 alone, or plus P4, significantly (P<0.05) increased Mif mRNA expression at all time points. Assessment of Mif protein revealed that in contrast to the effect on transcript expression, E2 significantly decreased Mif protein expression by approximately 35% and 65% at 4h and 8h post steroid treatment, respectively, while at 24h, Mif protein levels were approximately 20% below 0h values. Mif localized primarily to luminal and glandular epithelial cells with low stromal expression. The discordant pattern of expression between transcript and protein suggested to us that uterine Mif expression may be regulated by microRNAs (miRs). miR451 is a putative regulator of MIF expression which we have shown to be regulated by E2 within the uterus in a pattern that is inversely correlated to the pattern of Mif uterine protein expression. To test the hypothesis that miR451 regulates Mif protein expression, we transfected human endometrial epithelial cells (HES), with pre-miR-451 precursor, non-targeting precursor (pre-miR-NT; negative control) or transfection buffer alone and assessed miR451 transcript expression by qPCR while MIF protein expression was assessed by Western analysis. Transfection with pre-miR-451 precursor resulted in a significant increase in HES cell expression of mature miR451 which was associated with a significant reduction in MIF protein expression. To further verify that miR451 targets the MIF 3'UTR, HES cells were co-transfected with a Renilla-MIF 3'UTR vector containing the wild-type miR451 seed sequence or a Renilla-MIF 3'UTR mutant vector (mutant miR451 seed sequence), a control vector containing firefly luciferase, and either pre-miR-451 or pre-miR-NT precursors. HES cells co-transfected with constructs which contained the wild-type 3'UTR and pre-miR-451 exhibited significantly less luciferase activity (Renilla normalized to firefly) compared to cells co-transfected with the mutant 3'UTR and pre-miR-451. Cells co-transfected with the mutant 3'UTR and either the pre-miR-451 or pre-miR-NT precursors did not exhibit a significant change in luciferase activity compared to controls. Collectively, these data are interpreted to suggest that miR451 is capable of binding to the 3'UTR of MIF in vitro and suppressing luciferase reporter activity. These observations, coupled with the MIF Western blot data strongly suggest that MIF expression is regulated by miR451. In summary, we demonstrate for the first time that E2 regulates uterine Mif expression and that this regulation appears to be mediated in part by miR451.

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Estrogen Action Is Required for Expression of MMP-26 in the Secretory Phase of the Menstrual Cycle in Rhesus Macaques.

Camila Contin Diniz de Almeida Francia, Christopher S Keator, Kunie Mah, Lindsay Ohm, Ov D Slayden 1

Matrix metalloproteinase 26 (MMP-26) is a novel matrilysin-like endopeptidase that may play a role during embryo implantation, but our knowledge on MMP-26 regulation remains inadequate. In women, endometrial MMP-26 is up-regulated by progesterone (P) during the early and mid secretory phase. However, MMP-26 expression is refractory to P stimulation in the late secretory phase and the mechanism underlying MMP-26 down regulation is not known. In this study we examined hormone regulation of MMP-26 in ovariectomized artificially cycled rhesus macaques. The samples were obtained from animals assigned to other studies at the Oregon National Primate Research Center. The animals were treated with a controlled estradiol (E2) and P regimen to mimic the menstrual cycle. To stimulate the artificial cycles an E2-releasing capsule was placed subcutaneously (SC) to induce the proliferative phase and after 14 days of E2 priming, a P-releasing capsule was placed SC to induce the secretory phase of the cycle. Removal of the P implant on day 28 completed the cycles and stimulated menstruation on days 2-4 of the next cycle. Endometrium was collected on cycle day 3 (menstruation), days 7 and 14 (proliferative phase), day 17 (early secretory phase; E2 +3 days of P), day 21 (mid secretory phase; E2 +7 days of P), and day 28 (late secretory phase; E2 +14 days of P). In 3 animals, E2 was withdrawn at the onset of the secretory phase and samples were collected after 14 days of P treatment alone. Samples were frozen in liquid nitrogen for RNA isolation and TaqMan Real-time PCR analysis of MMP-26. Similar samples were embedded in Tissue-Tek OCT (Miles Laboratories), frozen in liquid propane, cryosectioned, and subjected to in situ hybridization (ISH) with macaque-specific [35S]-labeled riboprobes to MMP-26. Samples were also fixed in 4% paraformaldehyde, embedded in paraffin and analyzed by immunocytochemistry (ICC) for MMP-26 with goat polyclonal anti-MMP-26 IgG or with monoclonal antibodies to estrogen receptor alpha (ESR1) and progesterone receptor (PGR). Real-time PCR confirmed that MMP-26 transcript was minimal during the mid and late proliferative phase (n=9) and then increased >50 fold (P<0.001; n=4) by the mid secretory phase of the cycle. MMP-26 mRNA levels significantly declined by >10 fold (P<0.001) on day 14 of P treatment. Mean MMP-26 levels further declined to baseline during menses (P<0.01; n=8). ISH revealed that these striking changes in MMP-26 mRNA were localized solely to the endometrial glands. Immunocytochemistry showed that MMP-26 staining was absent in the glandular epithelium during proliferative phase. By day 3 of P treatment (early secretory phase), MMP-26 staining was strong in glands of the functionalis zone. MMP-26 staining was maximal on day 7 of P treatment and then declined in the late secretory phase. At this time MMP-26 was minimal by ICC and ISH in 2 of 3 animals (n=6 total). Secretory phase glands that retained MMP-26 immunoreactivity also retained staining for ESR1 and PGR. Animals with E2 removed at the onset of the secretory phase showed no immunoreactivity for ESR1, PGR, or MMP-26. We conclude that both E2 and P action is required for expression of MMP-26, and that down-regulation of glandular ESR1 by extended P action results in suppression of MMP-26 in the late secretory phase. Supported by NIH grants HD18185 and RR000163 and the NIH Fogarty International Center grant TW/HD-00668 (P. Michael Conn P.I.).

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Anti-Proliferative Effects of Evodiamine and Rutaecarpine on Human Ovarian Cancer Cell Line SKOV3.

Ching-Han Yu 1, Ru-Cui Lin 2, Paulus S Wang 2

Ovarian cancer is the fifth most common cancer among women in the United States, and it causes more deaths than any other type of female gynaecological malignancies. The human HER-2/neu-overexpressing ovarian cancer cells are resistant to chemotherapeutic agent (Taxol). Therefore, there are almost no efficacious therapeutic modalities for malignant ovarian cancer. It is important to develop some new effective therapies. Evodiamine and rutaecarpine are quinazolinocarboline alkaloids extracted from a kind of Chinese herb named Wu-Chu-Yu and has been demonstrated to be effective in preventing the growth of a variety of cancer cells, including downregulating the estrogen receptor of breast cancer. In the present study, the mechanism by which evodiamine and rutaecarpine inhibited the HER-2/neu-overexpressing ovarian cancer cell line SKOV3 was examined. Based on 3-(4,5-dimethylthiazol-2-yle)2,5-diphenyltetrazolium bromide (MTT) assay, cell proliferation rate was reduced dose-dependently by evodiamine and rutaecarpine. According to the flow cytometric analysis, evodiamine treatment resulted in G2/M arrest in SKOV3 cells, but not rutaecarpine. Furthermore, by using the annexin V assay, evodiamine- and rutaecarpine-induced apoptosis was observed at 48 hours and extended to 72 hours. These results suggested that evodiamine and rutaecarpine inhibited the growth and induced the cell apoptosis of the HER-2/neu-overexpressing ovarian cancer cells, SKOV3, via arrested them at G2/M phase or through other signaling pathway.

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Production of Aldosterone and Corticosterone in Polycystic Ovary Syndrome (PCOS) Rats.

Ru-Lian Hsu 1, Chi-Hong Ho 2, Chia-Jung Chan 1, Han-Wei Lin 1, Cai-Yun Jian 1, Yu-Chen Chang 1, Paulus S Wang 1

Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in women, which is characterized by hyperandrogenism, anovulation, and susceptibility to the metabolic syndrome. Altered levels of serum aldosterone and cortisol have been reported in human PCOS. However, the mechanisms of production of adrenocortical steroid hormones in PCOS are still unclear. The aim of this study was to investigate the cellular mechanisms of the release of adrenocortical steroid hormones in PCOS rats. PCOS model in female rats had been established in this study. Prepubertal female rats were randomly divided into three groups treated daily with sesame oil (1 ml per kg body weight, group 1), testosterone propionate (TP, 10 mg per kg body weight, i.p., group 2), and dihydotestosterone (DHT, 10 mg per kg body weight, i.p., group 3). Vaginal smears of both TP and DHT groups showed continuous diestrous phase rather than normal 4~5-day estrous cycle in the control group. The body weight of DHT treated group was significantly higher and the ratio of parametrical fat weight to total body weight was also higher in DHT groups than in the other groups. The adrenocortical cells were isolated and separated into zona glomerulosa (ZG) cells and zona fasciculata-reticularis (ZFR) cells. ZG cells were challenged with or without agiotensin II (Ang II) and adrenocorticotropin (ACTH). The release of aldosterone was reduced in TP-treated group with or without Ang II and ACTH, but increased in DHT-treated group after ACTH stimulation. ZFR cells were stimulated by ACTH, forskolin (adenylyl cyclase activator) and 8-bromo-adenosine cyclic monophosphate (8-Br-cAMP, permeable cAMP analogue). The release of corticosterone was increased in DHT-treated group following stimulation, but decreased in TP-treated group without stimulation. These results suggested that PCOS induced by DHT in female rats increased the production of aldosterone and corticosterone at least in part via a mechanism associated with cAMP-pathway.

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

WITHDRAWN.

WITHDRAWN. (Presentation replaced with Abstract 489.)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

PABP Interacting Protein 2 (Paip2) Regulates the Translation of Key Proteins Involved inSpermiogenesis.

Geraldine Delbes, Akiko Yanagiya, Nahum Sonenberg, Bernard Robaire 1

During spermiogenesis, several proteins that are needed for the proper male germ cell differentiation are under translational control. Such proteins are expressed at very specific steps in elongating spermatids. We have recently shown that Poly(A) Binding Protein (PABP) interacting protein 2 (Paip2) is a major translational regulator involved in the maturation of male germ cellsand male fertility. Paip2A is highly expressed in the testis; it is present only in the cytoplasm of spermatids from steps 7-8 of spermiogenesis until the sperm are released. In order to identify which protein are under translational control of Paip2 regulation, we characterized the proteomic profiles of elongating spermatids from wild type (WT) mice and double knock-out (DKO) transgenic mice in which both Paip2A and Pai2B genes were abrogated. Populations containing elongating and elongated spermatids were obtained after separation on a BSA gradient with a minimum purity of 90%. Proteins were extracted and loaded on a 7% to 15% gradient acrylamide gels. After electrophoresis, the gels were cut into 15 bands and proteins were digested with trypsin. The peptides obtained were identified by mass spectrometry and analyzed using Scaffold 2.0 software.We generated a list of 632 proteins for which we could identify at least two peptides and for which we have 95% confidence. 286 proteins were consistently detected in the three WT replicates and 289 proteins in the three DKO replicates. Of these consistently detected proteins, 9 and 13 proteins were never detected in the DKO and WT, respectively. Moreover, 17 proteins present in both samples showed a significant difference with at least a 1.5 fold change in the DKO compared to the WT; 4 proteins of them were down-regulated in the DKO and 13 proteins were up-regulated. Overall, 39 proteins were differentially expressed in the DKO when compared to the WT. These proteins are involved in various cellular functions such as translation (60S ribosomal protein L13,eukaryotic translation initiation factor 4H and 4G1, elongation factor 2), metabolism (hexokinase1,hydroxyacyl-coenzyme A dehydrogenase, protein kinase C substrate 80K-H, pyridoxal-dependent decarboxylase domain-containing protein 1) and proteolysis (dipeptidylpeptidase 3, dipeptidase 3, ubiquinol cytochrome c reductase core protein 2, proteasome 26S subunit, non ATPase (PSDM1)). Interestingly, all of the proteins involved in proteolysis were down regulated in DKO; this is consistent with the abnormal maintenance of expression of PABP in late spermiogenesis. Moreover, the PSDM1 has been suggested to be present in the perinuclear-arranged manchette of rat sperm and to be responsible for the degradation of testis-specific histone in the process of chromatin packaging. This protein cannot be detected in the DKO, in which the chromatin of elongated spermatids shows abnormal vacuolization and bubble-like structures, suggesting low compaction level. Further analysis of such candidate protein will provide new insight into chromatin compaction mechanism. Supported by CIHR.

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Sperm Messenger RNA Isolation from the Whole Sperm (Live and Dead) Population in Frozen Bull Semen Straws.

Christopher Card, Becky L Sartini 1

Transcriptionally-inert spermatozoa contain a pool of messenger RNA (mRNA) that is a remnant from untranslated transcripts during spermatogenesis. Several studies have reported specific sperm mRNA that are correlated with fertility in humans and bulls, although the function of these transcripts is unknown. Development of a fertility assay based on sperm mRNA has been hindered by a lack of isolation procedures that yield enough mRNA for further analysis. Current protocols outline methods to isolate sperm mRNA from live sperm only, although assaying the total live and dead sperm population in frozen bull semen straws would more accurately represent inseminated populations and could be correlated with other fertility assays, including % DNA fragmentation, that utilize whole semen straws. We have developed a sperm mRNA isolation procedure, with no contaminating mRNA, that yields approximately 5 μg of total RNA from two bull semen straws. Sperm from two straws (n = 3 bulls), approximately 8-10 million cells, were thawed at 37°C, pooled and washed twice with (4 ml) sperm-TALP (10 min each at 600 x g). After removal of the sperm-TALP from the sperm pellet, total RNA was isolated using the RNeasy isolation procedure (Qiagen; Valencia, CA) according to manufacturer's protocol with the following modifications: 1) time of sperm lysing was extended to 10 min, 2) sperm were homogenized through a 26-gauge needle three times, 3) RNase-free water was heated to 37.5°C then pipetted onto isolation column, and 4) after an 8-min incubation and centrifugation, elution water was reapplied to isolation column for a second elution. The total RNA isolated using this method routinely has an A260/A280 of 1.84 (Nanodrop; Wilmington, DE) indicating a relatively pure RNA isolation. To expand the mRNA population and increase yields, the total sperm RNA was amplified linearly using SMART mRNA Amplification (Clontech; Mountain View, CA). Bioanalyzer analysis (Agilent; Santa Clara, CA) of the amplified sperm mRNA indicated an abundance of shorter length transcripts that is consistent with reports from other sperm isolation procedures. This amplified sperm mRNA population was not contaminated with mRNA from testicular germ cells or leukocytes as intron spanning primers to c-kit (germ cells) and CD45 (leukocytes) did not amplify these transcripts. Protamine 1 was amplified in the sperm mRNA using intron spanning primers, also indicating lack of genomic DNA contamination, and is consistent with previous experiments that reported protamine 1 in human and bull sperm mRNA from live sperm. These results demonstrate that sperm mRNA can be isolated from the whole (live and dead) sperm population from two bull semen straws and should be further tested in function and fertility experiments.

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Comparative Changes on Stem Cell Potential of Bovine Male Germ Cells During Testicular Development from Neonatal to Adult.

Mayako Fujihara, Sung Min Kim, Naojiro Minami, Masayasu Yamada, Hiroshi Imai 1

The initial step of spermatogenesis is the transition from male primitive germ cells (gonocytes) to undifferentiated spermatogonia in the neonatal testis. Undifferentiated spermatogonia have a potential to self-renew and support spermatogenesis thorough a life time. In vitro culture system for these male germ cells is valuable for preservation and improvement of genetic resources especially in domestic species, and for understanding the mechanisms of mammalian spermatogenesis. However, a long term culture of germ cells in domestic animals has not been established and the stem-cell potential of these germ cells remains largely unknown. In this study, we have characterized bovine germ cells in developing testes from the neonatal stage to adult, and analyzed their stem-cell potential in in vivo and in vitro. The testes were collected from 2-day to 5.5-month-old calves (Bos taurus) and 2- to 12-year-old adult bulls to characterize the expression pattern in germ cells during spermatogenesis and to culture germ cells. The part of testis tissues was fixed with Bouin solution and stained with germ-cell markers, a lectin Dolichos biflorus agglutinin (DBA), ubiquitin C-terminal hydrolase 1 (UCHL1) and VASA, and stem-cell markers, NANOG and OCT3/4 (POU5F1). The remaining testis was digested by 2-step enzyme treatments, and gonocytes or undifferentiated spermatogonia were isolated from the neonatal testis or the adult testis by Parcoll density gradients. Collected cells were cultured in vitro in Dulbecco Modified Eagle Medium/F12 (DMEM/F12) supplemented with 10% FBS. Culture media were changed every other day and cells were passaged every 5 to 7 days using 0.25% trypsin-1 mM EDTA. Cultured cells were characterized by immunohistochemical analysis and Western blotting analysis using germ-cell and stem-cell markers. The pluripotencial ability of cultured cells was examined by transplantation assay using immunodeficient mice. The immunohistochemical analysis indicated that the expression of mouse germ-cell markers VASA along with DBA and UCHL1 restricted on gonocytes in the neonatal testis. Gonocytes also had stem-cell characteristics as evaluated by NANOG and OCT3/4 expressions. These expressions were continuously seen on germ cells at the age of 5.5 month, when gonocytes migrate from the center to the basal part of the tubules and differentiate to spermatogonia. In adult bovine testis, however, although DBA and UCHL1 expressions were detected only in undifferentiated spermatogonia, VASA, NANOG and OCT3/4 expressions were mainly in differentiating spermatic cells, such as spermatcytes and round spermatids, and rarely in undifferentiated spermatogonia. We subsequently utilized these markers to characterize gonocytes and spermatogonia after cultivation in vitro. Undifferentiated spermatogonia, which were collected from the adult testis, formed colonies in vitro, but could not passage thereafter. On the other hands, gonocytes from the neonatal testis could proliferate and form colonies at every passage for 1.5 months in culture, retaining undifferentiated states of gonocytes as confirmed by the expressions of germ-cell and stem-cell markers. The transplantation assay confirmed that long-term cultured cells had colonizing ability in the recipient testis, indicating that they could maintain stem-cell activity in vitro culture condition. Our findings provide that gonocytes could be a feasible target for establishing germ-cell lines in cattle.

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

A Multiclass Support Vector Machine Model for the Identification of Sperm Motility Patterns in Heterogeneous Populations.

Summer G Goodson 1, Zhaojun Zhang 2, James K Tsuruta 1, Wei Wang 2, Deborah A O'Brien 1

Sperm motility and the capacity to undergo hyperactivation in the female reproductive tract are requirements for normal fertilization in mammals. Quantitative analyses of motility in response to perturbations that modulate sperm metabolism or key signaling events would be facilitated by a method that quickly and accurately distinguishes different types of motility in large populations of sperm. To that end, we generated a multiclass support vector machine (SVM) model that identifies hyperactivated sperm and four other distinct patterns of sperm motility within a heterogeneous population. Our multiclass model is based on 2,434 sperm tracks from twelve CD1 mice that were captured using a Hamilton-Thorne CEROS instrument for computer-assisted sperm analysis (CASA) and visually classified into one of six distinct motility groups: progressive, intermediate, hyperactivated, slow, weakly motile, and bad (noise). The CASA parameters from these classified tracks were used to develop a series of SVM equations using established algorithms, yielding a model that identifies uncharacterized tracks with an overall accuracy of 86%. The model incorporates these equations into a binary decision tree that sequentially sorts tracks into distinct populations. Five standard CASA measurements are factored into each equation, including average path velocity (VAP), straight-line velocity (VSL), curvilinear velocity (VCL), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF). The first equation identifies all CASA tracks as either vigorous or non-vigorous. Vigorous tracks are then classified as progressive, intermediate or hyperactivated, while non-vigorous tracks are classified as slow, weakly motile, or bad. We tested our multiclass model by comparing motility profiles of sperm incubated for 2 h in HTF medium with and without bicarbonate. After capacitation for 90 min in the presence of bicarbonate, 20-30% of sperm tracks were classified as hyperactivated and ~10% as intermediate. Without bicarbonate, >65% of sperm tracks remained progressive throughout the incubation and <2% were classified as hyperactivated or intermediate, as expected. The model also accurately categorized tracks from mice with defects in sperm motility. For example, sperm from mice that lack GAPDHS, an essential sperm glycolytic isozyme, showed no progressive motility and >96% were classified as slow or weakly motile. We also assessed the motility profiles of C57BL6/J, 129S1/SvlmJ, and PWK/PhJ sperm over a 2 h in vitro capacitation period and found significant differences among these strains, both in the proportions of progressive and slow sperm and in the percentage of sperm that undergo hyperactivation. By enabling detailed, quantitative comparisons of motility throughout capacitation, this model will be a valuable tool for assessing potential genetic, biomolecular, and pharmaceutical effects on sperm motility in large, heterogeneous populations. Supported by the Eunice Kennedy Shriver NICHD/NIH through U01 HD60481 and cooperative agreement U54 HD35041 as part of the Specialized Cooperative Centers Program in Reproduction and Infertility Research.

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Characterization of the Spermatogonial Stem Cell Niche in the Seminiferous Tubule of the Mouse.

Kyle C Caires, Derek J McLean 1

A balance between self-renewal and differentiation of spermatogonial stem cells (SSCs) is required to maintain sperm production throughout adult life in males. The mature seminiferous epithelium is organized into small clusters (or stages of spermatogenesis) based on the presence and precise complement of germ cells types within a tubular section of the testis. These clusters exist in close physical proximity, but foster diverse phases of germ cell development despite exposure to a similar endocrine milieu. Thus, the seminiferous tubule microenvironment is under strict local regulation by Sertoli cells to provide nutrients and extracellular cues for coordinated spermatogenesis and SSC self-renewal to occur. Currently, little is known regarding the seminiferous tubule distribution and location of SSCs in the testis. We hypothesize that SSC populations are randomly distributed across the mature seminiferous epithelium in the mouse testis. Therefore, this study was designed to isolate and characterize specific regions of the mature seminiferous tubules to determine if a microenvironment enriched for SSCs is associated with particular stages or regions of the seminiferous epithelium in the testis. To achieve this objective, we used gene expression analysis of SSC niche associated factors and stem cell transplantation of germ cells from specific stages. We isolated clusters of seminiferous tubules using transillumination-assisted microdissection to obtain segments of tubules that represent areas of high responsiveness to FSH (IX-I), androgen (II-IV) and retinoid (V-VIII) signaling. Tubule segments from all stages (I-XII) in normal and busulfan-treated (germ cell depleted) mice were also collected as controls. To confirm the accuracy of tubule dissection (n≥3) we assayed the expression of fshr, ar and stra8 mRNA transcripts as stage-specific biomarkers in isolated cell preps using quantitative real time RT-PCR, in addition to morphological evaluation. The expression of bcl6b was considerably enriched (p<0.01) in stages IX-I when compared to other tubule clusters. Gdnf and ret expression was highest (p<0.05) in stages IX-IV and lowest in V-VIII. No stage-specific differences in the expression levels of gfrα1 or ngn3 were detected. When compared to normal adult mice (I-XII), busulfan-treatment diminished (3-fold or greater) the expression of bcl6b, gfrα1 and ngn3; but enriched (6-fold or greater) the expression of Sertoli cell transcripts. Stem cell transplantation analyses to functionally determine the relative abundance and tissue localization of SSC populations in the testis are ongoing. Investigation of factors produced by the niche microenvironment that regulate SSC homeostasis and novel approaches to obtain a pure SSC cell preparation will be the focus of future experiments.

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Differentiation of Stem/Progenitor Spermatogonia into Prostatic Epithelium: Direct or Indirect?

Paul S Cooke, Liz Simon, Gail C Ekman, Marie-Claude Hofmann 1

The urogenital sinus (UGS), which forms prostate in males, consists of urogenital sinus mesenchyme (UGM), which gives rise to prostatic stromal cells, and urogenital sinus epithelium (UGE), which forms prostatic epithelium. UGM is an instructive inducer, and can induce UGE or other tissues (e.g., bladder epithelium) to become prostatic epithelium. We have demonstrated that when stem/progenitor spermatogonia were recombined with UGM and grown as tissue recombinants in vivo, they differentiated into functional prostatic epithelium. The critical question now is to understand how spermatogonia differentiate into different cell types. This could involve dedifferentiation of stem/progenitor spermatogonia into embryonic stem cell-like cells and then into prostatic epithelium. Conversely, since spermatogonial cells are relatively undifferentiated (multipotent), a direct differentiation of spermatogonia into prostatic stem cells and/or prostatic epithelium may occur under the instructive influence of the UGM. UGS obtained from fetuses at day 16.5 of gestation were trypsinized and separated into UGM and UGE. Stem/progenitor spermatogonia were isolated from 5- to 6-day-old male pups and recombined with UGM on agar plates (1% agar in DMEM with fetal bovine serum). Twenty four hours later, the tissue recombinants were grafted under the renal capsule of adult syngeneic male mice and harvested at day 2, 4 and 8 of grafting. To track cell lineages, transgenic mice expressing green fluorescent protein ubiquitously were used. NKX3.1 is a transcription factor expressed early in prostatic development that drives prostatic epithelial morphogenesis; NKX3.1 was used as the primary indicator of prostatic development. Our results indicated that NKX3.1 was expressed within the first week of grafting and in some grafts as early as 4 days, although mRNA expression of NKX3.1 was not detected after 24 h of tissue recombination or 2 days after grafting. Some tissue recombinant grafts developed histologically identifiable prostatic epithelium by day 8. mRNA expression for pluripotency markers (SSEA-1, Oct3/4, Nanog and Sox2) was not detected in the grafts at any time points tested. mRNA expression of p63, a marker of basal prostatic cells, was detected by day 4 in tissue recombinants. Basal cells are prostatic stem cells and p63 is required for prostatic development. Thus, these results are consistent with the hypothesis that stem/progenitor spermatogonia are directly differentiating into prostatic epithelial cells expressing NKX3.1 and p63 without transiting through an embryonic stem cell-like stage and expressing pluripotency markers. Supported by The Billie A. Field Endowment, University of Illinois (PSC).

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Direct Evidence That GDNF Regulates Numbers of Undifferentiated Spermatogonia in Normal, Mature Testes.

Dolly M Singh 1, Joseph M Savitt 2, Liang-Chin Chen 1, William W Wright 1

Male fertility requires a careful balance between replication of stem spermatogonia that creates more stem cells and replication that creates A paired (Apr) and, subsequently, A aligned (Aal) spermatogonia. It has been hypothesized that in normal, mature males, this balance is regulated by the inhibition of stem cell differentiation by the Sertoli cell secretory product, glial cell-line derived neurotrophic factor (GDNF). However, to date, it has been impossible to directly test this hypothesis because of a lack of experimental models by which to specifically manipulate GDNF signaling. We now describe a chemical-genetic approach that allows us to directly test this hypothesis. This approach uses mice in which valine 805 in the ATP binding site of Ret, the tyrosine kinase subunit of the GDNF receptor, is replaced by alanine. While this replacement has no detectable effect on Ret function, it substantially increases the affinity of Ret for the ATP antagonist, NaPP1-HCl. To determine if GDNF is an acute regulator of numbers of stem spermatogonia, mice homozygous for Ret (V805 to A) were injected for 11 or 22 days with NaPP1-HCl or with vehicle (n=4 or 5 per group). We then measured the densities on whole mounts of seminiferous tubules of cells that had two fundamental morphological characteristics of stem spermatogonia, they were A single (As) spermatogonia that expressed the transcription factor, PLZF. To evaluate the effects of inhibiting GDNF signaling on the differentiation of stem spermatogonia, we also measured the densities of PLZF+ Apr and Aal spermatogonia. Eleven days of inhibition of GDNF signaling had a significant effect on As, Apr and Aal spermatogonia; their densities were reduced by 88%, 81%, and 94%, respectively as compared to densities in vehicle-treated mice. Additionally, consistent with the hypothesis that GDNF inhibits differentiation of stem spermatogonia, the ratio of Apr to As spermatogonia was significantly higher in NaPP1-HCl-treated mice. In contrast, treatment of wild-type mice with NaPP1-HCl mice had no effect. Injection of vehicle for 22 days had no effect on the densities of PLZF+ spermatogonia. However, PLZF+ As, Apr and Aal spermatogonia were not found on seminiferous tubules obtained from any of the 5 mice injected for 22 days with NaPP1-HCl. To determine if this result reflected a loss of cells or of a specific loss of expression of PLZF, we compared Ret mRNA/18S rRNA in testes of mice treated for 22 days with vehicle or with NaPP1-HCl. Results showed that inhibition of GDNF signaling produced an 80% decrease in testicular Ret mRNA levels. Finally, we examined the densities of spermatogonia that express the ligand binding subunit for GDNF, Gfralpha1. Densities of those cells were reduced by approximately 50% in treated mice. In summary, inhibition of GDNF signaling in mice homozygous for Ret (V805 to A) leads to a rapid loss of PLZF+ As spermatogonia and their immediate progeny. This marked decrease is mirrored in the decrease in Ret mRNA expression. However, the fact that GFRalpha1+ spermatogonia are still found, albeit in reduced numbers, after 22 days of inhibition of GDNF signaling supports the conclusion that this inhibition causes loss with different time courses of different genes required for "stemness" and subsequently to depletion of As spermatogonia and the stem cells within this pool of cells. Supported by U54 HD055740-01.

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Sertoli Cells Dictate Stem Cell Niche Number Within Mammalian Testes.

Karen E Racicot, Melissa J Oatley, Jon M Oatley 1

Interaction of stem cells with specific niche microenvironments that support their self-renewal and differentiation is essential for tissue homeostasis. Impaired and aged niches lead to stem cell failure and degenerative disease; whereas, niches within tissues of young individuals can rejuvenate stem cells from aged and degenerative tissues. These specialized microenvironments are comprised of growth factors and microarchitecture provided by so-called niche support cells. In mammalian testes, self-renewal and differentiation of spermatogonial stem cells (SSCs) is supported by a niche microenvironment to confer long-term fertility in males. Many somatic cell populations are known to aid in germ cell development including Sertoli, Leydig, myoid, and immune cells. Yet, the key support cell population that orchestrates SSC niche formation has not been unequivocally identified. In this study we tested the hypothesis that Sertoli cells dictate formation of SSC niches within mammalian testes. We devised an experimental strategy to test directly whether the number of Sertoli cells is linked to the number of SSCs and accessible niches within mouse testes. Sertoli cell numbers were artificially increased in male mice by inducing transient hypothyroidism during neonatal development with propylthiouracil (PTU) treatment. In agreement with previous studies, this treatment resulted in an increase of both testis weight and Sertoli cell numbers by 40% at adulthood compared to non-treated control mice. Next we determined if an increase in Sertoli cell numbers corresponded with increased numbers of SSCs. The SSC-containing THY1+ cell fraction from LacZ-expressing ROSA donor mice treated with PTU was transplanted into testes of immunologically compatible recipients. The number of THY1+ cells recovered from PTU-treated males was 90% greater than controls. Examination of recipient testes two months after transplantation revealed that the number of colonies of donor-derived spermatogenesis generated by THY1+ cells from PTU-treated donors was significantly greater by 2-fold compared to non-treated control donors. These results indicate that increased numbers of Sertoli cells cause an increase in the number of SSCs. Next, to determine if an increase in Sertoli cell numbers correspond to increased numbers of niches, PTU-treated mice were prepared as recipients for transplantation of SSCs from normal ROSA donors. Colonization analysis revealed that testes of PTU-treated recipients contained greater than 3-fold more colonies compared to non-treated control recipient males indicating that Sertoli cell numbers dictate the number of accessible SSC niches within testes of adult mice. Finally, we examined whether an increase in numbers of Sertoli cells is associated with increased vascularization of testes. Examination of cross-sections from PTU-treated mice revealed a significant increase of 80% in the number of blood vessels compared to non-treated control mice. In comparison, blood vessel number in cross-sections of liver from PTU treated mice was not different from controls, indicating that increased vascularization of testes was not a result of enhanced angiogenesis of organs caused by inducing hypothyroidism during neonatal development. Collectively, these results are the first to provide direct evidence that Sertoli cells are the main orchestrating support cell population for formation of SSC niches within mammalian testes.

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Inducible Transgene Expression in a Cloned Dog Using Tetracycline Vector System.

Min Jung Kim 1, Hyun Ju Oh 1, Jung Eun Park 1, So Gun Hong 1, Ji Eun Kim 1, Goo Jang 1, Mo Sun Gwon 2, Bon Chul Koo 2, Teoan Kim 2, Byeong Chun Lee 1

Controllable transgenic expression systems in transgenic animal model are valuable to the development of therapeutic approaches in human medical fields. The aim of this study was to 1) produce a transgenic cloned dog using inducible tetracycline vector system, and 2) investigate whether the transgenic cloned dog could be induced the transgene expression using doxycycline (Doxy). Canine fetal fibroblasts were infected with retroviral vectors designed to express the enhanced green fluorescent protein (eGFP) gene under the control of tetracycline-inducible promoter. For somatic cell nuclear transfer (SCNT), nucleus of an in vivo matured oocyte was removed and an eGFP expressed cell cultured with 1 μg/ml of Doxy was injected. After electrical fusion and chemical activation, the reconstructed embryos were transferred to a recipient and pregnancy diagnosis was performed by ultrasonography. Experiment I evaluated the mean fluorescence intensity (MFI) of infected cells while the cells were cultured in the presence of 1 μg/ml of Doxy for 5 days, and then in the absence of Doxy for 7 days using fluorescence-activated cell sorter. Experiment II was designed to produce an eGFP controllable transgenic cloned dog via SCNT. For verification of transgenic dog, experiment III was performed Southern Blot analysis and observation in vivo regulation of eGFP expression in the cloned dog treated with 100 mg/kg of Doxy every 2 days for 2 weeks under ultraviolet light. In experiment IV, western blot was used to detect eGFP increase and decrease in skin tissues of transgenic dog under the presence or absence of Doxy. In the results of experiment I, the MFI for infected cells was rapidly increased to approximately 42.3 times after 3 day-treatment compared to pre-treatment and quickly decreased 3 days after ceasing the treatment. In experiment II, a total of 203 embryos were transferred to nine recipients and three pregnant delivered three pups (GT0, GT1, and GT2) by C-sec and GT2 among them is still alive. All cloned pups were genetically identical to the donor cell. GT2 showed an apparent in vivo eGFP expression on her body after Doxy administration in experiment III. The result of Southern blotting showed that the transgene insertion was detected from the three cloned dogs and all organs of GT1. Experiment IV indicated that a robust eGFP expression in skin tissue of GT2 rapidly increased after Doxy treatment and gradually decreased to basal level on 9 weeks after ceasing the treatment. In conclusion, we report here for the first time an inducible transgenic system in canine species and it can stably induce the transgene expression at intended time. This study has demonstrated the capacity to generate transgenic model dog which could regulate the transgene and it would contribute to human medical research fields. This study was supported by Korean MEST through KOSEF (grant # M10625030005-09N250300510) and BK21 program, RNL BIO, and Natural Balance Korea.

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Embryonic and Induced Pluripotent Stem Cells Analogous to Inner Cell Mass-Derived LIF-Dependent Mouse Embryonic Stem Cells Established from the Domestic Pig, Sus scrofa.

Bhanu Prakash VL Telugu, Toshihiko Ezashi, Andrei P Alexenko, Lee Spate, Randall S Prather, R Michael Roberts 1

Authentic embryonic stem cells (ESC) may never have been successfully derived from the inner cell mass (ICM) of pig and other ungulates, despite over 25 years of effort. Recently, porcine induced pluripotent stem cells (piPSC) were generated by reprogramming somatic cells with a combination of four factors OCT4, SOX2, KLF4 and c-MYC (OSKM) delivered by lentiviral transduction. The piPSC resembled FGF2-dependent human (h) ESC and are likely to advance swine as a model in biomedical research, since grafts could be matched to the animal that donated the cells for re-programming. The piPSC also share similarities with murine ‘epiblast stem cells’ developed from advanced epiblast, which like hESC are characterized by their flattened morphology, dependence on FGF2 and TGF-beta/Activin/Nodal signaling for maintenance of their pluripotency, inactivation of one of the X chromosomes in female cell lines, intolerance to passage as single cells, and lack of competency for producing germ-line chimeras. Indeed, two emerging concepts are, first, that the FGF- and LIF-dependent ESC are not limited to rodents, and, second, that only LIF-dependent lines are competent to contribute to germ-line chimeras and are, therefore, intrinsically valuable for genetic modification. Recent successes in the establishment of LIF-dependent ESC and iPSC from hitherto "difficult" species such as rat, and non-permissive mouse strains such as NOD, suggested possible means for establishing analogous lines in pig and other domestic ungulates. The underlying principle behind such approaches is either to augment the endogenous levels of c-MYC and/or KLF4 ectopically or to supplement the culture medium with kinase inhibitors to influence endogenous signaling pathways. Of the latter, one compound CHIR99021 (CH) inhibits GSK3B and activates WNT signaling, thereby substituting for c-MYC, while a a second, Kenpaullone (KP), inhibits both GSK3B and CDK1 and replaces KLF4 function. The objective here has been to (1) to develop LIF-dependent porcine ESC (pESC) by over-expression of KLF4 in dissociated pig ICM and subsequently culturing the transfected cells in the presence of KP/CH medium, and (2) generate LIF-dependent piPSC by reprogramming somatic cells derived from umbilical cord mesenchyme with OSKM factors and also culturing the transfected cells in presence of the same inhibitors. The lentiviral vectors employed for introducing the re-programming genes were modified for doxycycline-mediated induction of expression, and floxed for Cre-mediated recombination and removal of transgenes following complete reprogramming. Two LIF-dependent cell lines have been derived from the ICM cells of late d 5 in vitro-produced blastocysts and four from umbilical cord mesenchyme recovered from fetuses at d 35 of pregnancy. The derived piPSC had been completely reprogrammed as evidenced by activation of their endogenous pluripotency genes and do not require doxycycline to maintain their undifferentiated phenotype. The LIF-dependent pESC and piPSC express markers consistent with pluripotency, are alkaline phosphatase positive and bear a striking resemblance to ICM-derived mESC in colony morphology, culture characteristics, and short cell cycle time. Currently, the transcriptome of the pESC and piPSC is being analyzed by microarray, and the ability of these cells to give rise to teratomas and chimeras is under investigation. Supported by MO Life Sciences Board Grant 00022147 and NIH grant HD21896.

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Identification of an Invasive Cell Population Within BMP4-Treated Human Embryonic Stem Cell Colonies.

Jessa Schlitt, Bhanu Telugu, R Michael Roberts, Laura Schulz 1

In humans, the trophectoderm forms the outer epithelial layer of the blastocyst and mediates attachment and subsequent penetration of the embryo into the maternal uterine endometrium. These trophectoderm cells give rise to the more advanced and specialized cell types of the placenta cytotrophoblast, syncytiotrophoblast, and extravillous trophoblast (EVT). Defective trophoblast (TR) invasion and development are one of the leading causes of infertility and many pregnancy disorders like preeclampsia. Frustratingly, studies of normal and pathological human TR development have been hindered by the lack of an appropriate model. However, human embryonic stem cells (hESC), when treated with BMP4 in the absence of FGF2, differentiate exclusively into TR cells, and this differentiation is promoted by a high oxygen (20%) environment. The differentiated cells exhibit a cobblestone morphology and express markers characteristic of cytotrophoblast and syncytiotrophoblast. Our goal in this study is to determine whether BMP4-treated hESC colonies (BMP4-hESC) also contain EVT, the invasive component of TR. To achieve this, we have conducted Matrigel invasion assays three times each on two hESC lines (H1 and H9) by using a 2x2 factorial design with either BMP4, which promotes differentiation, or FGF2, which sustains pluripotency and inhibits differentiation, in the presence of either ambient (20%) or low (4%) oxygen conditions. BMP4 treatment resulted in a ten-fold increase (p=0.0008, p=0.0003) in the size of the invaded cell population compared to FGF2. The number of invaded cells was increased 3-fold (p=0.03) when the hESC were maintained in high versus low oxygen conditions. Currently immunocytochemistry experiments are being performed on the invaded BMP4-hESC with a panel of antibodies indicative of trophoblast stem cell (cdx2, msx2), mature trophoblast (cytokeratin 7), syncytiotrophoblast (hCGα, β), EVT (HLA-G, integrin α5β3, CD9), and mesendoderm (vimentin, MIXL1) lineages to further characterize the invaded cells. The objective is to verify whether the invasive population from BMP4-hESC is homogenous and analogous to its in vivo counterparts, or represents a mixture of different cell types. More than 50% of the invaded cells in BMP4-hESC are KRT7 positive and a significant majority are CDX2 positive by immunocytochemistry, and approximately 40% are HLA-G positive by flow cytometry. Efforts will be dedicated to modifying the culture conditions which include altering BMP4 and oxygen concentrations as well as the choice of substratum (Matrigel/collagen) in which the invasion experiments can be performed to obtain more or less homogeneous HLA-G positive cells. This model system may have significant utility for identifying the intrinsic and extrinsic factors that influence the origins and development of EVT, and thereby the predisposing factors for pregnancy disorders such as preeclampsia. Supported by undergraduate fellowship from the Endocrine Society (JMS), Preeclampsia foundation Vision Grant Award (BT), NIH HD21896 (RMR), and HD055231 (RMR, LCS).

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Negative Regulation of Human Granulosa Tumor Cell Survival by TGF-Beta.

Kaye L Stenvers, Maree Bilandzic, Yao Wang, Simon Chu, Peter Fuller, Jock K Findlay 1

Disruption to the regulatory pathways that govern granulosa cell proliferation, differentiation, and death during ovarian development and folliculogenesis can result in a variety of disorders, including granulosa cell tumor (GCT) formation. Candidate growth regulatory molecules produced within the ovary include several TGF-beta superfamily members, but the specific stimuli and intracellular signalling pathways which determine human granulosa cell proliferation and survival are still poorly understood. We have used a human ovarian cancer cell line of GCT origin, the KGN cells, to study TGF-beta superfamily actions. We have recently reported that this GCT line exhibited very little expression of the TGF-beta accessory receptor betaglycan, a TGF-beta and inhibin co-receptor which determines cellular sensitivity to these ligands. We hypothesized that GCTs would be refractory to the growth-inhibitory effects of TGF-beta and/or inhibin and that overexpression of betaglycan would restore responsiveness to TGF-beta mediated growth inhibition. To test this hypothesis, KGN cells were stably transfected with a wildtype betaglycan expression construct or the empty vector as a control. Overexpression of betaglycan in the KGN cells restored sensitivity to TGF-beta and inhibin in luciferase reporter assays but had little effect on cell proliferation, cell cycle progression, or death in either GCT cell line as determined by flow cytometry. Furthermore, treatment with either exogenous TGF-beta2 or inhibin A, INHA knockdown, or neutralisation of endogenous TGF-betas also did not significantly affect cell survival and proliferation. As the GCT cells are tumour cell lines which exhibit constitutively active NF-kappaB, which is a pro-survival factor, we pre-treated the cells with the chemical NF-kappaB inhibitor, BAY11-7082. Under these conditions, overexpression of betaglycan resulted in a decrease in proliferation (15%; p<0.05) and an increase in apoptosis (300% p<0.05). The addition of TGF-beta1 and TGF-beta2 dose-dependently decreased cell viability, and this effect was enhanced by the presence of betaglycan. However, knockdown of INHA expression did not affect cell viability even in the presence of BAY11-7082. Following treatment with exogenous TGF-beta2, INHA gene knockdown, or a combination of the two, total cell lysates were extracted from betaglycan- and vector-transfected GCT cells. The relative levels of activation of specific pathways were determined by western blot analyses using activation-specific antibodies against SMAD2, SMAD3, and SMAD1/5. This analysis revealed that overexpression of betaglycan in the GCT cells increased both the basal and TGF-beta2-induced levels of activated SMAD3, with a smaller effect observed on activated SMAD2. Collectively, these data establish the TGF-beta-SMAD2/3-betaglycan signalling pathway as an important regulator of granulosa cell viability and suggest that deficiency of this pathway and/or inhibition by NF-kappaB contributes to dysregulated granulosa cell growth and cancer. Supported by the NHMRC of Australia (RegKeys 494802; 441101; 388904) and Victorian Government Infrastructure funds.

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Mouse Oocytes Promote the Mechanistic Target of Rapamycin (Serine/Threonine Kinase, MTOR) Pathway in Cumulus Cells by Suppressing the Expression of DNA-damage-inducible Transcript 4-like (Ddit4l).

You-Qiang Su, Koji Sugiura, Karen Wigglesworth, John J Eppig 1

Mechanistic (also known as mammalian) target of rapamycin (MTOR) is an evolutionally conserved kinase that plays an essential role in the control of cell growth and development by integrating and transmitting multiple extracellular cues, such as nutrient supply and signals of growth factor and stress. We reported previously that mouse oocytes promote several key metabolic processes including glycolysis, cholesterol biosynthesis and uptake of some amino acids in cumulus cells. This suggests that mouse oocytes promote MTOR activity in cumulus cells, but this implication had not been tested previously. Here, we show that mouse oocytes promote the MTOR pathway in cumulus cells by suppressing expression of DNA-damage-inducible transcript 4-like (Ddit4l) mRNA, a negative regulator of MTOR pathway. In situ hybridization of Ddit4l mRNA revealed its robust expression in mural granulosa cells, whereas expression in cumulus was significantly lower. Removal of oocytes from cumulus-oocyte complexes (COCs) by oocytectomy (OOX) caused dramatic up-regulation of Ddit4l mRNA in OOX cumulus cells, suggesting that the lower level of expression of Ddit4l in cumulus cells is due to suppression by oocytes. Coincident with up-regulation of Ddit4l expression, levels of phosphorylated MTOR (p-MTOR) in OOX cumulus cells was reduced. Up-regulation of Ddit4l mRNA and down-regulation of p-MTOR in wild type (WT) OOX cumulus cells was prevented by co-culture of these OOX cumulus cells with WT, but not Bmp15-/- or Gdf9+/-Bmp15-/-, fully-grown, germinal vesicle stage oocytes. This suggests that the regulatory effect of oocytes on MTOR pathway in cumulus cells is mediated by oocyte-secreted factors, presumably, GDF9 and/or BMP15. Indeed, Ddit4l mRNA was significantly up-regulated in Bmp15-/- and Gdf9+/-Bmp15-/- cumulus cells; and treatment with recombinant GDF9 and/or BMP15 suppressed expression of Ddit4l mRNA in WT OOX cumulus cells. Recombinant GDF9 and/or BMP15 were also found to promote the elevated level of p-MTOR in OOX cumulus cells. Treatment with inhibitors of SMAD2/3 but not of SMAD3 dramatically up-regulated the level of Ddit4l mRNA in WT COCs, and blocked GDF9-induced suppression of Ddit4l mRNA expression in WT OOX cumulus cells, indicating that a SMAD2-dependent pathway is involved in the process of oocyte and GDF9 regulation of MTOR pathway in cumulus. In conclusion, mouse oocytes, via GDF9 and BMP15, promote the MTOR pathway in cumulus cells, at least in part, by suppressing expression of Ddit4l, a negative regulator of this pathway. (Supported by NIH grant HD42137 and HD23839).

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Prohibitin (Phb1) Inhibits Apoptosis in Rat Granulosa Cells Through the Extracellular Signal-Regulated Kinase 1/2 (ERK1/2) and the Bcl Family of Proteins.

Indrajit Chowdhury, Minerva Garcia-Barrio, Kelwyn Thomas, Winston E Thompson 1

Prohibitin (Phb1) is a highly conserved, ubiquitous protein that is abundantly expressed in granulosa cells (GCs) and associated with GC differentiation and apoptosis. The present study supports new evidence that prohibitin (Phb1) is involved in GCs survival through activating the extracellular signal-regulated kinase 1/2 (ERK1/2) resulting in increase in the levels of Bcl2 and Bclxl proteins. We used a primary rat GC model to investigate the molecular mechanisms of Phb1 dependent survival of GCs. GCs were isolated from immature 23-25 days old female Sprague Dawley (SD) rats, were infected with adenovirus-GFP (Ad-GFP, control) or adenovirus Phb1-eGFP (Ad-Phb1-eGFP). Twenty-four hours later, cells were treated with the protein kinase C (PKC) inhibitor staurosporine (STS) for 2h. We found that the Ad-Phb1-eGFP infected GCs acquired a remarkable resistance to apoptosis. Using Affymetrix Gene-Chip System following STS treatment, we uncovered significant changes (50-80%) in several genes that are strong anti- or pro-apoptotic regulators, with forced-expression of Phb1 in GCs resulting in a profile of expression consistent with attenuated apoptosis when compared to control infected cells. Forced-expression of PHB1 in GCs increased more than two fold Bcl2, Bclxl and Erk transcription, whereas it decreased transcription of Bax, Bak, Mycs and caspase-3, results that were further confirmed by Western blot analysis. Forced-expression of PHB1 in GCs inhibits apoptosis associated with increased levels of the anti-apoptotic proteins Bcl2 and Bclxl, and reduced release of cytohrome c and stimulation of caspase-3 activity. Moreover, silencing of PHB1 expression using adenoviral micro interfering RNA (AdmirPHB1) or inhibiting Erk1/2 phosphorylation with the MEK-inhibitor PD-98059 blocked the protective effects Phb1 on STS-induce apoptosis in GCs. These findings shed new light on the PHB1-mediated apoptotic resistance mechanism of GCs through a Phb1-Mek-Erk1/2-Bcl/BclxL pathway and may have important clinical implications. This study was supported in part by National Institutes of Health Grants 1RO1HD057235-01A2, HD41749 and RR03034.

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

CCAAT/Enhancer Binding Protein (C/EBP)-alpha and -beta Are Essential for Ovulation and Luteinization by Regulating the Expression of Novel Target Genes.

Heng-Yu Fan 1, Zhilin Liu 1, Peter F Johnson 2, JoAnne S Richards 1

Luteinizing hormone (LH) via the EGF-like factors activates the RAS/MEK1/ERK1/2 pathway that, in murine preovulatory follicles, is essential for globally reprogramming granulosa cells and cumulus cells. As a consequence, oocyte maturation, cumulus cell-oocyte complex (COC) expansion, follicle rupture and luteinization are induced. Biochemical and genetic evidence suggests that the basic leucine zipper transcription factor C/EBP-beta might be a critical target and mediator of ERK1/2 functions in granulosa cells of preovulatory follicles. Granulosa cell-specific Cebpb knockout (Cebpbfl/fl;Cyp19-Cre) mice exhibit reduced, but not totally impaired, ovulation and luteinization, suggesting other factors are involved. Because C/EBPa is also induced significantly by hCG in granulosa cells of preovulatory follicles of wild type and Cebpbfl/fl;Cyp19-Cre mice, we hypothesized that C/EBPa might exert some overlapping functions with C/EBPb. Indeed, when C/EBPa was over-expressed in undifferentiated granulosa cells in culture, it enhanced the expression of known LH and EGF-factor target genes in a manner similar to that of C/EBPb. These results suggested that both C/EBPa and C/EBPb might be essential for regulating key events of ovulation and therefore, we generated additional conditional knockout mice.To overcome the neonatal lethality caused by germ-line depletion of Cebpa and to study the ovarian function of both C/EBPa/b, we generated the granulosa cell-specific, Cebpa knockout (Cebpafl/fl;Cyp19-Cre) and Cebpa/b double knockout (Cebpafl/fl;Cebpbfl/fl;Cyp19-Cre, termed Cebpa/bgc-/-) mice. The Cebpagc-/- females showed moderately reduced ovulation in superovulation studies but were completely fertile in normal mating paradigms. In contrast, the Cebpa/bgc-/- females failed to ovulate and were completely infertile, with adult ovaries devoid of corpora lutea. When Cebpa/bgc-/- mice were treated with eCG and hCG, preovulatory follicular development, oocyte maturation and COC expansion occurred normally. However, follicle rupture was completely blocked and, therefore, mature oocytes were not released into the oviduct. Furthermore, the luteinization process was impaired in Cebpa/bgc-/- ovaries, as shown by reduced expression of luteinization marker genes (Cyp11a1, Lhcgr and Star) and low serum progesterone levels at 48-72h after hCG injection. Hence, ovaries of Cebpa/bgc-/- mice display more severe defects than either of the individual knockouts. To identify additional specific C/EBPa/b-target genes in ovulatory follicles, microarray analyses were performed using RNA prepared from granulosa cells isolated from wild-type and Cebpa/bgc-/- mice at 4, 8, and 24h after hCG treatment. The gene profiling results showed that C/EBPa/b regulate a novel subset of LH target genes that are associated with matrix remodeling and vascularization. In addition, C/EBPa/b regulate genes associated with lipid metabolism, steroidogenesis and luteal cell function. The C/EBPa/b mutant mice document that these two transcription factors are not critical for the early ovulatory events (oocyte maturation and COC expansion), but are essential down-stream targets of LH and ERK1/2 that are required for regulating the expression of novel genes required for follicle rupture and luteinization. U54-HD-07495 (SCCPIR), NIH-HD-16229 and NIH-HD-07165.

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Synergistic Activation of Akt by IGF-1 and cAMP Is Correlated with Altered Expression of Paracrine Factors That Regulate Follicle Progression and Ovulation in Murine Granulosa Cells.

Elizabeth M Mack, Jacqueline E Smith, Jill G Kerl, Jennifer R Wood 1

Folliculogenesis and granulosa cell function are regulated, in part, by gonadotropin stimulation of cAMP-dependent signal transduction. For example, follicle stimulating hormone (FSH) regulates granulosa cell steroidogenesis, oocyte maturation, and the expression of paracrine factors which coordinate oocyte-granulosa cell growth and development. IGF-1 also contributes to follicular development via regulation of granulosa cell proliferation, apoptosis, and steroid production. These actions of IGF-1 are mediated through its interaction with the IGF-1 receptor, and in some cases, requires simultaneous gonadotropin-dependent signaling. Recent epidemiological studies demonstrate that IGF-1 levels decrease with increasing age, body mass index, and waist circumference. Given that advanced maternal age and obesity are the most common cause of anovulatory infertility, the objective of this study was to identify mechanisms by which IGF-1 regulates the expression of genes in granulosa cells which are crucial for follicle growth, follicle maturation, and ovulation. To achieve this objective, granulosa cells were isolated from the ovary of 23-day-old CF-1 mice and maintained in short-term culture. After 5 days, cells were exposed to no treatment, 1mM cAMP, 100ng/mL IGF-1, or a combination of cAMP and IGF-1 for 2 or 8 hours. The mRNA abundance of several paracrine factors which are produced by the granulosa cells and contribute to the coordination of oocyte and follicular growth and regulate the process of ovulation were examined using quantitative real-time PCR (QPCR). Genes associated with transzonal projections, which facilitate bi-directional communication between the granulosa cells and oocyte, were also examined. IGF-1 increased cAMP-dependent stimulation of kit ligand (Kitl), growth differentiation 9 (Gdf9), amphiregulin (Areg), and interleukin-6 (Il6) expression in both the 2 and 8 hour treatment groups. IGF-1 also increased cAMP-dependent stimulation of epiregulin (Ereg) expression 8 hours post-treatment. Conversely, anti-mullerian hormone (Amh) was downregulated upon combined treatment of granulosa cells with IGF-1 and cAMP for 2 or 8 hours. IGF-1 and cAMP alter gene expression through activation of the downstream signaling proteins Akt and Erk1/2. To assess the activity of these proteins, granulosa cells were maintained in short-term culture and exposed to no treatment, 1 mM cAMP, 100 ng/mL IGF-1, or a combination of cAMP and IGF-1 for 30 or 60 minutes. Western blot analyses demonstrated that cAMP alone or in combination with IGF-1 increased Erk1/2 phosphorylation compared to untreated cells 30 and 60 minutes post-treatment. IGF-1 or cAMP treatment alone also increased Akt phosphorylation in the granulosa cells. Interestingly, the combined treatment of IGF-1 and cAMP resulted in a synergistic increase in Akt phosphorylation indicating cross-talk between the cAMP and IGF-1 signaling pathways. Taken together, these data suggest that IGF-1 and cAMP may have an additive effect on paracrine factor gene expression due to the synergistic activation of Akt. Given that these genes are associated with follicle development and ovulation, these data also provide a plausible mechanism for age and obesity-dependent anovulatory infertility.

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

hCG-Induced Reduction in PPARγ and Liver X Receptor Expression Promotes Progesterone Synthesis by Luteinizing Rhesus Macaque Granulosa Cells.

Muraly Puttabyatappa 1, Catherine VandeVoort 2, Charles L Chaffin 1

In primates, the mid-cycle gonadotropin surge induces the rapid and dramatic synthesis of progesterone by granulosa cells. This increased progesterone production is associated in part with increased expression of mediators of cholesterol mobilization such as lipoprotein receptors (scavenger receptor BI [SR-BI] and low-density lipoprotein receptor [LDLR]) and StAR. Additionally steroidogenic enzymes such as P450 cholesterol side chain cleavage (CYP11A1) and 3β-hydroxysteroid dehydrogenase (HSD3B) are important for the synthesis of progesterone. While various transcription factors are up-regulated and participate in positive regulation of steroidogenesis, relatively little is known about potential repressors of steroidogenesis such as PPARγ and Liver X receptors (LXR). These transcription factors can reduce intra-cellular cholesterol levels through the regulation of multiple genes, including lipoprotein receptors and ATP-binding cassette (ABC) A1 and G1, which can promote cholesterol efflux, representing potentially key players in the earliest steps leading to LH/CG-mediated progesterone synthesis. The goal of this study was to test the hypothesis that an ovulatory stimulus rapidly reduces PPARγ, leading to the reduction in LXRα/β and the subsequent shift in gene expression to favor cholesterol ester uptake and metabolism. Granulosa cells were obtained from rhesus monkeys undergoing controlled ovarian stimulation protocols before (0 hr) or 3, 6, 12, and 24 hr after an hCG bolus in vivo. The mRNA expression of PPARγ, LXRα, ABCA1 and ABCG1 were dramatically reduced 3 hr post-hCG (9-, 9-, 5-, and 95-fold, respectively; p<0.05), while LXRβ mRNA was reduced 6 hr after hCG (2.5-fold; p<0.05). All five genes remained at low levels thereafter. Non-luteinized granulosa cells obtained prior to an hCG bolus were cultured in the presence of FSH or FSH+hCG ± the PPARγ agonist Rosiglitazone (0-1 μM) or LXR agonist T0901317 (0-10 μM) for 24 or 48 hr. Rosiglitazone dose-dependently increased the expression of LXRα (up to 4-fold; p<0.05) and not LXRβ, increased the expression of ABCG1 (8-fold; p<0.05), and attenuated the hCG-induced expression of StAR and SR-BI. Treatment of non-luteinized granulosa cells with hCG increased progesterone synthesis (45-fold; p<0.05), as well as STAR, CYP11A1 and SR-BI mRNA levels (28-, 12- and 670-fold, respectively; p<0.05). Addition of the LXR agonist T0901317 with hCG decreased (p<0.05) media progesterone levels, increased ABCA1 and ABCG1 mRNA relative to hCG alone (12- and 18-fold; p<0.05), dose-dependently attenuated hCG-induced expression of StAR, CYP11A1 and SR-BI mRNAs (p<0.05). These data indicate that PPARγ up-regulates LXRα, which in turn suppresses the expression of genes involved in periovulatory progesterone synthesis and increases genes involved in cholesterol efflux. One of the earliest steps in progesterone synthesis by luteinizing primate granulosa cells is thus the reduction of PPARγ and LXR expression, resulting increased cholesterol ester uptake and metabolism to progesterone. Supported in part by NIH HD043358 (CLC), RR13439 (CAV), RR00169 (CNPRC).

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Increased Beta-Oxidation, ATP Levels, and Improved Oocyte Developmental Competence in Response to L-Carnitine.

Kylie R Dunning, Kara Cashman, Darryl L Russell, Jeremy Thompson, Robert Norman, Rebecca L Robker 1

The metabolic rate of mammalian oocytes and embryos are closely associated with their developmental potential. Lipids are a rich source of energy; however their utilization by the oocyte and early embryo is poorly understood. The generation of ATP from lipids occurs in mitochondria via β-oxidation. We have found that β-oxidation increases in the cumulus oocyte complex (COC) during oocyte maturation and is essential for subsequent embryo development. We also found that carnitine palmitoyl transferase (Cpt1), the rate limiting enzyme in β-oxidation, is hormonally regulated in the COC. L-carnitine is a circulating metabolite required for Cpt1 activity which we hypothesized would be a key regulator of lipid metabolism in the COC and promote oocyte developmental competence. To better understand the importance of lipid metabolism during oocyte maturation and determine whether L-carnitine can be used to manipulate this process we 1) determined the effect of L-carnitine on β-oxidation and ATP levels in the COC, 2) assessed the effect of L-carnitine supplementation on oocyte developmental competence and 3) assessed whether L-carnitine could promote utilization of intracellular lipid stores and enable zygote survival in the absence of exogenous metabolic substrates. To determine the effect of L-carnitine supplementation on β-oxidation in COCs from d21 CBA/C57Bl6 F1 female mice, metabolism of 3H-palmitate by COCs in vitro was measured by the production of 3H2O in the media. The inclusion of L-carnitine (1mM) in the culture media resulted in a significant increase in β-oxidation in immature COCs and in COCs undergoing in vitro maturation. To determine whether increased β-oxidation in response to L-carnitine was associated with increased ATP production, ATP levels were measured in COCs following a 90 min culture period by bioluminescence assay. In a dose dependant manner, L-carnitine supplementation (0-10mM) led to increased ATP levels in the COC. As L-carnitine supplementation was found to increase β-oxidation and ATP levels in the COC we determined what effect this had on oocyte developmental competence. Following in vitro maturation, with or without L-carnitine (1mM), COCs were washed and fertilized in vitro and on-time embryo development assessed. L-carnitine supplementation resulted in a significant increase in the number of hatching blastocysts on day 5 compared to control. To determine whether L-carnitine improves oocyte developmental competence by promoting the utilization of intracellular lipid stores, presumptive zygotes were isolated from CBA/C57Bl6 F1 female mice and cultured in the absence of glucose, lactate, pyruvate and fatty acids for 24h. L-carnitine supplementation, in a dose dependant manner, led to increased numbers of 2-cell embryos, with a significant 2.1-fold increase in the cleavage rate of zygotes cultured in 5mM L-carnitine compared to 0mM. Thus fatty acids are a vital energy source for oocyte and embryo development and the physiological metabolite L-carnitine is an important co-factor for the utilization of these substrates. We have shown that inclusion of L-carnitine during oocyte maturation in vitro is able to increase lipid metabolism, raise ATP levels and improve embryo development. The inclusion of L-carnitine in culture media formulations is therefore expected to better mimic the normal in vivo environment and lead to improvements in oocyte and embryo quality in human clinics and in agriculturally important species.

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Conditional Ablation of the Progesterone Receptor Specifically in the Uterine Epithelium Demonstrates Its Necessary Role in the Regulation of Uterine Function.

Heather L Franco 1, Cory A Rubel 1, Jae-Wook Jeong 1, Grant D Orvis 2, Richard R Behringer 2, Rodrigo Fernandez-Valdivia 3, John P Lydon 1, Francesco J DeMayo 1

Uterine progesterone receptor (PR), expressed in both the epithelium and stroma, mediates epithelial-stromal crosstalk during embryo implantation and pregnancy. Ex vivo tissue reconstitution studies have pointed to stromal PR as being critical for the regulation of uterine epithelial gene expression (i.e., Indian hedgehog (Ihh)) and inhibition of mitogenic estrogen (E2) action whereas the role of epithelial PR has been largely unexplored. Here, we investigated the vivo role of epithelial PR during pregnancy and in the regulation of epithelial gene expression using compartment-specific ablation of PR. Using the Wnt7a-Cre mouse model, PR was ablated specifically in the uterine epithelium. This ablation rendered female mice infertile, demonstrating that epithelial PR is critical for female fertility. We examined uterine function by assessing the ability of these mice to undergo the decidual response. The epithelial PR conditional knockout mice failed to display a decidual response indicating that, despite the presence of PR in the stroma, decidualization is unable to occur without epithelial PR. We next wanted to determine if epithelial PR may regulate the expression of progesterone (P4)-target genes. Epithelial PR is critical for P4-induction of epithelial P4-target genes (Ihh, Areg, Gata2, Cyp26a1), but not stromal P4-target genes (Il13ra2). Transfection analysis revealed that epithelial PR can transactivate Ihh gene expression in Hec1a cells further confirming its critical role in P4-target gene expression. Therefore, epithelial PR plays a critical role in epithelial P4-target gene expression which differs from previous observations using tissue reconstitution experiments. In the uterus, P4 inhibits E2 actions such as uterine weight gain, epithelial proliferation, and E2-target gene expression. Normal P4-inhibition of estrogen-induced weight gain was observed in these mice. However, examination of P4-inhibition of specific E2-target genes demonstrated that epithelial PR plays a gene-specific role in P4-inhibition exhibiting inhibition of Ltf and Clca3 expression, but not Lif expression. We are currently studying its potential role in P4-inhibition of E2-induced epithelial proliferation. In summary, using an in vivo mouse model, we have shown that epithelial PR is necessary for female fertility as a mediator of decidualization, P4-target gene expression, and the P4-inhibition of E2 action. This work was supported by NIH Grant RO1CA077530 (to J.P.L.) and R01HD042311 (to F.J.D.).

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

A Potential Role for Mesenchymal-to-Epithelial Transition During Endometrial Regeneration.

Amanda L Krull 1, Ling Zhang 2, Nelson A Arango 2, Jose Teixeira 2, James K Pru 1

The uterus is an extremely plastic organ that has tremendous regenerative capacity. The mechanisms coordinating uterine regeneration during the menstrual/estrous cycle and following parturition are not well understood. It has been described in both humans and mice that bone marrow derived cells contribute to the regenerative process through engraftment into stromal and epithelial tissues of transplant recipients. In contrast, recent parabiosis studies in our lab involving the establishment of vascular anastomoses between wild type (WT) and ubiquitin-GFP transgenic (Tg) female mice revealed the unlikely architectural or functional contribution of circulating cells to the stromal or epithelial tissue compartments of the endometrium. Although GFP positive cells were found within the endometrium of WT parabiots, these cells expressed either endothelial or immune cell markers. Based on this experiment, we concluded that the endometrium harbors an endogenous population of cells that contribute to maintenance of the endometrial stromal and epithelial tissues in postnatal life. Pulse-chase label-retaining experiments from other labs, as well as our own support this notion. In the current study, we hypothesized that epithelial tissue regeneration in the endometrium is accomplished, at least in part, by mesenchymal-to-epithelial (MET) transition. MET occur during organogenesis and is thought to play a prominent role in tumor metastasis. To test this hypothesis, fate mapping studies were completed using Amhr2-Cre; Rose26-EYFP (i.e., flox-stop EYFP report) double Tg mice. As expected, EYFP expression was observed in mesenchymal (i.e., stroma and myometrium), but not epithelial tissue in prepubertal and nulliparous female Tg mice. No EYFP expression was detected in Rosa26-EYFP control mice. However, mosaic EYFP expression was observed in endometrial epithelia of Tg female mice following parturition or oil-induced decidualization. To ensure that the observed EYFP epithelial expression was not due to leaky Amhr2 promoter activity which could result in aberrant Cre expression, we used transgenic mice that express LacZ under the control of the Amhr2 promoter (Amhr2-LacZ) to monitor beta-galactosidase (beta-Gal) activity within the uterus. Beta-Gal activity was assessed during several time points including pre-pubertal days 14 and 25, as well as in adult nulliparous (1.5 and 6 months) and previously pregnant female mice. Additionally, an artificial decidualization model was used to maximize uterine remodeling. Beta-Gal activity was not detected in either the luminal or glandular epithelia regardless of age, reproductive status or the degree of damage incurred within the uterus. These findings confirm that Amhr2 promoter activity is restricted to the adult uterine stroma and myometrium and suggest that a daughter cell derived from the Mullerian duct mesenchyme contributes to epithelial regeneration in the murine uterus. To our knowledge, this is the first report of cellular transdifferentiation in the adult female reproductive tract under non-pathological conditions. These findings reveal a previously unappreciated role for MET in endometrial regeneration and therefore have important implications for endometrial cancer and other proliferative diseases of the endometrium such as endometriosis. This research is supported by Vincent Memorial Research Funds and the NIH 5R01HD052701.

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Hormad1 Mutation Disrupts Chromosomal Segregation in Oogenesis.

Yonghyun Shin 1, Youngsok Choi 2, Serpil Uckac 3, Svetlana A Yatsenko 3, Fang Yang 4, Jeremy Wang 4, Aleksandar Rajkovic 1

Meiosis is a unique biological mechanism that reduces the chromosome complement from diploid to haploid, and meiosis is a critical step in spermatogenesis and oogenesis. During the meiosis 1, homologous chromosomes pair, form chiasmata and recombine. Synaptonemal complex is a critical component necessary for chromosome pairing, synapsis, and recombination. We previously identified a novel germ cell-specific HORMA domain-encoding gene, Hormad1. Hormad1 is essential in mammalian gametogenesis as knockout male and female mice are infertile. In situ hybridization with anti-sense Hormad1 riboprobe revealed that Hormad1 expression was confined to germ cells. Also antibodies against HORMAD1 recognized HORMAD1 protein at E14.5 to E18.5 oocytes. Like testes, HORMAD1 co-localizes with SYCP3 and SYCP2, but does not co-localize with SYCP1. Hormad1-/- ovaries show normal histology between 2 and 30 weeks of life, with abundant corpora lutea in the Hormad1-/- ovaries indicating that normal process of oocyte maturation was not disrupted. We performed superovulation in knockout and wild type mice to determine whether ovarian defects contributed to infertility in Hormad1-/- females. Hormad1-/- females superovulated 28±11 eggs (n=22), while wild type animals superovulated 29±14 eggs (n=13). We therefore did not detect significant difference between the number of eggs superovulated from wild type versus knockout mice. We also tested whether blastocysts derived from Hormad1-/--fertilized eggs implant. We did not detect implantation of Hormad1-/--fertilized eggs. In order to determine the ploidy of Hormad1-/--fertilized eggs and embryos, we utilized fluorescent in situ hybridization (FISH) using BACs specific for chromosomes 19, 18, and X. We did not detect aneuploidy in GV oocytes. However after fertilization, embryos generated from Hormad1-deficient oocytes showed hyper- and hypodiploidy at the 2 cell to 8 cell stage embryos. Embryos arrested developmentally at the blastocyst stage. Moreover, The M2 stage in Hormad1-/- oocytes is grossly disrupted with mis-orientation of chromatid attachments to the spindle. HORMAD1 is therefore a critical component of spindle formation and chromosome segregation during oocytes maturation.

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Macrophages Are Essential for Maintenance of Corpus Luteum Function in Early Pregnancy.

Alison S Care, Melinda J Jasper, Wendy V Ingman, Sarah A Robertson 1

Macrophages are abundant within the ovary, and have been identified in the corpus luteum (CL) of most species studied, including in rodents and human. Through their secretory products, macrophages are thought to be involved in ovarian tissue remodelling, including luteinization, and in regulating steroidogenesis. Macrophages co-cultured with granulosa or luteal cells act to stimulate progesterone secretion in vitro. To determine the impact of macrophage ablation during early pregnancy, we utilised CD11b-DTR transgenic mice to elicit transient systemic ablation of macrophages by administration of diphtheria toxin (DT). The effects of macrophage ablation during the pre-implantation phase of pregnancy were evaluated. Ablation of macrophages on day 1 pc or day 4 pc caused complete pregnancy loss in all DT-treated CD11b-DTR mice, while DT-treated wild-type mice or PBS-treated CD11b-DTR mice maintained viable pregnancies. Macrophage ablation on day 3 pc significantly reduced serum progesterone (P4) levels to 40% of control levels when measured 24 h later. Administration of exogenous P4 on each of day 4-7 pc prevented fetal loss in DT-treated CD11b-DTR mice, and pregnancy progressed with viable pups at late gestation, while no pregnancies remained viable in DT-treated mice administered vehicle only. Immunohistochemical evaluation with anti-CD31 mAb showed that macrophage ablation in the CD11b-DTR mice resulted in disappearance of endothelial cells from within the CL. This suggests that macrophages are essential for support of endothelial cells within the CL, either by provision of trophic support and/or by macrophage transdifferentiation into endothelial cells. In conclusion, these data indicate a critical role for macrophages in corpus luteum development and steroidogenic function in early pregnancy. Given that various nutritional and immune stressors can profoundly affect macrophage numbers and behaviour, it seems reasonable to speculate that macrophage-regulated CL development is a vulnerable event and may contribute to some forms of unexplained infertility.

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

TSPY Gene Copy Number and Levels of mRNA for TSPY and Cell Cycle, Meiosis and Early Male Germ Cell Marker Genes in Holstein Bulls.

Christine K Hamilton 1, Laura Favetta 1, Patrick Blondin 2, W Allan King 1

Testis-specific protein, Y-encoded (TSPY) is present in varying gene copy number in both human and cattle. Copy number variation of TSPY has been linked to spermatogenesis and is hypothesized to be a potential indicator of male fertility although there is limited data on TSPY expression in testicular tissue. Mice have only a single, non-functional TSPY gene whereas cattle have multiple functional copies and since slaughterhouse testicular material is readily available, cattle are a better model species for TSPY analyses. This study aims to compare TSPY copy number and mRNA transcript levels with that of various genes related to cell cycle and meiosis in Holstein bulls. To do this we measured TSPY copy number in DNA extracted from blood (n=51) and TSPY expression in total mRNA extracted from testicular tissue (n=26) with real time polymerase chain reaction (PCR). Our results show that TSPY copy number is negatively correlated to TSPY mRNA expression in the testis (r=-0.694, p<0.0001). We also found negative correlations of TSPY copy number and positive correlations of TSPY mRNA expression with various cell cycle genes (CCNB1, CCNB2, CDK1), meiotic genes (RAD51, SYCP3 and MLH1) and markers of early germ cells (UCHL1, TRPC2). Based on our results, it appears that TSPY expression may represent a good marker of early male germ cells. The negative correlation that we found between TSPY copy number and TSPY expression may have useful applications; TSPY copy number could be measured in blood to predict the amount of early germ cells in individual bulls. Supported by National Sciences Engineering and Research Council grant 364747-08; Canadian Research Chair program; and L'Alliance Boviteq Inc.

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Estrogen Receptor Subtypes ERalpha and ERbeta May Have Overlapping Roles in Oocyte Development.

Fangjie Tang, Melissa E Pepling 1

The formation of the pool of functional primordial follicles is critical for reproduction and is conserved among species. Female primordial germ cells undergo incomplete mitotic division and form clusters called cysts. Shortly after birth, the cysts break down into individual oocytes that become surrounded by somatic pre-granulosa cells to form primordial follicles. In this process, a subset of oocytes undergo programmed cell death and only one third of the initial oocytes survives and represents a life-time pool of oocytes. Estrogen treatment of neonatal mouse ovaries results in multiple oocyte follicles in adults which are likely cysts that did not break apart. Supporting this, previous studies have shown that estrogenic compounds can disrupt cyst breakdown in neonatal mice in vitro and in vivo. However, the regulative mechanisms of estrogen signaling in this process are still unknown. Estrogen functions via estrogen receptors (ERs), which are members of the steroid receptor superfamily. In mammals, there are at least two estrogen receptor subtypes: ER alpha (ERalpha) and ER beta (ERbeta). And both ERs are present during cyst breakdown and primordial assembly. However, single knock-outs of either ERalpha or ERbeta did not affect the cyst breakdown process. These results suggest a more complicated interaction between estrogen and its receptors. The objective of this study was to analyze cyst breakdown and primordial follicle development in ERalpha, ERbeta, and ER double knockout mice. Wild type, ERalpha single knockout, ERbeta single knockout, and ER double knockout ovaries were collected at PND 7, a time when most oocytes have completed cyst breakdown. Oocyte survival, cyst breakdown, and follicle development were assessed via immunochemistry using an oocyte marker and confocal microscopy. ER double knockout mice had similar oocyte numbers compared to wild type and ER single knockout mice. However, the double knockout mice had delayed cyst breakdown compared to wild type and ERbeta knockouts. Moreover, a larger proportion of ER double knockout oocytes were in advanced follicle stages and indicating a faster rate of follicle development. These results show that the estrogen receptor subtypes might have overlapping roles and might compensate for each other in oocyte development. Further investigation of expression level of one estrogen receptor in the other knockout strain might provide insight into the specific function of ERalpha and ERbeta in oocyte development.

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Effect of Omega-3 Fatty Acids on Prostaglandin (PG) F2alpha-Induced Mitogen-Activated Protein Kinase Signaling in Bovine Luteal Cells In Vitro.

Robert D Cheatham 1, Nicole R White 1, Patrick D Burns 1, Erik S Chestnut 1, John S Hickman 1, Jayme KK Michishima 1, Daniel Suh 1, Jason E Bruemmer 2, Terry E Engle 2

The Mitogen-Activated Protein (MAP) kinases appear to play a key role in mediating PGF2alpha-induce cell signaling in bovine luteal tissue. The omega-3 fatty acids eicosapentaenoate (EPA) and docosahexaenoate (DHA) have been shown to reduce prostaglandin synthesis in several tissues including bovine endometrial tissue. The objective of this study was to determine the effects of omega-3 fatty acids on PGF2alpha-induced MAP kinase signaling in bovine luteal cells in vitro. Eleven non-lactating mature Angus cows were housed in individual pens and fed a corn silage-based diet for approximately 60 days. Diets were supplemented with fish meal at 5% dry matter intake (a rich source of omega-3 fatty acids; n = 6 cows) or corn gluten meal at 6% dry matter intake (n = 5 cows). Estrous cycles were synchronized using two injections of PGF2alpha administered at 14 day intervals. The ovary bearing the CL was surgically removed at mid-cycle (between days 10-12) after synchronized estrus which corresponded to approximately day 60 of supplementation. The ovary was transported to the laboratory and prepared for in vitro incubation. The CL was digested with collagenase and luteal cell concentration determined using a hemocytometer. Cell viability was determined using a propidium iodide exclusion assay. Six-well culture dishes were seeded with 5x105 viable luteal cells in Hams F-12 culture medium and treated in triplicate with 0, 0.1, 1, 10, 100, 1000 nM PGF2alpha analog (cloprostanol). Cells were cultured in a humidified atmosphere of 95% air and 5% CO2 at 37°C for 15 min. Incubations were terminated and protein cell lysates prepared and subjected to western blot analysis. Membranes were probed with antibodies for phosphorylated extracellular regulated kinase (ERK), c-Jun n-terminal stress kinase (JNK), and p38 MAP kinase, stripped and probed with antibodies for total ERK, JNK, and p38. A ratio of phosphorylated to total kinase was calculated to correct for loading differences. Prostaglandin F2alpha induced phosphorylation of p46 JNK (P < 0.05) and p38 MAP kinase (P < 0.07) in a quadratic manner. Abundance of phosphorylated p46 JNK and p38 MAP kinase in luteal cells increased (P < 0.05) when incubated in the presence of 0.1 to 10 nM PGF2alpha and then decreased (P < 0.05) at higher doses. This response was attenuated in cells obtained from cows supplemented with fish meal (P < 0.05). Prostaglandin F2alpha had no effect on phosphorylation of ERK (P > 0.10). In conclusion, the stress MAP kinases (p46 JNK and p38 MAP kinase) appear to mediate PGF2alpha-induced cell signaling in bovine luteal cells and omega-3 fatty acid supplementation mitigates this response. This project was supported by National Research Initiative Competitive Grant no. 2008-35203-19099 from the USDA Cooperative State Research, Education, and Extension Service and Omega Protein Corporation.

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Dynamic Expression of the SLIT-ROBO Pathway in the Periovulatory Follicle and Corpus Luteum in Primates.

Fuhua Xu 1, Amanda Hurliman 2, Richard L Stouffer 1

The Slit-Robo pathway was initially identified in Drosophila and plays a critical role in axon guidance during development of the nervous system. Recently, three Slit (1-3) ligands and four Robo (1-4) receptor isoforms were identified in mammals, in both non-neuronal and neuronal systems where they may function in tissue growth, remodeling, and development. Members of the SLIT-ROBO system were detected in human and nonprimate ovary, but their regulation and functions are unclear. As an initial step in considering its importance in the primate ovary, experiments were designed to investigate the expression of SLIT-ROBO family members in the pre- and peri-ovulatory follicle and corpus luteum (CL) of rhesus monkeys during the menstrual cycle. Using our previously reported Controlled Ovulation (COv) model, the dominant follicle (n=4/interval) was collected at 0, 12, 24, and 36 (ruptured, or unruptured) hours after injection of an ovulatory dose (1000 IU) of hCG. CL (n>4/stage) were collected at early, mid, mid-late, late, or very late (days 3-5, 6-8, 10-12, 14-16 post-LH surge, and menses respectively) luteal phase. These stages encompass the entire lifespan of the CL, from development (early luteal phase) through functional (late) and structural (very late) regression. Total RNAs were extracted from individual tissues, real-time PCR performed, and mRNA levels analyzed by Sigma Stat software using 18S rRNA as an internal control. The mRNAs for all three SLIT ligands and four ROBO receptors were detected in each tissue sample. Appreciable mRNA levels for SLIT1 were present in the preovulatory follicle at 0 hour before hCG injection, followed by a transient decline (p<0.05) at 12 hours post-hCG. Elevated levels returned at 24-36 hours post-hCG prior to follicle rupture, but then declined (p<0.05) to low-levels in the ruptured follicle, which remained invariant in the CL from early to very late luteal phase. The mRNA levels for SLIT2 were appreciable, but unchanging within the periovulatory follicle or CL, until increasing (p<0.05) in the very late luteal phase. Levels of SLIT3 mRNA were low and not significantly different during the periovulatory interval or luteal phase. In contrast, ROBO1 mRNA levels increased (p<0.05) in the preovulatory follicle within 12 hours post-hCG, and remained elevated through follicle rupture, as well as in CL throughout the luteal phase. The levels of ROBO2 or ROBO4 mRNA did not change in the periovulatory follicle or CL during the menstrual cycle, except for a modest, transient increase (p<0.05) in ROBO4 transcript at mid luteal phase. Remarkably, ROBO3 mRNA levels increased from 12 to 36 hours post-hCG in the periovulatory follicle, and again throughout the CL lifespan to peak at late to very late luteal phase (p<0.05). Thus, the presence and dynamic expression of several members of the SLIT-ROBO family in the preovulatory follicle and corpus luteum suggests that this ligand-receptor system plays a role in ovulatory processes and/or luteal development and regression during the ovarian cycle in primates. Supported by NICHD HD18185 (Proj 3), HD053648, RR00163

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Biol Reprod. 2010 Nov;83(1 Suppl):4.

Administration of a Gonadotropin-Releasing Hormone Agonist Affects Corpus Luteum Development, Angiogenesis, and Induces Apoptosis in a Rat Model of Ovarian Hyperstimulation Syndrome.

Leopoldina Scotti, Griselda Irusta, Dalhia Abramovich, Marta Tesone, Fernanda Parborell 1

Moderate to severe ovarian hyperstimulation syndrome (OHSS) has been calculated to occur in 0.2% to 2% of all ovarian stimulation cycles. Risk factors include low body weight, high follicle count, polycystic ovarian syndrome, previous OHSS and elevated serum estradiol. Gonadotropin-releasing hormone analogs are thought to be effective in preventing this complication and several clinical trials have found lower incidence of OHSS in patients treated with these molecules. Our objective was to analyze the in vivo effect of GnRH-I agonist on corpus luteum (CL) development, angiogenesis, luteal regression and cell proliferation in ovaries from rat OHSS model. Group OHSS (hyperstimulated immature Sprague Dawley rats) received excessive doses of pregnant mare serum gonadotropin (PMSG, 50 UI/day) injected for 4 consecutive days (from the 21th day to 24th of life), followed by human chorionic gonadotropin (hCG, 25 UI, 25th day of life). Group OHSS + GnRH-I a (GnRH-agonist treated hyperestimulated immature rats) received the same doses of gonadotropins than in group OHSS and, in turn, received injections of GnRH-I a for 5 consecutive days (Leuprolide Acetate, 100 µg/kg/day, from the 21th to the 25th day of life; twice a day), followed by hCG (25 UI, 25th day of life). Immature female Sprague Dawley rats were hyperstimulated and treated with GnRH-I agonist from the start of pregnant mare serum gonadotropin (PMSG) until the day of hCG (human chorionic gonadotropin) for 5 consecutive days. Blood and tissue samples were collected at 48 hours after hCG injection. VEGFA levels were evaluated in the peritoneal fluid by ELISA. Serum progesterone and estradiol were measured by RIA. Histological features of sectioned ovaries were assessed in hematoxilin and eosin stained slides. Cell proliferation and apoptosis were assessed by immunohistochemistry for PCNA and TUNEL, respectively. FLK-1 (VEGFA receptor) and PCNA protein levels were evaluated by western blot. In the OHSS model, treatment with GnRH-I agonist resulted in a significant decrease in ovarian weight as well as a decrease in progesterone and estradiol serum levels. Moreover, this agonist significantly decreased the concentration of peritoneal VEGF. GnRH-I agonist significantly decreased the number of development CLs and mature CLs in the OHSS group compared to the OHSS group without treatment. The treatment with GnRH-I agonist significantly decreased the levels of FLK-1 in the OHSS rats. GnRH-I agonist treatment reduced the diameter of CLs, as well as decreased CL cell proliferation in OHSS model. Finally, GnRH-I agonist increased apoptosis in CLs from OHSS group. In conclusion, in the OHSS model, the administration of GnRH-I agonist interferes with luteal steroidogenesis, angiogenesis and CL development associated with a decrease in luteal proliferation and an increase in apoptosis process. Therefore, the treatment with a GnRH-I agonist decreased the severity of OHSS in an immature rat model stimulated by PMSG/hCG. Supported by: Roemmers Foundation, CONICET (PIP 1223) and ANPCYT (PICT 2004/5-26047).

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Different Impact of Endogenous Endothelin-1 and Angiotensin II Inhibition Within the Corpus Luteum During Prostaglandin F2alpha-Induced Luteolysis in the Cow.

Akio Miyamoto 1, Wataru Nakae 1, Ayumi Irie 1, Kiemi Sasahara 1, Motozumi Matsui 1, Masayuki Ohtani 2, Takashi Shimizu 1, Koumei Shirasuna 1

The corpus luteum (CL) secretes progesterone (P) that is necessary to establish pregnancy. If pregnancy does not occur, the CL regresses rapidly by prostaglandin F2alpha (PGF) released from the uterus. Endothelin-1 (EDN1) and angiotensin II (Ang II) are considered as luteolytic mediators that are converted from big-EDN1 by endothelin converting enzyme-1 (ECE-1) and from angiotensin I by angiotensin converting enzyme (ACE) in the bovine CL. It has been reported that EDN1 and Ang II have an ability to inhibit P secretion in vitro, but there are a few in vivo information addressed on the physiological roles of EDN1 and Ang II within the CL in the luteolytic cascade in the cow. Therefore, the objective of the present study was to investigate the physiological impact of EDN1 and Ang II within the CL during PGF-induced luteolysis in the cow by using ECE-1 inhibitor and ACE inhibitor. Cows on days 10-12 of the estrous cycle (Day 1= ovulation) were classified into 2 experimental groups ; EDN1-inhibited group (EDN1-EXP) and Ang II-inhibited group (Ang II-EXP). Cows were subjected to 7 intraluteal injections of ECE-1 inhibiotor or NEP inhibitor (control of ECE-1 inhibitor) in the EDN1-EXP and ACE inhibitor or saline (control of ACE inhibitor) in the Ang II-EXP at -0.5, 0.5, 2, 4, 6, 8 and 10 h after PGF administration (= 0 h). In the experiment 1, the CLs were observed by color Doppler ultrasonography with blood collection. In the experiment 2, the CLs were collected by ovariectomy at 12 h after PGF adiministration to determine the effect of ECE-1 inhibitor or ACE inhibitor on the mRNA expression, peptide expression and number of immune cells in the luteal tissue. EDN1-EXP : ECE-1 inhibitor treatment did not affect the decrease of plasma P after PGF administration, but delayed the decrease of CL volume and blood flow area surrounding the CL compared to those of control (experiment 1). ECE-1 inhibitor treatment suppressed the EDN1 peptide level in the CL as examined by immunohistochemistry (IHC). Furthermore, the mRNA expressions of Bax/Bcl-2, caspase-8 and caspase-3 (indexes of apoptosis) and numbers of immune cells within the CL were suppressed by ECE-1 inhibitor treatment compared to those of control (experiment 2).Ang II-EXP : ACE inhibitor treatment did not affect the decrease of plasma P and blood flow surrounding the CL after PGF administration, but delayed the decrease of CL volume compared to those of control (experiment 1). ACE inhibitor treatment suppressed the Ang II peptide level in the CL as examined by IHC. Furthermore the mRNA expressions of CD68 (indefx of macrophage) and TNF-α as well as number of immune cells within the CL were suppressed by ACE inhibitor treatment compared to those of control (experiment 2). The results of the present study suggest that EDN1 and Ang II contribute to structural luteolysis through different pathways in the cow. EDN1 may induce apoptosis by shutting-off the blood flow with an inflax of immune cells, while Ang II may actively stimulate immune responses including recruitment of immune cells and secretion of cytokines to accelarate CL regression. Supported by Grobal COE and JSPS programs.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Comparison among Different Doses of Prostaglandin F2alpha (PGF) on Luteal Function of the Day 5 Corpus Luteum (CL) in Nonlactating Holstein Cows.

Anibal B Nascimento, Alexandre H Souza, Abdelkadir Keskin, Jerry N Guenther, Milo C Wiltbank 1

The early CL (before Day 6) does not regress after a single PGF treatment. We hypothesized that increasing PGF dose or number of PGF treatments would allow PGF to regress the early CL (Day 5). Non-lactating Holstein cows (n=22) were synchronized (100mg GnRH; 7d later 25 mg PGF2alpha; 2d later 100 mg GnRH) and ovarian ultrasonography was used to select cows that ovulated to the second GnRH treatment for this study. This allowed precise timing of cows on Day 5 of the cycle. On Day 5 of the cycle, cows were randomly assigned to one of four treatments: 1) control (n=5): no further treatment; 2) 1PGF (n=6): one dose of 25mg PGF; 3) DPGF (n=6): double dose of 25mg (50mg) given once; 4) 2PGF (n=5): two doses of 25mg PGF (50mg) given 8 h apart (First PGF 8 h before, second PGF at same time as other PGF treatments to eliminate confounding by time with double PGF treatments). Blood samples were collected in order to perform two separate studies, one study during the first 24 h, starting at 0 h, prior to PGF treatment, then at 2, 4, 6, 12 and 24 h, to observe effects of treatments during the first day. The second study period was from d 5 to d 15 (d 5, 6, 7, 8, 11, 12, 13, 14 and 15) to evaluate results of circulating P4 concentration during the complete estrous cycle. In the first study (0 to 24 h), there were effects of treatment (T1, P=0.01), time (t1, P<0.001) and an interaction of T1Xt1 (P=0.02). The group 1PGF vs. control was different only at 12 h (P=0.02) and had a trend to be lower at 24 h (P=0.06). Similarly, cows treated with DPGF were different than controls at 4 h (P=0.04), 12 h (P=0.0006) and 24 h (P=0.0003). Only cows treated with 2PGF had lower P4 than control during the whole period and had a low P4 concentration of 0.37 ± 0.17 ng/mL at 24 h, consistent with our definition of luteolysis. In the second study (d 5 to 15), were observed effects of treatment (T2=0.009), time (t2<0.0001) and an interaction of T2Xt2 (P=0.002). In this second analysis, all groups receiving PGF were not different from control on d 6 (P=0.46). The group 1PGF was not different from control at any time from d 5 through d 13 and it was even higher on d 14 (P=0.01) and d 15 (P=0.007). Cows receiving DPGF were lower than control from d 7 (P=0.046) through d 12 (P=0.0008), however, after d 13 (P=0.53) until d 15 (P=0.26) no differences were observed. Likewise, there were differences between control and 2PGF from d 7 (P=0.016) through d 13 (P=0.79), but not on d 14 (P=0.98) and d 15 (P=0.79). On Day 15, all PGF treated groups had normal circulating P4 concentrations, consistent with an active CL: Control (2.69 ± 0.79 ng/mL) 1PGF (5.51 ± 0.71 ng/mL), DPGF (3.94 ± 1.15 ng/mL); 2PGF (3.47 ± 0.67 ng/mL). Ultrasound evaluation confirmed that no CL from any group completely regressed during the experiment and no new ovulations occurred to account for functional CL later in cycle. In summary, the double dose of PGF (50mg) either in a full dose given once or divided in two doses 8 h apart, can produce a dramatic decrease in circulating P4, consistent with classical definitions of luteolysis; however, these CL recover and become fully functional. Thus, even two doses of PGF prior to Day 5 are insufficient to produce complete luteolysis in Day 5 CL.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Transforming Growth Factor-beta1 (TGFB1) Disrupts the Angiogenic Potential of Microvascular Endothelial Cells of the Bovine Corpus Luteum.

Dulce Maroni 1, John S Davis 2

Cyclical formation and regression of the corpus luteum is required for maintenance of female reproductive cycles. Luteolysis is characterized by angioregression, luteal cell apoptosis and remodeling of the extracellular matrix characterized by deposition of type 1 collagen. During regression of the bovine corpus luteum transforming growth factor beta1 (TGFB1) is produced in response to prostaglandin F2alpha, a primary signal for corpus luteum regression. Although TGFB1 can reduce progesterone secretion by steroidogenic cells, the action of TGFB1 on other cells of the corpus luteum is unknown. Endothelial cells comprise 50% of the cells present in the corpus luteum and during regression the microvasculature is extensively disrupted. We hypothesized that TGFB1 participates in the disassembly of the microvasculature during corpus luteum regression. We isolated microvascular endothelial cells from the bovine corpus luteum (CLENDO cells) in order to study the effects of TGFB1 on the ability of CLENDO cells to proliferate, form capillary-like structures, migrate and maintain the integrity of a monolayer of cells. CLENDO cells were isolated using BS1 lectin-coated magnetic beads and further purified by FACS with VE-cadherin antibody, a specific endothelial cell marker. Characterization of the purified CLENDO cells by western blot showed that these cells express the endothelial cell markers VE-cadherin and eNOS, as well as receptors for TGFB1. Less than 1% of CLENDO cells stained positive for alpha-smooth muscle actin. Western blot analysis revealed that Smad2 and Smad3 were phosphorylated within 5 min following treatment of CLENDO cells with TGFB1 (1 ng/ml). [3H]-thymidine incorporation studies showed that TGFB1 (0-10 ng/ml) caused dose-dependent reductions in DNA synthesis. At physiologic concentrations TGFB1 (1 ng/ml) significantly reduced (p < 0.05, 50%) the stimulatory effect of 5% fetal calf serum on DNA synthesis. However, TGFB1 did not reduce CLENDO cell viability when plated on plastic. The effect of TGFB1 on capillary morphogenesis was studied by plating CLENDO cells on Matrigel. CLENDO cells produced capillary-like structures that were disrupted by TGFB1 after 6 hr. In contrast, CLENDO cells plated on fibrillar collagen I did not form capillary-like structures and cell viability was significantly reduced after treatment with TGFB1 as evidenced by an increase (p < 0.05) in caspase 3 activity. The ability of CLENDO cells to migrate was tested in a Boyden chamber. Directional migration was reduced (50%) by TGFB1 after 6 hr. Immunofluorescence analysis using a VE-cadherin antibody revealed that TGFB1 caused loss of cell-cell contacts, although western blot analysis showed that the expression of VE-cadherin was not altered. As determined by the transfer of fluorescent dextran across confluent CLENDO monolayers, TGFB1 increased CLENDO monolayer permeability by eight fold. Each of the actions of TGFB1 on CLENDO cell function (proliferation, migration, capillary formation and maintenance of cell-cell adhesion) was prevented by treatment with SB431542, a selective TGFBR1 receptor kinase inhibitor. These studies demonstrate that TGFB1 acts directly on CLENDO cells to limit endothelial cell growth and angiogenic activity. These studies suggest that TGFB1 induces the disassembly of luteal capillaries thereby contributing to the regression of the corpus luteum. Supported by USDA 2006-35203-17249, the DVA, and Olson Center for Women's Health.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Selective Increased Luteal Prostaglandin E2 Compared to F2 Alpha Biosynthesis and Signaling Are Critical for Luteal Protection and Resistance During Establishment of Pregnancy in Sheep.

JeHoon Lee 1, Sam D Stephen 1, John A McCracken 2, Sahkila Banu 1, Thamizh K Nithy 1, Joe A Arosh 1

In ruminants, pulsatile secretion of prostaglandin F2 alpha (PGF2a) by the endometrium is locally transported to corpus luteum (CL) and stimulates intraluteal PGF2a biosynthesis and induces luteal regression/luteolysis. Interestinlgy, luteolytic PGF2a fails to accomplish regression of CL of early pregnancy. This increased resistance of the CL to PGF2a may suggest existence of luteal protective mechanisms. Early physiological studies proposed prostaglandin (E2) PGE2 as luteal protective mediator because PGE2 counteracted the effects of PGF2a and protected the CL from regression. However, the molecular and cellular aspects of resistance of the CL to luteolytic PGF2a are largely unknown. The objective of this study is to determine biosynthesis, transport, and signaling of PGF2a and PGE2 in CL during the estrous cycle and early pregnancy in sheep. Suffolk cross-bred ewes were ovariohysterectomized on days 12, 14 and 16 of the estrous cycle (ECY) or pregnancy (PNY), the CLs were collected, and temporal expression of PGF2a and PGE2 biosynthetic, transport and signaling proteins were determiend by western blot. Blood samples were collected from the ovarian artery and ovarian vein and transport of PGF2a, PGFM, PGE2, and PGEM were determiend by ELISA. Results indicated that: (i) COX-2 protein was abundantly expressed on days 12-16 of ECY and PNY and not affected by the ECY or PNY status, and COX-1 protein was expressed at very low level; (ii) PGES-1 protein was abundantly expressed on days 12-14 of ECY and PNY, and then decreased (4 fold) on day 16 of the ECY compared to that of PNY, expression of PGES-2 protein was not modulated by ECY and PNY, and expression of PGES-3 protein was decreased (5 fold) on days 12-16 of ECY compared to that of PNY; (iii) expression of PGFS-AKR1C1 protein was not modulated by ECY and PNY, and PGFS-AKR1C3 protein was expressed at steady sate level on day 10-16 of ECY but decreased (several fold) on days 12-16 of PNY; (iv) expression of FP, EP1 and EP3 proteins were not modulated by ECY and PNY, but interestingly, expression of EP2 and EP4 proteins were decreased (4 fold) on day 16 of ECY compared to that of PNY; (v) PGT protein was abundantly expressed on days 12-16 of ECY and PNY and its expression was decreased on day 16 of ECY compared to that of PNY; and (vi) PGDH protein expression was increased on days 14-16 of PNY compared to that of ECY. (vi) Ovarian venous plasma concentrations of PGF2a, PGFM, PGE2 and PGEM were 1500-2000 pg/ml, 400-500 pg/ml, 1000-1250 pg/ml, and 2-5 pg/ml on days 14-16 of the estrous cycle, respectively, and 400-500 pg/ml, 2000-2500 pg/ml, 3000-3500 pg/ml, and 3-5 pg/ml on days 14-16 of pregnancy, respectively. These results together suggest that the luteal PG biosynthetic and signaling machinery is selectively directed towards PGF2a at the time of luteolysis, by contrast; towards PGE2 at the time of establishment of pregnancy. In addition, CL of pregnancy has innate capacity to catabolize PGF2 to inactive PGFM but not PGE2 to PGEM and favors intraluteal PGE2 production. Thus, increased intraluteal PGE2 production and action are the critical mechanisms that resist/protect CL of early pregnancy to luteolytic PGF2a. Selective inhibition of PGES-1 or EP2 and EP4 along with PGF2a could be the potential future therapeutic strategy to improve reproductive efficiency in ruminants. Supported by USDA award 2008-35203-19101 to JAA.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Isolation of Bovine Luteal Microvascular Endothelial Cells: High Yield Method and Functional Interactions with T Lymphocytes.

SS Walusimbi, JL Pate 1

Endothelial cells are structurally and functionally an integral part of the corpus luteum and play multiple roles in luteal formation, development and regression. Luteal endothelial cells synthesize and secrete luteotropic and luteolytic products and facilitate trafficking of immune cells into the corpus luteum. To study the behavior and interaction of endothelial cells with other luteal cells, sufficient numbers of purified populations of endothelial cells have to be isolated. Current technologies and methods used to isolate endothelial cells are expensive and result in low yields of endothelial cells and thus require subsequent subculture. Here we describe a simple method for isolating luteal endothelial cells, characterization and interactions with T lymphocytes. Endothelial cells were physically or magnetically separated from corpora lutea of midcycle (n=4). Luteal cells were enzymatically dispersed into a single cell suspension using collagenase. The single cell suspension was serially and sequentially washed to sediment larger cells. All supernatants from serial washes were centrifuged at 250xg to pellet small cells. Both fractions of larger and small cells were either filtered through a10 micron sieve to enrich endothelial cells or labeled with CD31 antibodies for magnetic separation. The number of cells, viability, and purity were assessed. Contaminating red blood cells in the filtrate were lysed using a lysis buffer. Enriched cells were cultured to observe their morphology or labeled with von Willebrand factor VIII and observed by fluorescence microscopy. Fresh cells were labeled with BS-1 lectin-FITC and the proportion of positively labeled cells was determined by flow cytometry. Contaminating steroidogenic cells in the filtrate were quantified by labeling for luteinizing hormone receptor (LHR). Polymerase chain reaction was also performed to detect steroidogenic acute regulatory protein (StAR) in enriched and mixed cells. Peripheral T cells (γδ +CD2 +) were cocultured with endothelial cells for 72hrs. Changes in the proportion of γδ +and CD8 + and their functional phenotypes were determined. Larger numbers of endothelial cells of high viability were consistently recovered using the sieve method when compared to the magnetic method. The percentage of cells positive for BS-1 lectin in the enriched endothelial cells ranged between 88-95%. Less than 10% of the cells in the filtrate were positive for LHR. Endothelial cells activated γδ +CD2 + by increasing the proportion of T cells positive for the early activation marker, CD25, but did not alter the proportion of γδ + T cells. The proportion of CD8 αβ + and total CD8 + T cells tended to decrease when T cells were cocultured with endothelial cells. These data suggest that large numbers of highly viable endothelial cells can be isolated from the corpus luteum using 10 micron sieves. This isolation method is rapid, cost-effective and yields a sufficient number of endothelial cells of relatively high purity that can be used without the need for subculture. Endothelial cells isolated this way will enable the study of their interactions with other luteal cells in in vitro systems. This project was supported by National Research Initiative Competitive Grant no. 2008-00551 from the USDA Cooperative State Research, Education, and Extension Service to JLP.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Enhancing Teaching Effectiveness in Animal Histology by Taking Reproductive System as an Example.

Xueming Zhang, Xu Zhou, Zhanpeng Yue, Ziyi Li 1

As the microscopic extension of Animal Anatomy, Animal Histology is the type of course that enables the students of Veterinary medicine to tie structure and function together. However, the trivial histological terminology may make students boring and frequently forgetting how to verbally describe the various tissues and organ systems. The aim of this project is to improve teaching effectiveness in Animal Histology by employing a comprehensive strategy with Animal Reproductive System as an example. To this end, the following strategies were adopted. 1) Bilingual teaching style: For better academic communication and obtaining leading edge information from the internet, the basic terminology and concept in Animal Reproductive System were delivered both in Chinese and English. Especially the ability to pronounce the terminology of various reproductive organs, tissues and cells correctly was trained. We also published Listening Comprehension for Animal Science and Veterinary Medicine as the support material. 2) Laboratory practice: The students were encouraged to have an anatomic practice of mouse reproductive system, followed with the preparation of paraffin sections of reproductive organs (usually testis and ovary) and hematoxylin-eosin (HE) stain. The animals were sacrificed by competent staff and the procedures were approved by Jilin University Institutional Animal Care and Use Committee. 3) Make the course fun: The teaching effectiveness would be improved remarkably if the students had fun and enjoy the Histology class from beginning to the end. Therefore, scientific stories such as Nomenclature of Sertoli cells by Italian physiologist Eponym Enrico Sertoli, key discoveries in reproductive biology by Regnier de Graaf, etc, were told in the lecture or discussion. Additionally, in order to give the students the opportunity to apply his or her new knowledge and skills to reinforce learning and build confidence, competing game of Answering and asking Questions about reproductive system was organized frequently in the lecture. 4) Use general principles for good teaching: A designed interesting introduction, begin with what the student knows, move from simple to complex, tell the students how they are progressing, etc, were employed for each lecture planning and implementation. By using above combined strategies, the teaching effectiveness of Animal Histology was enhanced remarkably and the students successfully developed the following abilities: a) a visual understanding of how the four basic tissues interrelate to construct the various organs of animal reproductive system, b) a firm understanding of how structure and function of animal reproductive system relate histologically, c) a mental picture of all of the specimens studied in the laboratory, d) a comprehensive understanding of histological terminology in reproductive system, and e) effective international communication.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

How to Isolate Polysomal RNA to Study Mammalian Early Development.

Sara Scantland, Jean-Philippe Robert-Grenon, Marie-Helene Desrochers, Edward W Khandjian, Marc-Andre Sirard, Claude Robert 1

Pre-hatching development, which encompasses oocyte maturation until blastocyst escape from the zona pellucidae, can be boldly described as two distinct developmental windows, according to the transcriptional potential of the cells. The first window is characterized by transcriptional silence, where cells support protein synthesis by recruiting stored maternal RNAs, whereas in the second interval, the embryonic genome is active and thus capable of producing de novo mRNAs. The presence of large amounts of stored transcripts destined to be either transcribed later on during development or simply sent for decay creates a large background noise that hinders the potential to gain relevant knowledge on the making of pre-hatching development, either through low (real-time RT-PCR) or high throughput (microarray, deep sequencing) methods. We believe that the study of mRNAs in the process of being translated might offer a better perspective of the making of an early embryo. Consequently, we developed a method that enables the isolation of messenger RNAs found to be bound to ribosomes using sucrose gradient fractionation. The main constraint to achieve such an objective is the small amount of starting material. Here, we present the development of a reliable method that enables the survey of the sub-population of mRNAs found in the polysomal fraction using as little as 100 oocytes or embryos. The method utilizes carrier polysomes prepared from a distant species to provide a confirmation of the polysomal nature of the isolated mRNAs. With such an approach, a method is required to eliminate the contribution of carrier RNA on downstream steps. To do so, ribonucleoprotein complexes are cross-linked and the phylogenetic distance between the carrier and the species of interest was proven to almost completely prevent the potential contribution of the carrier on microarray results. The method was tested to confirm its high repeatability by performing a survey of GV stage polysomal mRNAs using microarray hybridization. The mean correlation value between technical replicates was 0.95. We tested the method in a physiological context by comparing the mRNAs found in the polysomal fractions of GV, GVBD and MII stage bovine oocytes. Survey of the identity of the mRNA found in the polysomal fractions was conducted using microarray and quantitative RT-PCR. Translation behaviour of candidates studied by quantitative RT-PCR (C-Mos, Cyclin B1 and CDK1) is correlated with the underlying physiology of oocyte maturation. Polysomal RNA abundance was shown not to correlate with the corresponding protein levels as polysomal synthesis is supplementary to the initial protein content. To our knowledge, this is the first successful isolation of mRNA confirmed to be of polysomal nature and allowing specific candidate studies. Polysomal fractionation provides a novel angle to study early development that may be more insightful than the study of the entire mRNA content.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Reduction of ER Stress Alleviates the Blastomere Apoptosis During Early Development of Porcine Embryos Fertilized In Vitro.

Ji-Su Kim 1, Bong-Seok Song 1, Sun-Uk Kim 1, Young-Hyun Kim 2, Seung-Bin Yoon 3, Seo-Jong Baek 4, Kyu-Sun Lee 5, Young-Kug Choo 6, Deog-Bon Koo 3, Kyu-Tae Chang 1

The efficient functioning of endoplasmic reticulum (ER) is essential for most cellular activities and survival. Recent evidences have shown that ER stress-mediated apoptosis is closely associated with a wide variety of diseases; however, the involvement of ER stress in early embryonic development is largely unknown. Thus, we explored the effect of modulation of ER stress on blastomere apoptosis during the preimplantation development of porcine embryos. After in vitro maturation and fertilization, presumptive porcine embryos were cultured in NCSU23 medium supplemented with 200 µg/ml tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor, or 1 µg/ml tunicamycin (TM), an ER stress inducer, for 6 d at 39°C, 5 % CO2 in air. Compared to control group, interestingly, TM treatment significantly decreased blastocyst formation rate of porcine embryos (27.4% vs. 8.2%, P<0.05), whereas addition of TUDCA into medium improve the blastocyst formation rate (22.2% vs. 32.8%, P<0.05), indicating the role of ES stress as a negative regulator of embryogenesis. From quantitative real time RT-PCR (qPCR), we also determined that TM and TUDCA positively and negatively regulate the expression of XBP-1 mRNA, which is closely associated with blastomere apoptosis of blastocysts. Thus, TUDCA treatment increase the total cell number of blastocysts (32.8% vs. 22.2%, P<0.05) and decrease the number of blastomeres undergoing apoptosis (3.2 vs. 6.0) compared to control embryos. Consistent with the results, qPCR data showed that the expression of anti-apoptotic Bcl-xl gene was greatly increased by treatment with TUDCA in blastocysts by TUDCA treatment, whereas the expression of pro-apoptotic Bax was decreased. Collectively these results suggest that ER stress is actively involved in porcine early embryonic development via modulation of apoptosis and that TUDCA is effective to improve the developmental rate of the porcine IVF embryos.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Relationship Between Embryo Energy Metabolism and Lipid Accumulation.

Mateus Jose Sudano, Daniela Martins Paschoal, Tatiana Da Silva Rascado, Luis Carlos Ona Magalhaes, Leticia Ferrari Crocomo, Joao Ferreira de Lima Neto, Fernanda da Cruz Landim-Alvarenga 1

Over the past decades, several studies aimed to establish a satisfactory media composition to provide in vitro development of bovine embryos. Nowadays, the in vitro production (IVP) of bovine embryo has a commercially successful application. However, the major obstacle associated with the extensive use of this technology is the high sensibility of IVP embryos to cryopreservation. In the literature, the reduced cryotolerance of IVP embryos is frequently associated with the high lipid content present in their cytoplasm. Although, until now is not clear how the lipid accumulation occurs, there is a common sense that it happens from the undefined culture media use which is supplemented with fetal calf serum (FCS); or due to embryo energy metabolism abnormalities that affect mitochondrial function. Both situations lead to a decreased in embryo quality and survival after cryopreservation. The objective of this study was to provide a balance of metabolic pathways and a reduction in lipid accumulation in IVP embryos by: 1) the use of a metabolic regulator, phenazine ethosulfate (PES), a reducer of NADPH electrons, which favors pentose - phosphate pathways and also inhibits the fatty acids synthesis; and 2) decreasing of FCS concentration in culture media. Slaughterhouse ovaries were used to obtain oocytes which were matured and fertilized in vitro. The presumptive zygotes were cultured on SOFaa under different FCS concentrations (0, 2.5, 5 and 10 %) and with the use or not 0.3 mM of PES at two different periods, 60 or 96 h after the beginning of embryo culture. Seven days after fertilization embryo development was assessed. In vivo produced bovine embryos were used as control group. In vitro and in vivo produced blastocysts were stained with Sudan Black B to quantify the lipid content. For statistical analysis, the arcsine transformation was applied to percentage data. If the ANOVA was significant, means were separated by Tukey test, P <0.05. The embryo production was not affected (P>0.05) by the use of PES at 96 h after the beginning of embryo culture. The decrease of FCS concentration to 2.5 % resulted in the same embryo development (P>0.05) as the 5 and 10 % of FCS concentration. The absence of FCS (0%) and the use of PES at 60 h of culture induced a significantly lower (P<0.05) embryo development. The addition of PES, independent of the period, and the decrease of FCS concentration reduced (P<0.05) the number of small, medium and large lipid droplets. But, the in vivo control group embryos showed the lowest (P<0.05) lipid accumulation. The results indicate a possible association of the lipid accumulation with an abnormality embryo energy metabolism; due to mitochondrial dysfunction from FCS supplementation and/or an inability of IVP embryos to metabolize lipid complex through beta-oxidation; which result in an imbalance of reduction-oxidation state with elevated reactive oxygen species. Further studies are underway in order to quantify the embryo oxidative stress and establish an association with lipid accumulation. FAPESP 07/57766-4

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Identification of Genes Associated with the Initial Proliferation of Primordial Germ Cells in the Gonads of Embryonic Rainbow Trout.

James J Nagler 1, Tim Cavileer 1, Sam Hunter 1, Robert Drew 2, Tomo Okutsu 3, Goro Yoshizaki 4

The proliferation of primordial germ cells (PGCs) is the first significant step in vertebrate gametogenesis. Fishes provide a useful model to study the molecular events that coordinate PGC proliferation because PGC mitosis does not begin until after they reach the genital ridge. All the PGCs are concentrated at this point and can be observed and collected for study. The purpose of this study was to quantify the initial proliferation of PGCs in both sexes of rainbow trout, determine whether a significant sex difference in timing of PGC proliferation is obvious, and use microarray data collected during this period to identify genes that could be involved in PGC proliferation. The experiments described in this study were facilitated by the use of pvasa-green fluorescent protein transgenic rainbow trout. The advantage of this transgenic fish is that the green fluorescent protein gene is uniquely expressed in germ cells and permitted PGC isolation by dissection and enumeration in embryos at a time when germ cells numbers were expanding. A steady increase was observed in the number of PGCs counted in the gonads of both female and male embryonic pvasa- green fluorescent protein transgenic rainbow trout, from 300 to 700 degree days (water temperature in °C x number of days). For both sexes, a statistically significant (p<0.05) increase in the PGC number was first noted at 350 degree days. The PGC number, in both sexes, continued to increase until 700 degree days when a 20-fold increase was apparent compared to 300 degree days. No sex-specific difference in the timing of the increase in PGC numbers was notable. A custom microarray based on cDNA libraries created from embryonic rainbow trout gonads (7,670 genes) was used with gonad mRNA samples obtained between 300-700 degree days for gene discovery. A novel bioinformatic approach was used that employed a target gene, gonadal soma-derived growth factor (GSDF; a member of the transforming growth factor- beta superfamily). GSDF, located in genital ridge somatic cells, is known to be involved in PGC proliferation in embryonic rainbow trout gonads. Five other genes were identified that closely correlated (>0.90) with the expression pattern of GSDF between 300-700 degree days, and were not sex-specifically related. These genes were guanine nucleotide binding protein, integral membrane protein 2B, transmembrane protein 47, tyrosine-protein kinase CSK, and the decorin precursor protein. The conclusions of this study are that: 1) the number of rainbow trout PGCs at the genital ridge in both sexes increases dramatically after 300 degree days, and 2) several genes represent strong candidates for involvement in the proliferation of PGCs that form the embryonic gonads of rainbow trout. This research was supported by NIH Grant Number P20 RR16448 from the COBRE Program of the National Center for Research Resources to JN.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Expression Profiling of Fibroblast Growth Factor Receptors (FGFRs) and FGF4, and Their Roles During Pre- and Peri-Implantation Embryonic Development in the Bovine.

Manabu Ozawa, Alan D Ealy 1

The fact that preimplantation development and blastocyst formation occurs in the absence of uterine-derived factors indicates that regulatory molecules controlling embryogenesis are produced by the embryo itself. Several FGFs regulate embryogenesis in various ways in mice and other species, but their functions during embryonic development in cattle are not well described. Four genes encode FGFRs (termed FGFR1-4), and two predominant spliced variant forms (b or c isoforms) exist for FGFR1, 2, and 3 but not FGFR4. The overall aims of this work were to profile expression of FGFR and one embryonic FGF, FGF4, throughout early development and describe the impact of providing a chemical inhibitor to FGFR kinase activity (PD173074) on embryonic development in vitro. The relative transcript abundance of each major FGFR was determined at various stages of embryo development using qRT-PCR. FGFR1, 3 and 4 mRNA concentrations remained low from the zygote to 8-16-cell stage, then increased (P<0.05) at the morula and blastocyst stages. FGFR2 mRNA was evident in zygotes, progressively decreased (P<0.05) in 2-4 and 8-16 cell stage embryos, and then increased (P<0.05) at morula and blastocyst stages. The relative abundance of alternatively spliced forms of FGFR1 and 2 isoforms were also examined. At the morula and blastocyst stages, FGFR1b and FGFR2b mRNA were more abundant (P<0.05) than the c-isoforms for each receptor. Confocal immunofluorescence microscopy indicated that FGFR2 or FGFR4 were present exclusively in trophectoderm (TE) whereas FGFR1 and FGFR3 were found in both TE and ICM. FGF4 mRNA abundance was very low between the zygote and 8-16 cell stages and then increased (P<0.05) at morula and blastocyst stages. Interestingly, FGF4 mRNA abundance decreased (P<0.05) thereafter and was not detected in ovoid and elongating conceptuses collected at day 14-15 post-estrus from superovulated cows. The importance of FGF4 and potentially other embryo-derived FGFs during early embryo development was examined by treatment with 1 uM PD173074 beginning on day 5 post-fertilization (i.e. early morula stage). Exposure to this chemical prevented an increase of interferon-tau expression but did not affect the progression of embryos to blastocysts by day 8. However, the ability of blastocysts to form outgrowths when incubated on Matrigel-coated plates was compromised (P<0.05) by inhibitor treatment. On day 14 post-IVF, only 4.2±2.2% of inhibitor treated blastocysts formed outgrowths whereas 18.3±3.8% of the non-treated blastocysts formed outgrowths. These findings indicate that the expression of each FGFR and FGF4 occurs at a similar time after embryonic genome activation and that some FGFRs are expressed preferentially in TE whereas others are expressed throughout blastocysts. FGFR-mediated signaling does not appear to be essential for blastocyst formation but is important for subsequent outgrowth formation. This project was supported by NRICGP number 2008-35203-19106 from the USDA-CSREES.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Cryotop Vitrification of Bovine Blastocysts after Artificial Shrinkage of the Blastocoele.

Sung-Hun Min, Myeong-Ju Son, Jin-Mo Park, Joo-Hee Hong, Humdai Park, Deog-Bon Koo 1

Freezing of bovine blastocysts has been proposed as a tool to improve the feasibility of cattle production by using embryo transfer technique. However, the low efficiency of frozen-thawed embryos survival and further development is a crucial problem. Thus, we examined the effect of artificial shrinkage before vitrification of bovine blastocyst on the survival rate and further development after thawing. Expanded blastocysts were vitrified after artificial shrinkage, which was performed by puncturing the blastocoele with a pulled pasteur pipet. Artificial shrinkage of the blastocyst was achieved after pushing a pulled pasteur pipet into the blastocoele cavity until it contrated. The shrunken and not shrunken embryos were exposed to cryoprotectant (15% ethylene glycol-15% DMSO and 0.5 M sucrose (Group A) or 20% ethylene glycol-20% DMSO and 0.5 M sucrose (Group B)) for 3 min, 4 min, and 5 min. They were placed in a small volume of vitrification solution and plunged into liquid nitrogen on a cryotop. Then, after thawing, cryoprotectants was diluted in 1.0 M, 0.5 M, 0.25 M, and 0 M sucrose for 1, 3, 5, and 5 min (Group C) or 0.5 M, 0.25 M, and 0 M sucrose for each 5 min (Group D), respectively. Difference in survival rate of blastocyst was found between Group A (67.9%) and B (48.4%). Furthermore, we also found that the survival rate of blastocysts in thawing Group C was higher than that of Group D (62.5% vs 47.3). Under the optimal conditions, overall efficiency of the survival rate of blastocysts in artificial shrinkage group was higher compared with non-artificial shrinkage group (76.7% vs 37.9%, P<0.05). Our results showed that survival rates in cryopreserved expanded blastocysts could be improved by reducing the fluid content. Therefore, we suggest that artificial shrinkage with pulled pasteur pipet is a effective pretreatment technique for the vitrification of expanded bovine blastocysts.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Effect of Heat Shock and Cysteamine During In Vitro Fertilization on Preimplantation Development of Bovine Embryo.

Miki Sakatani 1, Ahmed Zaky Balboula 2, Ken-ichi Yamanaka 1, Masashi Takahashi 1

Summer heat stress is well known to decrease the conception rate in domestic animals by affecting oocyte quality, maturation, fertilization and early embryo development. Under heat stress condition, both of sperm and oocytes are exposed to heat stress during the fertilization. But there are few reports about the effect of heat shock during fertilization. In this study, we investigated the effect of heat shock on membrane damage of sperm and stress levels in zygotes, fertilization rate, embryo development. In addition the effect of cysteamine, an antioxidant thiol, on sperm damage and embryo development under heat-schocked condition was also evaluated. Oocytes derived from the local slaughter house and matured at 38°C for 20 h were used for experiments. Frozen sperm were thawed and live sperm were prepared by 90% percoll and BO solution. Experiment 1: Oocytes and sperm were fertilized at 40°C for 6 h under 5% CO2 as heat shock group. After in vitro fertilization (IVF), putative zygotes removed cumulus cells were cultured at 38.5°C 5% CO2 5% O2 in air. Day 2 and day 7 after IVF, cleavage and blastocysts rates were evaluated, respectively. Sperm damage was evaluated by PI/FDA staining after IVF. Stress status of putative zygotes was analyzed by detecting the level of reactive oxygen species with DCHFDA and hsp70 mRNA expression level. Rates of fertilization and polyspermy were measured at 9 h after IVF by staining the pronuclei with Hoechst 33342. All results of heat shocked group were compared with control group fertilized at 38°C for 6 h. Experiment 2: Sperm and oocytes were fertilized at 40 °C for 6 h with 0, 1, 5 and 10 µM cysteamine to IVF medium (HS). After IVF, putative zygotes removed cumulus cells were cultured at 38.5°C. Sperm damage was evaluated after IVF. Cleavage rate on day 2 after IVF and blastocysts rates on day 7 were also evaluated. Total cell number of day 7 blastocysts was also counted. All results were compared with sperm and embryos fertilized at 38°C for 6 h. All data were analyzed by Student t-test. A P value of less than 0.05 denoted a statistically significant difference. Experiment 1: Heat shock significantly increased the number of PI positive sperm. Fertilization rate was not significantly different, but the rate of polyspermy tended to be increased by heat shock. The levels of reactive oxygen species and hsp70 mRNA expression of zygotes were significantly increased by heat shock. Besides, both cleavage and blastocysts rate were decreased significantly by heat shock. Experiment 2: Under heat shocked condition, supplementation of 10 µM cysteamine to IVF medium significantly decreased the number of PI positive sperm and improved the blastocysts rates compared to HS group without cysteamine. There were no significant differences of blastocysts total cell number. These results indicate that 1) heat shock during IVF inhibits the normal fertilization by increasing the membrane damage of sperm and oxidative stress of oocytes and 2) cysteamine protects the sperm and oocytes by possible reduction of heat-shock induced oxidative stress.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

NOT PRESENTED.

NOT PRESENTED.

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Developmental Impact of the Absence of Single Non-Essential Amino Acids During Mammalian Embryo Culture.

John Crocker 1, Blair McCallie 1, William B Schoolcraft 2, Mandy Katz-Jaffe 1

Amino acids act as energy substrates and biosynthetic precursors and have a role in minimizing stress within an embryo by buffering intracellular pH and regulating cell physiology. Optimal in vitro embryo development has been observed with the addition of non-essential amino acids during early cleavage stages, followed by growth to the blastocyst stage with all 20 amino acids. Amino acid turnover has been proposed as a non-invasive method for selecting embryos for transfer following IVF, with the overall pattern of amino acid depletion predictive of blastocyst development and pregnancy. However, to date, there is limited work describing the direct influence of a single amino acid on embryonic development. In this study, a chemically defined sequential culture medium was used to examine cleavage stage and blastocyst development, blastocyst cell counts, as well as key gene expression profiles following the removal a single non-essential amino acid. Mouse zygotes were collected from superovulated 6 week old outbred (CF1) females following mating. Zygotes were randomly allocated into control and treatment groups with a minimum of three replicate culture experiments performed for each of the 7 treatment groups. A single non-essential amino acid was removed from G1 and G2 sequential culture medium for each treatment group including: glutamate (Glu), glycine (Gly), alanine (Ala), aspartate (Asp), asparagine (Asn), proline (Pro) and serine (Ser). All zygotes were cultured in 20µl microdrops under oil at 37 oC in 5% O2, 7% CO2 with embryo grading performed at 24hr intervals. A total of 1199 embryos were either cultured without Glu (n=152), without Gly (n=160), without Ala (n=198), without Asp (n=236), without Asn (n=151), without Pro (n=104) or without Ser (n=198). Equivalent numbers of control embryos were also investigated with each treatment group. Results showed no significant differences in embryonic development during days 2, 3 and 4 for any single amino acid treatment group compared to controls. However, absence of Asp revealed a significant decrease in the number of embryos that progressed from morula to the blastocyst stage, as well as the total number of blastocysts compared to controls (P<0.05). In addition, the total number of good quality (>3BB) blastocysts was significantly reduced compared to control when Asn was removed from the culture medium (P<0.05). Day 5 blastocysts were fixed for differential staining of total, trophectoderm (TE) and inner cell mass (ICM) cell counts. Results revealed a significant decrease in the total number of blastocyst cells without Gly or without Ala (P<0.05), and a significant decrease in the number of TE cells only when Gly was removed from culture (P<0.05). In regards to ICM cell number, a significant decrease was observed without Glu, Ala, Asp or Asn in comparison with controls (P<0.05). In concordance, quantitative real-time PCR performed on individual day 5 blastocysts cultured either without Gly or without Asn showed a decrease in gene expression for Wnt3a (TE specific) and Oct4 (ICM specific), respectively (P<0.05). In summary, the removal of single amino acids showed no impact during the early cleavage stages of embryonic development. However, significant effects were observed at the blastocyst stage impacting developmental competence, cell numbers and gene expression, depending upon the removal of a specific non-essential amino acid.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Transcriptome Analysis of In Vitro- and In Vivo-Produced Preimplantation Porcine Embryos by 454 Pyrosequencing.

Stephen Tsoi 1, David Hulce 2, Megan Manion 2, Jonathan Liu 2, Walter Dixon 1, George Foxcroft 1, Michael Dyck 1

Currently, our understanding of the molecular events during the development of porcine preimplantation embryos is limited. Increased knowledge in this area will contribute to our understanding of basic reproductive biology, will allow for identification of molecular markers related to embryonic quality, and will facilitate improvement to in vitro embryo production and culture conditions. The objective of this study was to identify transcripts related to the development of in vitro- (IVT) and in vivo (IVV)-produced porcine embryos using the 454-sequencing platform. Two normalized cDNA libraries were generated from pools of IVT and IVV embryos at different developmental stages (oocyte, 2-cell, 4-cell, 8-cell, morula and blastocyst). In order to facilitate the identification of rare transcripts, TRIMMER kit (Evrogen, Moscow, Russia) was used to decrease the prevalence of abundant transcripts and increase the efficiency of 454 sequencing for discovery of rare copies. A preliminary sequence analysis of the IVT and IVV cDNA libraries from a 1/8 plate run on the 454-Genome Sequencer FLX system (Roche) using the Titanium reagent kit yielded 94,628 and 82,582 ESTs, respectively. NextGENe (SoftGenetics LLC) software was used to identify expressed genes from a variety of porcine sequence databases as references. These reference sequence collections were downloaded from Ensembl: Sus_scrofa.Sscrofa9.56.cdna.abinitio.fa, Sus_scrofa.Sscrofa9.56.cdna.all.fa, and Sus_scrofa.Sscrofa9.56.dna.chromosome.*.fa; and from NCBI: rna.gbk. The collection of chromosomes were indexed using the ‘Build Index for WGA’ (whole genome alignment) tool of NextGENe. An average of 20% of expressed genes matched to the above references in ESTs obtained from both normalized cDNA libraries. Differential expression levels for IVT and IVV embryos were compared using the RPKM (reads per kilobase of exon model per million mapped reads) method and mapped to two databases (the NCBI rna.gbk genome and a combined Ensembl abinitio and cdna-all reference). Some commonly expressed genes were identified, and also, unique genes expressed either in IVT or IVV embryos. The remaining uncharacterized transcripts had significant matches against the EST porcine database. We further annotated the transcripts using the non-porcine mammalian protein database and Gene Ontology terms. This newly found EST resource will be valuable for embryo specific microarray development and for comparative mammalian genome analysis. This research was supported by the Natural Sciences and Engineering Research Council (NSERC) and is a part of the activities of the EmbryoGENE NSERC Strategic Research Network.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Inheritable Model of Male Pseudohermaphroditism in Canine Somatic Cell Nuclear Transfer.

T Shin 1, YW Jeong 1, SW Park 1, JJ Kim 1, YW Choi 1, YC Park 1, CJ Moon 2, SH Hyun 3, EB Jeung 3, WS Hwang 1

Normal mammalian sex differentiation depends upon three genetically controlled steps: chromosomal sex determination, gonadal differentiation, and phenotypical sex development. However, two case studies have reported anomalies in the sexual differentiation of mouse and wolf that have occurred during SCNT procedure for unknown reasons. This evokes questions about the early event of reprogramming in SCNT when the chromosomal sex is clearly determined in genetic donor cells, which can never be overcome by technical inflexibilities of sex.During our canine somatic cell nuclear transfer (SCNT) using skin fibroblasts from a male adult as a genetic donor, we report here the birth of one sex reversed cloned dog out of 6 healthy live offspring. Chromosomal, genetic, and histological analyses revealed that the sex-reversed clone has a 78 XY, SRY positive with female external genitalia, which includes an oviduct and a uterus. Interestingly enough, the bilateral testis was in the shape of a hypotrophic ovary and was positioned in the location of normal ovaries. A complete bicornuate uterus was also present. Histological analyses showed that the testis consists of a gonocyte free semiferous tubule-like structure that was stained positive with vimentin, and inhibin alpha, the sertoli cell markers.To study further whether it can be reversible or not, we recloned him, and the result was that the sex reversal condition remained. Thus we believe that we have established the cell line for canine model of inheritable male pseudohermaphroditism and its sex differentiation mechanism. However, we have not observed any opposite sex reversal cases in female genotypes. Further molecular analyses on SRY, SOX-9, MISIIR, AR showed no mutations in their full or partial sequences, which were associated with disorders of sex development in previous reports. In summary, this report describes the first SRY positive XY sex reversal with an almost complete female phenotype including clinical signs; ultrasonographic findings in the abdomen; plasma concentrations of testosterone, oestradiol and progesterone before after administration of GnRH; cytogenetic analysis; PCR analysis for presence of the SRY gene and histology of the genital tract. The results will be discussed with regard to possible causes of the sex reversal.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Genetically-Induced Estrogen Receptor Alpha (ESR1) Overexpression Does Not Adversely Affect Fertility or Penile Development in Male Mice.

John Heath 1, Yazeed Abdelmageed 1, Tim Braden 2, Carol Williams 1, John W Williams 1, Tessie Paulose 3, Isabel H Ochoa 3, Rupesh Gupta 3, Jodi A Flaws 3, Hari O Goyal 1

Previously, we reported that ESR1 overexpression resulting from neonatal exposure to estrogens in rats and wild-type mice was associated with infertility and mal-developed penis characterized by reduced length and weight and abnormal accumulation of fat cells. The objective of this study was to determine if mutant male mice overexpressing ESR1 are naturally infertile or have reduced fertility and/or develop abnormal penis. The fertility parameters, including fertility and fecundity indices, numbers of days from the day of cohabitation to the day of delivery, and numbers of pups per female, were not altered from controls, as a result of ESR1 overexpression. Likewise, penile morphology, including the length, weight, and diameter and os penis development, was not altered from controls. Conversely, weights of the seminal vesicles and bulbospongiosus and levator ani (BS/LA) muscles were significantly (P < 0.05) lower as compared to controls; however, the weight of the testis, the morphology of the testis and epididymis, and the plasma and testicular testosterone concentration were not different from controls. Hence, this study concludes that the genetically-induced ESR1 overexpression in male mice does not adversely affect fertility, penis, testis or epididymis, but significantly reduces weights of the seminal vesicles and BS/LA muscles.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Potential Estrogenic Effect(s) of Parabens at the Neonatal Stage of an Immature Female Rat Mode.

Kyung-Chul Choi, Eui-Bae Jeung 1

This study was performed to examine the estrogenic effects of parabens on hormonal responsiveness and on the morphology of reproductive tissues during a critical developmental stage of female rats. Two hundred immature female Sprague-Dawley rats (n = 10/group) were orally treated with methyl-, ethyl-, propyl-, isopropyl-, butyl-, and isobutylparaben from postnatal day 21 to 40 in a dose-dependent manner based on our previous study [62.5, 250, and 1000 mg/kg of body weight (BW) per day]. A high dose of methyl- and isopropyl paraben (1000 mg/kg of BW per day) resulted in a significant delay in the date of vaginal opening and a decrease in length of the estrous cycle. In measurements of organ weight and body weight, we observed significant weight changes (i.e., decresed in ovaries, kidneys and increased in adrenal glands, thyroid glands, liver) conversely, body weight was not altered following paraben treatment. In all groups exposed to paraben treatment, histological analysis of the ovaries from the immature rats revealed interstitial cell disorders, decreased corpora lutea, an increase in the number of cystic follicles, and thinning of the follicular epithelium, which occurred in a dose-dependent manner. In addition, morphological study of the uterus revealed the myometrial dysplasia such as myometrial hyperplasia in the highest dose of propyl- and isopropyl paraben and in all dose of butyl- and isobutyl parabens. We also observed a significant decrease in serum estradiol and T4 concentrations in methyl-, ethyl-, propyl-, isopropyl-, and isobutylparaben-treated groups (P < 0.01 and 0.05). Taken together, long-term exposure to parabens can produce suppressive effects on hormonal responsiveness and can disrupt the morphology of reproductive target tissues during this critical stage of development in immature female rats.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Developmental Estrogen Exposure Alters Femur Length and Tensile Strength in Mice in Adulthood.

Katherine E Pelch, Stephanie M Carleton, Amy L Schroder, Charlotte L Phillips, Susan C Nagel 1

Estrogens are essential for normal bone growth during development and puberty and they impact and maintain the overall peak bone mass attained in adulthood. Peak bone mass is a major determinant of osteoporosis risk. Previous reports suggest that developmental exposure to diethylstilbestrol (2-3 μg/pup) results in decreased bone length in CD1 mice, but it is unclear how the geometrical changes affected overall bone strength. Additionally, it is unknown how developmental exposure to low doses of diethylstilbestrol or other xenoestrogens affects adult bone strength. To address this, C57BL6/J mice were time mated and implanted with mini-osmotic pumps designed to release a steady amount of diethylstilbestrol (0.1 μg/kg/day), bisphenol a (10 μg/kg/day), ethinyl estradiol (0.01, 0.1, or 1.0 μg/kg/day) or vehicle on gestation day 11. Exposed females were raised to adulthood and sacrificed at 8 or 13 weeks of age and males were collected at 23 weeks of age. Morphology, biomechanical and biochemical properties, were assessed by micro-CT analysis, torsional loading to failure, and hydroxyproline content, respectively. Developmental xenoestrogen exposure altered adult femur morphology. Developmental exposure to ethinyl estradiol (0.1 μg/kg) or diethylstilbestrol increased female adult femur length 2.54 or 3.32%, respectively. Exposure to bisphenol a or the low dose of ethinyl estradiol also tended to increase female femur length, although not significantly. Adult femur length also tended to be increased in males developmentally exposed to bisphenol a. Marrow diameter was decreased 12.9% and cortical bone width increased by 16.0% in diethylstilbestrol exposed males, further indicating that developmental xenoestrogen exposure alters adult bone morphology. Developmental xenoestrogen exposure also decreased femur strength. All doses of ethinyl estradiol tended to decrease female femur tensile strength, with the low dose being significantly different from vehicle. Furthermore, developmental diethylstilbestrol exposure decreased adult male femur tensile strength 26.3% relative to vehicle treated. Although there were no significant differences in collagen content as measured by the hydroxyproline assay, differences in other biochemical properties (e.g. trace minerals) cannot be ruled out. Thus, these results indicate that femurs from mice exposed developmentally to xenoestrogens have altered morphology and are weaker than those of vehicle treated mice. The decrease in strength could be a result of altered morphology or there could be material differences in the bones. Furthermore, these results may highlight the need for close monitoring of men and women that were exposed to diethylstilbestrol in utero as they reach the age when fractures and osteoporosis become prominent. Although diethylstilbestrol is no longer taken during pregnancy, the fetus is still exposed to numerous xenoestrogens. The findings from these small studies suggest that more research on the effects of developmental xenoestrogen exposure on bone is warranted.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Alterations in Gene Expression Levels in Estrogen Receptor Alpha Overexpressing Antral Follicles Treated with Methoxychlor.

Tessie Paulose, Isabel Hernandez-Ochoa, Jodi A Flaws 1

Antral follicles in the ovary are the only follicle type that can ovulate and this is critical for fertility. Besides this, antral follicles are capable of synthesizing and secreting sex steroid hormones including estrogen. When estrogen binds to estrogen receptors (ESRs) in antral follicles, it facilitates the proper development and function of antral follicles. However, estrogenic chemicals in the environment can bind to ESRs, blocking the normal functions of estrogen thereby affecting follicle development. Further, estrogenic chemicals can induce ESRs resulting in an overexpression of ESRs in antral follicles. Overexpression of ESRs has been proposed to result in an increased susceptibility of the antral follicles to estrogenic chemicals. To directly test whether overexpression of ESRs causes antral follicles to be more susceptible to estrogenic chemicals, we developed and validated a mouse model in which ESR alpha (ESR1) is overexpressed in several tissues, including the ovary. We then determined whether ESR1 overexpression increases the susceptibility of the mouse ovaries to the estrogenic chemical methoxychlor (MXC). MXC is an organochlorine pesticide which has been widely used as a model endocrine disruptor. When the antral follicles of control and ESR1 overexpressing mice (ESR1 OE) were treated in vitro with MXC (1-100 µg/ml) or vehicle dimethylsulfoxide (DMSO) for 96 hours, 10 and 100 µg/ml MXC inhibited growth in both genotypes compared to DMSO treatment. Interestingly, the growth of control follicles with 1µg/ml MXC was not different than DMSO treatment, whereas growth of ESR1 OE antral follicles was inhibited compared to controls. Gene expression analysis of Esr1, androgen receptor (Ar) and catalase (Cat) was done using mRNA from the cultured antral follicles. Esr1 and Ar were chosen because MXC is known to bind to ESR and AR while Cat was analyzed because it is an oxidative stress response gene and MXC is known to cause oxidative stress in antral follicles. The data indicate that Esr1 is significantly upregulated in ESR1 OE antral follicles compared to control follicles. However, in control follicles, MXC treatment caused a dose dependent decrease in Esr1 expression while in ESR1 OE follicles, MXC treatment caused a dose dependent increase in Esr1 expression (controls: DMSO=0.19±0.09, MXC1 = 0.12±0.00, MXC10=0.05±0.03, MXC100=0.03±0.03; ESR1 OE: DMSO=5.02±0.98, MXC1=4.43±0.42, MXC10=10.09±1.01, MXC100=29.06±7.08 genomic equivalents (ge); n=3, p≤0.05). Ar was significantly up-regulated in controls and ESR1 OE follicles in the highest treatment group (100 µg/ml MXC) compared to DMSO. However, Ar expression levels in ESR1 OE follicles at this dose were significantly higher compared to control follicles (controls: DMSO=26.89±4.31, MXC100=49.63±7.14; ESR1 OE: DMSO=5.53±2.59, MXC100 = 87.54±8.85 ge; n=3, p≤0.05). Cat was not different in the control follicles treated with DMSO or MXC, whereas in the ESR1 OE follicles, 100µg/ml MXC significantly increased Cat expression compared to DMSO (DMSO=18.86±3.01, MXC100=79.12±16.27 ge; n=3, p≤0.05). Collectively, these data suggest that ESR1 overexpression may increase the sensitivity of antral follicles to MXC-induced slow-growth by altering gene expression levels. Support: NIH R21ES13061, R01ES012893 and an Eli Lilly Fellowship in Toxicology.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Gold Nanoparticles Alter Sea Urchin Development to Pluteus Stage.

Jeremy K Larson, Reinhold J Hutz 1

Engineered nanoparticles have been investigated for potential application of their unique physicochemical properties due to their inherent size. Gold nanoparticles have gained considerable attention for potential medicinal drug delivery, bio-imaging, and diagnostic purposes. However, research exploring the potential untoward effects of gold nanoparticles on embryonic development has yet to be explored. The objective of the present study was to evaluate dose-dependent effects of gold nanoparticles (10 nm) on embryonic development via a sea urchin model (Arbacia Punctulata and Strongylocentrotus purpuratus). It is well-established that vertebrates share specific features of embryonic development with sea urchins (both deuterosomes) and thus the sea urchin has served as a fundamental model of developmental biology for over a century. Additionally, we are able to easily evaluate effects of environmental toxicants in vitro using this model. We hypothesized that gold nanoparticles would inhibit development to the larval pluteus stage. Sea urchins were injected with two aliquots of 100-300 µl 0.5 M KCl to induce gamete release. Thereafter, 100 µl each of sperm and oocyte suspension and 800 µl of artificial seawater (ASW; pH = 8, salinity = 36, and osmolarity = 800 mOsm/kg; Instant Ocean Aquarium Systems, Inc., Mentor, OH) were added to a 1-ml center-well culture dish (Falcon, Becton-Dickinson Labware, Franklin Lakes, NJ, USA) to allow for fertilization at 25 oC in ambient air. After 90 minutes of incubation, the 1 ml of 2-cell embryo suspension was diluted to 5 ml for further use. One-hundred microliter aliquots of 2-cell embryo suspension were added to 900 µl ASW to create either negative control or treatment conditions (2.85x104 particles/ml, 2.85x107 particles/ml, and 2.85x1010 particles/ml ASW). Embryos were incubated for 2, 20, and 48 hours. At each respective time-point, the number of embryos/plutei within three fields of vision of each plate was counted under an inverted microscope (Olympus America Inc., Center Valley, PA), and the relative percentage of each respective stage/total was calculated. A Kruskal-Wallis test revealed a significant decrease in the percentage of embryos attaining pluteus stage at 48 hours in nanogold-treated versus control cultures (n=6; p<0.05). Subsequent individual Mann-Whitney U tests revealed that gold nanoparticle concentrations of 2.85x104 (n=6; p<0.005) and 2.85x1010 (n=6; p<0.02) gold particles/ml ASW significantly inhibited development to pluteus stage relative to control. However, 2.85x107 particles/ml ASW (n=6; p = 0.055) did not significantly decrease development at 48 hours. These results suggest that gold nanoparticles can inhibit development to pluteus stage in A. punctulata and S. purpuratus. No other significant effects of gold nanoparticles on embryonic development (i.e., 2-cell through blastulae) were observed (n=6, p>0.05). Collectively, our studies point to sea urchin embryonic development as a target of gold nanoparticle action, and our model may serve to provide routine evaluation of toxicants in aqueous environments. This research is supported in part by the University of Wisconsin-Milwaukee College of Letters and Sciences.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

NOT PRESENTED.

NOT PRESENTED.

Biol Reprod. 2010 Nov;83(1 Suppl):4.

NOT PRESENTED.

NOT PRESENTED.

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Ionizing Radiation Mediates a Greater Mutagenic Response in Spermatogenic Cells from Middle-Aged and Old Mice Compared to Young Adult Mice.

Guogang Xu, Damon Herbert, Kim Hildreth, Christi A Walter 1

Spermatogenic cells maintain and package DNA that will direct the development of the next generation. A high level of genetic integrity is important for insuring successful reproduction. Genotoxic agents in the environment can exacerbate mutagenesis in the germline and increase the risk of siring children with a genetic disorder. The purpose of the present study was to determine if there is an age-related decline in the ability to respond to a genotoxin. LacI transgenic mice were sham irradiated or irradiated with 1.2Gy of ionizing radiation at 4-, 15- or 26-months-old. The lacI transgenic mouse model was designed to measure in vivo mutagenesis. Approximately 40 head-to-tail copies of lambda DNA are carried by hemizygotes and 80 copies by homozygotes. The lacI gene is inserted into the lambda DNA and serves as the mutation reporter. Spermatogenic cells were collected at 15 and 49 days post irradiation. Pachytene spermatocytes and round spermatids were separated using a StaPut apparatus. High molecular weight DNA was isolated from the cells and packaged with appropriate phage packaging extracts. The phage were then used to infect E. coli engineered to carry lacZ, the beta-galactosidase gene. E. coli was grown as a lawn on agarose containing X-gal which serves as a chromogenic substrate for beta-galactosidase. Mutations in the lacI gene result in expression of the lacZ gene and functional beta-galactosidase produces blue plaques. When the lacI gene is not mutated, it suppresses expression of lacZ and the plaques appear colorless because beta-galactosidase is not made. Blue plaques are cored and re-tested to confirm their mutant status. The infection of E. coli with lambda phage effectively produces single clones of mutated lacI genes that can then be sequenced to identify the mutagenic event. Mutant clones were sequenced by the University of Victoria sequencing facility. Spontaneous mutant frequency was increased in spermatogenic cells recovered from old mice and was particularly evident in the round spermatids obtained from sham irradiated 26-month-old mice. Ionizing radiation resulted in an increased mutant frequency in pachytene spermatocytes regardless of age at 15 and 49 days post irradiation. Mutant frequency was not elevated beyond the spontaneous level in round spermatids obtained from old mice, however there was an increase in mutagenesis in round spermatids obtained from middle-aged mice at 15 and 49 days after irradiation. In conclusion, middle-aged and old mice displayed a greater mutagenic response to ionizing radiation than did young mice. Environmental and therapeutic exposures to ionizing radiation may exacerbate naturally occurring increases in mutagenesis in the spermatogenic lineage as males age.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

NOT PRESENTED.

NOT PRESENTED.

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Effect of "Real-Life" Environmental Pollutant Exposures on Ovarian Development and Function in Sheep.

Beatrice Mandon-Pepin 1, Benoit Loup 1, Elodie Demondion 1, Paul A Fowler 2, Maria R Amezaga 2, Richard M Sharpe 3, Neils P Evans 4, Michelle Bellingham 4, Stewart M Rhind 5, Corinne Cotinot 1

European Union directives authorize the use of sewage sludge in agriculture and regulate its use in such a way as to improve the nutrient content of soils. Grazing animals allow access to grassland three weeks after the application of sludge. Sewage sludge is used as an organic fertilizer, however, it can contain potentially toxic elements, such as heavy metals and organic contaminants (PAHs, PCBs and PCDD/Fs), that can affect soil quality and be transmitted to grazing animals and then to the food chain. Consequently, processed sewage sludge provides a good model to investigate the effects of real-life exposure to complex cocktails of environmental chemicals (ECs), including endocrine disrupting compounds (EDCs). The aim of this study is to determine whether exposure of pregnant ewes and in utero and post natal life exposed adult ewes to theses environmental chemicals (via sewage sludge fertilizer by comparison to inorganic fertilizer), disrupts fetal ovary development leading to defects in the resulting adults. Fetal ovaries (110dpc) and adult ovaries (18 mouths) were collected and subjected to histological assessment and transcriptome analyses by using quantitative real-time RT-PCR on several key genes and 15K sheep oligo-microarray hybridization (Agilent). In the adult female offspring ovaries, the most striking effects were a decrease in numbers and health of ovarian follicles. From molecular biology, transcript levels of the germ cell-specific receptor cKIT was significantly increased (1.4-fold, p=0.032) and mRNA quantities for steroidogenic enzymes tended to be increased (1.8-fold StAR, 2.9-fold, p=0.014 CYP11A1, 2.72-fold CYP19). In the other hand, several transcripts were down-regulated like ESR2 (0.66-fold, p=0.036), the actin-modulator GSN (0.36-fold, p=0.04) and Cdc42EP5 (0.64-fold, p=0.022). Changes in GSN and Cdc42EP5 may have an incidence on establishment of oocyte maturation and developmental competence. Strikingly, switching the mother from control to EC-exposure pastures at conception caused different changes to the fetal ovary (110 dpc), such as a dramatic increase in follistatin in that group only. Whole of theses results show that in utero and/or post natal exposure to a complex cocktail of ECs affects ovarian gene expression that persists into adulthood and may contribute to alter the female reproductive function.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

BIX-01294, an Inhibitor of the Histone Methyltransferase EHMT2, Disrupts H3K9 Dimethylation in the Cleavage-Stage Porcine Embryo.

Kieun Park, Ryan Cabot 1

Dimethylation of the lysine 9 residue of histone protein H3 (H3K9), an epigenetic mark that is closely linked with transcriptionally inactive chromatin, has been shown to undergo a unique pattern of global remodeling during porcine cleavage development. Five enzymes have been shown to possess H3K9 methyltransferase activity. Of these enzymes, G9a (also called EHMT2) has been suggested to serve a key role in mediating dimethylation of H3K9. A small molecular weight molecule, BIX-01294 (Boehringer Ingelheim Pharmaceuticals), has been shown to be a reversible, specific inhibitor of the histone methyltransferase EHMT2. The goal of this study was to determine the role of EHMT2 in controlling H3K9 dimethylation status in cleavage stage porcine embryos. Based on the specificity of BIX-01294, we hypothesized that expose of porcine embryos to BIX-01294 would result in a reduced levels of global H3K9 methylation throughout cleavage development. To determine the effectiveness of BIX-01294 in inhibiting H3K9 dimethylation, in vitro matured porcine oocytes were fertilized and cultured in embryo culture containing a range of BIX-01294 concentrations (0, 0.5, 1.5 and 5 micromolar BIX-01294) medium for up to six days. Embryos were removed from culture at pronuclear, 2-cell and 4-cell stages of development (at 15, 30 and 48 hours post-fertilization, respectively) to assess the abundance of global H3K9 dimethylation by immunocytochemical analysis. After six days of culture total cell numbers in each embryos and embryo morphology were recorded for all embryos in each treatment group. The following data were obtained from three biological replicates. Following immunostaining for dimethylated H3K9, embryos from all treatment groups at pronuclear, 2-cell and 4-cell stages of development were classified as either "positive," "weak," or "negative" based on the amount fluorescent signal in the nuclei. At both pronuclear and 2-cell stages, embryos cultured in the presence of 5 micromolar BIX-01294 show dramatic reductions in the amount of dimethylated H3K9 as compared to controls (0% negative (control) vs 67% negative (5 micromolar) and 0% negative (control) vs 58% negative (5 micromolar) for pronuclear and 2-cell stages, respectively). Interestingly, at the 4-cell stage, all embryos examined possessed some dimethlyated H3K9 (96% positive and 4% weak for control embryos and 69% positive and 31% weak for embryos cultured in 5 micromolar BIX-01294). With regard embryo development six days after fertilization, no embryos cultured in the presence of 5 micromolar BIX-01294 formed blastocyst stage embryos, as compared to 28% of control embryos reached the blastocyst stage (n=138 and 140, for control and 5 micromolar BIX-01294, respectively). The average number of blastomeres was significantly lower in embryos cultured in the presence of 5 micromolar BIX-01294 as compared to controls, with embryo containing an average of 2.3 nuclei and 11.8 nuclei in embryos treated with BIX-01294 and control groups, respectively (control vs 5 micromolar, Dunnett's test, p<0.05). These data show that exposure of porcine embryos to BIX-01294 correlates with loss of H3K9 dimethylation and compromised cleavage development.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Characterization of Epigenetic Modifications Involved in Regulating The Expression of PBEF And TIMP-2 Genes in the Developing Mouse Placenta.

Hong Rye Kim, Rong Xun Han, Yun Fei Diao, Reza K Oqani, Chang Sik Park, Dong Il Jin 1

Our previous studies demonstrated aberrant expression of TIMP-2 and PBEF genes in the placenta of cloned animals. TIMP-2 protein is involved in extracellular matrix degradation and tissue remodeling processes, whereas PBEF is known to be associated with the induction of spontaneous labor. The functions of these two proteins are important for fetal and placental development. Epigenetic programming of the genome by DNA methylation and histone modification ensures appropriate gene expression during the development of the placenta and fetus. Errors in epigenetic reprogramming have appeared frequently in cloned animals, and are reflected in aberrant gene expression, and abnormal patterns of DNA methylation and/or histone modification. To examine the epigenetic modification features that dynamically regulate gene expression in the developing placenta, we analyzed correlations between the epigenetic modification status of PBEF and TIMP-2 genes, and their expression at the mRNA and protein levels. An analysis of DNA methylation using the sodium bisulfite-modified DNA/restriction digest method showed that CpG methylation of TIMP-2 and PBEF genes in the developing placenta (11.5, 13.5, 15.5, and 18.5 dpc) was unrelated to levels of mRNA or protein encoded by either gene. To determine whether the status of histone acetylation determines the degree of gene expression, we conducted ChIP assays on mouse placenta using antibodies against acetylated H3-K9/K14 and H4K5 histones. These assays revealed that H3-K9/K14 acetylation in TIMP-2 and PBEF loci was correlated with gene expression levels at each stage of placentation (11.5, 13.5, 15.5, and 18.5 dpc), whereas H4K5 acetylation was not. These results indicate a role for the epigenetic status of genes in determining expression levels in the developing placenta and suggest that inappropriate epigenetic reprogramming might contribute to abnormal placentas in cloned animals. Moreover, differential expression levels of TIMP-2 and PBEF proteins were likely regulated by histone modification, particularly acetylation of H3-K9/K14, rather than by DNA modification.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

In Utero Exposure to Bisphenol A and Bisphenol A Plus Genistein Effects on Metabolism and Behavioral Responses in Viable Yellow (Avy/a) and Black (a/a) C57Bl6J Mice.

Paizlee T Sieli, Jiude Mao, Eldin Jasarevic, Cheryl S Rosenfeld 1

Bispenol A (BPA) is a compound used routinely to make many plastic items, and it is considered an endocrine disruptor that can act through estrogen and other steroid receptors. Rodents exposed in utero to this compound demonstrate marked behavioral changes and C57 females carrying viable yellow (Avy/a) offspring are more likely to birth yellow coat color offspring. In contrast, concurrent in utero exposure to BPA + genistein results in increased number of brown coat color offspring. Yellow coat color mice develop diabetes and obesity but their darker coat color siblings remain healthy. In these studies, we sought to determine the effects that in utero exposure to BPA has on serum glucose concentrations and behavioral responses in Avy/a and a/a offspring. Six-week-old C57Bl6J (a/a) females were placed on one of four diets: 1) control AIN-93G diet (7% Corn oil diet, Harlan Teklad Diets), or this diet supplemented with 2) 50 micrograms/kg BPA, 3) 50 mg/kg of BPA, 4) 50 mg/kg BPA + 250 mg/kg genistein. Pups were maintained on control diet after weaning and tested weekly for 14 min durations in Activity Pro Open Field Maze (Med Associates, St. Albans, VT), which quantifies spontaneous behavior and anxiety by measuring time mice spend around outer quadrants (thigmotaxis) relative to time spent in center of the maze in addition to measuring velocity and time distance traveled. Offspring were tested with the hole floor board adaptor, which permits assessment of time it takes the mice to acquire food concealed within one of the slots. Weight and serum blood glucose concentrations were measured weekly in offspring. Another aim was to confirm the previous report on epigenetic effects of BPA and BPA + genistein in Avy/a mice, but we currently do not have sufficient numbers of offspring born for definitive conclusions. Results indicate that Avy/a males exposed to 50 micrograms/kg BPA, 50mg BPA, and 50mg BPA + genistein demonstrate lower average serum glucose concentrations than control Avy/a males (50 micrograms/kg BPA: 142.1 ± 6.1; 50 mg/kg BPA: 138.0 ± 8.1; 50mg BPA + 250mg/kg genistein: 154.5 ± 6.6; Control: 172.7 ± 6.1 mg/dl, P<0.05). No differences in serum glucose concentrations were observed in female cohorts or a/a males. Behavioral assessments reveal that a/a offspring born to dams on 50mg/kg BPA diet spent less time at the corners compared to a/a control offspring. Conversely, a/a 50mg/kg BPA offspring spent more time in the center of the maze compared to a/a controls. In the earlier timepoint assessments (26 to 60 days of age), a/a BPA offspring were speedier than BPA + genistein a/a offspring (78.4 ± 13.0 versus 38.2 ± 14.1 cm/sec, P<0.05), but no differences in velocity between the groups were evident in later behavioral trials. In hole floor board behavioral trials, a/a and Avy/a offspring born to dams on 50mg/kg BPA diet exhibited increased time to recover food hidden within one of the holes compared to their counterparts born to dams on BPA + genistein and control diets. These data suggest that while offspring exposed in utero to BPA demonstrate less anxiety and accompanying decreased serum glucose concentrations, learning and memory responses might be hindered in these a/a and Avy/a offspring. Further studies are needed to confirm BPA-induced epigenetic and neurobehavioral responses, but these results provide evidence that in utero exposure to BPA can result in adverse phenotypic changes in mice. Research supported by RC1 ES018195.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Size-Dependent Acquisition of Global DNA Methylation in Oocytes Is Altered by Hormonal Stimulation.

Md Almamun, Rocio Melissa Rivera 1

The normal progression of acquisition of global DNA methylation in oocytes of adult female mice is not known. Assisted reproductive technologies (ART) are commonly used for the treatment of infertility problems. Several studies have shown that ART procedures alter the expression of genes, causing delayed embryonic development, increased abnormal blastocyst formation, pronounced fetal growth retardation and is associated with increased incidence of loss-of-imprinting syndromes. A previous study from our laboratory showed that the maternal pronuclei of one-cell mouse embryos formed after superovulation (SO) had 50% less methylation when compared to those formed after natural ovulation (NO). We hypothesize that oocytes in ovaries of females undergoing a SO scheme will have different levels of global DNA methylation than oocytes from naturally cycling animals. First, we determined the progression of global DNA methylation during oocyte growth in naturally cycling animals and then compared this to the levels of DNA methylation in oocytes from females undergoing a SO scheme. A group of 14 cyclic females were selected at diestrus and divided into seven groups. Two females were sacrificed and collected ovaries at diestrus (0h). Six females underwent a SO scheme while the remaining six did not. In brief, SO females were injected with 5 IU of eCG followed 44 h later by 5 IU of hCG. Ovaries were collected at 24- and 44 h post-eCG and 10 h post-hCG (data not discussed). Ovaries from their NO counterparts were collected at the same time as the SO females. Ovarian sections were stained with an antibody that recognizes methylated cytosines (5MeC). All oocytes in a section were photographed and the largest cross section was used for analysis. Measurements related to oocyte growth were; 1) oocyte size in micrometers, 2) area of germinal vesicle, 3) size and type of enclosing follicle. Global DNA methylation was determined using the background corrected density macro of ImageJ. The 5MeC staining intensity level of the GV was normalized to the staining of the surrounding granulosa cells. For analyses, correlations were made between oocyte size and level of methylation and GV size and level of methylation. These numbers were then compared between similar size oocytes from SO and NO females. Results show that at diestrus the level of methylation increases in a linear manner form 10 to 80 µm diameter. The curve becomes exponential at 24 and 44 h in the NO group but is sigmoid in both SO groups. Analysis of full-grown oocytes (>70 µm) show that global methylation increases from diestrus (0 h) to 24 h and again to 44 h in the NO group but not in the SO groups. Interestingly, the levels of methylation remain low until ~ 40 µm diameter and start increasing exponentially in oocytes > 41 µm. This is coincident with the initiation of the formation of the second layer of granulosa cells. At 44 h, levels of DNA methylation were different in full-grown oocytes of NO vs. SO groups (P < 0.02). These full-grown oocytes are expected to ovulate upon the LH surge. Our results show that acquisition of DNA methylation continues in full grown oocytes until the presumed LH surge and we speculated that this is necessary to attain full developmental competence. On the other hand, SO full-grown oocytes do not attained full methylation before ovulation and this may in part be responsible for the lower developmental competence observed in embryos produced from SO oocytes.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

The Effect of TSA Treatment on Gene Expressions in Porcine Cloned Embryos During Preimplantation Development.

CH Park 1, YI Jeong 1, HJ Lee 1, YW Jeong 1, SH Hyun 2, T Shin 1, WS Hwang 1

It has been proposed that nuclear reprogramming in somatic cell nuclear transferred (SCNT) embryos may be enhanced by after treatment with epigenetic modifiers, such as TSA, scriptaid, or sodium butyrate. The present study was conducted using experiments involving quantitative real-time PCR in order to determine the effect of TSA on the expression pattern of seven genes (OCT4, ID1, H19, NNAT, PEG1, CYTOKERATIN8 and 18) in nuclear transfer (NT) blastocysts treated with/without TSA and in vivo-derived blastocysts. The results showed that the pluripotent genes, OCT4 and ID1, were transcribed at a low level in NT blastocysts than these of in vivo counterparts. There appeared to be a slightly higher expression of these genes in NT blastocysts with TSA treatment compared with untreated ones, but this was not significant. However, the imprinting genes, H19 and NNAT, tended to be highly expressed in both NT blastocysts with and without TSA treatment than in in vivo blastocysts. PEG1 transcripts were nearly equally expressed in all samples. As a trophoblast marker genes, cytokeratin 8 and 18, were transcribed at a low level in NT blastocysts than these of in vivo counterparts. Moreover, these genes were expressed at a similar level in TSA treated and untreated NT blastocysts. Our findings revealed that transcriptional activity of the tested genes, except for PEG1, became high or low in NT blastocysts when compared with in vivo blastocysts. These result indicated these was the differences in gene expression patterns between in vivo and cloned blastocysts, regardless of TSA treatment. Nevertheless, the several genes in NT blastocysts after TSA treatment showed a slight tendency towards the expression patterns of in vivo blastocysts.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Relationship Between Surface Morphology of the Zona Pellucida and Oocyte Health in the Canine.

Matthew O Lunn, Shirley J Wright 1

The zona pellucida (ZP) is the interface between the oocyte/embryo and its environment, acting as a selective barrier that influences oocyte maturation and the surrounding cumulus cells. In contrast to the mouse ZP which is composed of ZP1, ZP2 and ZP3 proteins, the canine ZP has ZP2, ZP3 and ZP4 proteins as in the cow and pig. The difference in ZP composition could lead to structural ZP differences and thus functional differences in the ZP as a regulator of materials entering/leaving the oocyte, and in sperm interaction with the ZP. Information about ZP ultrastructure is limited in dogs. We have developed methods to investigate the surface morphology of the canine ZP using scanning electron microscopy. Previously, our studies showed that the ZP of canine oocytes is heterogeneous with four distinct morphologies: Type I (occurs in 3% of the population), smooth ZP with no or few small (0.5 µm) pores; Type II, fenestrated ZP with regularly spaced pores; Type III, rough and uneven ZP with irregular hollows and pores; and Type IV, rough and uneven ZP with irregular hollows and pores that were filled with stringy filaments. The objective of this study was to determine whether ZP surface structure is correlated to oocyte health. Cumulus-oocyte complexes were isolated by slicing ovaries collected from four dogs of different breeds aged 7 months - 3 years at random stages of the estrous cycle. Only oocytes with a dark homogenous lipid center and at least three layers of cumulus cells were used for the study. After isolation by mechanical stripping, oocytes were immediately stained with propidium iodide (PI) for assessment of the integrity of the plasma membrane. With this technique, an oocyte with a disrupted plasma membrane fluoresces red with fluorescence microscopy. The oocytes were separated into PI-positive and PI-negative categories, and prepared for scanning electron microscopy by fixation in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4 for 1 hr, dehydrated with a graded series of ethanol, critical point dried and sputter coated. All oocytes (n=46) were viewed by scanning electron microscopy, measured to determine their diameter, and categorized by ZP type. Our results showed the diameter of PI-positive oocytes (n=21) was smaller (p<0.05) than PI-negative oocytes (n=25). Three ZP types were observed in the pool of oocytes collected from one dog indicating that the ZP heterogeneity was not due to an artifact of processing for microscopy. The percentage of PI-positive oocytes in each ZP type (I=0%, II=23.8%, III=47.6%, IV=28.6%) was similar to that of PI-negative oocytes (I=0%, II=20.0%, III=48.0%, IV=32.0%). The results indicate that ZP surface morphology under the conditions tested did not correlate to the health of the oocyte from the time of collection. Moreover, since three ZP types were observed in both PI-positive and PI-negative oocytes, it indicates that the oocytes were not degenerating. Taken together, these observations are important because they imply that heterogeneity in ZP structure occurs in vivo. These structural differences may reflect functional differences in the ZP to meet the changing needs for materials entering/exiting the oocyte during meiotic maturation.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Comparison of Vitrification Methods on the Viability of Bovine Immature Oocytes.

Bohsuk Yang 1, Flavia Tuany 2, Rodolfo Rumpf 2, Margot A N Dode 2

Cryopreservation of the female gamete is an important tool for the assisted reproductive technologies. However, because of their large size and marked sensitivity to cooling, cryopreservation of immature oocytes has been very difficult and no suitable results have been reported. The aim of this study was to compare the developmental competence of bovine immature oocytes vitrified by two different vitrification methods. Bovine oocytes obtained from slaughter house ovaries, were select and immediately allocated to three groups: vitrification in open pulled straw (OPS), vitrification in Cryotop (CyT) and non vitrified as a control group (CG). Immature oocytes were vitrified in 20% ethylene glycol, 20% DMSO and 1M sucrose solution and vitrified-thawed oocytes were matured, fertilized and cultured in vitro. Cleavage rate was evaluated on D2 after insemination (pi) and blastocyst rates on D7 and D8 pi. Chi-square analysis was used to compare cleavage and blastocyst rates. Both of CyT and OPS groups presented lower (P<0.05) cleavage (82%, 59% and 43%) and blastocyst rates on D7 and D8 compared to the control group. Although CyT group showed a higher (P<0.05) cleavage rate (59%) then the OPS (43%), the percentage of oocytes that developed to blastocyst stage was not significantly different on D7 (5.5% and 4.5%) and and D8 (5.5% and 5.3%). The results suggest that both vitrification method, OPS and Cryotop, had a deleterious effect on immature oocytes compromising their embryo development. However, even with low rates, cryopreserved immature bovine oocytes were capable of been fertilized and develop to blastocyst.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Effect of Estradiol on Expression of Maternal Determinant Genes During In-Vitro Maturation of Porcine Oocytes.

Nikki Leavins 1, Kosala Rajapaksha 2, Carl Lessard 1

In-vitro produced porcine embryos have limited success when compared to in-vitro produced bovine embryos. A critical step in the in-vitro production of embryos is in-vitro maturation, which allows immature oocytes to become competent. Follicular fluid is an important factor in the maturation of the oocytes which is high in estrogen. Interestingly, estrogen has been shown to both negatively and positively affect oocyte maturation in mice, cows, rabbits, and pigs. This investigation analyzes estradiol's effects on the expression of maternal determinant genes BNC1, NPM2, ZAR1, and TRIM24 on in-vitro matured porcine oocytes. Abattoir derived oocytes were matured for 44 hours at 38.5°C in follicular fluid free TCM-199 maturation medium with either 0 ng/ml (control), 50 ng/ml, 100 ng/ml, or 1000 ng/ml of estradiol or 10% follicular fluid. Each biological replicate is comprised of 40 oocytes collected and denuded from immature (not matured through in-vitro maturation), control group, and a test group. Nuclear maturation rate was evaluated using Lamin/Dapi staining and statistically analyzed with a binomial test for proportions. RNA expression was determined by quantitative real-time PCR (polymerase chain reaction) (Stratagene Mx3005P Real-Time PCR System), using Gapdh as a normalizing gene. BNC1, NPM2, ZAR1, and TRIM24 values of control and test groups were compared to immature groups of the same biological sample to give a fold change value (2-delta delta Ct).The values of the test and control groups from each biological replicate were compared using a Wilcoxon's rank test. There was no significant differences on the nuclear maturation rate between the control and 10% follicular fluid media (89% and 91% respectively; p=0.5), suggesting our system devoid of follicular fluid stimulated the nuclear maturation of porcine oocytes. When different concentrations of estradiol were added in the system without follicular fluid, none of the maternal determinant genes (BNC1, NPM2, ZAR1, and TRIM24) showed a difference the gene expression levels from the control (p≥0.1). Also, these genes did not reveal a change in their expression when follicular fluid was added in the medium (p≥0.2). Neither estradiol nor follicular fluid had an effect when compared to control media, implying that follicular fluid or estradiol is not necessary for upregulation of these maternal determinant genes. Future gene expression studies will investigate if follicular fluid or estradiol affect other genes within the oocyte and also if estradiol may influence other aspects of oocyte competence. This study was funded by the Canadian Animal Genetics Resource Program, Agriculture and Agri-foods Canada.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Global Analysis of Maternal mRNA Translation during Mouse Oocyte Meiotic Maturation.

Jing Chen, Collin Melton, NaYoung Suh, Robert Blelloch, Marco Conti 1

Fully-grown mouse oocytes are transcriptionally silent during meiotic maturation and fertilization. Transcription of the zygotic genome resumes mostly at the two-cell stage of the embryo. Therefore, selective translation of stored maternal mRNAs is critical for oocyte meiotic maturation and early embryonic development. To obtain a genome-wide profile of translational regulation at the oocyte to zygote transition, we have investigated the pattern of mRNA translation by microarray analysis of oocyte polysome-bound mRNAs during critical steps of meiotic maturation. Polysome-bound mRNAs were extracted from GV, MI and MII stage oocytes. After purification, the mRNAs were reverse transcribed and hybridized to Mouse Genome 430.2 array chips. Using this approach we have identified three classes of mRNA translation patterns in maturing oocytes. The largest group includes transcripts which are constitutively translated during the final stages of oocyte maturation (Class I = constitutive transcripts). A second group of transcripts decreases polysome recruitment during maturation (Class II = repressed transcripts). A third class of transcripts includes mRNAs progressively recruited to the polysomes (Class III = activated transcripts). A massive destruction of transcripts occurs during oocyte maturation. The relationship between transcripts stability and translation was assessed by comparison of the current polysome-bound mRNA data with the transcriptome data deposited in the Gene Expression Omnibus (GEO) repository. Among the repressed transcripts, most of them are degraded, suggesting that the stability of transcripts could be a rate limiting step to control translation. However, there are 25% of the transcripts remaining stable while the translation was repressed suggesting there are mechanisms terminating translation without affecting the stability of the transcripts. The mRNA for Cyclin B1, a major component of MPF, is recruited to the polysome during oocyte maturation as measured by both microarray and qPCR analysis, a recruitment that parallels the protein levels. Thus, these findings suggest that usable information on protein synthesis is obtained with this strategy. The behavior of the different classes of mRNAs was linked to enrichment of specific 3'UTR elements in the maternal transcripts. CPE (cytoplasmic polyadenylation element, 5'-UUU(U/A)AU-3'), a well-known translation regulation element, is enriched in the 3'UTR of activated transcripts. Interestingly, in spite of the fact that transcription is silent during oocyte maturation, transcripts coding for proteins involved in chromatin modification and transcription regulation are recruited to the polysome fractions. The recruitment of these transcripts is confirmed by quantitative PCR. In addition, the translation of transcript coding for RNA binding proteins is activated during oocyte maturation. To test the role of these transcripts at the egg to embryo transition, we have downregulated the translation of the protein by specific morpholino antisense oligonucleotide (MO) injection. Preliminary data suggest that translation of these proteins plays an essential role in oocyte maturation. These data demonstrate that components necessary for embryo development are synthesized early during oocyte maturation.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Assessment of Daily Menstrual Blood Loss in Rhesus Macaques.

Shayna Rothlein, Lindsay Ohm, Ov D Slayden 1

Rhesus macaques exhibit 28-day menstrual cycles similar to those of women. Menstruation can be experimentally induced by progesterone (P) withdrawal in estradiol (E2) plus P primed macaques and women. However, little is known regarding daily menstrual blood loss (MBL) in hormone controlled subjects. To fill this gap, we tested the effect of artificially short and extended menstrual cycles on daily MBL in macaques. Our goal was to establish baseline daily MBL in macaques for future studies on novel therapies to suppress menses. Twelve ovariectomized rhesus macaques were used in this study. Animal protocols were reviewed and approved by the ONPRC Institutional Animal Care and Use Committee. The animals were treated with an E2-filled Silastic capsule that produced 100-130 pg E2 /ml in serum. After 14 days of E2 priming the animals received a P-filled capsule that produced 5-7 ng P/ml in serum. Removing the P capsule (leaving the E2 capsule in place) on day 28 induced menses (“normal length” cycles). Short 21-day cycles were created by treating animals with E2 for 14 days followed by E2 + P for 7 days. Extended 35 day cycles were induced by treating animals for 14 days with E2, followed by E2 plus one P implant for 7 days and then E2 plus two P implants for 14 days (producing 10-14 ng P/ml). Daily MBL was quantified by a modified alkaline haematin assay. Menstrual blood was collected daily by tampon. On light menstrual flow days Tampex Pearl "Lite" tampons (1.2 cm diameter), cut transversely at 2.5 cm in length, were placed in the vagina to collect menstrual blood. On heavy menstrual flow days Tampex Pearl "Regular" tampons, 3 cm in length, were used. The cut end of the tampons was sealed with Dow Corning Type A Silastic Medical Adhesive and the original tampon applicators were used to insert the tampons into the animals. The tampons were replaced twice each day on normal flow days and 3 times each day on heavy flow days. After collection the tampons were vacuum dried and mixed with 5% sodium hydroxide in a Stomacher Lab Blender. The resulting alkaline haematin extract was quantified spectrophotometrically at 564nm on Elisa plates. MBL was determined by comparing the alkaline haematin values with levels from similar extracts of whole blood standards (20-600 µl blood). Results were expressed as milliliters (ml) of blood lost. The alkaline haematin technique was highly reproducible with intraassay and interassay coefficient of variation estimates at 4.3 and 6.7, respectively (n=8 assays). MBL values were log transformed to correct for heterogeneity of variance and then subjected to ANOVA. The average total MBL during the menstrual cycle in 11 animals was 5.87 ± 0.6 ml (over 6 days). Menstruation peaked in all the animals on day 3 after P withdrawal. Short menstrual cycles had no significant effect on total MBL compared to normal-length cycles. However, treatment with artificially amplified secretory phases resulted in greater MBL (8.8 ± 2.2 ml/cycle). These animals had a 1 day delay of frank menses and then compensated with significantly heavier MBL on days 3, 4, and 5 after P withdrawal (P<0.05). We conclude that very accurate daily MBL measurements can be obtained from hormone controlled, artificially cycled rhesus macaques and that these animals can provide a useful animal model for evaluating factors that modulate menstrual bleeding. Supported by NIH grants HD18185 and RR000163.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Expression of Calbindin-D28k in Human Endometrium During Menstrual Cycle.

Hyun Yang, Eui-Bae Jeung 1

Endometrium plays a role in embryo implantation during the window of receptivity. A change in endometrial gene expression is required for the development of receptivity. The uterine calcium balance is crucial for physiological function, including smooth muscle contraction and embryo implantation. The location of a cytoplasmic calcium binding protein, calbindin-D28k (CaBP-28k), pertains to a large group of eucaryotic proteins that bind to calcium (Ca2+) via a specific helix-loop-helix structure. Recently, this protein is mainly expressed in brain, kidneys, and pancreas. However, at this time little is understood about the potential role (s) of this calcium binding protein in the human uterus during menstrual cycle. In this study, we demonstrated the expression of CaBP-28k in the human endometrium in different phases of menstrual cycle. During menstrual cycle, significant increased levels of uterine CaBP-28k transcription and translation were observed at proliferative phase and its level fluctuated in the endometrium of uterus. In addition, spatial expression of CaBP-28k was detected by immunohistochemistry. Uterine CaBP-28k was abundantly localized in the cytoplasm of the luminal and glandular epithelial cells during menstrual phases. Taken together, these results indicate that uterine CaBP-28k is abundantly expressed in the uterus, and this calcium binding protein may be involved in reproductive function during the cycle in human.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Nursing During the First 48 h of Life Is Important for Expression of Proteins Involved in Neonatal Porcine Cervical Development at Postnatal Day 14.

Amy-Lynn Frankshun 1, Joseph Chen 1, Lauren A Barron 1, Kathleen M Ferio 1, Dori J Miller 2, Frank F Bartol 2, Carol A Bagnell 1

The first two weeks of neonatal life in pigs is a critical period for reproductive tract programming. Data for the uterus indicate that disruption of gene expression between birth (postnatal day = PND 0) and PND 14 changes the developmental trajectory of these tissues. Colostrum, or first milk, may play a role in maternal programming of neonatal development. The lactocrine hypothesis was proposed as a mechanism whereby bioactive milk-borne factors delivered to nursing offspring affect tissue development. Compared to nursing animals, expression of markers of cervical development in neonatal gilts fed a hormone-free milk replacer from birth is altered by PND 2. The extent to which this 48 h window of sensitivity to milk-borne factors influences subsequent cervical development is unknown. In addition, whether effects of the absence of nursing on the cervical phenotype at PND 2 can be reversed by returning gilts to nursing has not been investigated. Objectives were to determine effects of age at first nursing (birth or PND 2) and both duration and period of nursing on expression of molecular markers of cervical growth, remodeling and apoptosis at PND 14. Targeted proteins included estrogen receptor-alpha (ESR1) and vascular endothelial growth factor (VEGFA), both mediators of porcine cervical growth, matrix metalloproteinase 2 (MMP2), involved in tissue remodeling, and the anti-apoptotic marker BCL2. Gilts (n = 5-11/group) were assigned randomly to one of four treatment groups at birth, in which they were: 1) allowed to nurse ad libitum; 2) pan-fed a hormone-free milk replacer ad libitum; 3) nursed for 48 h from birth and switched to replacer; or 4) fed replacer for the first 48 h of life and switched to nursing. Cervices were collected on PND 14. Protein expression was evaluated by immunoblotting, using actin as a loading control, and quantified by densitometry. Expression of cervical ESR1 and BCL2 proteins, undetectable at PND 0, was induced in PND 14 gilts nursed from birth, but remained undetectable in gilts fed replacer during the first 48 h of life. VEGFA protein, undetectable at PND 0, was detectable on PND 14 in all groups, but was markedly reduced (p < 0.01) in PND 14 gilts fed replacer from birth. Latent MMP2 was detectable at similar levels at birth and on PND 14 in nursed and replacer-fed gilts. Active MMP2, undetectable at PND 0, was induced in both nursing and replacer-fed gilts by PND 14. There were no differences in cervical ESR1, VEGFA or BCL2 protein levels in gilts allowed to nurse continuously for two weeks or for only 48 h from birth. Data support the lactocrine hypothesis for maternal programming of neonatal development, indicating that milk-borne factors are required to support protein expression patterns important for cervical development in the neonatal gilt. Results indicate that the first 48 h of neonatal life constitutes a potentially critical period for lactocrine signaling in porcine cervical tissues. This idea is supported by the fact that effects of disruption of lactocrine signaling on cervical development between birth and PND 2 persisted to PND 14 even when replacer-fed gilts returned to nursing after PND 2. Thus, in the absence of lactocrine signaling from birth, altered expression patterns for morphoregulatory proteins, as observed here, could affect the cervical developmental trajectory with long-term consequences for reproductive performance and health. (Support: USDA-NRI 2007-35203-18098; NSF-EPS 0447675)

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

The Role of pkdrej in Sperm Transport.

Keith A Sutton 1, Melissa K Jungnickel 1, Susan S Suarez 2, Harvey M Florman 1

In mammals the number of sperm introduced into the female tract is orders of magnitude greater than the number of eggs available for fertilization. By the time spermatozoa reach the site of fertilization the ratio of sperm to egg has fallen due to regulation of sperm transport by the female tract. Male mice homozygous for a targeted mutation in pkdrej (pkdrej(tm/tm)) show normal fertility in unrestricted matings but sperm from these animals compete poorly with those of wild type males in vivo. Pkdrej is a member of the polycystin-1 family of genes which encode large 11 pass transmembrane proteins. Mutations in another member of the family, Pkd1, underly the majority of cases of autosomal dominant polycystic kidney disease. Unlike other family members pkdrej expression is limited to the male germline in both mouse and human. In vitro analysis showed that while motility and other aspects of the capacitation program are normal, there is a delay in the acquisition of the potential to undergo a zona pellucida induced acrosome reaction. To examine the basis for the under performance of sperm from these mice, we observed the migration of sperm within the oviducts of hormonally superovulated females mated at the time of ovulation. Using trans-illumination of oviducts to observe the behavior of sperm within, we found apparent differences between the behavior of sperm from wild type males and pkdrej(tm/tm) males. At 1.5 h after mating, sperm from pkdrej(tm/tm) mice showed reduced colonization of the extramural uterotubal junction and lower isthmus, which comprise the site of the sperm storage reservoir. The mutant sperm in this region were mostly hyperactivated and free swimming; whereas, most sperm from wild type males were bound by their heads to the oviductal epithelium. A few wild type sperm were seen beyond the reservoir in the upper isthmus and ampulla (oviducts from 6 mated females), but no sperm from pkdrej(tm/tm) males were found beyond the region of the resevoir (oviducts from 5 mated females). These observations indicate that sperm from pkdrej(tm/tm) males are defective in colonization of the storage reservoir and subsequent movement up the oviduct, which could explain the reduced competitiveness of the mutant males with wild type males for fertilization of oocytes. Supported by NIH 1R03HD062471-01 (S. Suarez), 5R03HD060034-01 (K. Sutton)

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Biochemical Comparison of Follicular Fluid From Mature and Nonmature Follicles in Pigs and Cows.

Stepan Ren 1, Pavla Postlerova 2, Jiri Liberda 2, Tomas Drab 2

Reproduction is complicated process with many crucial steps. One of the most important is the meeting of spermatozoa with oocyte. Oocyte is transported with follicular fluid to the oviduct after ovulation. Follicular fluid (FF) surrounding the oocyte during its follicular development in the ovary contains similar components as plasma. Many enzymes are also present in FF. Their activity depends on the developmental follicle stage and they are involved in the rupture of follicle during oocyte ovulation. We isolated FFs from mature and non-mature follicles of pigs and cows and determined their protein concentration. Protein and glycoprotein compositions of mature and non-mature FF were compared after SDS-PAGE. Moreover, we investigated saccharide structures using lectins on the blots and measured amount of free sugars and sugars bound in glycoproteins. We determined protease and hyaluronidase activities by the substrate zymographies and specific activity of several glycosidases by colorimetric methods. We confirmed that the concentration of FF proteins and sugars depended on the maturation level of the follicle. We found differences mainly in some proteins and glycoprotein saccharide structures between FF of distinct mammalian species. However, no differences in FF components were found between mature and non-mature follicles. Similarly, activity of proteases and hyaluronidases in FF from pigs and cows were only interspecies varied, not dependent on the follicular maturation. At determination of specific activity of glycosidases under physiological conditions, we found increasing activity of specie-specify glycosidases, like alfa-mannosidase in pigs and alfa-fucosidase in cows during their follicular maturation. These higher activities could correlate and participate on sperm release from the oviductal reservoir immediately after oocyte ovulation in specific mammalian species. (This work was supported by grants GACR 303/09/1285, 1M0601 MSMT and 50520701 AVOZ.)

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Urine Sediment Microbes as Part of Reproductive Status: Can PCR be Made More Portable?

Keith Ferguson 1, Kenneth L Campbell 1

As part of an effort to simultaneously assess ovarian, insemination, and microbial status using noninvasively collected urine sediment DNA we explored microbial PCR. To speed the screening process and to begin to move it toward applications in field health evaluations we also tried to multiplex for several microbes and to run amplifications on matrices such as filter paper. Organisms were cultured from pure strains (ATCC) and DNA isolated. Neiserria gonorrhoeae, Lactobacillus acidophilus, Gardnerella vaginalis, Escherichia coli, and Candida albicans were assayed using published and designed primers targeting the: cppB gene, 16S ribosomal gene, 16S ribosomal gene, beta-glucuronidase gene, and heat shock protein (HSP) 90 in the respective species. Both simple and multiplex PCR of known mixed concentrations of genomic DNA were run on a conventional thermocycler. PCR for N. gonorrhoeae, L. acidophilus, G. vaginalis, E. coli, and C. albicans detected the microbes at DNA masses of 500 fg, 100 fg, 3 pg, 5 pg, and 1 pg, respectively. When all species and primers were run as a complete multiplex, cross reactivity was observed; L. acidophilus primers were most promiscuous reacting with E. coli, C. albicans, and human female DNA. E. coli and N. gonorrhoeae primers gave consistent specific signals. New primers will be needed if multiplex assays are to be used. Simple PCR for N. gonorrhoeae was also run on media including cellulose, fiberglass, plastic foam, and on slides with depressions or silicon isolators. The reactions on cellulose, fiberglass and foam were first run in tubes and then run sealed in PCR microplate sealing film. PCR done on glass slides was sealed with mineral oil and a cover slip. The various membranes containing PCR product or 1kb DNA ladders were directly inserted into agarose gels for electrophoresis; DNA easily migrated from sponge, cellulose, and fiberglass. The reactions done on the glass slide or plastic foam showed no signal, probably due to inadequate liquid containment. PCR run on fiberglass in tubes using 10 ng of intact N. gonorrhoeae DNA produced a weak signal. Those on cellulose showed signal with 10 pg of DNA. In contrast, similar PCR done with DNA predigested with HpaII endonuclease which leaves the target sequence intact but cleaves other DNA at multiple points yielded much stronger signals. PCR on cellulose and fiberglass is promising for future field use of these or related membranes. Ideally, these could be run in flat format using a battery operated Peltier thermocontroller. The predigestion of genomic DNA with restriction enzymes prior to amplification is also promising as it makes PCR more efficient and reduces background both of which will increase assay sensitivity. (A Healey Grant (KLC) and an undergraduate research award (KF) from UMass Boston funded this work.)

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Cell-Type Specificity of Interleukins 1alpha and 1beta on Prostaglandin and Plasminogen Activator Production in Bovine Endometrial Cells.

Park Sung-Jai 1, Kim Hyeon-Shup 1, Baek Kwang-Soo 1, Lim Hyun-Joo 1, Kim Tae-Shun 2, Park Choon-Keun 2, Lee Dong-Seok 3

Interleukin (IL)-1alpha is a potent stimulator of prostaglandin production in bovine endometrium, and IL-1 affects plasminogen activator (PA) activity in several types of cells. In this study, we determined the effects of IL-1alpha and IL-1beta on production of the prostaglandins PGF2alpha and PGE2 and on PA activity in cultured bovine endometrial epithelial and stromal cells. We also determined the effects of PGE2 and PGF2alpha on PA activity in these cells. Finally, we used RT-PCR to examine the expression of IL-1alpha, IL-1beta, and IL-1 receptor type 1 (IL-1R) mRNA in cultured bovine endometrial cells. This analysis revealed that IL-1alpha mRNA was present only in the stromal cells, whereas IL-1beta and IL-1R mRNAs were present in both cell types. When cultured cells were exposed to IL-1alpha and IL-1beta at concentrations ranging from 0.006 to 3 nM for 24 h, IL-1alpha and IL-1beta were found to dose-dependently stimulate PGE2 and PGF2alpha production in stromal cells (P < 0.05) but not in epithelial cells. On the other hand, exposure to IL-1alpha and IL-1beta dose-dependently increased PA activity in the epithelial cells, whereas neither stimulated PA production in the stromal cells. When cells were exposed to IL-1alpha and IL-1beta at concentrations ranging from 0.06 to 3nM for 24 h, the two IL-1s differed in their effects on both PGE2 and PGF2alpha production in stromal cells and had significantly differed in their effects on PA activity in epithelial cells. Exposure to PGE2 and PGF2alpha did not affect PA activity in either stromal or epithelial cells (P > 0.05). Taken together, these results suggest the possibility that both IL-1alpha and IL-1beta are produced by the stromal cells, that IL-1beta is produced by the epithelial cells, and that IL-1beta is a far more potent stimulator than IL-1alpha of prostaglandin and PA production in cultured bovine endometrial epithelial and stromal cells.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Molecular Cloning of Porcine Chemokine CXC Motif Ligand 2 (CXCL2) and Mapping to the SSC8.

Jong Gug Kim 1, Gary A Rohrer 2, Dan Nonneman 2, Jeffrey L Vallet 2

Maternal recognition of pregnancy is accompanied by inflammatory responses with leukocytosis and increased levels of cytokines and chemokines. Human trophoblast cells secrete chemokine CXC motif ligand 1 (CXCL1)/Gro-alpha and other chemotactic proteins, while monocytes co-cultured with trophoblast cells secrete very high levels of chemokines including CXCL1. Seminal plasma elicits increased expression of pro-inflammatory cytokines and increased infiltration of inflammatory cells in pigs. CXCL1, CXCL2 and CXCL3 were originally cloned as growth-regulated genes. Due to their role in inflammatory response and growth regulation, these CXCLs may affect fetal survival and development during early pregnancy in pigs. Uterine capacity is a component trait contributing to litter size in pigs. Gene mapping analyses revealed a quantitative trait locus (QTL) for uterine capacity located on chromosome 8. Comparison to the human gene map suggests that the CXCL2/Gro-beta gene may be located near the region of the uterine capacity QTL. Its role in inflammatory response and growth modulating activity makes it a good candidate gene for the uterine capacity QTL. Complete coding sequence of porcine CXCL2 has not been reported previously. The objective of this study was to 1) clone CXCL2 cDNA, 2) map the gene and 3) obtain the gene structure. By RT-PCR using total RNA from the pig placenta, we obtained 948 bp of porcine CXCL2 cDNA. Then by iterative screening of a porcine reproductive tissue cDNA library, we obtained 1086bp CXCL2 cDNA containing the start and stop codons, and poly A tail (GenBank acc. No. AY577905). The predicted 107 amino acid sequence of porcine CXCL2 is 74 % identical to human CXCL2. Using two informative microsatellite markers, SB68 (GenBank accession No. AY577903) and SB69 (GenBank accession No. AY577904), developed from the RPCI-44 male porcine bacterial artificial chromosome (BAC) genomic library, CXCL2 gene was mapped to 65 cM on chromosome 8 within the same region as the previously identified uterine capacity QTL. The positive subclone containing the CXCL2 gene from the porcine BAC library was sequenced. The porcine CXCL2 gene (GenBank accession No. AY578786) is consisted of 4 exons and 3 introns. This is the first report of full coding region of porcine CXCL2 and the gene structure. The finding that CXCL2 mRNA is expressed in the placenta and the gene is mapped within the unterine capacity QTL suggests CXCL2 may play a role during early pregnancy in the pig.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Tumor Necrosis Factor alpha Mediates Luteinizing Hormone Stimulation of Prostaglandin Synthesis in Trout Preovulatory Follicles.

Diego Crespo, Arjan P Palstra, Josep V Planas 1

In vertebrates, ovulation is a complex, multistep process triggered by luteinizing hormone (LH), initiating a cascade of events that result in follicle rupture and oocyte release from the ovary. In line with the notion that ovulation is an inflammatory-like process in mammals, we tested the hypothesis that the pro-inflammatory cytokine tumor necrosis factor alpha (TNFalpha) could affect ovarian function and, specifically, mediate the effects of LH during the preovulatory period in a teleost fish, the brown trout (Salmo trutta). We examined the in vitro effects of coho salmon LH (sLH) on trout preovulatory ovarian follicles and showed that sLH significantly increased follicle contraction and that this effect was blocked by indomethacin (a prostaglandin synthesis inhibitor) and TAPI-1 (an inhibitor of TNFalpha converting enzyme or TACE/ADAM17, that blocks TNFalpha secretion). Furthermore, sLH treatment increased the expression of tnfalpha, tace/adam17 and prostaglandin synthases 1 and 2 (cox-1 and cox-2), as well as the production of prostaglandin F2alpha (PGF2alpha) into the culture medium. In order to further study the possible involvement of TNFalpha as a mediator of sLH in the trout ovary, we incubated trout preovulatory follicles in the presence of recombinant trout TNFalpha (rtTNFalpha). First, rtTNFalpha caused a significant increase in follicle contraction and indomethacin blocked this effect, suggesting a possible involvement of prostaglandins in rtTNFalpha action. Second, rtTNFalpha stimulated the expression of cox-1 and cox-2. Third, rtTNFalpha stimulated the in vitro production of prostaglandin F2alpha (PGF2alpha). Interestingly, PGF2alpha directly stimulated follicle contraction, confirming the results from other studies. In view of these results we propose that TNFalpha is a potential mediator of the effects of sLH in the ovulatory process in trout through its stimulation of the production of PGF2alpha.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Loss of Hoxa-10 Reveals Aberrant Upregulation of H19 Gene Expression During Decidualization: Evidence for a Direct Gene Regulation by Hoxa-10.

Xinghong Ma, Fei Gao, Jennifer Hemingway, Alicia Blackwell, Allison Russie, Sanjoy K Das 1

Uterine decidualization, a prerequisite for successful implantation, is critically controlled by stromal cell proliferation and differentiation. Hoxa-10, a member of AbdB subclass of Hox genes, is highly expressed in decidualizing stromal cells. Female mice lacking Hoxa-10 have severe decidualization defects, primarily due to impaired stromal cell responsiveness to progesterone with respect to cellular proliferation. However, the underlying molecular mechanisms by which Hoxa-10 regulates these events are not well understood. Here, we provide evidence for the first time that the loss of Hoxa-10 causes aberrant upregulation of expression for H19, a developmentally regulated non-coding imprinting gene, in the mouse uterus during decidualization. Our studies revealed that H19 gene expression was undetected in the preimplantation uterus on days 1-4 of pregnancy, either by the wild-type or Hoxa-10 null mice. However, this gene expression was robustly detected at the sites of implantation primarily in the invading trophoblast cells in the wild-type on days 6-8 of pregnancy, while on day 5, a subset of cells in the proliferating endometrial stroma was also positive for the expression. These results are consistent with the lack of H19 gene expression in the decidual bed, as examined by the artificial decidualization on days 7 and 8 of pregnancy. Strikingly, the mice lacking Hoxa-10 exhibit dramatic upregulation of H19 gene expression in the decidual bed, primarily in the antimesometrial location either at the implantation sites or artificial decidualization. Further studies by chromatin immunoprecipitation analysis revealed that Hoxa-10 may regulate H19 gene expression via direct interactions on distinct regions of the H19 gene promoter. One of the regions appears to be interesting since this area also occupies by CTCF, a well recognized regulator for maternal H19 gene expression. Studies are underway to examine further the mechanism of Hoxa-10 mediated H19 gene regulation in the uterine decidual bed. Overall, the induced expression of H19 in Hoxa-10 null mice during decidualization may contribute to the compromised stromal cell proliferation leading to the failure of decidualization. Collectively, our studies provide a novel evidence to indicate that H19 could be a target of Hoxa-10 during decidualization in mice. (Supported by NIH grants HD056044 and ES007814).

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Cytochrome P450 1B1 Genetic Polymorphisms and Menopausal Hot Flashes.

Ayelet Ziv Gal 1, Lisa Gallicchio 2, Jonathan E Beever 1, Jodi A Flaws 1

Menopausal hot flashes (HFs) are experienced by millions of women all over the world. In the US alone, it is estimated that during the next five decades, 27-37 million women may experience menopausal HFs. Despite the impact of HFs on women lives, little is known about the etiology and risk factors for menopausal HFs. Previous studies demonstrated that low estrogen levels, high body mass index (BMI>30 kg/m2), race/ethnicity, smoking, family history and molecular variation in genes encoding the cytochrome P450 (CYP450) enzymes of the estrogen biosynthesis and metabolic pathway are associated with an increased risk of HFs. The documented family history of HFs and associations between some single nucleotide polymorphisms (SNPs) and HFs, led us to further examine whether other SNPs were associated with HFs. Specifically, we examined whether a SNP in the cytochrome P450 1B1 gene (CYP1B1; rs1800440), which results in amino acid substitution of asparagine with serine (N453S), was associated with ‘ever experiencing HFs’. We also examined whether the selected SNP was associated with severity, frequency and duration of HFs. Additionally, we examined whether there was a potential additive effect of combined SNPs either on the same gene (CYP1B1) or other genes and the risk of experiencing any HFs. To complete the study, we genotyped 625 DNA samples (isolated from peripheral blood lymphocytes) that were collected in a cross-sectional study of midlife women from the greater Baltimore area using polymerase chain reactions followed by restriction enzyme digestion and agarose gel electrophoresis. Associations between the CYP1B1 N453S polymorphism, other SNPs and HFs then were statistically analyzed using regression analysis. The results indicate that the polymorphism in CYP1B1 was not significantly associated with having any HFs or the frequency, duration and severity of HFs. However, though the individual genotypes of CYP1B1 and 3beta-hydroxysteroid dehydrogenase (3betaHSD) were not significantly associated with HFs, a combination of genotypes from both loci produced a significant result suggesting a potential additive effect between the loci. More specifically, women who were homozygous and/or heterozygous for the non-ancestral alleles at both SNPs (CYP1B1 and 3betaHSD) had a higher risk of experiencing any HFs compared to women who did not carry both SNPs (OR 1.83; 95% CI 1.04-3.24 after adjusting for race, BMI and smoking). Additional combinations of the selected CYP1B1 polymorphisms with other SNPs in CYP1A1, CYPc17alpha MspA1, CYP19 and another SNP in CYP1B1 (rs1056836; L432V) did not result in any significant association with HFs. These data suggest that the N453S polymorphism in CYP1B1 is not associated with HFs, but it does increase the risk of HFs when a SNP in 3betaHSD is present as well. Supported by NIH R01 AG18400.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Members of the Hippo Pathway Are Regulated in the Uterus During the Menstrual Cycle.

Zuzana Strakova, Szilvia Kruss, Kirsten Morris, Jen Reed 1

The Hippo signaling pathway (referring to the overgrowth phenotype of mosaic flies, also known as Hpo) was originally discovered in Drosophila, but its components are found in mammals, including humans. The role and molecular mechanisms of this new signaling pathway are not yet fully understood. The pathway is important in the regulation of cell contact inhibition, organ size control, and cancer development. It is widely believed that the Hippo pathway is activated to inhibit cell proliferation and to promote apoptosis when an organ approaches its final size, or when cells enter the differentiation phase of development. We have recently reported that TAZ (transcriptional coactivator with PDZ binding motif, also known as WWTR1) is present in the uterine differentiation program during pregnancy. TAZ shares sequence and structural homology with Yes-associated protein (YAP). Both YAP and TAZ were identified as downstream targets of the Hippo pathway. As yet there is no knowledge about the Hippo pathway in the uterus. During the menstrual cycle, uterine endometrium undergoes cyclic changes connected with proliferative, secretory, and apoptotic activities. To determine whether the Hippo pathway is involved in the regulation of these changes, we investigated mRNA of its core members in baboon (Papio anubis) endometrium tissues isolated during proliferative and secretory phases of the menstrual cycle by real-time quantitative PCR (qPCR). We observed that mRNA for kinases Mst1 (also known as Hippo, STK4), Mst2 (also known as STK3), large tumor suppressor (Lats) 1, and Lats2 were significantly up-regulated during the secretory phase of the menstrual cycle compared to the proliferative phase. In contrast, there was no significant mRNA change for up-stream regulator merlin (also known as NF2 or schwannomin), adapter proteins WW45 (also known as Salvator, Sav1) and Mob1 (also known as Mats), or YAP. We also extracted total cell lysates from human uterine epithelial (ECC1, HES) cell lines and human stromal fibroblasts (HuF). Proteins were separated by SDS-PAGE and visualized by Western Blots probed with specific antibodies for members of the Hippo pathway; we detected appropriate molecular weight sizes for merlin, Mst1, Mst2, Lats1, Lats2, TAZ and YAP. The presence of these Hippo pathway members was also confirmed by immunofluorescence, indicating that these cell lines are suitable models for the in vitro study of the Hippo pathway. In summary, our results confirm that members of the novel Hippo signaling pathway are present in the uterus. Moreover, the expression of some of these members changes with the menstrual cycle, suggesting the involvement of the Hippo pathway in the regulation of uterine physiology. (Supported by ARRA NIH HD044713 grant to ZS)

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Endometriotic-like Lesion Formation in Estrogen Receptor Deficient Mice.

Katherine A Burns 1, Karina F Rodriguez 1, Casey E Reed 1, Sylvia C Hewitt 1, Steven L Young 2, Kenneth S Korach 1

Endometriosis, a gynecological disease resulting from the ectopic invasion of endometrial tissue within the peritoneal cavity, affects approximately 10% of reproductive-aged women and is a significant source of pelvic pain, subfertility and/or infertility. Estrogen is an important factor in stimulating the growth of endometriosis, as evidenced by the aberrant levels of the estrogen receptor (ER) α and β in women with this condition. Interestingly, immune responses also appear to be involved as patients with endometriosis often have a higher incidence of autoimmune disorders and have macrophages as the predominant cell type in their peritoneal fluid. In this study, we sought to generate a laboratory model of endometriosis that reflects human pathobiology to facilitate the investigation into the roles of estrogen in causing endometriosis and to study lesion development and progression in an immune-competent host. In our homologous mouse model, uterine tissue from a syngeneic donor mouse expressing a GFP transgene or a Luciferase transgene is dispersed into the peritoneal cavity of a recipient host. Wild-type, αERKO or βERKO mice were used as recipient hosts. Lesions were established under vehicle or estradiol treatment conditions for three weeks (GFP donor) or for three months (Luciferase donor). Mice injected with GFP donor tissue were visualized at necropsy under a fluorescence dissecting scope to observe short term lesion formation, while mice injected with Luciferase donor tissue were imaged weekly for three months for luciferase activity to quantitate lesion growth using the IVIS Spectrum system. Upon necroscopy, similar to what is observed in women, endometrial lesions were found outside the uterine cavity, including the peritoneal wall, rectovaginal septum and intestinal mesentery. Histological examination of these lesions revealed that they are cystic, have extensive blood supplies, and have the appearance of glandular epithelium and stroma, similar to human endometriotic lesions. WT, αERKO and βERKO mice injected with WT uterine tissue all demonstrate lesion establishment indicating establishment of lesions does not require host ERα or ERβ. The size and proliferation of the lesions was dependent on estrogen treatment indicating a potential role for ERα or ERβ in the lesion growth. In comparison to their vehicle-treated counterparts, endometriotic lesions removed from estrogen-treated mice exhibit more extensive glandular structures, are more fluid filled, and often contain more blood and macrophages. Immunohistochemistry confirmed that the removed lesions were donor-derived and also demonstrated that the lesions express ERα and progesterone receptor (PR) in a manner responsive to hormone treatment. Chemokine and cytokine analysis is underway to examine the immune milieu from these mice under the endometriosis-like disease condition. In sum, this syngeneic mouse model of endometriosis established lesions similar to human pathobiology in an immune-competent environment and suggests that lesion formation is independent of host genotype.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

NOT PRESENTED.

NOT PRESENTED.

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Endometrial Expression of Calcium Exchangers, NCKX3 and NCX1, During Human Menstrual Cycle.

Eui-Bae Jeung 1

Plasma membrane sodium/calcium exchangers are an important component of intracellular calcium homeostasis and electrical conduction. NCKX3 (gene SLC24A3) and NCX1 (gene SLC8A1), a potassium-dependent or/and sodium-/calcium exchangers, play a critical role in the transport of intracellular calcium across the cell membrane in exchange for extracellular sodium ions. NCKX3 and NCX1 transcripts are mostly abundant in the brain and smooth muscle, but many other tissues, in particular, the uterus, aorta and intestine also express this gene at lower levels. However, the expression and physiological roles of NCKX3 and NCX1 are largely unknown in human endometrium during menstrual cycle. Thus, we examined the endometrial expressions of NCKX3 and NCX1 at transcriptional and translational levels at different phases of menstrual cycle. Our findings revealed NCKX1 and NCKX3 were differentially expressed during the menstrual cycle. The endometrial expressions of NCKX3 mRNA and protein were enhanced up to 1.5-fold to 2.5-fold at early proliferative phase (EP), mid-proliferative phase (MP) and early secretory phase (ES) compared to other phases, whereas we did not observe a significant alteration in NCX1 expression during human menstrual cycle. A subsequent immunohistochemical analysis revealed that a large number of uterine NCKX3 and NCX1 protein were detected in the cytoplasm of luminal and glandular epithelial cells throughout the menstrual cycle. Taken together these results suggest that human endometrial NCKX3 is abundantly expressed in the endometrium and NCKX3 may be involved in reproductive function during the menstrual cycle in human endometrium.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Expression of TRPV6 (Transient Receptor Potential Cation Channel Subfamily V, Member 6) in the Human Endometrium During Menstrual Cycle.

Yu-Kyung Kim, Eui-Bae Jeung 1

Two highly selective calcium channels at the apical sides of cells, members of the transient receptor potential (TRP) superfamily of ion channels (TRPV6 and TRPV5), are the main calcium ion entry channels. Previously, the location of TRPV6 has been described in the intestine in several species, including human. It is located in the apical brush-border membrane of the intestinal enterocyte where regulates calcium entry into the cell. It is most abundant in the proximal small intestine (duodenum and jejunum) where calbindin and the calcium-pumping ATPase are also found. TRPV6 calcium transporter is also found in the human placenta, pancreas and prostate gland and in some species. However, TRPV6 expression and its potential roles remain to be clarified in the endometrium of human during menstrual cycle. In this study, we employed a human endometrial model to examine the expression of TRPV6, and its potential roles in human menstrual cycle. A significant increase (1.5 fold) in TRPV6 transcript and protein was observed in human uterus at proliferation phase compared to other phases. In addition, the spatial localization of TRPV6 in human uterus was determined by immunohistochemistry. TRPV6 protein was localized in the glandular epithelial cells in menstrual phases and highly expressed in the cytoplasm of the luminal. Overall, these results demonstrate that TRPV6 is abundantly expressed in the uterine human, suggesting that this protein may be involved in reproductive function during the cycle in human.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Roles for Natriuretic Peptide Receptor 2 and a Novel Gene in the HPG Axis and Skeletal Development.

Krista A Geister, Michelle L Brinkmeier, Sally A Camper 1

Development and function of the reproductive system can be affected in individuals with skeletal dysplasia, as is the case in Turner Syndrome and Campomelic Dysplasia. Skeletal dysplasias occur ~1/4000 births, and mutations in ~140 genes account for only about half of the 372 known subtypes. We mapped and characterized two recessive, spontaneous mouse mutants with skeletal dysplasia and fertility problems, peewee (pwe) and chagun (cha). Pwe maps to mouse Chr 4qA5-4qB1 and is caused by a frameshift and premature stop codon in exon 3 of Npr2 causing loss of function of natriuretic peptide receptor 2. Npr2 is a guanylyl cyclase that is activated by C-type natriuretic peptide (CNP), which stimulates endochondral ossification. CNP regulates penile erection and uterine changes during the estrus cycle and pregnancy, and Npr2 is expressed in multiple tissues of the HPG axis, including the brain, pituitary, ovary, uterus, and testis. Thus, reduced male fertility and female infertility of pwe mice may be attributable to requirements for Npr2 in multiple organs. Pwe females exhibit ovarian hypoplasia. Npr2 has been proposed to be a GnRH responsive gonadotroph-specific factor and to regulate follicular atresia in the ovary. Studies are underway to define the role of Npr2 in pituitary gonadotropin production and ovulation. The chagun critical interval maps to mouse Chr 9qF1, which corresponds to human 3q22.2, and contains only 3 known genes that we are screening for mutations. Cha males are infertile due to progressive loss of germ cells and present vacuolated seminiferous tubules similar to Sertoli cell-only syndrome. Identification of the causative mutation in mice will provide a candidate gene for spondyloepiphyseal dysplasia and male infertility of unknown etiology in humans. In addition, these mouse models are invaluable for understanding the cellular mechanisms underlying skeletal dysplasia and infertility and for exploring therapies.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

WITHDRAWN

WITHDRAWN.

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Effect of Relaxin on Bcl2 and Bax Expression in Porcine Oocytes and In Vitro-Produced Embryos.

Jean Magloire Feugang, Scott T Willard, Peter L Ryan 1

Balance between cell death and survival is crucial for cell homeostasis. Its maintenance is challenging in in vitro embryo culture systems, and relaxin hormone which is found in the vicinity of mammalian gametes and embryos has been reported to promote cell proliferation and inhibit apoptosis in reproductive tissues. Thus, the objective of this study was to investigate the ability of relaxin to affect the expression levels of anti- (Bcl2) and pro- (Bax) apoptotic genes in porcine oocytes and embryos. Immature cumulus-oocyte complexes were aspirated from porcine ovaries collected at a local abattoir. Oocytes surrounded with several layers of cumulus cells were selected, washed three times in TL-Hepes-polyvinyl-alcohol (PVA), and rinsed once in maturation medium (TCM199 supplemented with 0.1% (w/v) PVA, 2.8 mM glucose, 0.57 mM cysteamine, 0.91 mM pyruvate, 10 ng/ml EGF, 400 ng/ml FSH) before their transfer to the final maturation medium containing or not 10% (v/v) porcine follicular fluid (pFF), or 40 ng/ml porcine relaxin. After 44h of in vitro maturation (IVM), a subset of matured oocytes was fertilized (IVF) and the presumptive zygotes were cultured (IVC) until the blastocyst stage in a culture medium (NCSU-23+0.4% BSA) containing 0 or 40 ng/ml relaxin. All incubations occurred at 39°C under 5% CO2 in a humidified atmosphere. For gene expression, pools of 20 immature and mature denuded oocytes and corresponding cumulus cells, and 3 blastocysts were collected and snap-frozen for total RNA isolation (microRNAeasy kit, Qiagen). Pure RNA samples were reverse-transcribed to cDNA which were submitted for real-time PCR amplification (qPCR-SYBR GreenER for iCycler, Invitrogen) of Bcl2-xl1 and Bax gene transcripts. The internal control consisted of beta-actin gene, used for the relative expression of data. Sampling was processed in three independent collections and each sample was run in triplicate for qPCR. Mean values were expressed as ratios of Bcl2 on Bax and compared using the Student t test. The threshold of significance was fixed for P values less than 0.05. The results revealed that all samples expressed Bcl2 and Bax gene transcripts. The Bcl2/Bax ratios were always higher in oocytes compared to cumulus cells, irrespective of their maturation stage (P<0.05). The presence of relaxin during IVM significantly decreased the Bcl2/Bax ratio in the cumulus cells (P<0.05), but did not significantly affect this ratio in the oocyte (P>0.05). However, the addition of pFF rich in various components including relaxin, significantly increased the Bcl2/Bax ratio in matured oocytes (P<0.05). Moreover, the presence of relaxin during either IVM or IVC, did not significantly affect the balance of Bcl2 and Bax expression levels (P>0.05). Neither the presence of relaxin during both periods had a significant effect (P>0.05). We concluded that, under current experimental conditions, 40 ng/ml relaxin has a moderate effect on oocyte survival, but appears not to significantly affect the Bcl2/Bax ratio in embryos. This ratio remained in favor of cell survival (higher level of Bcl2 transcripts) having potential benefit on total cell number of blastocyst stage embryos. Research supported by the USDA-ARS Biophotonics Initiative project number 58-6402-3-0120 and the Mississippi Agricultural and Forestry Experiment Station (MAFES).

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Transcriptional Regulation of Lpar3 by Progesterone Receptor.

Fei Zhao, Honglu Diao, Shuo Xiao, Xiaoqin Ye 1

Lpar3 is a gene encoding the third G protein-coupled lysophosphatidic acid receptor. It plays a critical role in embryo implantation, specifically uterine receptivity and embryo spacing. Lpar3 is mainly expressed in the uterine luminal epithelium and is upregulated in the preimplantation uterus. We have further demonstrated that uterine Lpar3 expression is upregulated by progesterone in the ovariectomized mice. In addition, the upregulation of Lpar3 in the preimplantation uterus can be blocked by progesterone receptor (PR) antagonist RU486, but not estrogen receptor (ER) antagonist ICI 182 780, indicating the critical role of PR in regulating Lpar3 expression in the preimplantation uterus. To determine the molecular mechanism of PR in regulating Lpar3 expression, we have examined the Lpar3 promoter (~5kb) using Matlnspector and found five putative progesterone response elements (PRE). PCR primers with restriction sites have been designed for subcloning different Lpar3 promoter regions into a homemade luciferase expression vector, pIRES-LUC2-AcGFP (provided by Dr. Sylvain Bourgoin). Luciferase assay, supershift assay, and chromatin immunoprecipitation are among the techniques that will be employed to explore the molecular mechanism of PR in regulating Lpar3 transcription. This study will provide information about how PR-mediated hormonal signaling regulates a uterine local factor Lpar3 in the establishment of uterine receptivity. (Supported by startup funding from The University of Georgia and funding from the Interdisciplinary Toxicology Program at The University of Georgia)

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Identification of Developmental Competence Related Genes in Mature Porcine Oocytes.

Ye Yuan, Melissa Paczkowski, Rebecca L Krisher 1

Oocyte competence impacts the developmental ability of the subsequent embryo, which contributes to fetal development, as well as long-term health of the offspring. The fact that many in vitro matured oocytes can complete meiosis but fail to produce viable embryos has been explained as poor oocyte cytoplasmic maturation, although determination of the likely molecular mechanisms is lacking. We know that oocyte competence is progressively obtained as females approach puberty. The poor quality of oocytes derived from prepubertal females, compared to adults, suggests that essential processes of cytoplasmic maturation have not yet been fulfilled. The objective of this study is to examine the molecular mechanisms of oocyte cytoplasmic maturation, by comparing candidate gene expression profiles in metaphase II oocytes derived from prepubertal (poor developmental competence) and adult (good developmental competence) pigs. Candidate genes that were examined in this study are related to cholesterol synthesis ( HMGR, SREBP), fatty acid oxidation (ACSL3, ACADL), pentose phosphate pathway (G6PD, PGD), glycolysis (HK1, 4ALD, PGK1, ENO1, PK3, LDH-C, PDHA1), citric acid cycle (IDH3B, ATP5G1) and calcium homeostasis (ATP2B1). Cumulus-oocyte complexes collected from 2-6mm follicles from prepubertal or adult porcine ovaries were matured in vitro in defined Purdue Porcine Medium (PPM; containing 2mM glucose, 6mM lactate, 0.5mM cysteamine, 100ng/ml EGF, 0.01units/ml LH, 0.01units/ml FSH and supplemented with 1% recombumin (Vitrolife, Kungsbacka, Sweden) and 0.2% fetuin) in 5% CO2 in air at 39°C for 44h. Oocytes with a visible polar body were frozen at -80°C in pools of 20. mRNA from 3 pools of oocytes in both age groups (prepubertal and adult) were used to quantify the relative expression of the candidate genes by reverse transcription followed by linear amplification using the MEGAscript High Yield Transcription Kit (Ambion), and real-time PCR was performed in duplicate. Relative quantification of the transcripts was analyzed by REST 2005 version 1.9.12 software (Corbett Research); GAPDH expression was used as a reference. The cholesterol synthesis related gene HMG-CoA reductase (HMGR) was more highly expressed in adult derived oocytes (P < 0.05). Fatty acid oxidation genes acyl-CoA synthetase long-chain family member 3(ACSL-3) and long-chain acyl-CoA dehydrogenase (ACADL), and glycolysis genes fructose 1,6 bisphosphate aldolase (4ALD) and lactate dehydrogenase C (LDH-C) were more highly expressed in prepubertal derived oocytes (P < 0.05). No differences were found in the remaining analyzed transcripts. The differential expression patterns of genes involved in cholesterol synthesis, fatty acid oxidation and glycolysis in good and poor quality porcine oocytes suggest that activities of these metabolic pathways may be important mechanisms involved in oocyte competence.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

The Roles of Syntaxin2/Epimorphin (Stx2/Epim) in Progression of Meiosis During Spermatogenesis.

Yasuhiro Fujiwara 1, Kouyou Akiyama 1, Yuka Asano 1, Takehito Tsuji 1, Junko Noguchi 2, Tetsuo Kunieda 1

repro34 is an ENU-induced mutation in mice showing male-specific infertility caused by defective spermatogenesis. The homozygous mice (repro34/repro34) show abnormal spermatogenesis with multinucleated germ cells, and no mature spermatozoon nor elongated spermatid was observed in the seminiferous epithelium. We have previously identified Stx2/Epim as the gene responsible for repro34. In the present study, we performed detailed phenotypic analysis of the homozygotes in order to reveal the function of Stx2/Epim in germ cell differenciation of mice. Since several types of cells showed multinucleation in the seminiferous epithelium of the homozygotes, we first performed immunohistochemical staining of the testis using gammaH2AX, IZUMO, and HSC70T antibodies to identify the cells which multinucleated. It was clear that multinucleation occurred in various types of cells including pachytene spermatocytes, spermatocytes at MI/MII and round spermatids. Histological analysis of the homozygotes during the first-wave of spermatogenesis confirmed these three types of multinucleation. The increase of Stx2/Epim expression in the testes of mice at about day 18 of the first-wave of spermatogenesis, which we have previously revealed, was in concordance with the timing of the onset of multinucleation at pachytene stage. Since multinucleation occurs in pachytene spermatocytes, in which homorogous chromosome paring occurs, we examined to find any abnormalities in chromosome synapsis. Surface spread chromosome preparation of pachytene spermatocytes (not multinucleated ones) was stained with gammaH2AX and SCP3 antibodies and was observed under a fluorescence microscope. However, normal synapsis of homologous chromosomes was observed in the repro34 homozygotes. Next, we examined Giemsa stained chromosome preparation of metaphase spermatocytes and found abnormal karyotype, containing aneuploidy. Similar abnormality was detected in the surface spread chromosome preparation in the same way as above. Furthermore, immunohistochemical staining of the metaphase spermatocytes with alpha-TUBULIN antibody revealed more than one pair of spindle bodies and abnormal shape of metaphase plates, suggesting that the homozygous germ cells cannot undergo normal cell division at meiosis I and possibly meiosis II as well. In addition, TUNEL assay revealed some multinucleated metaphase spermatocytes, but neither multinucleated pachytene spermatocyte nor round spermatid, were apoptotic, suggeting that multinucleated spermatocytes at metaphase might be excreted from the seminiferous epithelium. On the other hand, some spermatocytes might go through normal meiotic progression, resulting in multinucleated round spermatids. Together, it was revealed that the loss of Stx2/Epim causes a series of abnormalities in meiotic progression, most significantly during metaphase, resulting in a formation of multinucleated germ cells in various stages of spermatogenesis. Thus, Stx2/Epim plays an important role in meiosis during spermatogenesis.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

P53 Is Involved in Developmental Arrest of In Vitro Cultured Porcine Zygotes.

Keon B Oh 1, Ji-Su Kim 2, Ju-Yun Choi 1, Nayoung Ko 1, Deog-Bon Koo 3, Seong-soo Hwang 1, Gi-Sun Im 1, Jae-Seok Woo 1, Soo-Bong Park 1

In vitro culture of porcine zygotes results in marked reduction in developmental competence at pre-implantation stage. The underlying molecular mechanism still remains to be defined, whereas the developmental fail of early embryos is expected to be caused by a number of cellular stresses resulting from in vitro culture. In somatic cells, p53 is a best known protein responding a wide of variety of cellular stress signals, consequently leading to cell cycle arrest and apoptosis. In this study, we investigated whether p53 is involved in developmental arrest of porcine zygotes in vitro fertilized. We performed RT-PCR with a single embryo and found that p53 transcripts were faint in the early stage of embryo, while were detectable in a metaphase II oocyte and a blastocyst. Next, we examined expression level of p53 in arrested embryos at 4-cell stage, which is known as a transition stage initiating zygotic gene expression, essentially required for beyond its stage. Western blot showed that p53 expression was upregulated in arrested 4-cell embryos, compared with normally developing 4-cell embryos. Subsequently, we demonstrated that supplementation of an inhibitor of p53 nuclear translocation, pifithrin-alpha into culture medium resulted in increased development of zygote to blastocyst, and total cell numbers of a blastocyst. Our observations suggest that p53 might regulate early arrest of porcine embryos in response to in vitro culture stress signals. This research was supported by Grant from the Korean Rural Development Administration (BioGreen 21 Program, PJ0071872010).

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

NOT PRESENTED.

NOT PRESENTED.

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Transactivation Function of A-MYB Regulates Testis Specific Ldhc Expression via the CRE Cis Element.

Huanghui Tang 1, Michael Griswold 2, Erwin Goldberg 1

The mechanism regulating gene expression is largely unknown for primary spermatocytes. In spermatids however, there is a specific co-activator ACT (Activator of CREM in testis) directing expression. In our model system a 100-bp core ldhc sequence proved to be sufficient to drive spermatocyte-specific reporter gene expression. The core promoter is composed of two essential elements, a GC-box and CRE site, both conserved in murine and human testes. Here we report that transcription factor A-Myb acts as a co-activator directing tissue-specific expression via the CRE cis element in primary spermatocytes. AMYB was localized in the same cell type as the reporter gene driven by ldhc promoter. AMYB was able to induce Ldhc promoter activity 4-8 folds. However a gel shift assay showed that A-Myb did not bind to the myb consensus sequences in the ldhc promoter. Instead, A-myb interacted with a synthetic promoter containing CRE elements only. A co-activator CBP was able to enhance promoter activity in a dosage-dependent manner. A ChIP experiment confirmed the binding of AMYB to the CRE-containing promoter in vivo. A testis-specific PGK2 gene containing CRE promoter element was also activated 3 folds by A-Myb. These observations support a novel mechanism that the interaction of A-Myb with CREB directs a tissue-specific activation in the primary spermatocyte.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

The Effect of Duration of the Preovulatory Period on Expression of ISG-15, SPP1, and Mx1 mRNA in Endometrial Tissues Collected on Day 17.5 in Pregnant and Cyclic Beef Cattle.

Leandro H Cruppe 1, Lucas A Souto 1, Martin Maquivar 1, Fernanda M Abreu 1, Martin L Mussard 1, Alexandre V Pires 2, Troy L Ott 3, Joy L Pate 3, Michael L Day 1

The aim of this study was to determine the impact of the length of the preovulatory period and consequent influences on preovulatory plasma estradiol concentrations, on expression of mRNA for ISG-15, SPP1 and Mx1 by endometrium collected from beef cows slaughtered 17.5 d after GnRH-induced ovulation. Twenty-one non-lactating cows in 2 replicates (Rep 1, n = 10; Rep 2, n = 11) were pre-synchronized to a common day of estrus and ultrasonographic-guided follicle aspiration was performed 6.2 ± 0.4 d after estrus (d -7 of the experiment). Cows were assigned to receive prostaglandin F2α on either d -3 (high estradiol group, n = 9; HiE), or d -2 (low estradiol group, n = 12; LoE). All cows received GnRH (100 µg) on d 0 (h 0), resulting in a preovulatory period of either 3 (HiE) or 2 (LoE) days. On d 7, cows received no further treatment (Cyclic = C) or were implanted with an embryo (Pregnant = P) resulting in 4 treatments (HiE-P, n = 5; HiE-C, n = 4; LoE-P, n = 8; LoE-C, n = 4). Ovarian ultrasonography was performed on d -7, -3, 0, 2 and 6 to monitor emergence and maximum diameter of the preovulatory follicle and formation of a new corpus luteum after GnRH. Blood samples collected at 12 h intervals from h -72 to h -12 and at h -6 and 0 were used to determine preovulatory concentrations of estradiol. Progesterone concentrations were assessed in samples collected on d -3, -2, 0, 2, 4, 6, 9, 11, 13 and daily to d 17.5. All cows were slaughtered on d 17.5 and endometrium was collected and snap frozen for later analyses. Number of animals in the HiE-P and LoE-P treatments reflects those from which embryos were recovered and for the HiE-C and LoE-C treatments, those cows with luteal phase progesterone concentrations at slaughter. Gene expression was determined using RT-qPCR for ISG-15, Mx1, SPP1 and RPL19. Statistical analyses were conducted using mixed procedures of SAS with appropriate models that included treatment, pregnant/cyclic, Rep and interactions for gene expression, with the addition of d as a repeated measure for hormone concentrations. Estradiol concentrations differed between the HiE and LoE treatments (treatment x h, P <.05), being greater (P<.05) in the HiE than LoE treatment at h 48 (3.2 ± 0.32 and 1.6 ± 0.26 pg/ml, respectively). Concentrations of progesterone from d 0 to d 17 did not differ between the HiE and LoE treatment. Relative amount of mRNA for Mx1 was greater in P than C females, but did not differ between the HiE-P (10.3 ± 2.9) and LoE-P (9.4 ± 2.8) treatments. ISG-15 mRNA was undetectable in 5/8 cows in the C treatments and did not differ between the HiE-P and LoE-P treatments (4.8 ± 1.1 and 5.9 ± 1.5, respectively). For SPP1, amount of mRNA did not differ between the HiE-C and HiE-P treatments, but was was greater (P < 0.05) in the LoE-P (2.8 ± 0.3) than LoE-C treatment (1.3 ± 0.03). In Rep 1, relative amount of SPP1 was greater (P < 0.05) in the HiE-P than LoE-P treatment (4.2 ± 0.5 and 2.5 ± 0.4, respectively) whereas in Rep 2 the inverse tended (P = 0.06) to occur (treatment x Rep, P < 0.05). In conclusion, altered concentrations of estradiol during the preovulatory period did not influence amounts of endometrial mRNA for ISG-15 and Mx1 in pregnant cows, and had variable effects on amounts of mRNA for SPP1.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Role of MSY2 in Global Stability of Maternal mRNA in Mouse Oocytes.

Sergey Medvedev, Hua Pan, Jun Ma, Norman Hecht, Richard Schultz 1

We previously demonstrated that MSY2, an RNA-binding proteins expressed exclusively in oocytes in females, confers stability to mRNAs during the growth phaseand that CDK1-mediated phosphorylation of MSY2 triggers the transition from mRNAstability to instability. To understand better the function of MSY2 in oocytedevelopment, we characterized further oocytes in which the Msy2 gene has been deleted;this deletion results in infertile females. A decrease of ~30% in the number of oocytesobtained from Msy2-/- mice 22-days of age was observed when compared to wild-type. Msy2-/- -deficient oocytes undergo germinal vesicle breakdown, but display severedeficiencies in spindle formation and chromosome segregation, which likely accounts forthe observed infertility. Oocytes from Msy2 -/- mice are slightly smaller in diameter, butreach the same diameter as oocytes in wild-type mice around day 27. The total amount of mRNA is reduced by about 25% in oocytes from Msy2 -/-mice suggesting that the mRNA is less stable. Moreover, co-injection of a Luc mRNA and Egfp mRNA into wild-type and Msy2-/- oocytes oocytes reveals that Luc mRNAstability, as assayed by measuring luciferase activity, is reduced about three-fold in Msy2-/- oocytes. However, Luc mRNA stability is marked enhanced when Msy2 -/- oocytes areco-injected with Msy2 mRNA. This rescue effect is not observed when the ooctyes are injected with an mRNA encoding a mutant form of Msy2 that cannot bind RNA.Transcription profiling of Msy2 -/- oocytes reveals that ~one third of the transcripts are down-regulated in abundance and the affected genes are implicated in transcription, cell cycle, and protein modification. Interestingly, in contrast to wild-type oocytes that are transcriptionally quiescent, transcription persists in Msy2 -/- oocytes, as assayed by BrUTP incorporation, despite a 4-fold increase in Hdac1 expression. This increase in Hdac1 expression is accompanied by a concomitant decrease in global histoneacetylation using acetylation of histone H4 as a proxy, thereby uncoupling histoneacetylation, which is a mark for permissive chromatin, with the global inhibition of transcription that occurs during oocyte growth. The persistence of transcription in Msy2 -/- oocytes may be linked to the observation that cumulus cells are loosely attached to oocytes obtained from Msy2 -/- mice. Results of these studies provide further evidence for a critical role of MSY2 in stabilizing maternal mRNAs in mouse oocytes during the growth phase.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Association Between Single Nucleotide Polymorphisms and Endometriosis and Endometriosis-Associated Infertility.

Lynnette A Ruiz 1, Julie Dutil 1, Abigail Ruiz 1, Jessica Fourquet 1, Sonia Abac 1, Idhaliz Flores 1

Endometriosis is considered a multifactorial disease influenced by genetic predisposition, deficient immune system and environmental factors. The most common symptoms are chronic pelvic pain and infertility. The pathophysiological mechanisms underlying endometriosis and endometriosis-associated infertility are still not well understood. Preliminary studies in our laboratory using gene expression profiling and genetic linkage have resulted in the identification of candidate genes that may play a role in these conditions. The aim of this study was to identify single nucleotide polymorphisms (SNPs) that may be associated to endometriosis and/or infertility in a Puerto Rican population. SNP genotype and allelic frequencies were evaluated in 216 patients and 168 controls, all with surgically confirmed diagnosis. Participants were divided into three groups; GROUP 1- patients with endometriosis vs. no endometriosis, GROUP 2- infertile patients vs. fertile patients and GROUP 3- infertile patients with endometriosis and fertile patients with endometriosis. Allelic frequencies, genotype distribution and multiple regressions were performed. Odd ratios (OR) were adjusted by age. The results show a significant difference between patients with endometriosis and controls with regards to two SNPs (p=0.011, OR=1.3; p=0.026, OR=0.7, respectively). Regarding the infertility status, we found significant differences in two SNPs (p=0.02, OR=2.6 and p=0.005, OR=1.5, respectively). Also, there was a significant difference in another SNP comparing infertile patients with endometriosis vs. fertile patients with endometriosis (p=0.021, OR=1.9). These data suggest that genetic polymorphisms are associated with increased the risk for endometriosis and/or infertility and that a genetic assay could be used to identify patients at risk who could benefit from prevention strategies and close clinical monitoring.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

ADP-ribosylation Factor-Like Protein 4C (ARL4C) Interacts with Galectin-3 During Oocyte Development and Embryogenesis in Rainbow Trout (Oncorhynchus mykiss).

Xiaoming Zhang 1, Caird E Rexroad 2, Jianbo Yao 1

ADP-ribosylation factor-like protein 4 (ARL4) is a GTP-binding protein which belongs to the ADP-ribosylation factor protein (ARF) superfamily of small GTPases. ARL4 has been shown to be mainly related to the development of male germ cells and embryogenesis in mouse. To investigate the role of ARL4 in oocyte and embryonic development in rainbow trout, we cloned the cDNA for rainbow trout ARL4C (RtARL4C), determined its cellular localization, and attempted to identify its interacting proteins in oocytes/embryos. The predicted RtARL4C protein contains 192 amino acids which is highly homologous to other ARL proteins found in mammals and fishes. Biochemical study of purified recombinant RtARL4C protein showed that RtARL4C has GTPase activity without the assistance of GTPase-activating protein. Fluorescence analysis demonstrated that GFP-tagged RtARL4C is primarily localized in the plasma membrane and cytoplasm of RTG cells and predominantly present in the plasma membrane of Hek-293 cells. Analysis of in vivo expression of RtARL4C mutants showed that localization of RtARL4C at the plasma membrane is N-terminal myristoylation dependent. To identify proteins that mediate ARL-dependent signaling in fish development, yeast two-hybrid screening was performed with a rainbow trout oocyte/embryo library. An interaction between RtARL4C and Galectin-3 (RtGal3) was identified. Galectin-3 is a beta-galactoside-binding animal lectin and has pleiotropic biological functions such as cell growth, cell adhesion, cell proliferation, apoptosis and mRNA processing. The interaction between RtARL4C and RtGal3 was validated by GST pull down assay, co-immunoprecipitation and co-localization in mammalian cells and fish cells. The domains necessary for RtGal3 binding were subsequently determined using different RtARL4C mutants. The analysis revealed that interaction of RtARL4C and RtGal3 requires two GTP-binding sites and the N-terminal myristoylation site. The data suggest that RtARL4C may be involved in a signal transduction pathway that regulates membrane trafficking during oocyte development and embryogenesis in rainbow trout.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Effects of VEGF on Porcine In Vitro Fertilized and Somatic Cell Nuclear Transfer (SCNT) Preimplantation Embryos.

Dibyendu Biswas, Yu-Byeol Jeon, Eui Man Jung, Eui Bae Jeung, Sang-Hwan Hyun 1

Present study was conducted to effects of VEGF on porcine IVF and SCNT embryos at different developmental stages. Four set of experiments were done. In first experiment, supplementation of 5ng/ml of VEGF in IVC medium was suitable for porcine IVF embryos development and the blastocyst development rate was significantly (p<0.05) higher (57.73 ± 6.78) compared to control (43.21 ± 10.22) and other groups. The total cell number was significantly (p <0.05) higher (151.85 ±39.77) than other groups and also from the control group (100.00 ± 34.43). In second experiment, when VEGF was added along with different developmental embryonic stages, early stage (day 1-3) and late stage (day 4-7), the blastocyst formation rate and total cell number were significantly (p <0.05) higher (47.71 ± 9.13 and 131.5 ± 20.70, respectively) at late stage compared to control (34.32 ± 7.44 and 85.50 ± 20.41, respectively) and early stage (33.60 ± 5.78 and 86.75 ± 25.10, respectively). There was no significant (p>0.05) difference of blastocyst development and total cell number between whole culture period (day 1-7) and late stage culture period with supplementation of VEGF. In experiment 3, cleavage rate (63.56 ± 15.52) was significantly (p<0.05) higher when SCNT embryos were cultured with VEGF during the whole culture period compared to late stage (4-7 days), but no significant (p>0.05) different between control and early stage of culture period. The Blastocyst formation rate was significantly (p<0.05) higher at late stage culture period (34.40 ± 15.06) with VEGF compare to early stage (16.07 ± 5.01) culture period. There was no significant (p>0.05) different of total cell number among the groups. In experiment 4, using real-time PCR, the expression of VEGF mRNA was detected in all developmental stages of IVF and SCNT embryos but the expression level was varied according to different developmental stages. This data suggested that, VEGF supplementation at late stage of embryonic development might be improve the embryo developmental ability in both IVF and SCNT of porcine preimplantation embryos.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Essential Role of Mammalian Target of Rapamycin (mTOR) in LH-dependent Testosterone Production in Rat Leydig Cells.

Ana Cecilia M Millena 1, Xiaoying Hou 2, John S Davis 2, Shafiq A Khan 1

Development and steroidogenic function of testicular Leydig cells is dependent on luteinizing hormone (LH) secreted by the pituitary gland. The effects of LH on Leydig cells are primarily mediated via the activation of the adenylyl cyclase-cAMP-protein kinase A (PKA) pathway. However, several studies have indicated that LH activates other signaling pathways in Leydig cells which are involved in its actions. The present study was carried out to determine the effects of LH on activation of phosphatidylinositol 3-kinase (PI3K), mitogen-activated protein kinase (MAPK) and mammalian target of rapamycin (mTOR) signaling pathways and their role in steroidogenesis in immature rat Leydig cells in vitro. Leydig cells isolated from 10-day old rats were treated for 1 hr with LH (10 ng/ml) in the presence or absence of inhibitors of PKA (H-89, 10 µM), MEK1/ERK (PD98059, 25 µM), PI3K (LY294002, 10 µM) and mTOR (rapamycin, 20 nM). Based on western blot analysis we observed that LH induced phosphorylation of AKT, ribosomal S6 protein kinase (S6K), ribosomal protein S6, ERK and CREB proteins indicating that LH induces the activation of the PI3K and MAPK pathways in addition to PKA, resulting in the down-stream activation of mTOR signaling. LH induction of mTOR pathway, as indicated by S6K/S6 phosphorylation, was abolished by prior exposure to rapamycin and LY294002. H-89 and PD98059 did not significantly suppress LH-stimulated S6K phosphorylation which suggests that PI3K activated by PKA independent pathway is primarily responsible for mTOR activation. Pretreatment with LY294002 or rapamycin resulted in >80% inhibition of LH stimulated testosterone production indicating an essential role of mTOR in steroidogenesis. To determine the intracellular targets of mTOR that may be responsible for the inhibition in testosterone production, we analyzed the expression of StAR, 3βHSD and other steroidogenic enzymes after treatment with LH in the presence of rapamycin for 4h and 48h. There were no significant changes in the expression of StAR and several other steroidogenic enzymes. However, basal and LH stimulated 3βHSD levels were significantly decreased upon treatment with rapamycin. The site of mTOR-induced inhibition of steroidogenesis (3βHSD) was further confirmed when cells were incubated with 22R-OH-cholesterol or progesterone. Rapamycin blocked conversion of 22R-OH-cholesterol into testosterone but failed to block conversion of progesterone into testosterone. The results indicate that rapamycin decreased androgen production by targeting specifically conversion of cholesterol into progesterone (P450scc and/or 3β-HSD) and not the other members of the steroidogenic pathway. We demonstrate for the first time that PI3K/AKT/mTOR pathway may be required for LH-dependent 3βHSD expression and steroid production in immature rat Leydig cells. Acknowledgements: These studies were supported by the NIH/NCRR/RCMI grant #G12RR003062, NIH P20 grant #5P20MD002285-02, DOD grant W8IWH-08-1-0077, USDA 2006-35203-17249, and the DVA.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Long-Term Hematologic Characteristics of Korean Native Beef Cattle (Hanwoo) Derived from Somatic Cells Nuclear Transfer.

Byoung-Chul Yang, Yeoung-Gyu Ko, Hwi-Cheul Lee, Seongsoo Hwang, Gi-Sun Im, Dong-Kyeong Lee, Na-Yeong Gu, Sang-Hyun Han, Boh-Suk Yang, Soo-Bong Park 1

At birth or after birth in cloned animal health problems such as the large offspring syndrome, malformations and sudden death has already been reported. However, after 175 days did not encounter the same problems as above. Cloning animal in relation to safety and health conditions for long-term monitoring should be continued. The objective of this study was to determine the long term health status for the cloned bovine and their offspring. To collect basic data about the safety of cloned animals and their offspring the RBC, WBC, cytokine were scanned. The age of female clone cow were 83 ± 14 months (9 cow), male clone was 72 months (1) and their offspring were in the 51 ± 14 months (5) had blood tests. Red blood cells, HCT, Hb, MCV, MCH and MCHC levels were 6.93 ± 0.47 x 106 /µl 35.61 ± 1.64%, 13.41 ± 0.55 g / dL, 51.53 ± 2.44 fL, 19.42 ± 0.91 pg and 37.70 ± 0.47 g / dL in the clone cow, and 6.81 ± 0.86 x 106/µl, 35.20 ± 3.20%, 13.15 ± 1.10 g / dL, 51.88 ± 2.26 fL, 19.40 ± 1.08 pg, 37.4 ± 0.67 g / dL in the control. White blood cells, Platelet, S. Neutro, Lymphocyte, Monocyte, Eosinophil levels were 7.35 ± 1.1 ± 103/µl 260.4 ± 81.2 x 103/µl, 32.89 ± 8.99 %, 43.56 ± 8.3%, 11.11 ± 2.8 %, 12.44 ± 3.9% in the clone cow, and 7.47 ± 1.5 x 103/µl, 318.5 ± 93.2 x 103/µl, 28.0 ± 11.31%, 48.0 ± 16.3%, 11 ± 0.8%, 13 ± 5.7 % in the control. Cytokine such as TNF-alpha, IL-6 and IL-10 did not differ between the clone and control cow. In the above data between clone and control were not significant differences and male and their offspring, as well did not differ. Although, between clone and control was no difference in hematological characteristics, these and other factors, but also the long-term research will be needed. These results suggested that health status of cloned animal and their funder is not something to worry about until now.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Colostrum Is Important for Estrogen Receptor-α Expression in Male and Female Neonatal Porcine Reproductive Tissues.

Kathleen M Ferio 1, Amy-Lynn Frankshun 1, Joseph Chen 1, Teh-Yuan Ho 1, Dori J Miller 2, Frank F Bartol 2, Carol A Bagnell 1

Maternal influence on neonatal reproductive development extends beyond the womb into postnatal life. Bioactive factors present in colostrum (first milk) are delivered to offspring via nursing. The lactocrine hypothesis describes a mechanism whereby such milk-borne factors affect development of neonatal tissues. In support of this hypothesis, data for the uterus and cervix indicate an alteration in estrogen receptor-α (ESR1) protein expression in gilts fed a hormone-free milk replacer via gavage from birth when compared to gilts allowed to nurse through postnatal day (PND) 2. Nursing in pigs encompasses a series of discrete behaviors that include sucking the teat. Whether the absence of one component of nursing behavior in replacer-fed gilts (sucking) influences uterine ESR1 protein expression is unknown. The importance of the ESR1 system on growth and function of male reproductive tissues in animals including the pig is also recognized. However, the extent to which consumption of colostrum affects ESR1 expression in these tissues during early neonatal life is unknown. Objectives were to determine if: (1) sucking behavior associated with ingestion of colostrum during nursing influences uterine ESR1 expression in PND 2 gilts; and (2) nursing in neonatal male pigs influences the expression of ESR1 in the testis, epididymis, and prostate at PND 2. In study 1, gilts (n=4-5/group) were randomly assigned at birth (PND 0) to one of three treatment groups: (1) nursed ad libitum; (2) gavage-fed milk replacer (15ml/kg BW/2h); or (3) bottle-fed milk replacer. In study 2, boars (n=6/group) were assigned randomly at birth to be nursed normally or to be pan-fed milk replacer ad libitum. Uteri (study 1), testes, prostates, and epididymides (caput, corpus and cauda; study 2) were collected on PND 2. ESR1 protein expression was evaluated by immunoblotting using actin as a loading control. In study 1, an immunoreactive 51 kDa band corresponding to ESR1 was detected in uterine tissue extracts from gilts allowed to nurse that was not detectable in tissues from gilts consuming milk replacer, regardless of the mode of delivery (gavage or bottle-fed). In study 2, ESR1 was detected in testis, prostate and all three regions of the epididymis in males allowed to nurse normally, but was not detectable in tissues obtained from males fed replacer from birth. Results indicate that mode of milk replacer ingestion (sucking behavior vs orogastric gavage) is not a factor affecting ESR1 protein expression in the neonatal porcine uterus at PND 2. Thus, it is the absence of milk-borne, lactocrine-acting factors in replacer-fed gilts that accounts for changes in the neonatal uterine developmental program observed in milk replacer-fed animals. Data extend the scope of the lactocrine hypothesis by showing that milk-borne factors affect ESR1 expression patterns in reproductive tract tissues of males as well as females. Overall, this study reinforces the importance of lactocrine signaling to support normal developmental programming of neonatal reproductive tract tissues. (Support: USDA-NRI 2007-35203-18098; NSF-EPS 0447675)

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Chronic Hypoxia Enhances Human Endothelial Cell Proliferation via the MEK/ERK1/2 Pathway.

Yizhou Jiang 1, Kai Wang 1, Yan Li 1, Dong-bao Chen 2, Jing Zheng 1

Endothelial cells in vivo reside under physiological hypoxia (~2-8% O2) relative to normoxia at atmosphere (~20% O2). This chronic hypoxia is critical for endothelial function. Previously, we have observed that chronic hypoxia (3% O2, ~20-25 days) enhances VEGF-stimulated proliferation of human umbilical cord vein endothelial (HUVE) cells as compared with normoxia. In this study, we examined if chronic hypoxia-enhanced VEGF-stimulated HUVE cell proliferation was mediated via promoting activation of the MEK/ERK2/1 pathway and VEGFR2. After isolation from normal term placentas, HUVE cells were cultured under hypoxia (37°C, 5% CO2, 3% O2) and normoxia (37°C, 5% CO2, 95% air) respectively. Cell proliferation was assayed using the BrdU ELISA kit. Cells were pre-incubated with or without PD98059 (a MEK inhibitor) for 1 hr, and then treated with VEGF for 16 hr. Activation of ERK1/2 was evaluated by Western blot analysis. Activation of VEGF receptor 2 (VEGFR2) was evaluated by immunoprecipitation of phosphotyrosine-containing proteins, following by immunoblotting with an anti-VEGFR2 antibody. We further confirmed that hypoxia enhanced VEGF-stimulated HUVE cell proliferation. This hypoxia-enhanced VEGF-stimulated cell proliferation was inhibited by PD98059 (10 µM) to a level similar to that under normoxia. PD98059, whereas did not inhibit VEGF-stimulated cell proliferation in normoxia-derived cells under normoxia. The VEGF-induced ERK1/2 phosphorylation (5-10 min) in hypoxia-derived cells (13 fold of control) was much greater than that in normoxia-derived cells (6 fold of control). Total VEGFR2 protein levels were similar between hypoxia-derived and normoxia-derived cells; however, the VEGF-induced VEGFR2 phosphorylation in hypoxia-derived cells (8 fold of control) was much more robust than that in normoxia-derived cells (3 fold of control). In conclusion: 1) Chronic hypoxia greatly promotes ERK1/2 and VEGFR2 activation and 2) The chronic hypoxia-enhanced HUVE cell proliferation is mediated at least partially via the MEK/ERK1/2 pathway and is associated with an increase in VEGFR2 activation. These data indicate that angiogenic factor-induced endothelial function under normoxia and hypoxia could be mediated via different signaling pathways (NIH HL64703 to JZ).

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Dynamic Changes in Secretory Activity of Adrenocortical Cell During the Reproductive Cycle in Female Rats.

Lingmei Pan 1, Sukanya Jaroenporn 2, Gen Watanabe 1, Kazuyoshi Taya 1

During pregnancy and lactation, corticosterone secretory responses of adrenal gland are attenuated in the rats, and this has been explained by reduced responsiveness of corticotropin-releasing hormone and vasopressin neurons in the parvocellular paraventricular nucleus. To further clarify the hyporesponsiveness of the hypothalamo-pituitary-adrenal axis in pregnancy and lactation, the secretory ability of adrenocortical cell for corticosterone and progesterone in response to adrenocorticotropin (ACTH) were compared among cyclic, pregnant day (PD5, 10, 15 and 20) and lactating (LD5 and 15) rats using the primary adrenal cell culture system. The absolute weight of adrenal gland was not different among cyclic, pregnant and lactating rats. The basal secretion of corticosterone in culture adrenal cells was significantly high in PD10, but low in PD20, whereas the basal secretion of progesterone was significantly high in PD 20 as compared with cyclic rats. During lactation period, however, the basal progesterone was significantly high in LD5 than LD 15. Administration of ACTH (10-15 to 10-10M) resulted in a clear dose-dependent increase in corticosterone and progesterone secretion in cyclic rats. On the other hand, no responses of ACTH stimulation was observed in corticosterone and progesterone secretion by cultured adrenal cells from pregnant rats (PD5 to 20) and early lactating rats (LD5). Both the secretion of corticosterone and progesterone from adrenocortical cells in response to ACTH started to recover from LD15, while sensitivity was lower in progesterone compared with corticosterone. These results clearly demonstrated that adrenocortical cells showed a dramatic change in secretion of corticosterone and progesterone during pregnancy and lactation in the rats. These results also demonstrated at the first time that not only the hypothalamus and pituitary gland, but also corticosterone and progesterone secretion is reduced from adrenocortical cells of pregnant and early lactating rats, indicating an important mechanism responsible for minimizing excess amounts of glucocorticoid exposure to fetus and neonates in the peripartum period.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Effect of VEGF on In Vitro Maturation of Porcine Oocytes and Subsequent Developmental Competence after SCNT.

Yu-Byeol Jeon, Dibyendu Biswas, Eui-Bae Jeung, Sang Hwan Hyun 1

This study was designed to investigate the effects of vascular endothelial growth factor (VEGF) on in vitro maturation and subsequent development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). In first experiment, porcine cumulus oocyte complexes (COCs) were matured for 44hr in porcine IVM medium (M-199) supplemented with 0 (control), 5, 50 and 500 ng/ml) of VEGF. Maturation rate among these groups were not significance difference (81.13 ± 2.61; 83.93 ± 1.97; 82.14 ± 4.03; 75.24 ± 2.68, respectively). Total intracellular glutathione (GSH) concentration was significantly increased in case of VEGF (5 ng/ml) treatment group (12.68 ± 0.076) compared to control (10.19 ± 0.66). In second experiment, in vitro matured oocytes with different concentration of VEGF were subjected to electrical activation for parthenogenesis. The blastocyst formation rate was significantly increased in 5 and 50 ng/ml in vitro matured oocytes (50.15 ± 5.34% and 38.72 ± 1.91%, respectively) compared to control and 500 ng/ml group (20.84 ± 5.11% and 26.44 ± 3.18%, respectively) but no significant different between 5 and 50 ng/ml group. There was no significant different in regards to cell number between the treatments groups (83.21 ± 4.89, 78.16 ± 6.15, 78.48 ± 6.53) but there were significant difference than control (56.91 ± 4.78). In third experiment, in vitro matured oocytes with 5 ng/ml of VEGF were subjected to SCNT. The highest (P<0.05) blastocyst formation rate (9.35 ± 1.1) was observed in case of VEGF supplemented matured group compared to control (4.64 ± 0.78) after SCNT. In conclusion, addition of VEGF at 5ng/ml during IVM could improve the developmental potential of PA and SCNT of porcine embryos through increase the intracellular GSH level during oocytes maturation.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Global Methylation in Placental Tissues During Early Pregnancy in Sheep.

Anna T Grazul-Bilska, Mary Lynn Johnson, Pawel P Borowicz, Robert Wroblewski, Dale A Redmer, Lawrence P Reynolds 1

Normal placental development is critical for placental function and thus for normal embryonic and fetal growth and development. Numerous factors, including those from the environment, or from the application of assisted reproductive techniques (ART) are known to affect embryonic development. In fact, it has been shown that embryos from ART frequently exhibit decreased cell proliferation and/or altered methylation patterns that may contribute to their high rate of loss after transfer. DNA methylation plays an important role during normal and abnormal embryonic development. However, very little is known about global methylation of placental tissues during early pregnancy in any species. To determine global methylation [measured by expression of 5-methyl-cytosine (5mC) protein and DNA methyltransferase (Dnmt)1 mRNA] and cell proliferation (based on Ki67 protein expression, a marker of proliferating cells) in normal placenta, uterine tissues were collected on days 14, 16, 18, 20, 22, 24, 26, 28, and 30 after natural mating (n = 5-6/day) and on day 9-11 after estrus (n = 5; non-pregnant controls). In addition, the crown-rump length of each fetus was measured. Cross sections of uterine tissues were immersion-fixed in Carnoy's solution, embedded in paraffin, and sectioned. Uterine tissue sections were immunostained to detect 5mC and Ki67 followed by image analysis of fetal membrane (FM) areas. Moreover, caruncular (CAR, maternal placenta) tissue and FM were collected and snap-frozen for mRNA extraction followed by determination of Dnmt1 mRNA expression using quantitative real time RT-PCR. The length of the fetus increased (P<0.0001) ~3-fold (from 6 to 19 mm) from day 20 to day 30 of pregnancy. Throughout early pregnancy, 5mC and Ki67 were detected in placental tissues and localized to the cell nucleus. Labeling index (% of proliferating cells) was 20.7±1.5% (range = 17 to 26%), and percent of nuclear area occupied by positive 5mC staining in FM cells was 10.5±1.0% (range = 9 to13%); neither measurement changed in FM throughout early pregnancy. Regression analysis of LI in FM demonstrated a linear decrease (R2 = 0.110, P=0.055) from day 18 to day 30 of early pregnancy. Expression of Dnmt1 mRNA tended (P=0.12) to increase ~2-fold from day 16 to day 30 in FM and tended (P=0.06) to be greater ~0.5-fold on day 18 of pregnancy than in non-pregnant controls in CAR. Regression analysis of Dnmt1 mRNA expression in FM demonstrated a linear increase (R2 = 0.173, P=0.002) from day 16 to 30 of early pregnancy. These data indicate that: 1) 5mC is expressed in FM tissues but its expression is unchanged, 2) LI in FM and the rate of fetal growth is very high, and 3) expression of Dnmt1 does not change substantially in FM or CAR tissues during early pregnancy. Thus, a lack of substantial change in global methylation indicates that the pattern of methylation in placenta is already established and not changing throughout early pregnancy. This more complete description of early placental methylation and cell proliferation provides a foundation for determining whether placental development is altered in compromised pregnancies and causes inadequate embryonic development. Supported by USDA grant 2007-01215 to LPR and ATGB, NIH grant HL64141 to LPR and DAR, and P20 RR016741 from the INBRE program of the National Center for Research Resource.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Glucose, Amino Acids, and Their Transporters in Pig Uteri, Conceptuses, and Placentae.

Greg A Johnson, Haijun Gao, Xilong Li, Morgan L Halvorsen, Guoyao Wu, Robert C Burghardt, Fuller W Bazer 1

Embryonic and fetal development in pigs requires glucose and select amino acids, particularly arginine (Arg). We have observed a >10-fold increase of combined glucose and fructose in uterine flushings between Days 12 and 15, and Arg increases from 0.1 to 4.1 mM in allantoic fluid between Days 30 and 40 of pregnancy, an unusually high abundance of Arg for any biological fluid. Therefore, we examined glucose, amino acids, the facilitated glucose transporters SLC2A1-4, the sodium dependent glucose transporters SLC5A1 and SLC5A11, and the amino acid transporters SLC7A1-3, SLC7A8, SLC6A9, and SLC38A1 in a series of three experiments. In Exp 1, gilts were hysterectomized on Day 9, 12, or 15 of the estrous cycle, or Day 9, 12, 15, 20, 25, 30, 35, 40, 50, 60, or 85 of pregnancy. Glucose, Arg, leucine (Leu) and glutamine (Gln) increased in uterine flushings with day of the cycle and pregnancy, but only Arg was greater for pregnant than cyclic gilts on Days 12 and 15. SLC2A3, SLC5A11, SLC7A1, and SLC7A2 mRNAs were not detectable by in situ hybridization in conceptus or uterine tissues. Expression patterns for SLC2A1 and SLC2A4 were similar, but SLC2A4 was more abundant in conceptuses (embryo/fetus and associated extraembryonic membranes) and in uterine luminal epithelia (LE) from Days 15 through 80. SLC2A2 mRNA was abundant in conceptuses from Days 12 to 40, decreased to Day 50 and then increased and was maintained specifically in placental areolae to Day 85. SLC5A1 mRNA was only expressed in the LE between Days 9 and 15 of pregnancy. SLC7A3, an Arg transporter, increased in the entire chorion between Days 25 and 30, and expression was maintained to Day 85. In Exp 2, cyclic gilts were injected daily (Days 11-14) with 17beta-estradiol benzoate (i.m.) or vehicle and hysterectomized on Day 15 or 90 of pseudopregnancy. The flushings (109±24 ml) contained significant amounts (µmol) of glucose (14.01±3.95), Arg (9.05±1.96), Gln (4.95±1.45) and Leu (2.46±0.77). SLC2A1, SLC2A4, and SLC4A1 mRNAs were expressed in the LE of pseudopregnant gilts. In Exp 3, gilts were ovariectomized on Day 12, injected daily with (P4;i.m.) or vehicle for 28 days, and hysterectomized on Day 40. Values (mean ± SEM; µmol) were greater for P4-treated than control gilts, respectively, for glucose (4.96±2.53 vs 0.73±0.13), Arg (207±160 vs 7.41±2.88) and Leu (248±178 vs 14.0±5.23). SLC2A1 and SLC2A4 mRNAs were expressed in the LE of P4-treated gilts. Interestingly the small neutral amino acids, serine, glycine, threonine and alanine were all significantly increased in uterine flushings of P4-treated gilts. Further, the mRNAs of two transporters for these amino acids, SLC7A8 and SLC38A1, were more abundant in endometria from pregnant, pseudopregnant and P4-treated gilts as determined by slot blot hybridization. Novel results in pigs are: 1) high expression of SLC7A3 in the chorion when Arg transport across the placenta is maximal; 2) SLC5A1 expression is induced in LE by estrogens, likely from conceptuses; and 3) long-term effects of P4 increase uterine secretion of small neutral amino acids. Supported by USDA CSREES National Research Initiative Grants 2006-35203-17199, 2006-35203-17283 and 2008-35203-19120.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Retinoic Acid-Stimulation of VEGF Secretion from Human Endometrial Stromal Cells Is Mediated by Production of Reactive Oxygen Species.

Juanjuan Wu 1, Lijuan Hao 2, Jason Hansen 1, Taylor Robert 1, Neil Sidell 1

It is widely accepted that endometrial angiogenesis and vascular endothelial growth factor (VEGF) play a critical role in successful embryonic implantation. Previously, we showed that retinoic acid (RA), which is known to play a necessary role in early events in pregnancy, can combine with transcriptional activators of VEGF (12-O-Tetradecanoylphorbol-13-acetate, TPA; TGF-beta; and IL-1beta) to rapidly induce VEGF secretion from human endometrial stromal cells through a translational mechanism of action. To further investigate the molecular mechanism regulating this process, we have tested the hypothesis that stimulation of VEGF by RA is directly mediated through an increased production of cellular reactive oxygen species (ROS). Results showed that RA, but not TPA or TGF-beta, directly increases ROS production in endometrial stromal cells and that the co-stimulating activity of RA on VEGF secretion can be mimicked by direct addition of H2O2. Importantly, co-treatments of RA and other VEGF transcriptional activators further exacerbated ROS production in a fashion that positively correlates with levels of VEGF secretion. The antioxidants, N-acetylcysteine and glutathione monoethyl ester, inhibited both RA + TPA and RA + TGF-beta-stimulated secretion of VEGF as well as RA-induced ROS production. In contrast, addition of the peroxidase, catalase, did not alter the effects of RA on these parameters. These results suggest that RA-regulated signal transduction is mediated through the antioxidant capacity, including couples such as glutathione. Together, these findings predict a focal mechanism for retinoid regulation of localized VEGF secretion in the human endometrium that is necessary for the successful establishment of pregnancy.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Decorin Is a Novel VEGFR-2 Binding Antagonist for the Human Extravillous Trophoblast.

Gannareddy V Girish, Gausal Azam Khan, Neena Lala, Peeyush Kanti Lala 1

Extravillous trophoblast (EVT) cells of the human placenta invade the uterine decidua and its arteries to nourish the fetus. We identified two decidua-derived molecules TGF-β, and a TGF-β binding small leucine-rich proteoglycan decorin (DCN) which independently controlled EVT cell proliferation, migration and invasiveness. Multiple tyrosine kinase receptors EGF-R, IGFR-1 and VEGFR-2 were shown to mediate these DCN actions. Since DCN binding to VEGFR-2 has never been reported before, we explored the characteristics of this binding and the identity of VEGFR-2 binding sites of DCN in our human first trimester EVT cell line HTR-8/SVneo; and whether this binding antagonised VEGF-induced cellular proliferation and migration. DCN binding to VEGFR-2 in EVT cell lysate or pure recombinant VEGFR-2/Fc chimera was shown with far-western blotting and co-immunoprecipitation. VEGFR-2 lacking Human Embryonic kidney (HEK) cells were used as a negative control. The binding was abrogated with a VEGFR-2 blocking antibody indicating an overlap between the ligand-binding and the DCN-binding domains of VEGFR-2. 125I-labeled VEGF-E (a VEGFR-2 specific ligand) bound to intact EVT cells, with a Kd of 566 pM, and DCN displaced this binding with a Ki of 5.78nM, indicating a 10 fold lower affinity of DCN for VEGFR-2. DCN peptide fragments in the leucine-rich repeat (LRR)-5 domain that preferentially blocked DCN-VEGFR-2 binding in EVT cell proteins also blocked VEGF-induced EVT cell proliferation and migration, indicative of functional VEGFR-2 binding sites of DCN. Our novel findings of DCN as an antagonistic ligand for VEGFR-2 and its ability to block EVT cell migration has implications for pathobiology of preeclampsia, a trophoblast hypo-invasive disorder in pregnancy, and explains its anti-angiogenic role. (Supported by funds from the Canadian Institutes of Health Research to PKL)

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Vitamin C Promotes Placental Angiogenesis by Liberating Nitro Oxide from S-Nitrosothiols Independent of Endothelial Nitric Oxide Synthase Activation.

Wu-xiang Liao, Dong-bao Chen 1

Nitric oxide (NO) derived from endothelial NO synthase (eNOS) plays a critical role in regulating placental angiogenesis. Once formed, the majority of NO is reserved as S-nitrosothiols (S-NO) via reacting with free thiols in vivo. However, it is unknown if NO released from this source also promotes placental angiogenesis. NO liberated from S-nitrosothiols stimulates placental angiogenesis. Primary ovine fetoplacental endothelial (oFPAE) cells were used as the cell model. The thiol-preserving antioxidant vitamin C (ascorbic acid) was used to liberate NO from S-nitrosothiols. DAF2 fluorescence live cell imaging was used to determine NO liberation. Immunoblotting was used to determine if ascorbic acid stimulates eNOS phosphorylation on Ser1177 indicative of eNOS activation. In vitro angiogenesis was determined by cell proliferation, migration and tube-formation. Treatment with 500 µM ascorbic acid significantly stimulated NO release from oFPAE cells; however, ascorbic acid did not stimulate eNOS phosphorylation on Ser1177. Treatments with increasing concentrations (0, 100, 250, 500 µM) of ascorbic acid dose-dependently stimulated invitro angiogenesis (i.e., proliferation, migration and tube formation) in oFPAE cells. Ascorbic acid stimulated oFPAE cell proliferation, migration and tube formation were attenuated by the NO scavenger 2-(4-carboxyphenylalanine)4,4,5,5 tetramethylimidazoline-1-oxyl-3-oxide (cPTIO, 500 µM), but not by the NOS inhibitor L-NAME (1 mM). Vitamin C promotes placental angiogenesis by liberating NO from S-nitrosothiols independent of endothelial nitric oxide synthase activation. Our data implicate a critical role of NO reserved in the S-nitrosothiols in placental angiogenesis (HL74947, HL98746, and HL70562).

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

NOT PRESENTED.

NOT PRESENTED.

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Calbindin-D28k Plays a Role in Hydrogen Peroxide-Mediated Cell Death in Human Endometrial Ishikawa Cells.

Sang Hwan Hyun, Eui-Man Jung, Eui-Bae Jeung 1

Calbindin-D28k (CaBP-28k), a calcium binding protein, buffers intracellular Ca2+. CaBP-28k has anti-apoptotic properties in neuronal and osteoblastic cells. Endometrial cancer is the most common invasive gynecologic malignancy but CaBP-28k expression in the apoptotic signaling is poorly understood. In this study, we investigated whether CaBP-28k expression is regulated by hydrogen peroxide H2O2-induced cell death in human endometrial Ishikawa cells. Ishikawa cells were treated with H2O2 in a dose-dependent manner (0, 0.25, 0.5, 1.0, 1.5, 2 mM), and time-dependent manner (0, 15, 30, 60, 90, 120 min). Then the protein expressions of Bax, p53 and caspase 3 were determined by western blot analysis. Treatment with H2O2- induced an increase in Bax and p53 expressions at the translational level in 1 mM and 60 min. Interestingly, over-expression of CaBP-28k caused a decrease in Bax, p53 and caspase 3 on H2O2-induced apoptosis in the Ishikawa cells. These results suggest that expression of CaBP-28k blocked up-regulation of apoptosis-related genes. In addition, the knockdown of CaBP-28k using a small inhibitory RNA resulted in an elevation of H2O2-induced cell death, whereas cell survival was increased in CaBP-28k over-expressing cells, providing additional evidence that the induction of CaBP-28k expression may be associated with survival signaling in H2O2- mediated oxidative cell death.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Active Roles of Heparin-Binding EGF-Like Growth Factor and Its Receptor in Bovine Endometrium During Implantation.

Keiichiro Kizaki 1, Koichi Ushizawa 2, Miki Kimura 1, Kosuke Iga 3, Toru Takahashi 2, Kazuyoshi Hashizume 1

Heparin-binding EGF-like growth factor (HBEGF) participates in a wide range of physiological and pathological processes, including heart development, wound healing, tumor development and angiogenesis. HBEGF has also been highlighted as an early molecular marker of conceptus-endometrium cross-talk during implantation. Although expression and signaling system of HBEGF and its receptor (EGFR) have been identified in human and mice, there are still unclear in cattle. The aim of this study was to identify the expression of HBEGF and EGFR in bovine endometrium, and investigate the role of this system during implantation and placentation in cattle. The endometrial tissues were collected from Japanese Black cow from early to late gestation and estrous cycle. The gene expression of HBEGF and EGFR were measured by quantitative real-time RT-PCR (QPCR), and the localization was examined by in situ hybridization. In QPCR analysis, expression of HBEGF mRNA was detected in endometrium during the estrous cycle, and the abundance of mRNA increased in intercaruncular endometrium during gestation, but not in caruncular endometrium. On the other hand, EGFR mRNA also expressed in estrous cycle, and strongly increased in caruncle during gestation. In situ hybridization analysis showed that HBEGF mRNA was detected in endometrial epithelia and stroma during estrous cycle, and strongly increased in luminal and glandular epithelium at day 19 of gestation. EGFR was weakly detected in endometrial epithelia and stroma at estrus, and mainly observed in luminal epithelium at during early gestation. Further we examined the role of HBEGF-EGFR system in bovine endometrium using cultured endometrial epithelial and stromal cells. Both genes were expressed in cultured endometrial eplithelial and stromal cells. Exogenously added recombinant HBEGF (0.1-10 ng/mL) stimulated dose-dependently the proliferation of endometrial stromal cells in vitro. When the cells were cultured for 4 days on different culture condition; on collagen-coated dish, on collagen gel and in collagen gel, respectively, the expression of EGFR was not changed in any conditions, but HBEGF was markedly increased in collagen gel culture. These results demonstrate that HBEGF-EGFR signaling system presents in bovine endometrium, and exhibits proliferation potential to the endometrial cells, indicating that the system play crucial role in implantation and placental development in cattle. This research was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports Science and Technology, Japan.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Characterization of Hand2 Expression in the Mouse Endometrium During the Peri-implantation Period.

Huyen V Doan, Brent M Bany 1

The Hand2 gene encodes a basic helix-loop-helix transcription factor that plays well known roles in development. Previously we have shown that Hand2 mRNA levels increase in the mouse uterus as it undergoes decidualization during implantation. Currently the precise function of Hand2 in the uterus during implantation is not known. The purpose of this work was to fully characterize Hand2 expression at both the mRNA and protein levels during the peri-implantation period in mice. Quantitative RT-PCR showed Hand2 mRNA is detected in the uterus on Day 3.5 of pregnancy (Day 0.5 = day vaginal plug found) just prior to the onset of implantation. These levels dramatically increase in the implantation segments during Days 4.5 to 8.5 as the endometrium undergoes decidualization. In situ hybridization and immunohistochemistry analysis revealed that on Day 3.5 of pregnancy that Hand2 mRNA and HAND2 protein were localized to a subpopulation of endometrial stromal cells adjacent to the luminal epithelium. As decidualization proceeds between Days 4.5-8.5, the number of these endometrial stromal cells positive for the mRNA and protein increase as the endometrium undergoes decidualization. These cells appear to all strongly express nuclear progesterone receptors in adjacent sections. At no time was Hand2 mRNA or HAND2 protein found localized to the luminal or glandular epithelia. Overall, these results are consistent with the hypothesis that HAND2 plays a role in the process of decidualization. However, since Hand2 expression was seen in the uterus just prior to the onset of implantation we also hypothesize that this gene may play a role in uterine sensitization. Using an artificial model where the changes in endometrial gene expression of key genes involved in uterine sensitization (Lif, Hoxa10, Calca, etc) is similar to that of pregnancy, we found low basal levels of Hand2 mRNA until the animals were given progesterone. Within 6 h after injection of progesterone the expression of Hand2 dramatically increased in the subepithelial stromal cells. These cells have high levels of nuclear progesterone receptors and this response was not seen in progesterone receptor knockout mice. In a final set of experiments, mouse endometrial stromal cells were isolated form pregnant mice and treated with or without estradiol-17beta and progesterone. Within 24 h markers of decidualization (e.g. Prl8a2) as well as Hand2 significantly (P<0.05) increased in the cells treated with steroids. Currently we are determining if siRNAs targeting Hand2 expression can inhibit the decidualization of these cells in vitro. In conclusion, our findings suggest that Hand2 is a progesterone-responsive gene in the mouse endometrium which may have the roles in not only decidualization but also progesterone-dependent uterine sensitization. (This work was supported by a CRC Grant from SIU School of Medicine, SIUC)

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

STIM1 Plays a Role in Store Depletion-Operated Calcium Entry and Intracellular Store Refilling in Human Myometrial Cells.

Dilyara A Murtazina, Aida Ulloa, Barbara M Sanborn 1

Myometrial cells respond to extracellular signals such as G-protein coupled receptor stimulation and store depletion as a result of endoplasmic reticulum (ER) calcium ATPase (SERCA) inhibition with an increase in intracellular calcium, termed here signal-regulated calcium entry (SRCE). We have previously shown that SRCE stimulated by store depletion is dependent on extracellular calcium but independent of extracellular sodium or inhibition of voltage-operated calcium channels. We have also found that cation channels TRPC1, 4 and 6 are the predominant TRPC mRNAs in human myometrial cells and that knockdown of TRPC1 and 4 attenuate GPCR-stimulated SRCE, whereas TRPC6 knockdown attenuates diacylglycerol-mediated SRCE. Notably, none of these TRPC knockdowns affected SRCE stimulated by cyclopiazonic acid (CPA), a reversible SERCA inhibitor. STIM1, an ER transmembrane protein and calcium sensor, has been reported in other systems to function as part of a store depletion-operated calcium entry channel. We find that myometrial cells express STIM1 mRNA and protein. To address the role of STIM1 in store-depletion stimulated SRCE, we cloned a short hairpin RNA (shRNA) sequence targeting STIM1 into an adenoviral construct under the control of a CMV promoter. Infection of immortalized PHM1 human myometrial cells with this vector reduced STIM1 mRNA by 67 % and STIM1 protein by 50%. Primary uterine smooth muscle cells and PHM1-41 cells were differentially loaded with Fura-2 and Mag-Fluo4 to allow simultaneous measurement of changes in cytosolic and ER calcium, respectively (Shmigol, J Physiol 531:707, 2001). Responses from 10-25 cells/dish were averaged (n=19-20 dishes). In cells infected with scrambled control vector, CPA elicited an increase in cytoplasmic calcium in the absence of extracellular calcium, along with a simultaneous reduction in ER calcium. This is the response expected after inhibition of the SERCA pump. Upon washing out the CPA, addition of 1 mM extracellular calcium resulted in a rapid increase in intracellular calcium (SRCE) in 95% of the dishes and rapid ER store refilling in 90% of the dishes. In contrast, infection of myometrial cells with the STIM1 shRNA vector resulted in either attenuation or slowing of the SRCE response in 74% of the dishes and attenuation or slowing of ER store refilling in 79% of the dishes. These data indicate that STIM1 is important for store depletion-stimulated SRCE in human myometrium and that STIM1 attenuation affects both calcium entry and ER store refilling dynamics. These data have significant implications for understanding how STIM1 influences myometrial cytosolic and ER calcium dynamics, the signals that elicit these responses, and the control of the ability of the myometrium to maintain sustained contractions. Supported by HD38970.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Sustained Progesterone Receptor Expression in Day 4.5 LPA3-Deficient Luminal Epithelium.

Xiaoqin Ye, Honglu Diao, Shuo Xiao, Fei Zhao 1

Lysophosphatidic acid (LPA) is a small lipid-signaling molecule detected in various biological fluids. LPA3 (LPAR3/EDG7) is the third G protein-coupled receptor for LPA. Lpar3 is mainly localized in the luminal epithelium (LE) and Lpar3 expression peaks in the preimplantation day 3.5 uterus (mating night is defined as day 0 and implantation normally initiates ~day 4.0 in mice). The upregulation of Lpar3 in the preimplantation uterus can be blocked by RU486, a progesterone receptor (PR) antagonist, but not ICI 182 780, an estrogen receptor (ER) antagonist, indicating the involvement of progesterone receptor (PR) in regulating Lpar3 expression in the preimplantation uterus. In wild-type (WT) uterus, PR is highly expressed in the preimplantation LE but disappears in LE upon embryo implantation. Lpar3-deficient females have delayed embryo implantation. No significant difference in the PR expression levels between day 3.5 or day 4.5 WT and Lpar3(-/-) whole uterus was detected using realtime PCR. However, sustained PR expression level in isolated day 4.5 Lpar3(-/-) LE but not day 4.5 WT LE was observed. PR immunohistochemistry confirmed such spatiotemporal changes of PR expression in the WT and Lpar3(-/-) uterus. No significant difference in the expression levels of ERalpha, the main estrogen receptor in the uterus, was seen between day 4.5 WT and Lpar3(-/-) LE. Correspondingly, we found that several PR target genes but not ER target genes were differentially expressed in the day 4.5 Lpar3(-/-) LE compared to day 4.5 WT LE. These results indicate that PR signaling but not ER signaling is altered in the non-receptive day 4.5 Lpar3(-/-) LE.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

The Relationship Between Pregnancy Status and Production of IFN-Tau of Bovine Conceptus by Measuring Expression of Interferon-Stimulated Gene 15 (ISG15) of Peripheral Leukocyte in Cattle.

Shuichi Matsuyama 1, Takatoshi Kojima 2, Satoshi Kato 3, Shogo Shiratsuki 4, Hiroshi Tanaka 4, Keiichi Nagataki 1, Shoichi Hoshi 1, Koichi Muroi 1, Koji Kimura 1

Conceptus of ruminant ungulate synthesizes and secretes interferon (IFN)-tau, which has been involved in maternal recognition of pregnancy. However, it remains unclear whether the positive relationship between the ability of IFN-tau secretion of bovine conceptus during the period of maternal recognition of pregnancy and its establishment of pregnancy. Recently, in sheep, it has been demonstrated that IFN-tau is released into the uterine vein and consequently induces expression of IFN-stimulated gene 15-kDa protein (ISG15) in peripheral blood mononuclear cells. Therefore, in the first experiment of the present study, we examined the relationship between the amount of IFN-tau injected into the uterus and the expression level of ISG15 in peripheral leukocyte of cattle. On 16-17 days post estrus, recombinant bovine IFN-tau produced by E. coli (0 [control], 500 or 1000 μg) was injected into ipsilateral uterine horns of 6 cattle per group. Blood samples were collected at every 2-4 hours until 24 hours post injection, peripheral leukocytes isolated by standard methods, and their ISG15 expression measured by real-time RT-PCR. ISG15 expression was stimulated by IFN-tau and peaked 4 hours after the initiation of treatment. Moreover, the expression level of ISG15 was positively correlated with the amount of IFN-tau injected into the uterus (R=0.86, p<0.01). Secondly, we examined IFN-tau secretion from conceptuses until the period of maternal recognition of pregnancy in cattle by measuring ISG15 expression in peripheral leukocyte. Blood samples were collected 7, 16, 18, 21 and 25 days post estrus, and ISG15 expression measured in peripheral leukocyte via real-time RT-PCR. AI (n=50) or ET (n=59) was performed on the day of estrus or 7 days later, respectively. In this experiment, non-pregnant cattle (AI: n=23, ET: n=30) were classified into 2 groups, in which cattle showed estrus with prolonged estrus cycle (greater than or equal to 25 days) and normal estrus cycle (<25 days). ISG15 expression increased and reached to the peak on day 21 in pregnant cattle. In non-pregnant cattle showing prolonged estrus cycle, the ISG15 expression was also increased, but the degree of the increase was smaller than that in pregnant cattle. On the other hand, in non-pregnant cattle showing normal estrus cycle, there was no significant increase of ISG15 expression during whole experiment period. In the period of maternal recognition of pregnancy, there was significant difference in ISG15 expression among 3 groups (p<0.05). These results demonstrated the direct relationship between the amount of IFN-tau secretion during the period of the maternal recognition of pregnancy and conception. In non-pregnant cattle showing normal estrus cycle, undetectable level of IFN-tau production during the period of maternal recognition of pregnancy indicated that the conceptuses might be already dead before this period. In non-pregnant cattle showing prolonged estrus cycle, the conceptuses were survived and produced enough IFN-tau to inhibit CL regression during the period of maternal recognition of pregnancy. However, in these cattle, the production of IFN-tau production was lower than pregnant cattle, suggesting that the growth of their conceptuses was deteriorated and consequently died beyond the period of maternal recognition of pregnancy.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

The In Vitro Study of Resveratrol on Uterine Contraction and Ca2+ Mobilization in the Rat.

Shih-Min Hsia 1, Kai-Lee Wang 2, Chia-Jung Chan 2, Paulus S Wang 3

Dysmenorrhea is directly related to elevated PGF2alpha levels. It is treated with NSAIDs (nonsteroid antiinflammatory drugs) in Western medicine. Since NSAIDs produce many side effects, Chinese medicinal therapy is considered as a feasible alternative medicine. Many special physiological components (ex: Resveratrol) in Chinese medicine have been isolated and identified. Resveratrol have a lot of physiological functions like anti-oxidation and anti-cancer effects. However, the relationship between uterine smooth muscle contraction and resveratrol remains veiled. We studied the in vitro effects of resveratrol on uterine smooth muscle contraction. The uterus was separated from female SD rat and uterine smooth muscle contraction activity was measured and recorded. Resveratrol inhibited uterine contractions induced by PGF2alpha, Ca2+ channel activator Bay K 8644, and high K+ in a concentration-dependent manner in vitro; furthermore, resveratrol inhibited the Ca2+-dependent uterine contractions. Thus, resveratrol consistently suppressed the increases in intracellular Ca2+ concentrations ([Ca2+]i) induced by PGF2alpha and high K+. Thus, resveratrol probably inhibited uterine contraction by blocking external Ca2+ influx, leading to a decrease in [Ca2+]i. Thus, resveratrol may be considered as a feasible alternative therapeutic agent for dysmenorrhea.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Unilateral Effects of Pregnancy on Differential Expression of the Gap Junction Proteins Connexin 43 and 37 in Ovine Uterine Artery Endothelium.

Timothy J Morschauser, Jayanth Ramadoss, Jill M Koch, Gladys E Lopez, Ian M Bird, Ronald R Magness 1

Coordinated and sufficient uterine vascular endothelial adaptations facilitate the dramatic increases in uterine blood flow necessary for fetal growth during pregnancy. These endothelial adaptations may be modulated by coordinated intercellular interactions via gap junction proteins which regulate eNOS activation. We hypothesized that in uterine artery endothelium (UA endo), expression of the gap junction proteins connexin (Cx) 43 and 37 is elevated during pregnancy compared to nonpregnant luteal and follicular phases. We further hypothesized that restricting pregnancy to a single uterine horn will induce unilateral increases of connexin expression only in UA endo ipsilateral to the gravid horn. Restricting pregnancy to a single uterine horn (gravid unilateral) was established by surgically severing the intercorneal vascular connections and then ligating one horn (i.e. contralateral nongravid unilateral) 2-3 months before breeding. UA endo were isolated from nonpregnant sheep (luteal, n=5 vessels; follicular, n=5) as well as late pregnant (120-130d, term = 147d) sheep (nongravid unilateral side, n=8; gravid unilateral side, n=8, control pregnant n=8). Cx43 and Cx37 protein expression as well as eNOS expression was determined by Western analysis. No difference was observed between nonpregnant luteal, follicular, and nongravid unilateral Cx43 and Cx37 expression in UA endo. Cx43 and Cx37 were significantly elevated by 2086% and 786%, respectively in control pregnant (P<0.001) whereas Cx43 was significantly elevated by 1392% in gravid unilateral (P<0.001) compared to luteal. Additionally, Cx43 expression was significantly elevated by 295% (P=0.003) in the UA endo of the gravid unilateral horn compared to the nongravid horn whereas Cx37 was barely detectable in both the nongravid and gravid horns. Further analysis of eNOS expression indicated that unlike Cx37, the patterns of Cx43 expression mimicked UA endo eNOS expression as well as the dramatic physiologic rises in uterine blood flows, and uterine artery shear stresses. These data demonstrate that there is a distinct difference in uterine vascular programming of gap junction proteins Cx43 and Cx37 during pregnancy. Both Cx43 and Cx37 are lower in gravid unilateral compared to pregnant, suggesting that the local responses of fetal restriction to a single horn has a direct negative effect on connexin expression during pregnancy. It is also noteworthy that in the unilateral gravid and pregnant horns, blood flow and shear stress are substantially increased which appears to be associated with the observed increase in Cx43 expression. These data suggest that the regulatory mechanisms pertaining to gap junction proteins may delineate a greater understanding of local rather then the systemic vascular adaptations during pregnancy. NIH HL49210, HD38843, HL87144, HL79020.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Proteomic Profile of Uterine Luminal Fluid from Early Pregnant Ewes.

Jill M Koch, Jayanth Ramadoss, Ronald R Magness 1

Embryonic development and the maintenance of pregnancy is a time sensitive period requiring a synchronized uterine environment which is created by the secretion of proteins from both the developing embryo and the uterus itself. Numerous studies have identified uterine luminal proteins and related these proteins to the specific adaptations that must occur during early pregnancy for proper embryonic growth and development. However, no study has yet utilized high throughput proteomics to identify the signature profile of protein in the uterine lumen during early pregnancy. In this study, uterine luminal fluid from nonpregnant (NP; n=3) and early pregnant (EP n=3; gestational day 16) ewes were analyzed by nanoLC-MS/MS and validated by Western immunoblotting. We identified a unique signature profile for EP luminal fluid in which 12 of the top 50 most abundant proteins related to specific aspects of embryonic development including: immune system regulation (complement component family 4A and 5), growth and remodeling (A2M, transgelin, carbonic anhydrase II, BCL2-like 15, ACTN4, and alpha 1 B glycoprotein), oxidative stress balance (paraoxonase), trophoblast cells (placental protein 9), and nutrition (APO-AI and ceruloplasmin). These proteins were up regulated up to 65 fold in the EP profile compared to the NP profile except for ceruloplasmin which had a decrease in expression compared to the NP profile. Specific uterine remodeling proteins such as transgelin (P=0.008) and placental proteins including placental protein 9 (P=0.02) were only present in EP luminal fluid which was further validated by immunoblotting. Transgelin has been described as a biomarker for uterine arterial vascular remodeling and may play a key role in uterine adaptations during initial placental attachment. Additionally, placental protein 9 is produced by the trophoblast cells of the developing embryo and is a protein that catalyzes the conversion of glucose to fructose which at this point in embryonic development is important because fructose is the primary sugar in fetal fluids and the embryo is increasing in size exponentially. The data demonstrated that 38 out of the 50 abundant proteins including HSP90 and HSP60 were not altered by pregnancy status. Therefore proteins such as HSP90 and HSP60 were utilized as unaltered representative uterine luminal proteins to examine a direct correlation between proteomics data and immunoblotting. Specifically the data for transgelin showed a positive correlation (R2>0.8, P<0.05) between the two methods. These data provide important information on dynamic physiological processes associated with early pregnancy at the level of the uterus and conceptus and may demonstrate a signature biomarker profile associated with embryonic well-being. NIH HL49210, HD38843, HL87144.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Uterine Artery Endothelial Cell Caveolar Proteomics Underscores Subcellular Compartmentalization of Estrogen Receptors Alpha and Beta.

Benjamin C Hofeld 1, Jayanth Ramadoss 1, Mayra Pastore 1, Wu-Xiang Liao 2, Dong-bao Chen 2, Ronald R Magness 1

Estrogen elicits a spectrum of uterine vascular adaptations by its actions on the nuclear (classical) and membrane (nongenomic) Estrogen Receptors (ERs) alpha and beta. Estrogen binding to the more abundant nuclear receptors regulates gene transcription, whereas the less abundant membrane receptors control rapid activation of endothelial nitric oxide synthase (eNOS) and thus nitric oxide (NO)-mediated uterine vasodilation. Interestingly, in uterine artery endothelial cells (UAECs), numerous proteins that control estrogen-induced NO production are localized to specific regions of the plasma membrane called the caveolae. However, it is unknown if both types of membrane ERs are also compartmentalized to UAEC caveolar domains. We hypothesized that both ERalpha and ERbeta are present in the UAEC caveolar subcellular domain and will exhibit differential partitioning between caveolar and non-caveolar domains and that the caveolae are also home to a number of other important estrogen related proteins. Passage 4 caveolae were enriched from UAECs isolated from pregnant ewes using sucrose gradient ultra-centrifugation. Enriched caveolar membranes were analyzed by nano LC MS/MS and validated by Western blotting. Although mass spectrometric protein and peptide identification probability for caveolar ERs did not reach significance (P>0.05), Y and B ion peptide series assignment did confirm the presence of ERs in the UAEC caveolae and the major signals in the spectra were explained by the assigned sequences. Caveolar proteomic analysis also identified sequences for the first time in uterine arterial endothelial caveolae of other important proteins related to ERs including Estrogen-Related Receptor beta (ERRbeta - KLYAMPPPGMPEGDIKA), Modulator of Estrogen-Induced Transcription (RTVIIHDRPDTTHPRH), and breast cancer anti-Estrogen Resistance 1 (BCAR1- RGLLPGQCSQEVYDTPPMAVKG). Validation by gradient centrifugation followed by Western blotting demonstrated that UAECs abundantly expressed both ERalpha and ERbeta proteins. Further, 13.74% of the total UAEC ERalpha protein was confined to the caveolae, whereas the remaining 86.26% was located in the non-caveolar pool. Similarly, ERbeta was expressed in both the caveolar and non-caveolar domains although only 2.18% of the total UAEC ERbeta was localized to the caveolae and 97.82% of the receptor was located outside the caveolae. These data demonstrate that the ratio of ERalpha levels in caveolar to non-caveolar domains is significantly higher than that of ERbeta, suggesting ERalpha may play a more important role in regulating estrogen actions in UAEC caveolae. Furthermore, these caveolae are "hubs" for numerous ER-related proteins which are known to modulate their functions. We conclude that the stoichiometric differences in the ER distribution between subcellular compartments may be critical for understanding of estrogenic actions during pregnancy and may aid in delineating the etiologies of gestational diseases. NIH HL49210, HD38843, HL87144, and HL70562.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

The Expression Profile of Ovine Chemokine Receptor 4 (CXCR4) and Its Ligand, CXCL12, in Endometrium and Conceptus During Early Pregnancy: Implications in Implantation and Placentation.

Ryan L Ashley, Alfredo Q Antoniazzi, Russell V Anthony, Thomas R Hansen 1

The progression of implantation and placentation in ruminants is complex and is regulated by interplay between sex steroids and local signaling molecules, many of which have immune function. Chemokines and their receptors are pivotal factors in implantation and vascularization of the placenta. Chemokine receptor 4 (CXCR4) is up-regulated in the human endometrium during implantation and has only one recognized ligand, CXCL12. Activation of CXCR4 causes recruitment of leukocytes into the uterus of pregnant females and stimulates trophoblast proliferation and invasion. Using PCR, we detected CXCR4 mRNA in ovine and bovine endometrium, although studies investigating temporal changes in CXCR4 mRNA are lacking. Based on known critical roles for CXCR4 during early pregnancy in other species, we hypothesized that expression of mRNA for CXCR4 and CXCL12 in the endometrium and conceptus would increase during the time of implantation in ewes. The objectives of the current study were to determine if mRNA for CXCR4 and CXCL12 was differentially expressed using real-time PCR (qPCR) in: endometrium from pregnant and non-pregnant ewes on Days 12, 13, 14 and 15; ovine conceptuses on Days 13, 15, 16, 17, 21 and 30 after mating; and cotyledons, caruncles and intercaruncular tissue from ewes on Days 35 and 50 of gestation. Differences described are P < 0.05. In ovine endometrium, mRNA for CXCR4 increased on Day 15 of pregnancy compared to the estrous cycle, while expression of CXCL12 mRNA was detected on all days and did not differ. Expression of mRNA for CXCL12 and CXCR4 in the sheep conceptus exhibited a similar expression pattern across days tested with greater levels on Days 21 and 30 compared to earlier days collected. Additionally, CXCL12 mRNA was greater in cotyledon on Day 35 compared to Day 50, whereas CXCR4 did not differ between these two gestational ages. On Day 35 of gestation, expression of mRNA for CXCR4 was greater compared to Day 50 in both caruncle and intercaruncular tissue, while mRNA for CXCL12 did not differ in these tissues between the two gestational ages. The increase in CXCL12 and CXCR4 in trophoblast cells from Day 16-21 of pregnancy is intriguing as this correlates with time of apposition with luminal epithelium of the endometrium where adhesion complexes are formed by Day 21. A further increase in trophoblast CXCR4 and CXCL12 from Day 21-30 of gestation represents a time during which placentation occurs in sheep, which is not completed until Day 50 to 60 of pregnancy. We interpret these data to mean the CXCL12/CXCR4 pathway is activated during implantation and placentation in sheep and is likely playing a role in the communication between trophoblast cells and the maternal endometrium. Supported by USDA-NIFA-NRI grants 2009-65203-05717, 2006-35203-17258 and 2005-35203-15885.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Oxidative Metabolism Is Required for Sperm Motility and Viability in Felids, but May be Impaired in Cheetah (Acinonyx jubatus) Ejaculates.

Kimberly A Terrell 1, David E Wildt 1, Nicola M Anthony 2, Barry D Bavister 3, Stanley P Leibo 2, Linda M Penfold 4, Laurie L Marker 5, Adrienne E Crosier 1

Teratospermia (ejaculation of ≥ 60% structurally abnormal spermatozoa) is common in certain felid species or populations, especially in the presence of low genetic diversity. We recently determined that (1) sperm cellular rates of pyruvate uptake and lactate production are reduced in ejaculates from teratospermic cheetahs and inbred domestic cats and (2) spermatozoa from both species minimally utilize exogenous glucose regardless of ejaculate phenotype. Sperm metabolism of endogenous glucose (i.e., glycogen) has been documented in the domestic dog, and also could occur in the domestic cat and cheetah. However, there is no information to indicate which metabolic pathways support motility and viability in felid spermatozoa. In this study, we tested the hypotheses that oxidative phosphorylation, rather than glycolysis, is the primary energy source in felid spermatozoa and is impaired in teratospermic ejaculates. Our objective was to comparatively assess glycolytic and oxidative sperm metabolism in the domestic cat and cheetah in relation to ejaculate phenotype (normospermic vs. teratospermic). Electroejaculates (n = 31 total, 9-13 per group) were collected from: 1) normospermic domestic cats (3 males); 2) teratospermic domestic cats (4 males); and 3) cheetahs (13 males). Washed sperm samples were incubated (37°C) in a chemically-defined mouse tubal fluid medium (cMTF) in the presence/absence of exogenous 1 mM glucose and 1 mM pyruvate. A second set of ejaculates was incubated in cMTF containing both substrates as well with an inhibitor of glycolysis (50 mM alpha-chlorohydrin) or of oxidative phosphorylation (160 nM myxothiazol) to control for endogenous substrate metabolism. Cellular lactate production, percent sperm motility (%M), forward progression (FPS) and acrosomal integrity (correlated with sperm viability in felids) were assessed in all samples at 0, 1, 3 and 7 h of incubation. Overall, the absence of exogenous glucose/pyruvate did not (P > 0.05) influence sperm motility (%M and FPS) or acrosomal integrity in either domestic cat group or in cheetahs. Unexpectedly, spermatozoa produced lactate in the absence of glucose, but only when pyruvate was present. The influence of metabolic inhibition on sperm function was consistent across animal groups. Glycolytic inhibition resulted in decreased (P < 0.05) lactate production, %M and FPS after 1 h, but acrosomal integrity did not decline (P < 0.05) until 7 h of incubation. In contrast, oxidative inhibition severely (~70%) decreased (P < 0.05) %M and FPS and caused a ~20% loss (P < 0.05) of acrosomal integrity by 1 h of incubation. A 100x higher concentration of oxidative inhibitor was required to alter (P < 0.05) sperm function in the domestic cat (normospermic and teratospermic) compared to the cheetah. Results indicate that both oxidative and glycolytic pathways are required to maintain motility and viability of domestic cat and cheetah spermatozoa. However, a greater proportion of the energy supporting these processes is obtained from the more efficient oxidative pathway. The sensitivity of cheetah spermatozoa to extremely low concentrations of the oxidative inhibitor suggests that mitochondrial load/function is reduced in these cells compared to domestic cats. Importantly, comparison with the teratospermic domestic cat model reveals that this metabolic defect is related to species-specific physiology rather than ejaculate phenotype.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

The Effect of Raffinose and Methionine on Frozen/Thawed Angora Buck (Capra hircus ancryrensis) Semen Quality, Lipid Peroxidation, and Antioxidant Enzyme Activities.

Mustafa Numan Bucak 1, Purhan Barbaros Barbaros Tuncer 1, Serpil Sariozkan 2, Ahmet Atessahin 3, Deniz Yeni 4, Fatih Avdatek 4

The aim of the present study was to determine the effects of different doses of raffinose and methionine on post-thawed semen quality, lipid peroxidation and antioxidant enzyme activities of Angora buck (Capra hircus ancryrensis) sperm following cryopreservation. Ejaculates collected from three Angora bucks were evaluated and pooled at 37°C. Semen samples, which were diluted with a Tris-based extender containing the additives raffinose (2.5, 5, 10 mM) and methionine (2.5, 5, 10 mM) and an extender containing no antioxidants (control), were cooled to 5oC and frozen in 0.25 ml French straws to be stored in liquid nitrogen. Frozen straws were thawed individually at 37°C for 20 sec in a water bath for evaluation. The freezing extender supplemented with 2.5 and 5 mM methionine led to higher percentages of computer–assisted sperm motility analysis (CASA) motility (63.6±7.0; 63.4±3.1 %, respectively), in comparison to the controls (P<0.01) following the freeze-thawing process. The addition of antioxidants did not provide any significant effect on the percentages of post-thaw subjective and CASA progressive motilities as well as sperm motion characteristics (VAP, VSL, LIN and ALH), compared to the control groups (P>0.05). The freezing extender with raffinose (5 and 10 mM) and methionine at three different doses (2.5, 5 and 10 mM) led to lower percentages of acrosome abnormalities, in comparison to the controls (P<0.001). In the comet test, raffinose (5 and 10 mM) and methionine (10 mM) gave scores lower than those of the controls, and thereby reduced DNA damage (P<0.05). Malondialdehyde (MDA) formation was found to be lower (1.8 ± 0.1 (nmol/L) in the group of 5 mM raffinose, compared to the controls following the freeze-thawing process (P<0.01). The additives did not show any effectiveness on the maintenance of SOD, GSH-PX and GSH activities, when compared to the controls (P>0.05). In conclusion, methionine and raffinose play a cryoprotective role against sperm CASA motility, acrosome abnormality and DNA damage. Raffinose 5 mM exhibited antioxidative properties, decreasing MDA levels. Further studies are required to obtain more concrete results on the characterization of microscopic parameters and antioxidant activities in cryopreserved goat sperm with different additives.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Suppression of Mitochondrial Activity in Activated Bovine Sperm Advances Capacitation Status and Induces Normal Embryogenesis.

Yoku Kato 1, Rui Miyamoto 2, Janice L Bailey 3, Yoshikazu Nagao 2

We have previously shown that activated bovine sperm induce capacitation. Activated sperm can fertilize normally. However, our previous study also indicated that the selection and injection (ICSI) of activated and hyperactivated sperm into bovine oocytes induced chromosomal aberration during early stages of development. Activation of sperm motility is a highly energetic process. Mitochondria produce significant amounts of ATP and reactive oxygen species (ROS) in those motile sperm. There is a possibility that ROS produced by activated sperm affect embryogenesis following sperm incorporation, resulting in chromosomal aberration. Here, we examined sperm capacitation state, ROS production along with the developmental ability of embryos following intracytoplasmic injection of activated sperm in which mitochondrial activity had been reduced experimentally. In the first experiment, we used a luminometer to investigate ROS production by sperm treated with carbonyl cyanide m-chlorophenyl hydrazone (cccp), an uncoupler of cell respiration. In our second experiment, mitochondria integrity and activity was determined quantitatively using mitotracker red and 5, 50, 6, 60-tetrachloro-1, 10, 3, 30-tetraethylbenzimidazolyl- carbocyanine iodide (JC-1) in groups of normal motile sperm (control group), activated (Act group) sperm, and activated sperm in which mitochondrial activity had been reduced by cccp (MT group). The proportion of sperm exhibiting a characteristic pattern of capacitation was visualized by chlortetracycline staining and tyrosine phosphorylation in each experimental group. In our third experiment, we examined how sperm from each experimental group might affect developmental competence of an embryo produced by ICSI, including cleavage, development to the blastocyst stage, and chromosomal integrity of the blastocyst stage. ROS produced by mitochondria was decreased by cccp. Mitochondrial integrity, as assessed by mitotracker red was intact in all group and the proportion of active mitochondria as indicated by JC-1 was 82.4%, 83.3% and 33.3%, in the control group, Act group and MT group, respectively (P<0.05). The proportion of sperm exhibiting the characteristic pattern of capacitation, as determined by chlortetracycline staining, was 58.3%, 71.4% and 71.4% in the control group, Act group and MT group, respectively, whilst levels of tyrosine phosphorylation were 0.0%, 26.9% and 61.8% (P<0.05). There were no significant differences in the rate of embryo development to the blastocyst stage among the three groups (20.7% vs 25.0% vs 31.4% respectively, P>0.05). On the other hand, chromosomal integrity of the blastocyst stage in the MT and control groups were higher than in the Act group (78.6%:11/14 vs 86.7%:13/15 vs 7.1%:1/14, respectively, P<0.05). Our results indicate that suppression of mitochondorial activity in activated bovine sperm reduce ROS production but induce normal embryogenesis following ICSI.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Effects of Radiographic Contrast Dye on Domestic Cat Spermatozoa.

Serena A Barnes, Ana M Cepeda, Linda M Penfold 1

Laparoscopic artificial insemination has played an important role in felid conservation, but requires minor surgery. The less invasive technique of transcervical artificial insemination has not been heavily pursued because of concerns that sperm is not adequately transported through the uterine horns. The use of radiographic contrast dyes to visualize and verify gamete placement within the uterine horns could ensure the success rate of this less invasive procedure. Two radiographic dyes (Omnipaque and MD76) were diluted 1:1.1 (Omnipaque) and 1:4.4 (MD76) with ultra-purified water to a final osmolarity of 320-330±3 mOsm, consistent with the osmolarity of domestic cat spermatozoa. Each diluted dye was tested for radio-opacity by injecting aliquots into uterine horns recovered from spayed cats and radiographing the tissue. Gamete rescued freshly collected and frozen-thawed spermatozoa were extended 1:1 with feline in vitro fertilization media (control) or with diluted radiographic dyes. Motility, forward progression, and acrosomal integrity were recorded every 30 minutes for four hours to analyze felid sperm activity after the addition of the experimental media, while morphology data was taken to determine if radiographic dye induced abnormalities in the sperm cells. To assess sperm penetration abilities, spermatozoa (fresh: 2×105cells/100µl droplets; frozen: 5×105cells/100µl droplets) extended with treatment media were coincubated with conspecific in vitro matured oocytes for 18-20h. All presumptive embryos were fixed and stained three days post insemination to determine fertilization and cleavage rates based on DNA content. Morphology analysis confirmed radiographic dyes did not induce abnormalities in felid spermatozoa. Motility and forward progression were converted into sperm motility indices, and results demonstrated no significant differences between media for freshly collected spermatozoa. For frozen-thawed spermatozoa, a lower sperm motility index was calculated for Omnipaque vs control, but not compared with MD76. Motility index decreased over time for all treatments for both freshly collected and frozen-thawed sperm. Acrosomal integrity for freshly collected spermatozoa was significantly reduced when treated with Omnipaque vs MD76 but not compared with control media. Differences in acrosomal integrity were not seen within frozen-thawed treatment groups. Acrosomal integrity for freshly collected and frozen-thawed sperm cells also significantly decreased with time. Preliminary data also indicate sperm exposed to radiographic contrast media maintained their ability to penetrate and fertilize conspecific oocytes. These findings propose radiographic dye can be added to domestic cat spermatozoa with minimal reduction to sperm fitness, thus creating a foundation for developing improved transcervical insemination techniques for non-domestic felids.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

TSPY Gene Copy Number and Levels of mRNA for TSPY and Cell Cycle, Meiosis and Early Male Germ Cell Marker Genes in Holstein Bulls.

Christine K Hamilton 1, Laura Favetta 1, Patrick Blondin 2, W Allan King 1

Testis-specific protein, Y-encoded (TSPY) is present in varying gene copy number in both human and cattle. Copy number variation of TSPY has been linked to spermatogenesis and is hypothesized to be a potential indicator of male fertility although there is limited data on TSPY expression in testicular tissue. Mice have only a single, non-functional TSPY gene whereas cattle have multiple functional copies and since slaughterhouse testicular material is readily available, cattle are a better model species for TSPY analyses. This study aims to compare TSPY copy number and mRNA transcript levels with that of various genes related to cell cycle and meiosis in Holstein bulls. To do this we measured TSPY copy number in DNA extracted from blood (n=51) and TSPY expression in total mRNA extracted from testicular tissue (n=26) with real time polymerase chain reaction (PCR). Our results show that TSPY copy number is negatively correlated to TSPY mRNA expression in the testis (r=-0.694, p<0.0001). We also found negative correlations of TSPY copy number and positive correlations of TSPY mRNA expression with various cell cycle genes (CCNB1, CCNB2, CDK1), meiotic genes (RAD51, SYCP3 and MLH1) and markers of early germ cells (UCHL1, TRPC2). Based on our results, it appears that TSPY expression may represent a good marker of early male germ cells. The negative correlation that we found between TSPY copy number and TSPY expression may have useful applications; TSPY copy number could be measured in blood to predict the amount of early germ cells in individual bulls. Supported by National Sciences Engineering and Research Council grant 364747-08; Canadian Research Chair program; and L'Alliance Boviteq Inc.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Chemokines Enhance the Invasive Potential of SKI Knockdown NCCIT Testicular Cancer Cells.

Laura L Richardson, Aslam Chaudhry, Amy N Nash 1

Testicular cancer is the most common malignancy affecting young men ages 15 to 35. Although highly treatable if detected at an early stage, patients with metastases at the time of initial diagnosis have a poor prognosis for survival. Our previous studies of the role of the SKI gene in testicular cancer indicate that loss of SKI function promotes a metastatic phenotype in cell lines derived from testicular germ cell tumors. Germ cell tumors commonly metastasize to the retroperitonal lymph nodes and lung. Metastases can also occur in liver, brain and bone, and more rarely in kidney. In this study we hypothesized that cells in tissues associated with metastatic disease produce factors that promote the migration of testicular cancer cells to these sites. We used the Matigel invasion assay to determine whether the migration of our previously established SKI knockdown NCCIT cells (sh-SKI NCCIT) and control NCCIT cells (sh-NC NCCIT) is enhanced by factors secreted by cell lines of brain and kidney origin. C8D1A cells, derived from mouse cerebellum and exhibiting properties of astrocytes, normal rat kidney epithelial cells (NRK), and NIH-3T3 mouse fibroblasts were grown in six well culture dishes in low serum medium for 48 hours. Matrigel coated invasion chambers containing the sh-SKI NCCIT or sh-NC NCCIT cells were then placed in the wells. Wells containing only low serum medium were used as a control. After 24 hours, the numbers of NCCIT cells migrating through the Matrigel coated membrane were counted. SKI knockdown cells exhibited significantly increased migration when grown in the presence of C8D1A, NRK, and NIH-3T3 compared to medium alone. The migration of the control sh-NC NCCIT cell line was not significantly increased by the presence of any of the other cell lines. These data suggest that cells derived from brain and kidney, as well as fibroblasts, produce chemokines that can attract cancer cells to metastasize to these sites and that in the absence of SKI function, testicular cancer cells have an increased ability to respond to these signaling molecules. One candidate chemokine that has been shown to play a role in germ cell migration is stromal derived factor 1 (SDF1/CXCL12). We investigated whether SDF1 could increase sh-SKI NCCIT cell migration using the Matrigel invasion assay. SDF1 was added to the low serum medium in the lower chamber and migration of sh-SKI NCCITand sh-NC NCCIT cells was measured after 24 h. The migration of sh-SKI NCCIT cells increased slightly, but significantly, in the presence of SDF1 compared to cells not treated with SDF1. This effect was blocked by the simultaneous addition of AMD 3100, a specific inhibitor of CXCR4--the receptor for SDF1. The migration of the sh-NC NCCIT cells was not affected by either treatment. The data support the hypothesis that cells at sites distant from the primary tumors secrete factors that promote the metastasis of germ cell tumors to specific organs and suggest that SDF1 is at least one such factor. (This work supported by COBRE grant 1P20 RR020180 and a WV EPSCoR NASA Space Grant Consortium Graduate Student Fellowship to ANN.)

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Evaluation of Alternatives for Analysis of the Binding Capacity of Goat Sperm.

Ciro AA Torres, Madriano C Santos, Jose D Guimaraes 1

This study aimed to carry out the analysis using the simple binding of goat sperm to bovine oocytes and to the perivitelline membrane of the hen's egg yolk compared to binding to the goat oocytes, looking for an alternative to predict the buck semen quality without losing the precision and accuracy of their own oocytes. Sixteen doses of goat semen were collected from 04 breed bucks, 2 Saanen and 2 Alpine via artificial vagina, using an estrus female as dummy. The physical aspects of the semen (volume, sperm whirlwind, force and motility) and sperm concentration were evaluated. Three groups were formed, and 20 µL of semen from the same collection were thawed at a concentration of 5 × 106 spermatozoon/mL and used. In the 1st group goat oocytes (LIGCAP) and in the 2nd group bovine oocytes (LIGBOV) were inseminated and in the 3rd one the perivitelline membrane of the hen's egg yolk (MPVGO) were used. Two others groups were formed, in the first one and in the second one 16 doses of fresh (MPVGOF) and thawed (MPVGOD) semen from the same collection were used, respectively, to inseminate. Semen samples from both groups were used to inseminate perivitelline membrane from chicken egg yolk. The imposed treatment did not affect the animals variables studied (P>0.05). The average volume of semen collected was 1.1 ± 0.15 ml. The average sperm concentration observed was:1.94 ± 0.21 × 10,9. The average sperm whirlwind was 3.6 ± 0.23. The sperm motility was 77.5 ± 1.37 and 32.2 ± 1.37 for fresh and thawed sperm, respectively. No differences (P>0.05) among the binding assays using the bovine oocytes or hen's egg yolk in relation to the goat oocytes were showned. The binding assay using fresh and thawed semen did not differ between themselves. It is concluded that, both the MPVGO and the LIGBOV may be used to replace LIGCAP. However, the LIGBOV had a higher precision than the MPVGO as a test. The fresh or thawed semen may be used to evaluate the binding capacity of the semen through the MPVGO without compromise the results.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Germ Cell-Selective Ablation of Fgfr-1 or Fgfr-2 in Mice Does Not Affect Male Fertility.

Shengqiang Li 1, Jing Lin 1, Xian Li 1, Yoshihiko Araki 2, Zi-Jan Lan 1, Zhenmin Lei 1

Fibroblast growth factor (FGF) signaling is suggested to play diverse roles in the male reproductive system including sexual differentiation, testicular development, hormone production and spermatogenesis. Our previous study demonstrated the expression of FGF receptor (FGFR)-1 and FGFR-2 in both somatic and germ cells during postnatal testicular development and adulthood. To specifically investigate the role of FGFR-1 and -2 in germ cells, we first generated a transgenic mouse line (Tex101-Cre) expressing an improved Cre recombinase driven by the mouse Tex101 promoter. Using floxed ROSA reporter mice, we found that Cre recombinase was expressed in the testes, but not in the heart, liver, spleen, lung, adrenal gland, pancreas, thymus or pituitary. Cre activity was detected in the prospermtogonia within seminiferous tubules from eight-day-old mice. In adult mice, there were robust Cre activities in spermatocytes and spermatids and a weak Cre activity in spermatogonia. Breeding wild type females with homozygous floxed Fgfr-2 males carrying the Tex101-Cre transgene did not produce any progeny with the floxed Fgfr-2 allele, indicating that Tex101-Cre mice are capable of complete deletion of the floxed allele in male germ cells. Homozygous mutant animals, either Fgfr-1flox/floxTex101-Cre or Fgfr-2flox/floxTex101-Cre mice, were found to be viable with no developmental abnormalities in the testes and accessary sex organs. During a four-month fertility test, both genotype animals sired normally. Immunohistochemistry revealed that spermatocytes and spermatids avoided FGFR-1 staining in Fgfr-1flox/floxTex101-Cre and FGFR-2 staining in Fgfr-2flox/floxTex101-Cre mice, while the expression of these two proteins in testicular somatic cells of either genotype animals was not altered. Testicular histology in both Fgfr-1flox/floxTex101-Cre and Fgfr-2flox/floxTex101-Cre mice was indistinguishable from wild-type littermates. RT-PCR did not detect a potential compensatory upregulation of Fgfr-2 in Fgfr-1flox/floxTex101-Cre mice or Fgfr-1 in Fgfr-2flox/floxTe101x-Cre mice. Our results demonstrate that male germ cell-selective individual ablation of Fgfr-1 or Fgfr-2 does not affect mouse reproductive capability and suggest the presence of redundant FGF signal pathways in the male germ cells.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Molecular Expression of Ly6k, a Putative Glycosylphosphatidyl-Inositol-Anchored Membrane Protein on the Mouse Testicular Germ Cells.

Mayuko Maruyama, Hiroshi Yoshitake, Kenji Takamori, Yoshihiko Araki 1

Previous studies from our research group demonstrated that TEX101, a unique germ-cell marker molecule, is specifically associated with Ly6k, a mouse putative glycosylphosphatidyl-inositol (GPI)-anchored membrane protein, on the testicular gem cells using newly developed specific anti-Ly6k polyclonal antibody (pAb). Although a human homologue for mouse Ly6k, Ly6K is recently proposed as a new candidate member of cancer-testis antigen, the precise molecular nature of the molecules is still obscure, since molecular characterization of Ly6k (or Ly6K) reported is mainly based on RT-PCR analysis so far. The aim of this study is to characterize the molecular nature of Ly6k more precisely, especially, to focus on the Ly6k tissue distribution and the molecular expression during testicular development. For this purpose, we have performed RT-PCR including quantitative analysis, and developed a new monoclonal antibody (mAb) to Ly6k (named mk34) in addition to the anti-Ly6k pAb for further protein analysis. Data obtained from the present study have shown that: 1) Ly6k message was strongly observed in testis as previously reported, but faint expression was broadly noticed in other tissues by RT-PCR; 2) Like TEX101, Ly6k was weakly detected in the testis from dpc18 to dpp1, then reached plateau around 8 dpp; 3) Testicular Ly6k showed two-peak expression at around dpp14 and dpp 24, then expressed stably from 6-week after birth by quantitative analyses; 4) Ly6k was obviously detected in testes from adults, and clear but restricted immunoreaction was detected in the stomach, cerebellum, uterus and ovary using cryosections with the anti-Ly6k pAb; 5) Ly6k was detectable from dpc18 and the molecular expression was gradually increased by the stage of sexual maturation (~21dpp) in the mouse testis; and 6) Ly6k was not detected in the testicular cells before 10 dpp with the mAb. Western blot analyses using both the anti-Ly6k pAb and the mAb suggested that the mAb recognizes membrane bind (i.e., GPI-anchored) form of Ly6k, whereas the pAb can react with both GPI-anchored and water-soluble forms of the molecule. This knowledge is basic for the molecular classification of Ly6k, and will expect to provide some clues that hint at the physiological role of Ly6k in the fundamental molecular mechanism of gametogenesis, or cancer biology. (Supported in part by Grants-in-Aid for General Scientific Research, No. 21592206, No 21592111 and "High-Tech Research Center" Project for Private Universities: matching fund subsidy from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.)

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Immunolocalization of Lactoferrin in the Rodent Epididymis.

Christopher Pearl 1

Lactoferrin has been hypothesized to be a general defense protein of the male reproductive tract. In other biological systems, such as milk, it has been demonstrated to have antibacterial, antiviral, antifungal and antioxidant activity, as well as immuno-regulatory effects. If lactoferrin is important for sperm protection then it is likely expressed in the reproductive tract of multiple species. Previous work by me and others has demonstrated that lactoferrin is present in high concentrations and binds to sperm in the epididymis of boars and stallions. This study was designed to determine if lactoferrin is expressed in the epididymis of rats and mice. Epididymides and testes were collected from Sprague Dawley rats and C57BL/6 mice. All animals were retired breeders from Charles River Laboratories. Tissues were fixed in paraformaldehyde, embedded in paraffin, and sectioned at a thickness of five micrometers. Tissues were incubated overnight with goat anti-human lactoferrin, followed by biotinylated secondary antibody and an ABC reagent. Negative control sections were incubated with IgG instead of primary antibody. Immunostaining was visualized using NovaRed chromagen and evaluated by light microscopy. In rats, lactoferrin was expressed in the cytoplasm of principal cells in all three regions of the epididymis, but immunostaining was strongest in the cauda. Sperm in the cauda also appeared to be positive. In mice, lactoferrin was expressed in the cytoplasm of principal cells in all three regions of the epididymis with immunostaining strongest in the corpus. Interestingly, some principal cell nuclei in all three regions of the mouse epididymis appear positive for lactoferrin; this result is unique to mice. No immunostaining was seen in the testis or sections incubated with IgG. The lack of lactoferrin in the testis is consistent with results seen in other species. Additionally, lactoferrin is an estrogen regulated protein in the female reproductive tract and potentially regulated by estrogen in the male reproductive tract. The overall intensity of lactoferrin immunostaining appears greater in boars and stallions than in rats and mice. Since boars and stallions produce high concentrations of estrogen compared to rats and mice, the difference in intensity could be explained by the responsiveness of lactoferrin to differences in estrogen concentrations of these species. Together these results demonstrate that lactoferrin is present in the epididymis of multiple species and suggest that it may be a target for estrogen regulation. Future studies are planned to determine the extent of estrogen regulation and function of lactoferrin in the mammalian epididymis.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Antioxidant Effects of Codonopsis pilosula Reverse Glucose Tolerance Impairment-Induced Testicular Dysfunction.

Kuan-Hua Chiu, Hsiu Chuan Chang, Chih-Chien Chen, Shiow-Chwen Tsai 1

Glucose tolerance decline and insulin resistance often accompanied functional decline in multiple organs. The present study was to examine whether administration of Danseng (Codonopsis pilosula) root extract to adult male rats will reverse glucose tolerance impairment induced testicular dysfunction. The adult Sprague Dawley male rats were divided into three groups (n=6): control group, fructose alone group, fructose plus danseng group. In the fructose group, the tap drinking water was replaced by a 10% fructose solution for 8 weeks. All the rats administered with fructose got glucose tolerance impairment. In the fructose plus danseng group, after the adult male rats got glucose tolerance impairment, Danseng were added by 1 mg/kg body weight in their drinking water for another 4 weeks. Control rats were having standard chow and tap drinking water ad libitum. The rats were anaesthetized and blood was removed from the inferior vena cava and collected into heparinized tubes. The testis was excised and weighed, and used for biochemical analysis. Samples were kept frozen (-20°C) until assay. Activities of antioxidant enzymes in testis were measured by assay kit. The protein expression of steroidogenic acute regulatory protein (StAR), P450 side chain cleavage (P450scc), antioxidant enzymes, and protein kinase A (PKA) were detected by Western blot. Rats that received Danseng experienced a significant increase in blood testosterone level compared to the fructose group (p < 0.01). There were found no significant changes in weight of testis and the relative organ weight. No significant changes in testicular lipid peroxidation were observed between the three groups. The administration of fructose resulted in increased activity of superoxide dismutase (SOD) compared to control group (p<0.05). Combination of fructose and Danseng significantly enhanced the activity of SOD, catalase and glutathione peroxidase (GPx) compared to control group (p<0.05). A correspondent higher increase of testicular SOD expression was also observed in the fructose, and fructose plus Danseng group. The expression of StAR was also increased significantly in the fructose group compared with the control group (p<0.05). Combination of Danseng resulted in StAR expression recovered near the expression of control group. In conclusion, the obtained results strongly suggest that the consumption of fructose decreased the plasma testosterone level due to the declined testicular function, including imbalance activities of antioxidant enzymes, and resulted the compensatory increased StAR expression. The combination of Danseng has protective effects on testicular tissues and can reverse the effect of fructose on antioxidant enzymes.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

SERPINE2, a Serine Protease Inhibitor Extensively Expressed in Adult Male Mouse Reproductive Tissues, May Serve as a Murine Sperm Decapacitation Factor.

Sheng-Hsiang Li 1, Chung-Hao Lu 1, Robert Kuo-Kuang Li 1, Yuh-Ming Hwu 1, Ying-Jie Chen 1, Wei-Chao Chang 2, Shau-Ping Lin 3

SERPINE2, one of the potent serine protease inhibitor for modulating the activity of the plasminogen activator and thrombin, has been implicated in many biological processes. In the present study, we purified SERPINE2 from the mouse seminal vesicle secretion using liquid chromatography, identified by the liquid chromatography/tandem mass spectrometry, and showed a potent inhibitory activity to the urokinase-type plasminogen activator. SERPINE2 was expressed predominantly in seminal vesicles among murine male reproductive tissues. It was mainly detected in the mouse seminal vesicle secretion and mucosal epithelium of the seminal vesicle, epididymis, coagulating gland, and vas deferens. In the testis, SERPINE2 was found in spermatogonia, spermatocyte, spermatid, Leydig cell, and on spermatozoa. SERPINE2 was detected on the acrosomal cap of the testicular and epididymal sperm and suggested to be an intrinsic sperm surface protein. The purified SERPINE2 protein could bind onto the epididymal sperm. A prominent amount of SERPINE2 was detected on ejaculated and oviductal spermatozoa. Nevertheless, SERPINE2 was only detected on the uncapacitated sperm but not on the capacitated sperm, indicating that SERPINE2 is lost after the initiation of capacitation process. Moreover, SERPINE2 could inhibit BSA-induced sperm capacitation in vitro and prevent sperm binding to the egg thus block fertilization. It was through preventing the cholesterol efflux, one of the initiation events of capacitation, from the sperm. These findings suggest that the SERPINE2 protein may play a role as a sperm decapacitation factor.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Effects of Nonylphenol on the Production of Testosterone by Rat Leydig Cells.

Yu-Chen Chang 1, Kai-Lee Wang 1, Ru-Lian Hsu 1, Cai-Yun Jian 1, Shih-Min Hsia 2, Paulus S Wang 1

Nonylphenol (NP) is formed as a non-ionic surfactant that is used widely as industrial detergents. Previous studies have reported that NP impairs male reproductive functions. However, the action mechanism of NP remains unclear. In an in vivo study, male rats were exposed to NP by oral gavage at daily dosage of 100 µg per kg body weight for 7 days. After 7 days of NP exposure, the plasma testosterone level was significantly higher in NP treatment group than in the vehicle group. Moreover, as compared with vehicle group, treatment of NP in vivo could stimulate release of the basal testosterone in vitro from Leydig cells. The levels of testosterone in plasma and medium were measured by radioimmunoassay. In addition, after incubation of rat Leydig cells with human chorionic gonadotropin (hCG), 8-bromo-adenosine cyclic monophosphate (8-Br-cAMP, a permeable analogue of cAMP), or A23187 (an ionophore), testosterone release was significantly higher than in control group. In another study, the pregnant rats (F0 generation) were gavaged orally with or without NP (100 µg per kg body weight) once daily during the interval of pregnancy, then the male rats of the F1 generation were sacrificed at the age of 8 weeks. As compared with control group (without NP feeding in the F0 generation), administration of NP in the F0 generation stimulated testosterone release both in vivo and in vitro in male rats of the F1 generation. After incubation of Leydig cells with hCG, testosterone secretion from rat Leydig cells of the F1 generation was significantly higher than that in the control group. In another in vitro study, Leydig cells from normal male rats were challenged with NP at 13 µM, 43 µM and 128 µM for 60 min. A significant stimulation on the in vitro release of testosterone was observed in the groups treated with 128 µM of NP as compared to the basal level. To further explore the underlying mechanisms of NP on testosterone secretion, we found that administration of calcium-ATPase inhibitors (cyclopiazonic acid and thapsigargin) markedly inhibited NP-induced testosterone release. Neither adenylate cyclase inhibitor SQ22536 (10 µM) nor MEK1 inhibitor PD98059 (2 ìM) had any effect. The present results suggested that NP treatment led to increase testosterone secretion both in vivo and in vitro at least in part via a mechanism associated with disturbance of intracellular Ca2+ homeostasis in Leydig cells.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Expression of 5Alpha-Reductases and Androgen Receptor mRNA in the Female Prostate of Mastomys.

Erina Murao, Takashi Kato, Akihiko Ohta 1

Prostate growth is primarily dependent on androgen concentrations, the most abundant of which in circulation is testosterone (T). T is converted by 5alpha-reductase type 2 into the most active androgen, 5alpha-dihydrotestosterone (DHT), which interacts with androgen receptors in the prostate. The African rodent, Mastomys [Praomys (Mastomys) Coucha], has been used extensively for biomedical research in the areas of oncology, parasitology and epidemiology. One of the outstanding characteristics of this species is the presence of a well-developed prostate in females. Although the physiological significance of the female prostate in this species has not yet been clearly clarified, the histological characteristics of the gland suggest that it is actively secretory and functional. We previously demonstrated that T stimulates growth of the female prostate and that the DHT concentration in the female prostate is comparable to that observed in males, although serum androgen levels in females are markedly lower. To elucidate the mechanisms and significance of DHT production and accumulation in the female prostate, the mRNA expression of 5alpha-reductase isozymes (type 1, type 2) and androgen receptor in the prostate and other organs was examined in both sexes. 3-4 month-old CHAM strain mastomys were used in this study. cDNA sequences of 5alpha-reductase type 1, type 2 and androgen receptor were determined by 5'- and 3'- rapid amplification of cDNA ends. Real time PCR analysis was performed using a commercial PCR kit containing sybergreen florescent dye in the presence of specific primers. The primers were designed to be specific for the mastomys sequences that were determined in the present study. The highest level of 5alpha-reductase type 1 mRNA expression was observed in the female liver, with moderate levels of expression observed in the male liver, epididymis and ovary. Very low levels were detected in testis and uterus. The level of 5alpha-reductase type 1 expression was very low in the female prostate and was comparable to that observed in the male prostate. Expression of 5alpha-reductase type 2 mRNA was highest in the epididymis, moderate in the male prostate, and absent in the liver, testis, ovary and uterus. Interestingly, the 5alpha-reductase type 2 mRNA was expressed at moderate levels in the female prostate and was comparable to that detected in the male prostate. Androgen receptor mRNA was expressed at high levels in the epididymis, the male prostate and the ovary, with moderate expression observed in the testis and uterus. Also, expression of androgen receptor mRNA was highest in the female prostate, with levels twice that observed in the male prostate. These findings suggest that 5alpha-reductase type 2 is the dominant isozyme associated with the production of DHT in the prostates of both female and male mastomys, and that the high level of androgen receptor expression in the female prostate increases the sensitivity and growth of the female prostate in response to DHT.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Effect of H2-Gamendazole on Rapid HSF1 Expression and Interaction with Heat Shock Proteins in Rat Testis In Vivo and Primary Sertoli Cells.

Lesya M Holets 1, Anne Grissell 1, Sudhakar Jakkaray 2, Ramappa Chakrasali 2, Gunda Georg 2, Mary Zelinski 3, Joseph S Tash 1

H2-Gamendazole (H2-GMZ) is a non-hormonal male contraceptive lead that inhibits spermatogenesis and is reversible in rat fertility mating trials. H2-GMZ also demonstrates antispermatogenic activity in mice, rabbits, and in non-human primates. Recently we indicated that H2-GMZ rapidly activates mRNA and protein for HSP90beta and reduced HSP70-3 in rat testis and primary Sertoli cells. Heat shock gene regulation is mediated by activation of transcription activators, heat shock factors (HSF1 and HSF2). However, the molecular mechanism of this regulation in the testis in response to H2-GMZ remains to be determined. We investigated the effect of H2-GMZ on HSF1 expression and regulation of chaperons HSP90beta, HSP70-3, CDC37 and IL-1alpha (a known disrupting factor for Sertoli cell tight junctions) in rat testis and primary Sertoli cells. 70 day-old rats were given a single oral dose of 6 mg/kg H2-GMZ. RNA and protein were isolated from testes at 1, 3, 6, and 24 hr and subjected to quantitative real time RTPCR and western blot, respectively. Primary Sertoli cells from 15-days rats were cultured in the absence or presence 100 nM H2-GMZ for 0, 30, 60, 90, and 180 min, and real time RT-PCR was performed. Western blot revealed protein expression levels in Sertoli cells with 1000 nM of H2-GMZ for 3, 6, and 24 hr. Cellular HSF1 and HSF2 levels were reduced by transient transfection with specific siRNA. Transfected cells were treated with H2-GMZ for 1hr or 6hr for RNA or protein extraction appropriately. Our results establish that H2-GMZ significantly enhances (3 fold) HSF1 mRNA 1hr after H2-GMZ exposure in both in vivo and in vitro and continued high during the remainder of the 24hr time-course in vivo and the 3hr time course in Sertoli cells. H2-GMZ also up-regulated testicular CDC37 protein expression in 3-6 hours post-treatment (two-four fold). Using HSF1 and HSF2 siRNA transfection of primary Sertoli cells, we showed that H2-GMZ mediates HSF2-HSP90 and HSF2-CDC37 interactions by consistently down-regulating HSP90b and CDC37 protein level in siHSF2 transfected cells. HSF1 and HSF2 siRNA transfectants displayed reduction of HSP70-3 (four fold ) and IL-1alpha mRNA (eight fold) after H2-GMZ treatment. In conclusion, rapid differential expression of HSP70, HSP90beta and CDC37 in primary Sertoli cells triggered by H2-GMZ suggested the involvement of additional regulatory mechanisms other than HSF1 and support the hypothesis that Sertoli cells may be a target of H2-GMZ contraceptive effect. Our results also suggest that HSF-2 may play a more significant role in HSP90beta and CDC37 expression in testis than reported in other tissues. This research was supported by grant U54 HD055763.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Immunohistochemical Localization of Estrogen Alpha and Beta Receptors and Aromatase Cytochrome P450 in Adult Stallion Testicles.

Ian Martin 1, Cely Marini Melo 1, Denise Pereira Leme 2, Gabriel Augusto Monteiro 1, Priscilla Nascimento Guasti 1, Renee Laufer Amorim 1, Frederico Ozanam Papa 1

Steroid hormones like testosterone and estradiol are primary produced in the testes. Innumerous studies suggest the role of estrogens in the male reproduction via their specific receptors named estrogen receptor α (ERα) and estrogen receptor β(ERβ). In adult stallions, it have been found the presence of high levels of plasma estrogens, supporting the importance of the estrogen receptors and also of cytochrome P450 aromatase, the enzyme responsible for aromatization of androgens into estradiol. The present study aimed to confirm the presence of both ERs and cytochrome P450 aromatase enzyme via immunohistochemistry in adult stallion testes showing that this tissue is responsive to estrogen. Tissue specimens were collected after castration, washed in saline, placed in plastic cassettes, fixed in 10% buffered formalin for 24h and stored in 70% ethanol until they were embedded in paraffin. 4µm-tissue-section slides were deparaffinized with xylene, rehydrated in graded alcohol and then washed in tap water for 10 minutes. For antigen retrieval, sections were either microwaved for 3 periods of 5 minutes (ERβ) or in a water bath at 96°C for 30 minutes (cytochrome P450 aromatase) in sodium citrate solution 10mM (pH=6.0). Slides were allowed to cool for 20 minutes and then washed ten times in tap water. Endogenous peroxidase activity was quenched with 8% peroxidase solution for 20 minutes followed by 10 baths in tap water and incubated with a 3% milk solution for 1 hour at room temperature. Slides were washed in TRIS buffered solution 10 mM (pH=7.4), encircled using DakoPen (Dako, USA) and incubated with the primary mouse anti-human ERβ monoclonal antibody (clone PPG5/10, Dako, USA), diluted 1:100 in antibody diluent (Dako, USA) or rabbit anti-human cytochrome P450 aromatase (Hauptman Woodward Medical Research Institute Inc., USA), diluted 1:1000. Both were incubated in a humidified chamber for 18h at 4°C. Then, slides were washed in TRIS buffered solution and incubated with the secondary antibody (Advance, Dako, USA). Tissue sections were washed in TRIS buffered solution and DAB was added as chromogen staining substrate for 5 minutes. Tissue sections were rinsed in TRIS buffered solution, counterstained with Mayers hematoxylin for 30 seconds, dehydrated and preserved using Permount mounting medium. For negative controls, another section was incubated with either mouse or rabbit immunoglobulin (Dako, USA) instead of primary antibody. For the evaluation of immunoreactivity, stained sections were observed and photographed using a Leica DML optic microscope (DMLB, Germany) at 400x magnification. All slides were evaluated for positively stained nuclei (ERβ) or cytopmasmatic immunostainning (aromatase). It could be observed a strong immunostainning for cytochrome P450 aromatase in almost all interstitial area, demonstrating that the leydig cells' cytoplasm were positive for this enzyme. However, the nuclei of the cells remained negative. It was also observed for the nuclei of leydig, sertoli and germ cells an ERβ immunostainning. However, for leydig cells it could be visualized a mixed pattern, with some positive and other negative cell regions. The majority of sertoli cells were positive with strong immunostainning. The majority of round germ cells were also positive. However, elongated germ cells were negative. At the present moment we failed to find immunostainning for ERα and new protocols are yet to be performed. Research supported by Fapesp.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Proteins from Seminal Plasma from Tropically-Adapted Brazilian Rams.

Claudia Roberta de Andrade 1, Joao Paulo Arcelino Rego 1, Carlos Eduardo Azevedo Souza 1, Fabio Cesar Gozzo 2, Jose Tadeu Abreu Oliveira 1, Arlindo Alencar Moura 1

Seminal plasma is a mixture composed by fluids secreted from the testes, epididymides and accessory sex glands, and it is involved in various sperm functions and events preceding fertilization. The spermatozoa formed in the seminiferous tubules reach its complete maturation moving through the epididymis, and motility gain, changes in the composition of membrane lipids and proteins, ion exchange between the extra- and intracellular environments, and cytoskeleton rearrangement, among others, are events involved on spermatozoa maturation. Furthermore, the accessory sex glands and epididymis fluids contain proteins that determine important aspects of the fertilizing capacity of spermatozoa, despite the evidence linking these proteins to male fertility indexes been limited. In Brazil, the sheep that have no wool are the main genotypes because of their exceptional adaptability to the tropics. Considering the importance of these animals, our aim was to study aspects of reproductive physiology in Santa Ines rams, by studying the seminal plasma, focusing the analysis on the proteomics of ram seminal plasma. For this purpose, semen was collected from 10 yearling animals and centrifuged to obtain the seminal plasma. Samples with 350 µg of protein were electrophocused (13 cm IPG strips; pH 4 to 7), followed by SDS-PAGE (15%) and the analysis of Coommasie-stained gels was performed using PDQuest software. Proteins were separated by 2-D-PAGE, subjected to in-gel trypsin digestion and analyzed by CapLC HPLC. A Micromass Q-Tof API US mass spectrometer coupled with a Waters CapLC HPLC unit was used for the analysis of tryptic peptides in data-dependent acquisition mode for precursor ion selection using charge-state recognition and intensity threshold as selection criteria using MassLynx 4.0 SP1. Previously, we identified 322 ± 21 spots per gel, from 12 to 160 kDa, among them, spots that presented homology with clusterin and clusterin precursor, zinc-alpha-2-glycoprotein precursor and serotransferrin, proteins well documented in the male reproductive tract. However, some spots could not be identified due to the sensitivity of mass spectrometer technique (MALDI), and here, by using the Q-Tof mass spectrometer, we identified, for the first time in Santa Ines rams, proteins which showed homology with microseminoprotein-beta (gi 76656898; 12 kDa; pI 5.0), described only in human and mouse prostate cancer, and seminal vesicle protein precursor (gi 148225308; 18 kDa; pI 5.2), confirming their importance among proteins involved on the sperm function. The alpha-2-macroglobulin (gi 157954061; 169 kDa; pI 5.7) was described only in the immune system cells, suggesting an important role in protect the sperm during its transit on the female reproductive tract. In addition, phosphoglycolate phosphatase (gi 84000329; 35 kDa; 5.2), arylsulfatase A (gi 114053269; 54 kDa; pI 5.4) have not been described in the reproductive tract, and their importance in this system remains to be proven, since the expression of these proteins was detected in tissues not related to the reproductive tract. Study of the seminal plasma proteins from tropically adapted rams could contribute to a better understanding of reproductive physiology, as well as factors involved in the sperm maturation and function, providing the basis for understanding aspects of these animals and their fertility potential. This study was supported by grants from Brazilian Research Councils, CNPq and CAPES (to AAM).

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Reproductive Criteria of Tropically-Adapted Morada Nova Rams from Brazil.

Joao PA Rego 1, Fabiane Sousa 1, Carlos EA Souza 1, Aletheia L Souza 1, Fabio Cesar Gozzo 2, Jose TA Oliveira 1, Airton A Araujo 1, Erika B Menezes 1, Rodrigo V Oliveira 1, Arlindo A Moura 1

Morada Nova is a breed of hair sheep, native from the Northeast of Brazil. These are unique genotypes because they are highly adapted to tropical environments. The present work describes basic reproductive parameters of Morada Nova rams. We evaluated the biometry of the reproductive tract, aspects of spermatogenesis, seminal parameters, as well as the protein maps of seminal plasma, accessory sex gland and cauda epididymal fluids. Animals (n = 15) were evaluated at 42 weeks of age and semen samples collected by electroejaculation. One week later, rams were slaughtered to obtain samples of the reproductive tract. Seminal plasma, CEF and VGF were assayed for total protein concentration and a sample containing 500 µg of proteins, subjected to 2D electrophoresis, using IPGs (4-7L; 13 cm) and 15% SDS-PAGE gels. The gels were stained with Coomassie blue and analyzed with PDQuest. The major spots from seminal plasma were cut, digested with trypsin and identified by LC-MS/MS and Mascot search. Pearson correlations (p<0.05) were calculated. The average scrotal circumference and testicular weight of rams were 27.5 ± 0.5 cm and 109.5 ± 6.0 g. Seminiferous tubule diameter and epithelial height were 188.3 ± 4.0 and 58.0 ± 3.0 µm. Each testis contained (x109) 1.9 ± 0.1 Sertoli cells, 0.3 ± 0.02 A spermatogonia, 5.8 ± 0.5 pachytene spermatocytes and 17.5 ± 1.3 round spermatids per testis, respectively. Each Sertoli cell supported 0.1 ± 0.01 A spermatogonia, 3.0 ± 0.2 pachytene spermatocytes and 7.7 ± 0.5 round spermatids. Daily sperm production reached 5.6 million cells per gram of testicular tissue. Ejaculates averaged 48.9% of motile sperm, 0.7 billion sperm/mL of ejaculated semen and 83.2% sperm cells with normal morphology. We detected, on average, 89.2 ± 10.4, 145.8 ± 8.4 and 90.9 ± 9.6 spots for CEF, VGF and SP gels, respectively. The four major spots from seminal plasma were identified as RSVP14, RSVP22 and bodhesin 2, representing, respectively, 7.6%, 5.8% and 3.0% of the total intensity of the valid spots of the gels. RSVPs belong to the BSP (Binder of Sperm Proteins) superfamily. Such proteins are putatively associated with sperm capacitation. In ruminants, spermadhesins play a role in sperm metabolism and protection of membranes. However, their concentration in the AGF protein maps have been negatively related to dairy bull fertility. We recently described that RSVPs and spermadhesins are also the major proteins secreted in the seminal plasma of Santa Ines rams, another native breed in Brazil. Testicular weight was related to all other testicular (r=0.73 to 0.98) and epididymal (r=0.59 to 0.64) measurements. Testicular diameter (TD) was indicative of seminiferous tubule (ST) diameter (r=0.94) and length (r=0.73), as well as the percentage volume of ST (r=0.94). Higher TD values were also associated with the population of pachytene spermatocytes (r=0.87), round spermatids (r=0.74) and Sertoli cells (r=0.53) per testis. Rams with havier testes, also had higher TD (r=0.97), daily sperm production (r=0.79) and more Sertoli cells/testis (r=0.50). This is the first work describing the basic reproductive characteristics of Morada Nova rams, as well as protein profiles of their reproductive tract fluids. These findings will allow a better understanding of their reproductive physiology, and may serve as a tool for the improvement of their reproductive efficiency.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

NOT PRESENTED.

NOT PRESENTED.

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Rosiglitazone Enhances Na+ Absorption Across Porcine Vas Deferens Epithelia.

Jacob Hull, Qian Wang, Florence Wang, Vladimir Akoyev, Bruce D Schultz 1

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) activators, including rosiglitazone, reportedly affect fluid and electrolyte transport across a variety of epithelia. However, the transporters affected, including the cystic fibrosis anion channel (CFTR) and the epithelial Na+ channel (ENaC), and the effects on net flux appear to be cell type or tissue specific. The goal of the current project was to determine the effects of chronic (2-4 day) rosiglitazone exposure on epithelial cells isolated from adult pig vas deferens, a tissue known to express CFTR and to express ENaC when exposed to glucocorticoids. Results showed that, in the absence of glucocorticoids, rosiglitazone had little effect on electrophysiological parameters including baseline short circuit current, baseline transepithelial electrical resistance, and the responses to amiloride (an ENaC blocker) forskolin (an activator of adenyll cyclace), and DASU-02, a CFTR channel blocker. However, when cells were cultured in the presence of dexamethasone, rosiglitazone was associated with a 2-fold increase in baseline short circuit current and a two-fold increase in the magnitude of the amiloride-inhibitable short circuit current. These results suggest that PPAR-gamma activation in vas deferens epithelial cells causes a profound increase in Na+ absorption that would likely change the luminal environment and reduce the luminal volume in the vas deferens, effects that would likely affect sperm viability or function. [Supported by NIH R01 HD058398]

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Aging Causes Alterations in the Base Excision Repair Pathway in Pachytene Spermatocytes in the Brown Norway Rat.

Catriona Paul, Makoto Nagano, Bernard Robaire 1

With a current trend of increasing paternal age, there is a concern about a higher risk for the development of complex multi-gene diseases in newborns. Recent studies have established increased incidences of autism and schizophrenia with advancing paternal age. There is a strong correlation between the age of the male partner and decreased sperm quality and structural aberrations of chromatin and DNA in spermatozoa. It is known that the production of reactive oxygen species (ROS) increases with age in the testis. The Base Excision Repair (BER) pathway is the main operator in the removal of oxidative DNA damage and removes aberrant bases in DNA induced by hydrolysis, ROS or other metabolites. We hypothesize that with age the ability to repair oxidative stress-induced DNA damage is impaired in male germ cells. To test this, pachytene spermatocytes and round spermatids were isolated from Brown Norway rats at 4 months of age (young) and 18 months (aged) using the STA-PUT velocity sedimentation technique, achieving >80% purity. RNA was extracted from these two cell populations and gene expression was assessed using Affymetrix rat 230 2.0 whole rat genome microarrays. Raw data were analysed using Genespring v10 (Agilent Technologies) and Pathway Studio. Of the probe sets with significantly altered expression there appeared to be changes in the oxidative stress response and DNA repair in the pachytene spermatocytes but not in the round spermatids. Further analysis in the pachytene spermatocytes emphasized that genes involved in the BER pathway were specifically altered. We found that Ogg1 was overexpressed and Ape1, Fen1 and Xrcc1 were down regulated. Quantitative RT-PCR analysis showed at least a 1.5 fold change in these genes in the old in comparison to the young. We also identified Ogg1 protein in the early spermatocytes, elongating spermatids and the peritubular myoid cells using immunohistochemistry. The Ogg1 protein appeared to be more highly expressed in those testes from the aged rats. In the BER pathway Ogg1 encodes the enzyme responsible for the initiation of the inscision of 8-oxoguanine, a mutagenic base byproduct resulting from exposure to ROS. In conclusion, aging causes altered expression of genes involved the BER pathway. The fact that Ogg1 is upregulated and the others downregulated suggests that the initiation of the pathway is enhanced but that the subsequent repair of the damaged site is impaired. This work was supported by a grant from CIHR.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Seasonal Localization and Expression of Activin Signaling Components in the Wild Ground Squirrel Testis.

Xia Sheng 1, Yi-Ting Jiang 1, Lin Li 1, Qiang Weng 1, Kazuyoshi Taya 2

The mechanism of seasonal spermatogenesis displayed by most wild animals is basically unknown. As our animal model, the wild ground squirrel is a typical seasonal breeder with a breeding season from April to May, followed by a long period of sexual dormancy from June to March. By acting through a heteromeric receptor complex at the cell surface and an intracellular signal transducing Smad complex, the critical role Activin played in the spermatogenesis was proved by increasingly accumulated evidences. Our previous works showed the arresting seasonal expression of inhibin/activin subunits (alpha, betaA, and betaB) in wild ground squirrel testis. We hypothesize that activin signaling components are dynamically regulated during seasonal spermatogenesis, thereby permitting activin to have distinct temporal regulatory actions. In this study, we investigated the localization and expression level of activin signaling components (ActRIIA, ActRIIB, Smad2/3, Smad4 and Smad7) in the testes of wild ground squirrel captured in breeding season (April) and non-breeding season (July). The methods include histology, immunohistochemistry and western blotting. Histological appearances exhibited the marked seasonal variation in spermatogenesis. All types of spermatogenic cells including mature spermatozoa were detected in the breeding season testicular tubules, but only spermatogonia and primary spermatocytes were observed in non-breeding season. Immunohistochemical results showed that in the breeding season, ActRIIA, Smad2/3, Smad4 and Smad7 were all strongly stained in the cytoplasm of Sertoli cells, whereas ActRIIB was not. Positive signals of Smad2/3, Smad4 and ActRIIA were also detected in the Leydig cells, while the signals of Smad7 and ActRIIB were weaker. Interestingly, specific and intense signals of ActRIIB and Smad7 were detected in the cytoplasm of round spermatids. Smad2/3 and ActRIIA were also expressed in the sperm. In the non-breeding season, however, the proteins were predominately detected in the cytoplasm of spermatogonia. Additionally, Smad4 and Smad7 were expressed in the Leydig cells. We further performed western blotting to determine the expression level of each protein in testis tissue. Bands of approximately 60 kDa for ActRIIA, 50 kDa for ActRIIB, 55 kDa for Smad2/3, 61 kDa for Smad4 and 51 kDa for Smad7 were identified in the lysates, in consistent with previously reported in mouse and rat. Meanwhile, all proteins showed higher expression levels in the breeding season than those in the non-breeding season. In conclusion, this study demonstrated, to our knowledge for the first time, the seasonal expression of activin signaling components in wild ground squirrel testis. The dynamic regulation of both activin type II receptors and Smad proteins suggested that activin played an important autocrine and paracrine role in the seasonal spermatogenesis of wild ground squirrel. Furthermore, the distinct localization and immunoreactivity of these proteins may indicate different functions of activin in the testis of breeding and non-breeding seasons. This study was supported by the Program for Changjiang Scholars and Innovative Research Team in Universities (IRT0607), P. R. China.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

The Activity of Ubiquitin Activating Enzyme UBA1 Is Required for Sperm Capacitation, Acrosomal Exocytosis, and Sperm-Egg Coat Penetration During Porcine Fertilization.

Young-Joo Yi 1, Shawn W Zimmerman 1, Gaurishankar Manandhar 1, John F Odhiambo 1, Vera Jonakova 2, Miriam Sutovsky 1, Chang-Sik Park 3, Peter Sutovsky 1

The ubiquitin-proteasome pathway has been implicated in the process of sperm-egg coat penetration during mammalian, ascidian and echinoderm fertilization. Ubiquitin is a highly conserved chaperone protein of 76 amino acid residues which binds covalently to substrate proteins through a multi-step enzymatic pathway, catalyzed by the enzymatic activities of the E1-type ubiquitin-activating enzyme (UBA1), ubiquitin-conjugating enzymes (UBE2), and a large number of substrate-specific E3-type ubiquitin ligases. Tandem ligation of ubiquitin molecules, resulting in the formation of a polyubiquitin chain, is necessary for substrate degradation by the 26S proteasome. Enzyme UBA1 is responsible for the first step of ubiquitin-protein ligation and is a critical component for the initiation of ubiquitination. The present study examined the requirement of UBA1 activity for sperm capacitation, acrosomal exocytosis and sperm-zona pellucida (ZP) penetration during porcine fertilization in vitro (IVF). Porcine ova were inseminated in the presence of various concentrations of a specific UBA1 inhibitor (4[4-(5-Nitro-furan-2-ylmethylene)-3,5-dioxo-pyrazolidin-1-yl]-benzoic acid ethyl ester), and evaluated for fertilization rates. Fresh boar semen was capacitated with or without UBA1 inhibitor, and incubated with FITC-conjugated peanut agglutinin (FITC-PNA) to monitor the capacitation-induced changes of the outer acrosomal membrane (OAM) by flow cytometry. The activity of UBA1 in the isolated sperm acrosomal fractions was tested by thiol ester assay with biotinylated ubiquitin. The presence of ubiquitinated proteins in capacitated and non-capacitated sperm acrosomes was evaluated by Western blotting. UBA1 was localized in the acrosomes of boar spermatozoa by immunofluorescence. During IVF, the rates of sperm-ZP penetration/fertilization were significantly decreased following IVF with addition of UBA1 inhibitor (27.2-81.6 vs. 96.3% in control, p<0.05). On the contrary, there were no significant changes in the sperm penetration rate during IVF in the presence of extrinsic, recombinant UBA1. Significantly lower fluorescence intensities of PNA-FITC, indicative of the suppressed remodeling of OAM/sperm capacitation, were observed by flow cytometry in spermatozoa capacitated in the presence of UBA1 inhibitor. While viable and motile, these spermatozoa showed significantly reduced fertilization rates during IVF. In Western blotting with anti-ubiquitin antibodies, a typical smear of polyubiquitinated proteins was observed above the 176 kDa marker in both the non-capacitated and capacitated sperm extract. Unusually high Mr bands, suggestive of posttranslational modification by ubiquitination, were detected in non-capacitated spermatozoa by Western blotting with antibodies specific to acrosomal proteins SPINK2 (acrosin-inhibitor) and AQN1 (spermadhesin). Thiol ester assays utilizing biotinylated substrate ubiquitin and isolated sperm acrosome fractions confirmed the enzymatic activity of UBA1 in the sperm acrosome, which was sensitive to UBA1 inhibitor. To our knowledge, this is the first report of UBA1-requirement for mammalian fertilization. It appears that UBA1 has an essential role in sperm capacitation and acrosomal function during fertilization. This work was supported by NRI Grant# 2007-01319 from the USDA-CREES and by the F21C Program of the University of Missouri.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Identification of Oog1 Promoter Regions which Function During the First Meiosis in Oocytes.

Eriko Okazaki 1, Satoshi Tsukamoto 2, Koji Kimura 3, Yuki Ohta 4, Seiji Kito 2, Hiroshi Imai 1, Naojiro Minami 1

We previously reported the characteristics of Oog1 in female germ cells and the phenotypes of knockdown mouse and the embryos using dsRNAi (double-stranded RNA interference) transgenic technology. Interestingly, Oog1 localizes the nucleus of 1- to 2-cell stage embryos at the time of zygotic gene activation although none of the phenotypes such as decrease of litter size and impair of developmental competence were observed in knockdown females and the embryos in spite of the complete knockdown of mRNA in GV oocyte. We produced transgenic mice that express double strand Oog1 RNA under the control of ZP3 promoter. Result of real-time PCR clearly shows that the amount of Oog1 mRNA remarkably reduced in transgenic GV oocytes; however, Western blotting analysis revealed that the amount of Oog1 proteins did not significantly decrease in these transgenic oocytes. The reason of the persistence of Oog1 proteins in the oocytes is unknown, but the protein translated before ZP3 expression might be retained in the GV oocytes. However, it is probable that the persistence of the protein causes normal phenotype in female fertility and embryonic development. ZP3 starts to express in the oocytes of primary follicles whose formation occurs after birth, however, in our previous study Oog1 expression starts in oocytes at E15.5. The gap of the expression between ZP3 and Oog1 would prevent the functional analysis of Oog1 during oogenesis and embryogenesis. In the present study, we confirm the protein expression of Oog1 during oogenesis using immunohistochemistry and analyze the Oog1 promoter regions that control the expression of Oog1. Immunohistochemistry reveals that Oog1 protein starts to be translated in oocytes of germline cyst and continues subsequently. Interestingly, Oog1 protein localizes in nucleus of oocytes at E15.5. This time coincides with a period in which the majority of oocytes enter the pachytene stage of the first meiotic prophase, suggesting that Oog1 is involved in the process of meiosis. To elucidate the function of Oog1 during meiotic prophase using dsRNAi transgenic mice, the promoter that is expressed at this stage is required. We selected 3 upstream regions of Oog1 from the information of 5 copies of the gene on chromosome 4 and 12, and produced 2 kinds (5 lines) of transgenic mice having 2 different Oog1 upstream regions followed by GFP gene. The observation of GFP signal revealed that both of 2 upstream regions functioned specifically in oocytes of germline cysts and oocyte of all stages of follicles, suggesting that Oog1 knockdown oocytes during meiosis can be obtained using these Oog1 upstream regions.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Zinc Requirement During Meiosis I-Meiosis II Transition in Mouse Oocytes Is Independent of the Mos-MAPK Pathway.

Miranda L Bernhardt 1, Alison M Kim 1, Thomas V O'Halloran 2, Teresa K Woodruff 1

Zinc is an essential cofactor in hundreds of molecules needed for many biological processes, including formation and maturation of gametes. Our laboratory has recently demonstrated an important role for zinc during oocyte meiotic maturation. The first meiotic division is normally asymmetric and produces a large oocyte and a much smaller polar body. However, the attenuation of intracellular zinc availability during in vitro maturation of mouse oocytes using the membrane permeable heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) causes formation of large polar bodies and failure to form a meiosis II spindle. Oocytes matured in the presence of TPEN display a peculiar spindle phenotype in which a telophase-I-like conformation of spindle microtubules is maintained while chromatin begins to decondense. Formation of the cortical granule free domain is also impaired in these zinc-insufficient oocytes. Features of this phenotype resemble those of Mos null oocytes. Mos protein normally accumulates after germinal vesicle breakdown and phosphorylates mitogen activated kinase kinase 1 and 2 (MAP2K1/2) which, in turn, phosphorylate and activate mitogen activated kinase 1 and 3 (MAPK1/3). In Mos null oocytes, this pathway is disrupted and MAPK1/3 does not become phosphorylated; the phenotype of these oocytes is attributed mainly to absence of MAPK activation. The goal of this project was to investigate the mechanism by which zinc insufficiency causes abnormal meiotic maturation, focusing on components of the Mos-MAPK pathway. Reduced levels of both Mos protein and phospho-MAP2K1/2 are observed in zinc-insufficient oocytes; however, these differences appear only after completion of the first meiotic division. In addition, activation of downstream effector of the Mos pathway, MAPK1/3, is unaffected by zinc-insufficiency, and reduced Mos levels are observed only with the presence of TPEN after first polar body extrusion. These data are inconsistent with the hypothesis that reduced Mos mediates the observed phenotype. Finally, Mos overexpression does not rescue the phenotype of zinc-insufficient oocytes. Combined, these data lead us to conclude that reduced Mos levels are not responsible for the failure of asymmetric division and spindle abnormalities observed in our model of oocyte zinc-insufficiency. Zinc-insufficient oocytes fail to increase maturation promoting factor activity following the first meiotic division, indicating a failure to establish cytostatic factor (CSF), rather than the failure to maintain CSF observed in Mos null oocytes. Although we have shown that zinc has a novel role as a key regulator of the meiotic cell cycle, this role is not mediated through the Mos-MAPK pathway. Further work will be necessary to determine what pathways are impacted by zinc insufficiency and the mechanism by which disruption of meiotic maturation is occurring. This research was supported by NIH/NICHD P01 HD021921 and T32 HD07068 and by the W. M. Keck Foundation.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Effect of Different Maturation Media on In Vitro-Matured Common Marmoset Oocytes.

Tsukasa Takahashi 1, Ikuo Tomioka 1, Akiko Shimada 2, Koji Yoshioka 3, Erika Sasaki 1

The common marmoset (Callithrix jacchus) is a nonendangered New World primate. Though genetically modified common marmosets have been produced, effective in vitro oocytes maturation method is not well established. Protein-free chemically defined maturation medium POM (porcine oocyte medium) is efficient in vitro maturation medium in porcine. Chemically defined medium is useful for analyzing the physical action of substances. However in mouse, oocytes maturation rate of serum-free medium showed significant reduction in sperm penetration as compared to oocytes maturation rate in the presence of serum. Necessity of serum depends on species. In this study, we investigated the effect of maturation media for in vitro maturation of common marmoset oocytes that better supports subsequent embryonic development. In experiment 1, oocytes collected from follicles were randomly divided into two media and cultured with modified Waymouth's MB 752/1 (mWM) medium (supplemented with 5% FBS, 1% ITS, 100IU/ml FSH, 1µg/ml estradiol, 0.5mM Na-pyruvate, 10mM sodium lactate, 4mM hypotaurine and 1mM glutamine) or POM (supplemented with 100IU/ml FSH). In experiment 2, POM or modified POM (mPOM; supplemented with 5% FBS, and 100IU/ml FSH). After 27h of the culture, remaining cumulus cells were removed. Then metaphase II oocytes were inseminated with ejaculated sperm in TYH medium and resulting embryos were cultured in ISM1 for the first 3days and then cultured further in ISM2 until development arrest. In experiment 1, although significant difference was not observed, POM medium showed higher maturation rate than mWM (36.4% vs 29.5%). Embryo development rate to blastocyst between POM and mWM (8.9% vs 6.8%) were similar. However fertilized rate of POM was tend to be lower than mWM (60.0% vs 72.7%). In experiment 2, maturation rate of mPOM was higher than POM (53.1% vs 45.2%). Fertilized rate of mPOM was significantly higher (p < 0.05) than POM (76.5% vs 61.2%). The mPOM increased the rate of blastocyst development than POM (18.6% vs 12.9%). The mPOM increased blastocysts developmental rate because FBS effect on sperm insemination for common marmoset. These results indicate that POM is suitable as a basic maturation medium and serum promoted fertilization.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

NOT PRESENTED.

NOT PRESENTED.

Biol Reprod. 2010 Nov;83(1 Suppl):4.

FSH Glycoform Abundance During The Menstrual Cycle.

George R Bousfield 1, Vladimir Y Butnev 1, Monica A Rueda-Santos 2

FSH plays a central role in regulating reproductive cycles by stimulating ovarian follicle growth, cell differentiation, and steroidogenic activity. FSH is a heterodimeric glycoprotein composed of two non-covalently associated, dissimilar subunits designated α and β. The α subunit is common to FSH and three other glycoprotein hormones--LH, TSH, and CG--and always possesses two N-glycans. The hormone-specific FSHβ subunit often lacks one or more glycans. Human FSHβ appears to be N-glycosylated in an all-or-none manner, producing two major variants, the classic 4-glycan, tetra-glycosylated hFSH and a novel, 2-glycan, di-glycosylated hFSH. In vitro receptor-binding assays indicate the latter is 10- to 25-fold more active than the former. The abundance of these glycoforms as determined by Western blot analysis of heterodimeric FSH isolated from individual pituitary glands appears to vary as a function of age and physiological state. In the present study, we examined day-to-day changes in urinary hFSH glycoform abundance. Accomplishing this required some changes in our experimental procedures. Western blots using anti-hFSHβ monoclonal antibody RFSH20 require 1 μg hFSH, which is suitable for the amounts available in human pituitary glands. However, only 100-200 ng FSH can be recovered from first void daily urine samples. Assessment with anti-hFSHβ peptide monoclonal antibody P03 or polyclonal antiserum W521, could detect 100 ng hormone, but the ratios of the 21- and 24-kDa bands differed from those determined with RFSH20. As glycoforms can be separated by size using high performance gel filtration, we developed a column-based assay system. When applied to the FSH heterodimer fraction isolated from a male pituitary, the same glycoform ratio was obtained by RFSH20 Western blotting and Superdex 75 gel filtration monitored at both 210 and 280 nm. Analysis of daily urinary FSH immunopurified from three subjects showed day-to-day variation in glycoform abundance. Previous pituitary FSH glycoform abundance determinations, which provide a snapshot of hormonal variation, had suggested changes in glycoform abundance occurred during the cycle. The glycoform abundance pattern in a 27-year-old woman consisted of low abundance of di-glycosylated hFSH early in the cycle, which changed to higher abundance on day 8, five days before the LH surge. This was followed by a pattern of progressive decrease in abundance over a 2- to 3-day period, an abrupt increase in abundance followed by progressive decline until the end of the cycle. In a 43-year-old woman, di-glycosylated hFSH was high early in the cycle, but progressively decreased until the day of the LH surge, when it abruptly rose, then became less abundant for the rest of the cycle. In a third woman, with no obvious LH surge, the abundance of di-glycosylated hFSH started low, rose to a maximum on day 12, and declined progressively until the last day of the cycle on day 19. These results confirm that day-to-day variations in FSH glycoform abundance take place and suggest the pattern of glycoform abundance may contribute to age-related changes in fertility. Supported by NIH P01 AG029531.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Cortisol Inhibits Sexual Receptivity, but Has No Effect on Attractivity or Proceptivity in Ovariectomized Ewes Treated with Different Doses of Estradiol Benzoate.

Melissa M Papargiris 1, Paul H Hemsworth 2, Elizabeth TA Rivalland 1, Adam D Morrissey 1, Alan J Tilbrook 1

Stress inhibits reproductive processes, including gonadotropin secretion and sexual behavior; however, the mechanisms and mediators involved are largely unknown. Recently, we have shown that stress and stress-like levels of cortisol can hinder sexual behavior but the mechanisms by which this occurs are also unknown. One possible mechanism that has yet to be explored is the ability of cortisol to interfere with the actions of estradiol to induce ovulation and estrus, a period of heightened sexual activity characterized by specific behaviors. We know that higher doses of estradiol to artificially induce estrus reduce the time to onset of estrus and that the administration of cortisol significantly delays this onset of estrus. We therefore propose that a higher dose of estradiol may overcome the inhibitory effects of cortisol on sexual behavior. We tested this hypothesis in ovariectomized ewes, induced into estrus by 12 days of intramuscular (i.m.) progesterone priming (20 mg/48h) followed by a normal (25 µg) and high (50 µg) dose of estradiol benzoate (EB) given i.m. The ewes (n=5/group) were randomly allocated to 4 groups; saline + 25 µg EB, saline + 50 µg EB, cortisol + 25 µg EB and cortisol + 50 µg EB. Saline or cortisol (250 µg/kg/h) was infused for 40h beginning at the time of the EB injection. Blood samples were collected and behavioral observations were conducted every 6h. The specific behaviors that were measured were attractivity, proceptivity, and receptivity. The infusion of cortisol significantly (p<0.001) elevated plasma concentrations of cortisol compared to controls, irrespective of the dose of EB. Plasma concentrations of LH were significantly (p<0.05) reduced in cortisol-treated ewes compared to saline-treated ewes, and this reduction was greater in the ewes treated with cortisol + 50 µg of EB (p<0.05). Furthermore, the reduction in plasma LH seen in the cortisol treated ewes was due to a reduction in the amplitude of the LH pulses (p<0.05). The onset of estrus was reduced in ewes treated with 50 µg EB compared to ewes treated with 25 µg EB, however, there was no change in the duration of estrus. In contrast, cortisol had no effect on the onset or the duration of estrus, regardless of the dose of EB. Cortisol suppressed receptivity, but had no effect on attractivity or proceptivity. Specifically, cortisol inhibited receptivity index (number of immobilizations by ewe/courtship displays by ram) (p<0.05), the number of times the ewe was mounted (p<0.001) and the number of times the ewe stood to be mounted by the ram (p<0.05), irrespective of the dose of EB administered. Collectively, these results suggest that the inhibitory actions of cortisol on sexual behavior cannot be overcome by increasing the dose of estradiol used to induce estrus. In conclusion, cortisol has no effect on the ability of the female to attract the male or the motivation of the female to be with the male, however, cortisol inhibits the consummatory phase of sexual behavior, which is necessary for successful reproduction to occur and this is independent of the dose of estradiol administered.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Kisspeptin Neurons in the Hypothalamic Arcuate Nucleus Are Potent Stimulators of GnRH/LH Secretion in Holstein Steers.

Satoshi Ohkura 1, Kenji Takase 1, Yoshihiro Wakabayashi 2, Ken-ichi Yayou 2, Yoshihisa Uenoyama 1, Hiroko Tsukamura 1, Kei-ichiro Maeda 1, Hiroaki Okamura 2

The present study aims to clarify the physiological roles of kisspeptin signaling in the bovine hypothalamus in the regulation of gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) secretion. First, we cloned the full-length sequence of Kiss1 cDNA from the hypothalamic tissue of a Holstein steer by 5'- and 3'-RACE techniques. Sequence analysis revealed that deduced Kiss1 peptide composed of 135-amino-acid residues and that processing of the peptide resulted in a putative cattle kisspeptin of 53 amino acids, which showed 91, 89, 81, 74, 74, and 68% sequence identities with goat, sheep, pig, mouse, rat, and human kisspeptin, respectively. The C-terminal 10-amino-acid residues, which are the minimal core sequence of kisspeptin, were identical among cattle, goat, sheep, pig, mouse, and rat. Second, distribution of kisspeptin-immunoreactivity in Holstein steers (n = 2) was examined using a polyclonal antibody (C2 antibody) against the synthetic peptide corresponding to [Cys41]goat kisspeptin (41-53), CSAYNWNSFGLRY, which is an identical to [Cys41]cattle kisspeptin (41-53). The head was perfused bilaterally through the carotid arteries with 10 mM phosphate-buffered saline, followed by fixative consisting of 4% paraformaldehyde, pH 7.4, and then the brain block containing the hypothalamus was removed and processed for immunohistochemistry. Cell bodies containing kisspeptin-immunoreactivity were found in the hypothalamic arcuate nucleus (ARC) in steers. Many fibers with kisspeptin-immunoreactivity were observed in the median eminence, where the majority of GnRH neuronal fibers projected. Third, effects of peripheral bolus injection of kisspeptin on LH secretion were examined in Holstein steers (n = 4 per treatment). We found that intravenous injection of active C-terminal peptide of human kisspeptin, kisspeptin-10 (Kp-10; 38 and 380 nmol), stimulated LH secretions. Statistical comparison revealed that mean area-under-the-curve of LH profiles 2 hours after Kp-10 (380 nmol) administration was significantly (P<0.05) greater than that after control treatment. The action site of kisspeptin administered peripherally could be GnRH neuronal terminals in the median eminence to stimulate GnRH release, because distribution of kisspeptin- and GnRH-immunoreactive fibers made intimate association in the median eminence.The present study suggests that kisspeptin neurons in the ARC send their fibers to the median eminence to facilitate GnRH release in steers. Kisspeptin signaling in the hypothalamus would be critical for the control of reproduction in cattle as in other ruminant species.This research was supported by PROBRAIN of Japan.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Production and Characterization of a Chimeric Recombinant Salmon Follicle-Stimulating Hormone Analog.

Penny Swanson 1, Jon T Dickey 2, J Adam Luckenbach 1, Michael P Bernard 3, William R Moyle 3

The first piscine follicle-stimulating hormone (FSH) was identified in salmon more than 20 years ago and has since been identified in numerous species of bony and cartilaginous fish. Yet, knowledge of FSH function in fishes is limited because of the lack of highly purified native protein and specific immunoassays for physiological studies in divergent species. The long-term aim of our research is to produce recombinant piscine FSH analogs that can be used to investigate FSH function and enhance reproduction of divergent fish species that are important for aquaculture. Initially we focused on production of recombinant salmon (s)FSH because of the availability of: native sFSH for comparison; cDNAs encoding sFSH, luteinizing hormone (sLH), and thyroid-stimulating hormone (sTSH) subunits and their receptors (R); and sFSH immunoassays and bioassays for characterization of the analog. Our strategy has been to produce recombinant single chain sFSH analogs in either COS-7, CHO, or HEK293T cells and screen them for their ability to induce cAMP production in transiently (HEK293T) or stably (CHO) transfected cells expressing recombinant sFSHR. Analogs that were active in the receptor assays were then tested for steroidogenic activity in vitro using salmon ovarian follicles and immunological cross-reactivity in the sFSH radioimmunoassay (RIA). We found that single-chain analogs in which the C-terminal end of the hCG beta-subunit was used as a spacer to link the sFSH beta-subunit to the alpha-subunit were not expressed well. This result was surprising because this strategy is commonly used to express mammalian FSH and LH; however, it is consistent with earlier studies showing that heterodimers having structures similar to sFSH having altered seatbelt latch sites are assembled differently from mammalian FSH. In contrast, a single-chain construct that encoded FLAG-tagged sFSH beta-subunit-glycine-human alpha-subunit was expressed very well by HEK293T cells. The FLAG tag and the human alpha-subunit were included to facilitate quantification of the material in sandwich assays employing a human alpha-subunit monoclonal antibody for capture and a radioiodinated M1 anti-FLAG antibody for detection. This FSH analog was a very potent stimulator of cAMP production by mammalian cells expressing the sFSHR, and concentrations that gave maximal cAMP induction did not elicit a response from cells expressing the sTSHR. Furthermore, this analog had similar potency to native sFSH in the in vitro bioassay and serial dilutions of the sFSH analog were parallel to the native sFSH in the RIA. In other structure/function studies, we found that FSH analogs in which the human alpha-subunit is replaced by the salmon alpha-subunit II or in which the C-terminal end of the sFSH beta-subunit and N-terminal end of the human alpha-subunit can be truncated without destroying its ability to initiate signal transduction in cells that express sFSHR. We have also prepared and tested several other analogs to characterize the structure and function of sFSH. Our results appear to be consistent with the notion that salmon and human FSH interact with different portions of their respective receptors, a phenomenon predicted on the basis of differences in the folding patterns of salmon and human FSH. Research supported by National Research Initiative Competitive Grant no. 2007-35203-18088 from the USDA CSREES.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

NOT PRESENTED.

NOT PRESENTED.

Biol Reprod. 2010 Nov;83(1 Suppl):4.

The Metabolic Syndrome and Ovarian Dysfunction in Mice.

Jennifer W Hill 1, Angela Burgess 1, Jens C Bruning 2, Bradford B Lowell 3, Joel K Elmquist 4, Nader G Abraham 1

Polycystic ovarian syndrome often presents alongside the metabolic syndrome: obesity, impaired glucose tolerance and type 2 diabetes, hyperlipidemia, chronic low-grade inflammation, and early-stage atherosclerosis. We have recently demonstrated that mice with impaired insulin and leptin signaling in POMC neurons exhibit a PCOS-like phenotype including systemic insulin resistance, elevated serum testosterone levels and ovarian abnormalities resulting in reduced fertility. In a separate study, we have recently shown that an HDL analog reduces cardiovascular risk factors independent of body weight, improves insulin sensitivity, reduces testosterone levels and increases estradiol levels in obese leptin-deficient female mice. We hypothesize that inflammation, insulin resistance, and elevated triglyceride levels contribute to the impairment of reproductive functioning in ob/ob and POMC-cre,LepRfloxIRflox mice. To test our hypothesis, we have administered L4-F, gemfibrozil, flutamide, metformin, and pioglitazone to separate cohorts to examine the ability of these drugs to improve fertility. Our results demonstrate that increased insulin sensitivity improves ovarian functioning in these mice and provide some insight into mechanisms involved. Thus, these mouse models offer a novel approach for examining the etiology and treatment of obesity-associated infertility. Research supported by NICHD HD056491 to JWH.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Does Maternal Diet Affect Phenotypic Outcomes in Offspring?

Denise A Warzak, Jiude Mao, Jessica A Keegan, Paizlee T Sieli, Cheryl S Rosenfeld, R Michael Roberts 1

Maternal diet might govern the risk of her offspring for adult-onset diseases, including obesity, diabetes, and behavioral changes. We have previously demonstrated that murine offspring born to dams fed a very high fat diet for an extended time (40 weeks) weigh more and exhibit adverse changes in behavioral patterns compared to those born to dams on a low fat or Purina 5015 Chow diet. Here, we sought to determine whether similar weight and behavioral outcomes are observed in offspring born to murine dams (NIH Swiss) maintained for a shorter duration on very high fat (VHF), low fat (LF), or a control diet intermediate in fat (Purina 5015). Females were placed on diets at 5 weeks of age and bred at 12 and 20 weeks of age. These experiments were also designed to determine if maternal diet alters pre-weaning and development responses, body fat content, and serum glucose concentrations in resulting offspring. Pre-weaning behavioral assessments measured motor skill development of the pups. These tests included negative geotaxis, cliff avoidance, and surface self righting tests. No significant differences were observed between the diet groups in any of these pre-weaning responses. Weaned pups (10 males and 10 females per group) were randomly selected (1 to 2/litter) from offspring of each dietary group of dams and fed a standard mouse chow diet (Purina 5001). Mice were weighed weekly, and their behavioral responses examined at three time points (6, 9, and 12 weeks of age) by using an ActivityPro open-field maze, which tracks movement to the corners of the maze, periods at rest, and other parameters. Mice were also placed in the maze with a hole floor board adaptor to evaluate learning and memory responses and latency to retrieve food concealed within the holes. While maternal diet did not alter serum glucose concentrations, marked sexual dimorphism in serum glucose concentrations was evident (130 ± 0.87 mg/dL for females and 200 ± .58 mg/dL for males, P<0.05), and there was strong correlation (r=0.8) between serum glucose concentrations and body weight. Gonadal fat pads were dissected and weighed for the offspring in each group. Males born to dams on the VHF diet had decreased gonadal fat pad weight compared to those born to dams on Control and LF diets (VHF Males: 1.4 ± 0.3g ;Control Males: 2.7 ± 0.3g; LF Males: 2.2 ± 0.3g; P<0.05). VHF male offspring also weighed less than their LF male counterparts (47 ± 2.6 versus 55.6±2.4g, respectively, P<0.05). By 16 weeks of age, daughters from the VHF dams weighed more than those born to dams on the 5015 Chow diet (38.1 ± 1.8 g and 32.3 ± 1.8g, respectively, P<0.05). However, no differences in weight were detected in the males at this later time point. While behavioral differences were not as pronounced in offspring derived from dams exposed to shorter durations of the VHF and LF diets, 9 week old males from VHF dams displayed more anxiety-like behaviors, as evidence by increased time spent clinging to the corners of the maze (thigmotaxis) compared to males from LF dams (682.2 ± 12.3s versus 647.5 ± 12.3s, respectively, P<.05). No differences in learning and memory were observed in the groups. In summary, the duration of a maternal diet enriched in fat versus carbohydrates can influence the body weight and behavioral responses, including increased anxiety, displayed by her offspring. Supported by NIH grant HD 44042.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

NOT PRESENTED.

NOT PRESENTED.

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Actions and Interactions of Thyroid Hormone and Follicle-Stimulating Hormone in the Regulation of Rat Granulosa Cell Apoptosis.

Cheng Zhang 1, Guo Liang Xia 2, Benjamin K Tsang 1

Thyroid hormone (T3) is important for normal reproduction function and dysregulation of TH support is associated with reproductive disorders, including impaired follicular development. Our preliminary studies indicate that FSH increases preantral follicle growth in vitro, a response markedly enhanced by T3. However, the nature of this hormonal interaction is poorly understood. This study is aimed to improve our understanding on the cellular and molecular mechanisms by which T3 regulates ovarian follicle growth, and specifically on the expression and actions of intracellular survival and death intermediates in the regulation of granulosa cell fate and follicle destiny. In the present study, we investigated the possible involvement of the Src and phosphatidylinositol 3-kinase (PI3-K)/Akt survival pathway in the regulation of granulosa cell fate. We have demonstrated by Western blot analysis that, while ineffective alone, T3 markedly enhanced FSH (100 ng/ml)-induced phospho-Src and phospho-Akt contents in granulosa cells in vitro. Although the expression of the anti-apoptotic protein Xiap and the pro-apoptotic ligand (FasL) were also not altered by T3 alone (10-10 to 10-7 M), the presence of T3 significantly increased Xiap content and down-regulated FasL expression induced by FSH. The effects of T3 were concentration-dependent, with maximal responses observable at 10-9 M. Interestingly, FSH and T3 together decreased granulosa cell Fas expression, although neither of the hormone alone elicited a significant response. In addition, addition of the cell death intermediate ceramide to granulosa cell cultures significantly induced apoptosis (as determined by TUNEL) which was attenuated by either FSH or T3. The present of both hormones did not elicit a greater protective effect than either hormone alone. Taken together, the findings of the present study demonstrate that thyroid hormone potentiates the cell survival action of FSH through up-regulation of Xiap and down-regulation of Fas and FasL which is associated with the activation of the Src&PI3K/Akt pathway (supported in part by grants from the National Natural Science Foundation of China and the Canadian Institutes of Health Research).

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Effect of Glucocorticoid Treatment to Induce Insulin Resistance on Follicle Development and Ovulation.

Katherine S Hackbart, Rudelle K Meyer, Pauline M Cunha, Robb W Bender, Hemanta K Shrestha, Patrick G Kusilek, Milo C Wiltbank 1

Polycystic ovary syndrome is a reproductive disorder in women that is characterized by excessive androgen, anovulation, and polycystic ovaries. Frequently these women also exhibit insulin resistance. In this work we studied the effect of glucocorticoid-induced insulin resistance on bovine follicle development and ovulation. Experiment 1: Cows were given dexamethasone (DEX; 15 mg, i.m.; T1; n=5) or saline (7.5 ml, i.m.; C1; n=4) once daily. T1 cows received recombinant bovine somatotropin (rBST; 500 mg, s.q.) at initiation of treatment and 1 week later. Estrous cycles were synchronized using an intravaginal progesterone (P4) releasing device (CIDR), PGF2alpha (25 mg, i.m.), and follicular aspiration (ASP) to remove follicles ≥5 mm to induce a new follicular wave. Blood was collected and ovarian structures measured daily. Treatment increased glucose (T1=105.0 mg/dl, C1=57.9 mg/dl; P<0.01) and insulin (T1=118.4 µU/ml, C1=19.1 µU/ml; P<0.01) and abolished ovulation (T1=0/5; C1=4/4; P<0.01), thereby decreasing P4 concentrations at days 12 (P=0.01), 14 (P<0.01), and 16 (P<0.01) after ASP. In a subsequent experiment insulin resistance and anovulation were generated in cows (n=3) treated with a lower dose of DEX (10 mg) without rBST. Experiment 2: To test the hypothesis that treatment affected ovulation through LH signaling, cows (n=7) were utilized in a cross-over design experiment. Cows were given DEX (10 mg, i.m.; T2) or saline (5 ml, i.m.; C2) once daily. Cows had their reproductive cycles synchronized with a CIDR, PGF2alpha, and ASP. Seven days after ASP, the CIDR was removed and 6.5 hrs later cows were administered GnRH (200 µg, i.m.). Blood was collected once daily and at 0, 1, 2, 3, and 4 hrs post-GnRH. Ovarian structures were measured daily. Treatment increased glucose (T2=85.4 mg/dl, C2=57.5 mg/dl; P<0.01) and insulin (T2=88.2 µU/ml, C2=21.1 µU/ml; P<0.01) but did not affect LH concentrations (P=0.735), the production of an LH surge (T2=7/7; C2=7/7) or ovulation (T2=7/7; C2=7/7) in response to exogenous GnRH, or P4 concentrations (P=0.329). Treatment decreased estradiol (E2) on days 6 (T2=1.47 pg/ml; C2=2.66 pg/ml; P<0.01) and 7 (T2=1.43 pg/ml; C2=3.05 pg/ml; P<0.01) after ASP. Experiment 3: To test the hypothesis that treatment abolished ovulation by affecting the positive E2 feedback, cows (n=8) were utilized in a cross-over design experiment. Treatments (DEX=T3, saline=C3) and hormone administration were similar to Experiment 2 except that at 12 hrs after CIDR removal, cows were administered E2-17beta (0.5 mg, i.m.). Blood was collected daily and at 0, 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 hrs post-E2. Cows underwent ovarian ultrasonography daily. Treatment increased glucose (T3=83.2 mg/dl, C3=54.9 mg/dl; P<0.01) and insulin (T3=106.9 µU/ml, C3=21.4 µU/ml; P<0.01) and abolished the E2-induced LH surge, or delayed it such that it was not observed during the period of blood sampling (T3=0/8; C3=8/8). Treatment decreased ovulation (2/8) in response to exogenous E2. However, some T3 cows had a late ovulation (2/8; 4-7 d after E2, past the physiologically relevant time in relation to exogenous E2). 4/8 T3 cows were anovular throughout the experimental period. C3 ovulation rate was 8/8. We conclude that treating cows with DEX increases circulating glucose and insulin, decreases E2 at days 6 and 7 of the follicular wave, and induces an anovular condition which is fully reversible by exogenous GnRH but only minimally reversible by exogenous E2.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

In Vitro Oocyte Maturation and Preantral Follicle Growth from Luteal Phase Baboon Ovary Produce Mature and Fertilizable Eggs.

Min Xu 1, Ariella Shikanov 1, Erin Jackson 1, Susan L Barrett 1, Jenny Hirshfeld-Cytron 1, Sarak E Kiesewetter 1, Lonnie D Shea 1, Asgerally T Fazleabas 2, Teresa K Woodruff 1

Female cancer patients who seek fertility preservation and cannot undergo regular ovarian stimulation before chemotherapy and/or radiotherapy may choose: 1) retrieval of immature oocytes followed by in vitro maturation (IVM); 2) ovarian tissue cryopreservation followed by transplantation or in vitro follicle growth (IVFG). Oocyte aspiration of the conventional IVM was carried out during the follicular phase of the menstrual cycle. There was not enough evidence showing that immature oocytes retrieved during the luteal phase could mature in vitro and be fertilized to produce viable embryos. While in vitro follicle growth has proved successful in rodents, further studies on nonhuman primates are required to translate the system to humans. Our objective was to investigate the meiotic and fertilization ability of baboon immature oocytes and the effect of fibrin-alginate-matrigel (FAM) and FSH on baboon preantral follicle growth and oocyte maturation in vitro. COCs were collected from small antral follicles (1-2 mm) of luteal phase ovaries and classified to three groups: 1) COCs containing at least three complete layers of compact cumulus cells (ML-COC); 2) COCs with only 1-2 complete layers of cumulus cells (1L-COC); 3) COCs without one complete layers of cumulus cells (0L-COC). After 46-48 hours in vitro maturation, the nuclear status of germinal vesicle (GV) oocytes and the spindle structure of metaphase II (MII) eggs were evaluated by using confocal microscopy. ICSI was performed to MII eggs from ML-COC group. Embryos were cultured up to 5 days to evaluate the developmental competence. Preantral follicles (120-250 μm) were enzymatically isolated, encapsulated in FAM matrices, and cultured in medium containing 0, 10, and 100 mIU/ml FSH for up to 2 weeks. COCs were dissected from the cultured follicles, and IVM was performed. Oocyte nuclear status and spindle structure were evaluated by confocal microscopy. More oocytes resumed meiosis in the ML-COC group, with 41.6%, 22.8%, and 2.9% MII rates in ML-COC, 1L-COC, and 0L-COC, respectively. Normal spindle rates were found no difference between ML-COC and 1L-COC groups. Eight pronuclear (2PN) embryos were obtained from 32 MII eggs after ICSI; four of them cleaved and two progressed to 8-cell-morula stage. Baboon preantral follicles cultured in FAM matrices for 2 weeks exhibited FSH dose-dependent increases in follicle size, with more than 60% of follicles developing to the antral stage. However, exogenous FSH negatively impacted follicle survival. No compact COCs were obtained from follicles cultured in 100 mIU/ml FSH, while 40%-50% of the oocytes cultured in the absence of FSH developed at least one layer of compact cumulus cells. After 60 hours IVM, over 90% of oocytes exhibited GVBD, and MII eggs were obtained with normal spindle structure. Baboon immature oocytes collected from luteal phase small antral follicles were matured and fertilized, and underwent future embryo development in vitro. FAM matrices supported baboon preantral follicle development to the small antral follicle stage in an FSH-independent manner, producing meiotically competent oocytes. The current study suggests that IVM of oocyte and in vitro growth of follicle obtained from luteal phase ovaries may represent additional fertility preservation options for female cancer patients who face immediate chemotherapy and/or radiotherapy. This work was supported by Oncofertility Consortium Grants NIH RL1HD058295 and PL1EB008542, and the Division of Fertility Preservation, Northwestern University.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Adrenomedullin in the Rat Ovary: Its Production, Interaction with Endothelin 1, and Actions on Progesterone Secretion During Gestation.

Wai-sum O 1, Lei Li 1, Fai Tang 1

Adrenomedullin (ADM), a novel vasorelaxant peptide, was found in human/rat ovaries and uteri. Plasma ADM level increases in pregnant women and pregnant rats. Our previous study in Sprague-Dawley rats showed that large antral follicles (diameter above 900 µm) and newly formed corpus luteum (CL) expressed higher levels of Adm and one of its receptor activity-modifying protein (Ramp2) than small antral follicles (diameter of 200-400 µm). EDN is luteolytic and is known to inhibit progesterone production. The aims of this study was to study the interaction of ADM and endothelin 1 (EDN1) in follicles and newly formed CL and the action of ADM on progesterone production in CL during pregnancy. Adult Sprague-Dawley rats were induced to ovulate by administration of eCG followed by hCG injection. Isolated large follicles were incubated with ADM/EDN1 for 6 h; newly formed corpora lutea were also incubated with ADM/EDN1 for 6 h with/without hCG. For the effects of EDN1, the gene expression of Adm and its receptor components in the large antral follicles and CL were measured by real-time RT-PCR. For the effects of ADM, the gene expression of Edn1 and endothelin receptor B (Ednrb) was measured. The effects of the ADM and CGRP receptor antagonists (hADM22-25 and hCGRP8-37) were also tested. CL were isolated from early (7-day), mid (12-day), and late (17-day) pregnant rats and were incubated for 6 h with ADM with or without an ADM or a receptor antagonist. Progesterone secretion from CL was measured by Enzyme Immunoassay. EDN1 was found to reduce the gene expression of Adm and Ramp1 in large antral follicles after 6 h incubation with ADM. ADM treatment upregulated Edn1 expression levels in large antral follicles. EDN1 also reduced the gene expression of Adm in the CL with/without hCG. ADM treatment increased Edn1 expression. In the presence of hCG, ADM treatment increased both Edn1 and also Ednrb expression. ADM suppressed progesterone production from the CL in early and late pregnancy while enhancing progesterone production in mid-pregnancy, which correlated well with the greater progesterone production at mid-pregnancy. The effects of ADM on Edn1 gene expression and progesterone production were blocked by the CGRP receptor antagonist. One of the roles of ADM in CL during pregnancy might be the regulation of progesterone production and this may be achieved via the interaction with END1. Acknowledgement: This work was substantially supported by a grant from the Research Grants Council of the Hong Kong Special Administrative Region, China (HKU7736/07M).

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Estrogen Receptor Subtypes ERalpha and ERbeta May Have Overlapping Roles in Oocyte Development.

Fangjie Tang, Melissa E Pepling 1

The formation of the pool of functional primordial follicles is critical for reproduction and is conserved among species. Female primordial germ cells undergo incomplete mitotic division and form clusters called cysts. Shortly after birth, the cysts break down into individual oocytes that become surrounded by somatic pre-granulosa cells to form primordial follicles. In this process, a subset of oocytes undergo programmed cell death and only one third of the initial oocytes survives and represents a life-time pool of oocytes. Estrogen treatment of neonatal mouse ovaries results in multiple oocyte follicles in adults which are likely cysts that did not break apart. Supporting this, previous studies have shown that estrogenic compounds can disrupt cyst breakdown in neonatal mice in vitro and in vivo. However, the regulative mechanisms of estrogen signaling in this process are still unknown. Estrogen functions via estrogen receptors (ERs), which are members of the steroid receptor superfamily. In mammals, there are at least two estrogen receptor subtypes: ER alpha (ERalpha) and ER beta (ERbeta). And both ERs are present during cyst breakdown and primordial assembly. However, single knock-outs of either ERalpha or ERbeta did not affect the cyst breakdown process. These results suggest a more complicated interaction between estrogen and its receptors. The objective of this study was to analyze cyst breakdown and primordial follicle development in ERalpha, ERbeta, and ER double knockout mice. Wild type, ERalpha single knockout, ERbeta single knockout, and ER double knockout ovaries were collected at PND 7, a time when most oocytes have completed cyst breakdown. Oocyte survival, cyst breakdown, and follicle development were assessed via immunochemistry using an oocyte marker and confocal microscopy. ER double knockout mice had similar oocyte numbers compared to wild type and ER single knockout mice. However, the double knockout mice had delayed cyst breakdown compared to wild type and ERbeta knockouts. Moreover, a larger proportion of ER double knockout oocytes were in advanced follicle stages and indicating a faster rate of follicle development. These results show that the estrogen receptor subtypes might have overlapping roles and might compensate for each other in oocyte development. Further investigation of expression level of one estrogen receptor in the other knockout strain might provide insight into the specific function of ERalpha and ERbeta in oocyte development.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Use of a Lentiviral Platform for Subcellular Localization of FIGLA, A Key Gene for Female Reproductive Capacity.

Ernesto Orozco-Lucero, Everardo Gonzalez-Rodríguez, Eduviges Burrola-Barraza, Felipe A Rodríguez-Almeida, Moises Barceló-Fimbres, Verónica Moreno-Brito, Irene Leal-Berumen 1

During germline establishment and its molecular control, the Figla gene, which codes a transcription factor, is preferentially expressed in oocytes, and is key for oogenesis/folliculogenesis, ovarian function, and female fertility. However, the specific subcellular localization (cytoplasmic or nuclear) of this transcription factor is unknown. We analyzed the FIGLA protein using the computer programs NucPred, SubCellProt, and NLStradamus to predict subcellular localization; also, we established a molecular platform for artificial expression of the murine Figla gene in cultured 293 FT cells to study its localization directly. This gene was cloned from mouse ovaries to constitute the vector pCR2.1/Figla, which was used to subclone Figla, producing the vector pENTR/GFP-Figla, which originated by construction of pLenti/GFP-Figla through recombination. This lentivector was used for artificial expression of the Figla gene in cultured 293 FT cells. The transfected 293 FT cells were analyzed by fluorescence microscopy for protein localization, and then harvested for characterization through RT-PCR and SDS-PAGE. Restriction, PCR, and sequence analysis confirmed the veracity of the developed vectors. The RT-PCR generated Figla, GFP, and GFP-Figla transgenic transcripts from the pLenti/GFP-Figla-transfected 293 FT cells, and the SDS-PAGE analysis revealed an expected band of ~52 kDa for the GFP-FIGLA recombinant fusion protein. The pLenti/GFP-Figla-lipofected 293 FT cells showed nucleoplasmic fluorescence using microscopy (FITC filter), which is in agreement with the predictions of the programs NucPred, SubCellProt, (probability ≥ 0.7 and 0.6 of nuclear localization, respectively), and NLStradamus (absence of classical nuclear localization signal), which strongly suggested the presence of this protein in both nuclear and cytoplasmic compartments. In conclusion pLenti/GFP-Figla cellular expression showed necloplasmic localization of this transcription factor in accord with in silico predictions. This research was supported by CONACYT, grant 45731, Mexico.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Follicle Size and Volume Is Less Indicative of Development of a Persistent Follicle In Beef Heifers.

Renee McFee 1, William Pohlmeier 1, Jacqueline Smith 1, Jill Kerl 1, Racheal Slattery 1, Debra Clopton 1, Jennifer Wood 1, Robert Cushman 2, Andrea Cupp 1

We have established that feeding 0.5 mg/h/d of melengestrol acetate (MGA) for 14 d (< 1 ng/ml of P4) develops persistent follicles with increased follicle size, follicular fluid volume, theca cell weight, and follicular fluid androstenedione concentration in mature cows. Therefore, we hypothesized that similar parameters would be developed in heifers that were 2 years of age or younger. Mature cows and heifers received either: 1) prostaglandin F2alpha (PG) on d0 and d7 and MGA for 14 d; or 2) GnRH on d 7 and a CIDR for 7 days. On d14, all cows received PG and CIDRs were removed. Ovariectomies were conducted 36 h post-PG and granulosa cells, theca cells, follicular fluid, and blood were collected for analysis. Data were analyzed using proc mixed of SAS with means separation by LSD to evaluate differences between treatment, age, and follicle status. Interestingly, MGA treatment did not result in a persistent (P) dominant (DOM) follicle diameter or follicular fluid volume in heifers (2-years-old or younger) that were as large as the P-DOM in mature cows. Mature cows had P-DOM and control (C) subordinate (SUB) follicles that were 40% (P < 0.006) and 45% (P < 0.009) larger in diameter than the same follicles in 2-year-olds. Furthermore, the mean follicular fluid volume of C-SUB follicles was 200% greater (P < 0.05) in mature cows and the mean diameter (P < 0.09) and the theca cell weight (P < 0.07) of C-DOM follicles tended to be larger in mature cows compared to the 2-year-olds. No differences were identified between mature cows and 2-year-olds in granulosa cell pellet weight or in the follicular fluid concentration of E2 and P4 for any of the follicle types; suggesting that even though the follicles were not the same diameter they still had characteristic steroid and theca cell weights of persistent follicles. Gene expression profiles for mRNA of genes involved in follicle development such as vascular endothelial growth factor A_164 (VEGFA_164) were also not different between mature cows and 2-year-olds. However, VEGFA_165B mRNA was more abundant (P < 0.03) in C-DOM follicles from 2-year-olds compared to mature cows. Furthermore, mRNA levels for both anti-Mullerian hormone (P < 0.08) and CART prepropeptide (P < 0.03) in SUB follicles were more abundant in mature cows than in 2-year-olds. Therefore, we can conclude from these data that follicle size may not be indicative of whether a follicle is persistent or not, especially in beef heifers. In addition, follicular fluid steroid hormone profiles and gene expression may be more accurate measures of whether a follicle is persistent.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Follicle Size and Volume Is Less Indicative of Development of a Persistent Follicle In Beef Heifers.

Renee McFee 1, William Pohlmeier 1, Jacqueline Smith 1, Jill Kerl 1, Racheal Slattery 1, Debra Clopton 1, Jennifer Wood 1, Robert Cushman 2, Andrea Cupp 1

We have established that feeding 0.5 mg/h/d of melengestrol acetate (MGA) for 14 d (< 1 ng/ml of P4) develops persistent follicles with increased follicle size, follicular fluid volume, theca cell weight, and follicular fluid androstenedione concentration in mature cows. Therefore, we hypothesized that similar parameters would be developed in heifers that were 2 years of age or younger. Mature cows and heifers received either: 1) prostaglandin F2alpha (PG) on d0 and d7 and MGA for 14 d; or 2) GnRH on d 7 and a CIDR for 7 days. On d14, all cows received PG and CIDRs were removed. Ovariectomies were conducted 36 h post-PG and granulosa cells, theca cells, follicular fluid, and blood were collected for analysis. Data were analyzed using proc mixed of SAS with means separation by LSD to evaluate differences between treatment, age, and follicle status. Interestingly, MGA treatment did not result in a persistent (P) dominant (DOM) follicle diameter or follicular fluid volume in heifers (2-years-old or younger) that were as large as the P-DOM in mature cows. Mature cows had P-DOM and control (C) subordinate (SUB) follicles that were 40% (P < 0.006) and 45% (P < 0.009) larger in diameter than the same follicles in 2-year-olds. Furthermore, the mean follicular fluid volume of C-SUB follicles was 200% greater (P < 0.05) in mature cows and the mean diameter (P < 0.09) and the theca cell weight (P < 0.07) of C-DOM follicles tended to be larger in mature cows compared to the 2-year-olds. No differences were identified between mature cows and 2-year-olds in granulosa cell pellet weight or in the follicular fluid concentration of E2 and P4 for any of the follicle types; suggesting that even though the follicles were not the same diameter they still had characteristic steroid and theca cell weights of persistent follicles. Gene expression profiles for mRNA of genes involved in follicle development such as vascular endothelial growth factor A_164 (VEGFA_164) were also not different between mature cows and 2-year-olds. However, VEGFA_165B mRNA was more abundant (P < 0.03) in C-DOM follicles from 2-year-olds compared to mature cows. Furthermore, mRNA levels for both anti-Mullerian hormone (P < 0.08) and CART prepropeptide (P < 0.03) in SUB follicles were more abundant in mature cows than in 2-year-olds. Therefore, we can conclude from these data that follicle size may not be indicative of whether a follicle is persistent or not, especially in beef heifers. In addition, follicular fluid steroid hormone profiles and gene expression may be more accurate measures of whether a follicle is persistent.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Six Genes, Including the IrxB Cluster (Irx3, 5, 6), Ftm, Fto, and Fts, Are Required for Germ Cell Progression in Developing Ovaries of Fused Toes Mutant Mice.

Bongki Kim 1, Youngha Kim 2, Joan S Jorgensen 2

Fused toes mutant mice lack six genes including the entire IrxB cluster, Irx3, 5 and 6, and three other genes, Fto, Fts, and Ftm. Homozygous embryos die early in development (E10.5-E14.5) because of severe malformation of the developing brain and loss of left-right asymmetry. Previously, we reported defective follicular development in Ft mutant ovaries. Wild type and Ft mutant ovaries were harvested before embryonic death and allowed to develop as a transplant under the kidney capsule of nude mouse hosts. While the transplanted wild type ovaries supported maturation of follicles up to the pre-antral stage, Ft mutant ovaries produced follicles that progressed no further than the primary stage with several retained germ cell cysts. Furthermore, follicles within Ft mutant ovaries demonstrated disintegration of the granulosa cell phenotype. Some oocytes within these follicles were able to enter meiotic division, but most failed to grow in size. Based on these findings, we hypothesized that the Ft mutation interfered with communication between somatic and germ cells early on in ovary development to cause defective follicle formation. To test this hypothesis, we first identified which cell types express the genes within the Ft mutation and then assessed the affect of the mutation on germ cell maturation within ovaries harvested at E12.5 and E12.5 plus two days in vitro culture. Quantitative PCR and immunohistochemistry (IHC) results indicate that all six genes are expressed within somatic cells of the XX gonad. Despite this finding, somatic cell markers, Foxl2 and Wnt4, maintained expression suggesting early somatic cell differentiation was not affected. However, analysis of germ cells at both time points determined a significant decrease in germ cell numbers due to a defect in germ cell proliferation without changes in apoptosis. While vasa expression was decreased in Ft mutant gonads, transcript levels of other factors that have the potential to contribute to germ cell survival and proliferation within gonads, including sdf, bmp2, 4, 5, 6, 7, were not different. To test whether this defect was inherent to the germ cell and exclusive to the gonadal environment, we investigated germ cell numbers and proliferation during hindgut migration at E9.5. Using whole mount IHC, we detected no difference in germ cell numbers between wild type and Ft mutant embryos suggesting their development and proliferation was normal up to and through migration. From these data, we conclude that germ cell development is normal during early germ cell differentiation and migration; however, once germ cells enter the gonad, somatic cells harboring the Ft mutation interfere with continued germ cell development causing a defect in proliferation and progressive follicular failure.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Effects of Exogenous Luteinizing Hormone on Development and Acquisition of Ovulatory Competence by Dominant Follicles During the Equine Anovulatory Season.

Stephanie N Schauer 1, Christine Briant 2, Monique Ottogalli 2, Francesc X Donadeu 1

The natural spring period of transition into the ovulatory season in mares is characterized by increased follicular growth and the development of up to three successive dominant-size anovulatory follicles. These transitional follicles are severely underdeveloped, which has been attributed to low secretion of pituitary LH. It can therefore be hypothesized that administration of equine (e) LH during a follicular wave in early transitional mares will promote the development of ovulation-competent follicles. To test this hypothesis, 14 seasonally anovulatory mares were used starting in January in the Northern Hemisphere. Once a follicle >25mm was first identified (early transition) all follicles >15 mm were ablated by transvaginal ultrasound-guided follicle puncture. When the largest follicle of the ablation-induced wave reached >20mm, mares were given a subovulatory dose of a purified pituitary eLH fraction (0.25μg or 1μg eLH/kg, n=3/group) or saline (n=8) i.v. every 12 h and ovarian activity was monitored by ultrasound daily. Once the dominant follicle reached a diameter of >32mm, 3000 IU of hCG were administered to induce ovulation. Some of the mares were also used as ovulatory-season controls (n=5) during June and July. In these mares, follicles were ablated 10 days after ovulation followed by i.v administration of saline beginning when the largest follicle reached 20mm and injection of hCG once the follicle reached 32mm. During both seasons, blood samples were collected daily throughout the experiment for determination of plasma hormone levels by RIA. Data from transitional mares treated with the two different LH doses was not significantly different (P>0.8) and was therefore combined into a single LH-treated group (n=6) for analyses. Data was analyzed by repeated measures ANOVA and is reported as mean±SEM. Treatment with eLH during early transitional waves stimulated the growth of the dominant follicle (Group×Day, P=0.003) with a maximum diameter (day five of treatment) of 32.9±1.8 mm and 26.4±1.8 mm in eLH-treated and saline-treated mares, respectively (P<0.05). Follicle profiles were similar (P>0.9) between LH-treated transitional mares and ovulatory season controls. Profiles for the largest subordinate follicle were not affected (P>0.8) either by LH treatment during transition or by season. However, more follicles >10mm developed during the experimental period in saline-treated transitional mares (8.0±0.7 follicles) than in LH-treated transitional mares (5.2±0.6, P=0.008) or ovulatory season controls (5.2±0.6, P<0.001). Double-dominant follicles did not develop in any mare. Five eLH-treated mares were injected with hCG and three ovulated (60%), at an average of 56±8 h post-hCG. All ovulatory season controls ovulated and did so at a mean of 54±8.6 h after hCG, an interval similar to that in transitional mares (P>0.1). In addition, an increase in circulating progesterone levels occurred as expected in the three transitional mares that ovulated in response to hCG although this increase tended to be of lower magnitude than that after ovulation in ovulatory-season controls (Group×Day, P=0.08). These results are consistent with a causal effect of deficient circulating LH on reduced follicle development during the equine anovulatory season and demonstrate that exogenous eLH administration can stimulate the development of dominant follicles and facilitate early ovulations followed by the development of a functional CL.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Global Gene Expression Analyses of Equine Follicle Development.

Somayyeh Fahiminiya 1, Dave Waddington 2, Nadine Gerard 1, Xavier Donadeu 2

Follicle development in the mammalian ovary involves the growth of cohorts of follicles or follicular waves at periodic intervals throughout a female's reproductive life. In monovular species only one follicle (dominant follicle) will emerge, through follicle selection, within each cohort to reach full maturation and become ovulation-competent, whereas all other follicles (subordinate follicles) of the wave will cease developing before they can become dominant. The molecular basis of the two key events during the development of a dominant follicle, emergence through selection and acquisition of ovulatory capacity, remain largely unknown, particularly in large animal species. Like humans, the horse is a monovular species. Moreover, follicle development patterns during equine reproductive cycles have been shown to be strikingly similar to that of humans. This, together with the relative large size and long developmental timeline of equine follicles, make the horse a unique model to study the development of dominant follicles. The aim of this study was to identify for the first time the precise gene expression signatures associated with different stages of dominant follicle growth in the horse. Ovaries were collected from 12 oestrus mares during a breeding season and follicles were isolated belonging to one of the following categories (n=5 follicles/category): Early Dominant (ED; the largest growing follicle of an ovulatory wave once it reached 25 mm in diameter), Late Dominant (LD; the largest follicle once it reached 33-35 mm) and Preovulatory (PO; the late dominant follicle 34h after an injection of an ovulatory dose of Crude Equine Gonadotropin). Granulosa and theca cells were separately collected from each follicle and total RNA was extracted with TRIzol. RIA measurements of progesterone and 17βestradiol in follicular fluid together with qPCR analysis of LH receptor in granulosa cells were used to more precisely establish the developmental status of each collected follicle. In addition, the expression of genes specific for theca (CYP17A1, BMP7) and granulosa (CYP19A1) was determined by PCR to rule out cell cross-contamination. A 44K equine gene expression microarray (Agilent Technologies Inc., CA, USA) was used separately on granulosa and theca cells by performing one-dye hybridization. Statistical analysis was performed with a Limma package using a false discovery rate (q-value <0.05) to identify differentially expressed sequences between ED and LD and between LD and PO follicle categories. Differences in gene expression were found during each of the two follicle category comparisons. Differences were particularly extensive between LD and PO, specifically, exposure of late dominant follicles to an ovulatory stimulus resulted in differential expression of 8349 transcripts in granulosa cells and 2338 transcripts in theca cells. In contrast, the number of transcripts differentially regulated between ED and LD was comparatively small, 1602 transcripts in granulosa cells and 8 transcripts in theca cells. Further analyses are currently being performed to identify in detail the specific genes and cellular pathways associated with each developmental stage. The results of this study, the first transcriptome analyses of equine follicles, demonstrate that development of ovarian follicles in mares is associated with profound changes in global gene expression, particularly in granulosa cells and during the follicular-to-luteal transition.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

NOT PRESENTED.

NOT PRESENTED.

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Mitochondrial Dysfunction and Apoptosis in Cumulus Cells of Diabetic Mice.

Qiang Wang, Antonina Frolova, Katie Boehle, Erica Schoeller, Maggie Chi, Tim Schedl, Kelle H Moley 1

Women with poorly controlled diabetes often suffer from reproductive problems, such as miscarriage and offspring with congenital malformations. Recently, emerging evidence has shown that oocytes from diabetic mice experience delayed maturation, abnormal cellular metabolism, mitochondrial dysfunction and meiotic defects. These changes in the oocyte may be manifested later as developmental abnormalities in preimplantation embryos and congenital malformations. However, the pathway(s) by which maternal diabetes exerts its effects on the oocyte remains ill defined. Cumulus cells are in direct contact with the oocyte via gap junctions and have long been known to play a nurturing role in supporting oocyte development by providing essential nutrients to oocytes. We therefore hypothesized that maternal diabetes adversely impacts the mitochondria in cumulus cells, which may be further transferred into the oocyte, contributing to poor oocyte quality. To test this assumption, we investigated mitochondrial status in cumulus cells from chemically induced diabetic mice. Using transmission electron microscope, we found that mitochondria in diabetic cumulus cells display small spherical structures with fewer and disarrayed cristae, and a decreased electron density of the matrix. These ultrastructural defects have been correlated with mitochondrial fission and metabolic disorders. By MitoPT-JC1 assay, we revealed a general drop of mitochondrial membrane potential in cumulus cells exposed to maternal diabetes. Mitochondrial mislocalization was observed in cumulus cells from diabetic mice, showing a decreased percentage of the perinuclear distribution relative to control and a corresponding increased proportion of an aggregated distribution. Quantitative real-time PCR analysis of mtDNA content and the mRNA levels of several genes implicated in mitochondrial biogenesis demonstrated a mitochondrial biogenic response in cumulus cells of diabetic mice. Notably, while mitochondrial biogenesis in diabetic cumulus cells was induced, their function was markedly decreased as evidenced by the lower ATP and citrate levels. TUNEL assay showed a significant increase in the incidence of cumulus cell apoptosis from diabetic mice as compared with controls. Importantly, these apoptotic cumulus cells from diabetic mice always display a diffuse staining of cytochrome c in the cytoplasm compared to a punctate distribution pattern in control cumulus cells, providing evidence that mitochondrial impairments are involved in apoptosis of cumulus cells induced by maternal diabetes. Some key findings mentioned above were also confirmed by using the Akita genetic mouse model as a control for the STZ-induced effects. In summary, molecular, cellular and biochemical analysis demonstrated structural, spatial and metabolic dysfunction of mitochondria in cumulus cells of diabetic mice, which lead, at least in part, to apoptosis in diabetic cumulus cells through the cytochrome c release. These deleterious effects of maternal diabetes on cumulus cells may disrupt trophic and signaling interactions with the oocyte, contributing to oocyte incompetence in diabetic females. Research supported by NIH-UO1 HD044691-03 (KHM) and NIH-R01 GM085150 (TS).

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

PGE2 Receptors in Subpopulations of Primate Granulosa Cells: Differential Expression During the Periovulatory Interval.

Siabhon M Harris, Diane M Duffy 1

The midcycle surge of luteinizing hormone (LH) initiates changes within the follicle which lead to ovulation. The LH surge increases production of prostaglandins (PGs) by granulosa cells, and PGE2 is a key mediator of LH action within the periovulatory follicle. Previous studies demonstrated that the four PGE2 receptors (PTGER1-PTGER4, also known as EP1-EP4) are expressed by granulosa cells. EP receptors couple to different G proteins to generate intracellular signals. Therefore, the specific EP receptors expressed by granulosa cells may lead to different intracellular responses to PGE2 and, ultimately, specialized functions in the process of ovulation. To determine if subpopulations of granulosa cells within the primate follicle express distinct subsets of all EP receptors during the periovulatory interval, cynomolgus monkeys received gonadotropins to stimulate the development of multiple preovulatory follicles; an ovulatory dose of human chorionic gonadotropin (hCG) was administered to initiate ovulatory events. Ovulation is anticipated about 40 hours after hCG, so follicular aspirates and whole ovaries were obtained before (0 hour), 24 hours, and 36 hours after hCG administration to span the periovulatory interval. Granulosa cells from follicular aspirates were separated into cumulus and mural subpopulations. Frozen ovarian tissue sections were used for collection of additional granulosa cell subpopulations by laser capture microscopy. EP mRNAs were quantified by real time PCR. A semiquantitative immunofluorescence microscopy strategy was used to compare EP protein levels in subpopulations of granulosa cells. Both cumulus and mural granulosa cells expressed EP2 and EP3 mRNA and protein throughout the periovulatory interval. Cumulus cells expressed higher levels of EP2 and EP3 mRNA compared to mural cells at 24 and 36 hours after hCG. While EP1 mRNA levels in cumulus cells increased between 0 hour and 36 hours after hCG, EP1 protein was only observed in mural, but not cumulus, granulosa cells. EP4 protein was low/nondetectable in cumulus and mural granulosa cells, despite detectable mRNA throughout the periovulatory interval. These data suggest that EP2 and EP3 may preferentially regulate the ovulatory functions of cumulus cells while EP1 may be involved in transducing the PGE2 signal primarily in mural granulosa cells. To determine if expression of EP receptor subsets may regulate aspects of follicle rupture, EP expression was compared between mural granulosa cells near the follicle apex and mural granulosa cells not at the apex. Both apex and non-apex granulosa cells expressed EP1 protein, with low EP1 protein at 0 hour hCG and increased EP1 24-36 hours after hCG. However, EP1 protein levels in non-apex granulosa cells were higher than in apex granulosa cells 36 hours after hCG. Expression of EP2, EP3 and EP4 were similar between apex and non-apex mural granulosa cells throughout the periovulatory interval. PGE2 has been implicated in the process of follicle rupture. Thus, lower expression of EP1 in apex granulosa cells may be involved in mediating this action of PGE2. Differential expression of the four EP receptors by subpopulations of granulosa cells may lead to cell-specific responses to the ovulatory PGE2 signal and contribute to the distinct function of each granulosa cell subpopulation during the periovulatory interval. This study was supported by NIH HD054691 and Schering-Plough, now Merck & Co.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Luteinizing Hormone Contributes to Androstenedione Production by Regulating Nuclear Movement of Steroidogenic Factor 1 (SF-1) in Bovine Theca Cells.

Chiaki Murayama 1, Hitoshi Miyazaki 2, Akio Miyamoto 1, Takashi Shimizu 1

During follicular development in bovine ovary, androstenedione (A4) that is essential for estrogen production in granulosa cells is produced in theca cells. A4 production in theca cells is regulated by steroidogenic acute regulatory protein (StAR) and CYP17. In addition, steroidogenic factor 1 (SF-1) and DAX-1 transcription factors contribute to the expression of genes associated with A4 production. Although luteinizing hormone (LH) plays an important role in A4 production in theca cells, whether LH involves in the increase in A4 production by enhancing StAR and CYP17 via SF-1 is unknown well yet. Therefore, we examined whether LH affect the manner of intra-cellular SF-1 when theca cell produced A4 at high levels by LH. Theca cells were isolated from large follicle (10-15 mm diameter) in bovine ovary obtained slaughterhouse. Theca cells were seeded in 12-well or 6-well culture plate for 1×105 cell/well or 4×105 cell/well, respectively, in medium containing 5% FCS, and cultured for 24 h at 37°C in 5% CO2, 95% air. After 24 h of culture, the culture medium was replaced with serum-free medium supplemented with 0 or 50 ng/ml LH (high LH), and the culture performed for 24h. After culture, the media was replaced with fresh medium (1% FCS) supplemented with 0 and 2.5 ng/ml (low LH) every 48 h for 6 days. We divided culture treatment into four groups as follows; 1) control, 2) low LH alone group, 3) high LH alone group, and 4) high LH + low LH group. The concentration of progesterone (P4) and A4 in culture medium each 48 h were analyzed by enzyme immunoassay (EIA). Total RNA was extracted from theca cells and the mRNA expressions of StAR and CYP17 were analyzed by the real-time PCR. The total protein expressions of SF-1 and DAX-1, and those expressions in cytoplasm and nuclear fractions of theca cell were analyzed by Western blotting. Intra-cellular localization of SF-1 and DAX-1 were visualized by using confocal microscopy with fluorescence immunostaining. The interaction between SF-1, and StAR and CYP17 promoter regions were shown by chromatin immunoprecipitation (ChiP assay). A4 production in theca cells at 48 h of culture were stimulated by low LH alone. The expression of StAR and CYP17 genes in theca cells with treated low LH alone were significantly higher than in control group, and the high levels maintained throughout culture period. The total protein expression of SF-1 and DAX-1 in theca cell in all groups showed a constant level throughout culture period. In nuclear fractions of theca cell, protein expression of DAX-1 did not change by low LH alone, whereas those of SF-1 are stimulated by low LH alone but not high LH alone and high LH + low LH group. Low LH alone enhanced the binding of SF-1 to the promoter regions for star and CYP17 than in other groups. These results suggested that low LH alone induces the movement of SF-1 to nuclear of theca cell, and consequently the expression of StAR and CYP17 genes were induced in theca cells. Such sequential mechanism is associated with the increase in A4 production in theca cells. Taken together, our data indicated that low LH, but not high LH, may be a necessary factor for A4 production in theca cells during follicular development in bovine ovary.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Expression of Matrix Metalloproteinase 10 (Mmp10) During the Periovulatory Period.

Lauren McCord 1, Eunsil Park 1, Misung Jo 1, Matts Brannstrom 2, Thomas E Curry 1

The matrix metalloproteinase (MMP) family is comprised of proteolytic enzymes that remodel the extracellular matrix (ECM). These MMP members reflect vast biological roles, including the extensive remodeling associated with follicular rupture. Mmp10, a member of the stromelysin MMP sub-family, has been shown to be highly expressed in numerous cancer types and to correlate with inflammatory responses. However, little is known about the role of Mmp10 in the ovary. In the present study, Mmp10 expression was analyzed in the periovulatory rat ovary, in vitro cultured rat granulosa cells and human periovulatory granulosa cells. In the periovulatory rat model, follicular stimulation was initiated in immature female rats (21 days old) by pregnant mare serum gonadotropin hormone (PMSG). Forty-eight hours after stimulation by PMSG, rats were then injected with human chorionic gonadotropin (hCG) to induce ovulation. In this experimental model, ovulation occurs at approximately 14 to 16h following hCG administration. Thus whole ovaries were collected at 0, 6, 12 and 24h after hCG to determine changes in Mmp10 mRNA throughout the periovulatory period. For rat granulosa cells, ovaries were collected 48h following PMSG injection; granulosa cells were then isolated and cultured in the absence or presence of hCG for 0, 9, and 24h. Ovaries and granulosa cells were frozen for analysis of Mmp10 mRNA expression. To analyze Mmp10 mRNA expression in human granulosa cells, granulosa cells were first collected from patients undergoing elective surgery at different periovulatory periods (preovulatory, early ovulatory and late ovulatory phase groups). Surgery was performed on the patients at the preovulatory phase, prior to the LH surge, without the administration of hCG. The remaining patients received an injection of 250 ug hCG to mimic the endogenous LH surge and underwent surgery after varying lengths of time following hCG injection: early ovulatory phase 12 to ≤18h and late ovulatory phase >18 to ≤34h. Granulosa cells were frozen and Mmp10 mRNA expression analyzed by microarray profiles. For the rat, RNA was isolated from ovaries or granulosa cells and quantitative Real-Time PCR analysis was carried out. Administration of hCG induced the expression of Mmp10 in the rat ovary prior to ovulation at 6h post-hCG and expression remained elevated through 24h post-hCG. In cultured granulosa cells, Mmp10 expression increased in both control (untreated) and hCG treated cells after 9h of culture before declining to 0h levels after 24h of culture. The observation that Mmp10 mRNA increases independent of hCG hormonal stimulation suggests the stress of cell isolation and/or culture induces Mmp10. Based on the induction of Mmp10 observed in the rat ovary, a microarray experiment was conducted to determine the expression in the human. Mmp10 expression increased 150-fold between the pre- and late ovulatory phase in human granulosa cells. Thus the increase in Mmp10 prior to ovulation in human granulosa cells is similar to findings in the rat ovary indicating an increased expression of Mmp10 mRNA after hCG stimulation. In summary, these results suggest that the induction of Mmp10 prior to ovulation, in both the rat model and human granulosa cells, may reflect a role of Mmp10 in both the late stages of follicular growth as well as the breakdown of the follicular wall during the periovulatory process.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Effect of FSH and its Glycosylation Variants on Proliferation, Steroid and Inhibin Production in a Human Ovarian Granulosa-Like Tumor Cell Line (KGN).

Nazareth Rosana Loreti 1, Griselda Irusta 2, Luz Andreone 1, Veronica Ambao 1, Marta Tesone 2, Stella Campo 1

KGN is a steroidogenic human ovarian granulosa-like tumor cell line that expresses functional FSH receptor; in these cells steroid production is stimulated by FSH. This glycoprotein is released from the pituitary gland as a mixture of glycosylation variants that act on the target cells inducing different biological responses in vitro and in vivo. The aim of the present study was to determine the effect of recombinant human FSH (rhFSH) and its glycosylation variants on proliferation and estradiol (E2), progesterone (P4), monomeric (Pro-alphaC) and dimeric inhibin (inhibin A and B) production. Preparative isoelectrofocusing was used to isolate rhFSH charge analogues in a pH range of 2.6-7.5. Two preparations were obtained by combining pH 3 to 4 (more acidic/sialylated, AC) and pH 5 to 7 (less acidic/sialylated, BA) fractions. Concanavalin-A chromatography was used to isolate rhFSH glycosylation variants on the basis of glycan complexity. Two preparations were obtained: unbound rhFSH isoforms (UB) bearing complex, highly branched carbohydrate chains and firmly bound rhFSH isoforms (FB) bearing hybrid type oligosaccharides. Cells were cultured on 24-well plate treated with or without rhFSH or its glycosylation variants (20 ng/mL) for 24h or 72h; E2 and P4 (24h) were determined by RIA and inhibins (72h) by specific ELISAs. [Methyl-3H]- thymidine incorporation was used as a measure of proliferative activity (24h). Under basal conditions KGN cells were able to produce E2 in the presence of androstenedione (100nM), P4 in the absence of a steroidogenic substrate (25-OH-Cholesterol), Pro-alphaC and inhibin A. Inhibin B was not detected. There was a direct correlation between basal P4 production and cell density. This condition did not affect either E2 or inhibin production. rhFSH significantly stimulated the production of E2, P4, Pro-alphaC, inhibin A and the incorporation of thymidine. Less acidic/sialylated rhFSH was more biopotent to stimulate the production of E2 and inhibins than its more sialylated counterpart. Progesterone production was not significantly affected by the degree of sialylation of the hormone. UB isoforms showed lower biopotency to stimulate steroids and inhibin production when compared to FB isoforms. These glycosylation variants, bearing hybrid type oligosaccharides, exerted a potent stimulatory effect on E2, P4, Pro-alphaC and inhibin A production. These results suggest that the degree of sialylation as well as the complexity of carbohydrate chains present in rhFSH molecules may be considered as additional factors that differentially regulate KGN cells function. (Supported by CONICET, PIP 5479 and FONCYT, BID-1728 OC/AR PICT 2004, N° 25365, Argentina)

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

The Nuclear Receptor Nr5a2 Coordinates Ovulation and Female Fertility in Granulosa Cells of Different Stages of Follicle Maturation in Mice.

Kalyne Bertolin 1, Raj Duggavathi 2, Jan Gossen 3, Kristina Schoonjans 4, Bruce Murphy 1

The nuclear receptor Nr5a2 (liver receptor homolog-1) is expressed in the ovary, exclusively in granulosa and luteal cells. Germline ablation of Nr5a2 causes embryo lethality, while conditional disruption beginning with the primary follicle stage by means of Nr5a2-floxed and Amhr2Cre/+ mice results in failure of ovulation and luteinization. We hypothesized that Nr5a2 modulates the events leading to development of a preovulatory follicle and its consequent ovulation in a temporal sequence. To test this, granulosa-specific mutants (hereafter characterized as Nr5a2gc-/-) were generated by crossing Nr5a2-floxed mice with mice expressing the Cyp19-Cre, thereby excising Nr5a2 at the antral (tertiary) follicle stage. Similar to Amhr2-Cre driven disruption of Nr5a2, no litters were born to Nr5a2gc-/- females, while Nr5a2gc+/+ females were fertile (six-month breeding trial; n = 5 per genotype). In contrast to Amhr2-Cre driven disruption of Nr5a2, the pattern of estrous cyclicity and serum progesterone appeared to be normal in Nr5a2gc-/- mice compared to Nr5a2gc+/+ mice (n = 5). Likewise, ovarian morphology indicated that antral follicles were present but that cumulus expansion was distinctly absent in Nr5a2gc-/- follicles. In further contrast to Amhr2-Cre driven disruption of Nr5a2, Nr5a2gc-/- ovaries in our study had corpus luteum-like structures that appeared to be comprised of luteinized theca cells surrounding degenerating granulosa cells. Entrapped oocytes were found within these structures. Thus, it was apparent that interdiction of Nr5a2 expression using Cyp19Cre/+ in antral follicles enabled further maturation of follicles than did excision by Amhr2 Cre/+. It also allowed the development of a luteal-like structure in the absence of ovulation. We conclude that Nr5a2 differentially regulates ovarian folliculogenetic and ovulatory processes in granulosa cells of small and antral follicles. Supported by CIHR grant FRN 11018.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Expression of PDGFs and Their Receptors in the Fetal and Adult Sheep Ovary.

Peter R Smith 1, Laurel Quirke 1, Jennifer L Juengel 1, Peter R Hurst 2

Platelet derived growth factors (PDGFs) and their receptors are key regulators of cell growth and angiogenesis, especially during fetal development. In rodents and humans, expression of PDGF mRNA was observed in specific ovarian compartments and the addition of PDGF to follicles in culture increased their size and enhanced the transition from primordial to primary follicles. Differences in the pattern of PDGF expression were noted depending on the species or age studied. However the effect of follicular health on expression has not been examined in depth. The objectives of this study were to determine the cell specific gene expression of PDGF ligands (PDGFA, B and C) and their receptors (PDGFRA and B) during ovarian and follicular development in sheep. Ovaries were collected from fetuses on days 55, 75 and 95 of gestation and from ewes at 4 weeks, 10 months, 2 years and 8 years of age and examined using in situ hybridization. During fetal life, PDGFA expression was sometimes associated with the vasculature and extra ovarian rete. Consistent expression was noted in the surface epithelium and in oocytes in both ovigerous cords and formed follicles. No expression was observed in granulosa cells of formed follicles or connecting rete. Fetal expression of PDGFB was similar to PDGFA at days 55 and 75; however no expression was apparent in the rete and by day 95 expression was largely confined to the surface epithelium. Expression of PDGFC was concentrated in the surface epithelium and cells surrounding the vasculature, with little or no expression observed in oocytes, follicles or in cells of the rete system. Postnatally, PDGFA and B were expressed in oocytes at all stages of follicular development from primordial to large antral follicles and in granulosa cells from the primary stage. Granulosa cell expression of PDGFC was evident in large preantral and small antral follicles. Thecal expression was not apparent until the large preantral stage and was initially restricted to PDGFB. At antral stages PDGFB was expressed predominantly in the theca interna while PDGFA was expressed in both theca interna and externa. PDGF receptors were not expressed in follicles until after antrum formation with PDGFRA expression in both theca interna and externa while PDGFRB expression was limited to theca interna. In general, atresia resulted in decreased expression of PDGFA and B in granulosa cells, increased expression of PDGFA and B in the thecal layers, increased expression of PDGFC in the granulosa and the appearance of PDGFC expression in the thecal layers. Only PDGFA expression was consistently noted in components of the ovarian rete system. Expression of PDGFA, B and C and PDGFRA and B was apparent throughout the CL, with receptor expression often associated with the vasculature. Cells associated with the vasculature showed expression of PDGFA, B and C, as well as PDGFA and B. Changes in expression patterns at different stages of follicular development along with changes associated with follicular atresia are consistent with a role for these factors in regulation of follicular development.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Structural Changes in Basement Membranes in the Gonad of the Frog Rana rugosa During Sex Differentiation.

Masahisa Nakamura 1, Tomohiro Saotome 1, Tomoko Isomura 1, Tatsunori Seki 2, Yoriko Nakamura 3

The sex of vertebrates is determined by either chromosomal factors (GSD) or environmental factors. In mammals, an individual having two X chromosomes is female, whereas one having an X and a Y is male. Sry on the Y chromosome acts as a developmental switch, initiating a pathway for the testicular differentiation. In some, but not several other species of vertebrates such as fish and reptiles, sex determination does not merely depend on the combination of the sex chromosomes alone, but is influenced by environmental and social factors, in particular temperatures (TSD). The determinants in GSD and TSD that initiate sexual differentiation in vertebrates may differ from each other. Of interest, similar morphological structures are observed among the testes and ovaries of vertebrates. Thus, whichever system GSD or TSG each species in vertebrates employs, vertebrates probably share common downstream mechanisms for the formation of the testis and ovary. However, structural changes in gonads during sex differentiation have not been examined in details in any species of vertebrates. Then, we examined the changes in gonadal basement membranes during sex differentiation in the frog R. rugosa using an antibody to its laminin component. Immunohistochemical staining showed that the first sexual dimorphism appeared in testicular cords and ovarian cavities in differentiating gonads of tadpoles at very early developmental stages. During development, as the testis enlarged, testicular cord partitions appeared to form by invagination of the testicular epithelium. Ovarian cavities also increased in volume. Laminin-positive basement membranes initially surrounded a partial surface of oocytes close to the ovarian cavity, and fully covered growing oocytes by St. X. Laminin-reactive signals were present in somatic cells outside seminiferous tubules in the testis and outside oocytes in one-year old frogs. BrdU-labeling revealed that the number of dividing germ cells increased continuously in male gonads but increased in females only up to St. V, declining at St. X and thereafter. The number of proliferating germ cells declined when the basement membranes had fully covered the oocytes. Taken together, these findings suggest that the first sexual dimorphism in the gonad of R. rugosa first appears as a structural change in the basement membranes. In addition, the basement membrane on the surface of oocytes may affect their proliferation in this species.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Abortive-Prone Matings Alters the Expression of A2V-ATPase in Mouse Spleen.

Mukesh Kumar Jaiswal 1, Christina Kwong 1, Alice Gilman-Sachs 1, Gerard Chaouat 2, Kenneth Beaman 1

The a2 isoform of vacuolar ATPase (A2V-ATPase) has immunosuppressive effects and is in part responsible for the survival of the fetal allograft. Pregnancy brings vital changes in the maternal environment that comprises reversible modifications of immune cells in response to both systemic and local mediators and signals. The spleen can be a useful organ to determine the systemic affects of pregnancy on multiple cell populations, including those of the immune system. By taking the well studied, allogeneic abortion prone (maleCBAX femaleDBA) model and other syngeneic and allogeneic mating models, the effects of pregnancy on murine immune cells of the spleen were studied. We assessed the expression of A2V-ATPase in the spleen of different mating combinations on different days of pregnancy. Our data shows that the splenic A2V-ATPase gene expression increased from day 8 to 16 in maleBALB/cXfemaileBALB/c and maleCBA XfemaleBALB/c matings established as successful pregnancy controls. However, in abortion prone mating (maleCBAX femaleDBA) its expression was similar as the successful pregnancy controls on day 8 and then decreased significantly on later days of pregnancy. Additionally, administration of LPS at different days of pregnancy significantly decreased the expression of A2V-ATPase in spleen. We also assessed the surface expression of A2V-ATPase on splenic lymphocytes at different days of pregnancy by using flow cytometry. We show that the expression of A2V-ATPase in CD4+ and CD8+ cells was significantly lower in both abortive prone mating as well as in LPS-administrated condition as compared to successful pregnancy controls on days 12 and 16 of pregnancy. Together, these findings indicate that the lower expression of A2V-ATPase corresponds with the dynamic changes in spleen cell populations of abortion-prone matings. This supports our hypothesis that A2V-ATPase participates in the establishment of a unique immune milieu that helps the fetus to survive and develop in the uterus until parturition. This research was supported by Clinical Laboratory, Rosalind Franklin University of Medical and Science.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

A Potential Immune Deviating Pathway that Protects Human Sperm and Aggressive Tumor Cells.

Gary F Clark, David R Lee, Erma Z Drobnis, Durwood E Neal, David E Lafrenz 1

The possibility exists that malignant tumor cells employ the same immune deviating pathways that are used to protect immune privileged sites. Glycoproteins associated with human sperm and seminal plasma express Lewis x and Lewis y carbohydrate sequences presented on N-glycans in a very unusual polyvalent fashion (poly-Lewis x/Lewis y type N-glycans). This observation is relevant because previous studies carried out with variants of the human gastric pathogen Helicobacter pylori confirm that lipopolysaccharides bearing Lewis x and Lewis y sequences bind to the dendritic cell-specific lectin designated DC-SIGN and trigger a signal transduction cascade that diverts the adaptive immune response directed against these variants. Therefore the addition of Lewis x and Lewis y sequences to glycoproteins present in sperm and seminal plasma glycoproteins could play a major role in inhibiting Th1 responses directed against autoantigens present in human semen. The Lewis y sequence is also a very well established human tumor associated carbohydrate antigen that is highly upregulated on >70% of all human cancers of epithelial origin but not on normal adult tissues with the notable exception of the male reproductive system. Until recently, the glycoproteins and the glycan types that carry Lewis antigens on aggressive tumor cells were unknown. A recent study confirmed that the major carrier of Lewis x and Lewis y sequences on human MCF-7 breast cancer cells is CD98hc. An intriguing linkage is that these sequences are also associated with poly-Lewis x/Lewis y type N-glycans that are similar to those found on human sperm and in seminal plasma. In this study, lectin affinity chromatography of human seminal plasma glycoproteins was employed to isolate the major glycoprotein carriers of Lewis x and Lewis y sequences within this fluid. A major band of 75 kDa glycoprotein with the same MW as CD98hc in MCF-7 cells was identified. Minor bands were also detected at 125 and 150 kDa. Glycoproteomic and functional analyses are now underway to determine the identity of these glycoproteins, define their glycosylation state, and analyze their immunological activities. It is well established that human seminal plasma also contains copious amounts of transforming growth factor-β. (TGF-β) and prostaglandin PGE2, two other very potent immunomodulatory factors. Therefore immune privilege in the human male reproductive system could rely primarily on the coordinated expression of these factors and Lewis x and Lewis y sequences in combination with physical boundaries like the blood:testis barrier. Many metastatic tumors express precisely this same combination of factors and Lewis antigens but lack any physical barrier. The absence of such barriers could enable tumor associated antigens to easily escape from malignancies and evoke adaptive responses, unlike autoantigens at immune privileged sites. These observations are consistent with the hypothesis that aggressive tumor cells employ the same immune deviating pathways that are used to protect human sperm in the human male reproductive system. These results are also consistent with the eutherian fetoembryonic defense system hypothesis. (This investigation was supported by a Pilot Study Fund from the Institute of Clinical and Translational Science at the University of Missouri-Columbia, the Breeden-Adams Foundation and the Mission Enhancement Program in Reproductive Biology and Medicine funded by the state of Missouri.)

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

NOT PRESENTED.

NOT PRESENTED.

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Caffeine Inhibits Aging of Mouse Oocytes: The Efficiency and Recoverability.

Jing-He Tan, Zhen-Hua Zhang, Yi-Liang Miao, Dong Han, De-Qiang Miao, Jun-Zuo Wang, Si-Yu Zhang 1

Inhibiting oocyte aging is important not only for healthy reproduction but also for the success of embryo techniques including cloning by nuclear transfer. Functional and morphological changes observed with oocyte aging include an increased susceptibility to activating stimuli, a decrease in the maturation-promoting factor (MPF) activity, abnormal distribution of cortical granules and abnormal development after activation/fertilization. However, changes in the MPF activity remain to be correlated with other aging alterations. Furthermore, while caffeine has been found to inhibit aging of in vitro matured (IVM) porcine oocytes by dephosphorylating T14 and Y15 of CDC2A, the conclusion must be confirmed in other species and in the in vivo matured (IVO) oocytes as well. We wanted to study the efficiency and recoverability of caffeine inhibition on aging of mouse oocytes. Freshly matured mouse IVO and IVM oocytes were cultured in CZB medium supplemented with different concentrations of caffeine for 6 or 12 h before examined for activation susceptibility, MPF activity or spindle and cortical granule morphology. Oocytes for controls were cultured for the same period in CZB without caffeine. Some of the caffeine-treated oocytes were cultured for 6 h in CZB without caffeine to observe recoverability. The results showed that caffeine prevented aging of both IVO and IVM mouse oocytes by maintaining MPF activities. However, while 1-mM caffeine efficiently inhibited aging of the IVM oocytes, aging of IVO oocytes was not prevented until caffeine concentration increased to 8-mM. When IVO oocytes that had been inhibited for 6 h with 6-, 8- or 10-mM caffeine were cultured in absence of caffeine, rates of activation increased with culture time and reached the same level as in control oocytes by 6 h of culture. While oocytes inhibited with 6-mM caffeine developed into blastocysts at a similar rate as the control oocytes, oocytes inhibited with 8- or 10-mM caffeine showed decreased blastocyst rates after recovery culture and activation. The activation competence of IVM oocytes was also recoverable but they could not develop into blastocysts after caffeine treatment. Laser confocal microscopy revealed that spindle morphology and cortical granule distribution were affected after IVO oocytes were treated with 8-mM caffeine for 12 and 6 h, respectively; however, while spindle morphology was recovered, cortical granule distribution were unrecoverable after recovery culture. It was concluded that caffeine recoverably prevented aging of both IVO and IVM mouse oocytes by maintaining their MPF activities, but protocols must be optimized according to oocyte types to increase the recoverability after inhibition. This study was supported by grants from the National Basic Research Project of the China Ministry of Science and Technology (Nos. 2006CB944003 and 2007CB947403) and the China National Natural Science Foundation (Nos. 30771556 and 30972096).

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

The Possibility of Storage at Room Temperature for Freeze-Dried C57BL/6J Mouse Sperm.

Yosuke Kawase, Naoko A Wada, Kou-ichi Jishage 1

Freeze-drying mouse sperm is a low cost, easily transportable method of maintaining genetic material. We previously reported that freeze-dried mouse sperm must be kept at lower than -80°C for long-term preservation (Kawase et al., 2005). Moreover, a pressure of 0.37 hPa at primary drying significantly improves both the rate of development to the blastocyst stage (Kawase et al., 2007) and the number of live term fetuses from internationally air-transported freeze-dried mouse sperm. Hence, we now consider freeze-drying mouse sperm to be a practical and for transportable method for the storage of mouse gene resources. However, storing the samples at such low temperatures over a long period of time has the risk of technical difficulties (e.g. power failure) and requires a relatively high initial investment; it is essential to assure against such risks. Therefore, we examined the stability over time of freeze-dried C57BL/6J mouse sperm having undergone a temporary temperature rise and transported at room temperature (RT). We analyzed freeze-dried sperm DNA fragmentation using sperm chromatin structure assay (SCSA) ( Kawase et al., 2009) of freeze-dried sperm after storage at RT for 0, 1, 3, 5, 7, 14 and 28 days and determined the developmental rate to the blastocyst stage of embryos from ICSI using sperm freeze-dried after storage at RT for 0, 5 and 14 days. DNA fragmentation index (DFI) values storage at RT for 0, 1, 3, and 5 days were 4.3%, 6.3%, 5.1% and 6.8%, respectively, and no sperm DNA fragmentation was found. However, we did find sperm DNA fragmentation at 7 days (8.6%), 14 days (15.1%) and 28 days (31.2%). The rate of development to the blastocyst stage of embryos from ICSI using sperm freeze-dried after storage for 0 and 5 days at RT was 60% (63/105) and 58% (53/91), respectively, and 38% (41/108) when stored for 14 days. There was no difference in the DFI and the rate at which embryos developed to the blastocyst stage between storage for 0 days or for 5 days at RT. We concluded that freeze-dried sperm can be exposed to RT for up to 5 days without affecting the rate of development to the blastocyst stage found with sperm kept at RT with no storage. After 14 days of storage at RT, the embryos from ICSI were able to develop to the blastocyst stage; however, sperm DNA fragmentation increased. Therefore, the functional integrity of freeze-dried C57BL/6J sperm was retained when exposure at RT was limited to 5 days. The freeze-dried sperm sustained transportation at RT for 14 days without loss of reproductive potential and thus assures against much of the risk of storing and transporting freeze-dried sperm. We suggest that freeze-drying sperm become more widespread as an effective method of maintaining gene resources.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Application of Equine Epididymal Sperm.

Cely M Melo, Frederico O Papa, Eduardo G Fioratti, Luis Carlos O Magalhaes, Marco A Alvarenga 1

Freezing epididymal sperm is a method to preserve germplasm from animals with not only high genetic potential but also endangered species. In the equine some owners have chosen this freezing possibility in cases of either severe illness or death of stallions. Some studies have been evaluating the fertility of equine epididymal sperm. However, the commercial application has not been described. The aim of the present study was to compare the fertility of samples obtained from ejaculate and epididymal cauda in an equine commercial artificial insemination program. A stallion aged 8-years-old that would be euthanized due to a large wound caused by Pythiosis was used. The samples from the ejaculate of a commercial program were frozen with INRA 82 extender associated with 2.5% of dimethyformamide and 2.5% of methylformamide. Epididymal samples were obtained from retrograde flushing. The epididymides were recovered during routine castration. A 200 μL pipette tip was attached to a 20 mL syringe and epididymal tails from both testicles of each colt were flushed using 40 mL of a skim milk based extender (Botu-Semen, Biotec, Botucatu, Brazil). The samples were centrifuged at 600×g/10 min. The supernatant was discharged and the pellet resuspended with Botu-Crio. The straws were maintained at 5°C for 20 min followed by another 20 min at 6 cm above liquid nitrogen before immersion. Two straws from each group, in each pool, were used for analysis. After thawing at 46°C for 20 seconds, the samples were analyzed by CASA (HTM-IVOS 12), and plasma membrane integrity (PMI) using fluorescent probes: CFDA and PI. Five inseminations using semen from ejaculate were performed with 2 straws containing 100×106/straw. The epididymal samples were evaluated by CASA. Total (TM), progressive motility (PM), and percentage of rapid cells (RAP) after centrifugation were 77%, 25% and 52%, respectively. The frozen-thawed values for TM, PM, RAP and PMI were 77%, 27%, 44% and 52% respectively. Three inseminations using equine epididymal frozen semen were performed using 2 straws containing 100×106/straw. The straws from the ejaculate were evaluated immediately before the insemination and presented 60% of total motility. No pregnancy was observed in the ejaculate samples. However, all mares became pregnant with epididymal semen. Seminal plasma can be detrimental to sperm motility and viability of equine semen. Similar results were obtained when the samples from the epididymis were incubated with other extenders such as Sperm and Fert-Talp, even for subfertile stallions. This result indicates that frozen of epididymal sperm is an option for those stallions that have died, being the last chance to preserve the germplasm from valuable animals. More studies using lower doses have to be developed aiming to optimize the use of epididymal frozen semen using different technologies, such as artificial insemination and intracytoplasmatic sperm injection. Acknowledges: Financial support FAPESP, Proc No. 2008/57504-2.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Improved Sperm Cryopreservation in the Critically Endangered Przewalski's Horse (Equus ferus przewalskii) Using Different Cryoprotectants.

Budhan S Pukazhenthi 1, Francesca Lapiana 1, Luis Padilla 1, Jenny Santiestevan 2, Marco Coutinho da Silva 3, Marco Alvarenga 4, David Wildt 1

There are approximately 500 critically endangered Przewalski's horse living in nature, and fewer than 1,500 managed in ex situ collections worldwide. Because the contemporary population was derived from only 14 founders, genetic management is a priority. The ability to preserve and transport gene diversity could be enhanced through the use of frozen-thawed sperm and artificial insemination. Our aim was to assess cryotoxicity of amides (methylformamide, MF; dimethylformamide, DMF) and glycerol (G) and its effects on sperm cryosurvival. Objectives were to evaluate: 1) toxicity of MF, DMF, MF + DMF versus G; and 2) effects of three cryodiluents, EQ (Lactose-EDTA + 20% egg yolk + 4% G), Botu-Crio (Base Medium + 20% egg yolk + 1% G + 4% MF; Biotech-Botucatu, Brazil) versus INRA-F (INRA 96 + 2% egg yolk + 2.5% MF + 2.5% DMF) on total motility (TM), progressive motility (PM) and acrosomal integrity (AI). Electroejaculates from four Przewalski's stallions were evaluated for TM, PM and AI. Ejaculates were diluted 1:2 with warm INRA 96, centrifuged and the supernatant discarded. For Study 1, sperm pellets were resuspended in INRA 96 and sub-divided into 16 aliquots. Cryoprotectants G (0, 4, 8 and 16%), MF (0, 2.5, 5 and 10%), DMF (0, 2.5, 5 and 10%) and equal concentrations of MF + DMF (0, 2.5, 5 and 10% total) were added to respective tubes, cooled in a water jacket and incubated at 4°C for 24 h. Aliquots were re-warmed (37°C) and assessed at 0, 4, 12 and 24 h for TM, PM and AI. For Study 2, washed sperm pellets were resuspended in EQ, Botu-Crio or INRA-F at room temperature, loaded in straws (0.5 ml), cooled to 4°C and placed over LN2 for 15 min before being plunged into LN2. Straws were thawed at 37°C (30 s), stored at 25°C and evaluated for TM, PM and AI at 0, 2, 4 and 6 h. Data were analyzed using ANOVA. Significance was defined as P<0.05. All stallions produced ejaculates with good initial TM (~71.4%), PM (~61.5%), AI (~88.4%) and low proportions of morphologically normal spermatozoa (~14.7%). In Study 1, although TM and PM declined over 24 h, AI remained unaffected by all cryoprotectants. Spermatozoa in 10% MF or a combination of 5% each of MF and DMF exhibited a significant (P<0.05) decline in TM and PM compared to 10% MF or 16% G. In Study 2, cryopreservation in INRA-F or Botu-Crio improved (P<0.05) post-thaw TM compared to EQ. At 6 h post-thaw, TM was higher (P<0.05) in INRA-F and Botu-Crio compared to EQ. Immediately after thawing, PM was unaffected in Botu-Crio, but declined (P<0.05) in INRA-F and EQ. At 6 h post-thaw, INRA-F and Botu-Crio maintained higher (P<0.05) PM compared to EQ. Cryopreservation had no effect on AI in EQ, but resulted in a decline (P<0.05) in INRA-F and Botu-Crio compared to Raw. At 6 h post-thaw, although AI in INRA-F and Botu-Crio was lower (P<0.05) than Raw, the proportion of AI spermatozoa was similar among INRA-F, Botu-Crio or EQ. Date demonstrated that: 1) Przewalski's horse spermatozoa tolerate a variety of cryoprotectants during liquid storage; 2) glycerol protected AI but compromised TM during cryopreservation; and 3) cryopreservation in diluents containing amides provided optimal post-thaw sperm viability and longevity.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Superovulated Oocytes from Mature Mice Are of Better Quality Than Oocytes from Prepubescent Mice.

Sadie L Murdie 1, Shannon L Byers 2, Michael V Wiles 2, Robert A Taft 2

Reproductive technologies such as in vitro fertilization (IVF) are integral processes for preserving and rederiving mouse (Mus musculus) strains. Superovulation is a crucial step for successful IVF and entails administering pregnant mare serum gonadotropin (PMSG) followed by the administration of human chorionic gonadotropin (hCG) 48 hours after PMSG administration to induce the growth and ovulation of a large cohort of follicles. Typically, prepubescent females are superovulated to produce oocytes for IVF as oocyte yield is often greater and more consistent than in mature females, which are often asynchronous in estrous stage and respond dissimilarly to superovulation. While the yield of oocytes from prepubescent females is greater, it is not clear how the quality of oocytes from prepubescent females compares to the quality of oocytes from mature females. In this study, the quality of oocytes from prepubescent C57BL/6J females was compared to the quality of oocytes from mature C57BL/6J donors in their capability to develop embryos and produce live offspring. Two groups of females at approximately 3 and 12 weeks of age were superovulated using 5.0 IU of PMSG and 5.0 IU of hCG. Thirteen hours post hCG administration, females were euthanized via cervical dislocation and oocytes were collected. IVF was performed using fresh sperm from C57BL/6J males. The resulting presumptive zygotes were cultured overnight in IVF medium and two-cell stage embryos were collected the following morning. Embryos were surgically transferred into one oviduct of 0.5 dpc pseudopregnant CByB6F1/J females (10 embryos per female). Animals were euthanized 15-18 days post transfer and the number of fetuses or resorption sites was counted and classified. Prepubescent donors ovulated more oocytes than mature donors, with the mean of 29 and 18 oocytes per female respectively (P<0.05). The mean fertilization rate was 71% for both groups. The proportion of live fetuses was greater (P<0.05) when using mature rather than prepubescent oocyte donors (61% and 47% respectively). Conversely, the proportion of resorption sites was greater (P<0.05) when using prepubescent rather than mature oocyte donors (29% and 17% respectively). There was no difference (P>0.05) in the proportion of dead fetuses, which were 0.7% and 1.1% for prepubescent and mature oocyte donors, respectively. Furthermore, 24% and 21% of the embryos from prepubescent and mature oocyte donors, respectively, were unaccounted for (not significantly different, P>0.05). In conclusion, even though prepubescent females produce increased numbers of oocytes in comparison to mature females, they have a decreased ability to produce live offspring. Hence these results suggest that the oocytes from mature females are of better quality than those from prepubescent females, and should be utilized as oocyte donors for IVF to increase the yield of live offspring. Research supported by NIH grant RR001262.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Effects of Assisted Reproductive Technologies and/or Fat-Based Hypercaloric Diet on Cardiovascular Performance in Mice.

Rocio Melissa Rivera, Sarah Rose Huffman, Guiling Zhao, Luis A Martinez-Lemus 1

Children conceived by the use of assisted reproductive technologies (ART) can account for as high as 3.9% of all births in some countries. ART may be affecting cardiovascular health in children conceived through these procedures. For example, it has been demonstrated that children conceived through these technologies have altered systolic and diastolic blood pressure. Similarly, in mice, an association exists between ART and a raised systolic blood pressure. Our study aims at determining outcomes of method of conception on cardiovascular parameters after birth. We produced mouse embryos by ART and will determine their cardiovascular health during early adult life (6-8 weeks) as compared to pups born after natural conception. We added a nutritional challenge to the mother in order to determine if high fat diets would affect the developmental competence of growing oocytes and/or affect the uterine environment which would result in offspring with abnormal cardiovascular health. Briefly, CF1 females were divided in two groups and fed either a high- (~35%) or low-fat diet (~5%; HFM or LFM, for high-fat and low-fat mother, respectively) for at least three weeks prior to and during the experimental period. Females were further divided in two groups, ART group and natural conception group (NCG). NCG was mated and left undisturbed to give birth and rear their pups. Male and female pups were separated at weaning and fed a HF or LF diet (HFO and LFO, O = offspring) until 6-8 weeks of age at which time they underwent analyses of cardiovascular parameters. The ART group received an injection of 5 IU of eCG followed 44 h later with 5 IU of hCG and mating. Two-cell embryos were collected and cultured in either KSOM augmented with amino acids or Whitten’s medium in an atmosphere of 5% O2, 5% CO2, and 90% N2 or 5% CO2 in air, respectively. Blastocyst-stage embryos were transferred to the uterus of pseudopregnant CF1 females that had been eating either the HF or LF diets. Surrogate mothers continued to eat their respective diets until pups were weaned. At weaning, pups were divided into groups same as for the NCG group. In all, we expect to analyze 8 possible permutations for the NCG group and 32 for the ART group. To date, 33% (12/36) of embryo transfers resulted in analyzable litters (i.e. those with enough pups to separate by sex and diet). Interestingly, any ART combination in which embryos from HFM were transferred to HF surrogates (6 transfers) did not yield any litters that survived more than a few hours. We have started to determine the effects of fat-based hypercaloric maternal and/or offspring feeding on offspring cardiovascular performance by measuring; 1) mean systemic blood pressure in anesthetized animals via carotid catheterization and 2) vasomotor responses to topical norepinephrine (10-8 to 10-5 M), acetylcholine (10-8 to 10-5 M), sodium nitroprusside (10-7 to 10-4 M) and calcium-free solution in isolated resistance arteries. Thus far we have analyzed a set of the NCG controls. Results show differences in body weight at the time of analyses between the LFM/LFO vs.HFM/HFO (P < 0.004) and LFM/LFO vs. LFM/HFO (P < 0.08) treatments. Further, blood pressure values show differences between groups. Weight and blood pressure differences are further differentiable by sex. Our results are consistent with the hypothesis that ART and intrauterine environment have profound consequences on the subsequent cardiovascular health of adult offspring.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

WITHDRAWN

WITHDRAWN.

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Indirect and Direct Effects of Increased Androgen Exposure In Utero on the Ovine Fetal Ovary.

Kirsten Hogg 1, Alan S McNeilly 2, W Colin Duncan 1

Exposure of a female monkey fetus to increased androgen concentrations in utero results in an adult phenotype that is remarkably similar to polycystic ovary syndrome (PCOS) in women. Similarly in an ovine model, fetal androgenization leads to hormonal, metabolic and ovarian features reminiscent of PCOS. We hypothesized that the enlarged polyfollicular ovarian phenotype was a consequence of the hormonal and metabolic changes and that androgens had no direct effect on fetal ovarian structure or function. We therefore investigated the in utero effects of androgens on the fetal sheep mid-gestation ovary to establish whether specific early ovarian changes are present. We studied: 1) The effects of indirect exposure to testosterone propionate (Tp) after maternal administration compared with exposure to Tp using direct fetal injection and 2) The effects of Tp on: a) fetal ovarian morphology, cell proliferation and germ cell volume; b) the expression of fetal ovarian androgen receptors (AR); c) the functional status of ovarian cells by the expression of the SLIT/ROBO pathway, reported to be involved in cell migration, cell death and ovarian development and also steroid regulated, and d) the differentiation of ovarian cells by their expression of inhibitor of differentiation (Id) transcription factors. Pregnant Scottish Greyface ewes were injected intramuscularly with Tp (100mg in vegetable oil) or vehicle control (C) twice weekly from d60 of gestation, and fetal ovaries were collected on d70 (C n=3, Tp n=6) or d90 (C n=6, Tp n=8). In a second cohort of sheep a single dose of Tp (20mg) or vehicle control was administered directly into the flank of the fetus at d60 under ultrasound guidance and fetal ovaries were collected at d70 (C n=4, Tp n=8). Ovarian morphology, assessed by histology, was not altered following indirect (maternal) or direct (fetal) androgen exposure. In addition a quantitative immunohistochemical and stereological analysis for ovarian cell proliferation (Ki67 immunostaining) and germ cell volume (Oct4 immunostaining) revealed no significant effects of Tp. Although AR mRNA expression was similar in each group, nuclear AR immunostaining was increased in fetally exposed d70 ovaries when compared with controls (P<0.05), whereas this did not occur in maternally exposed d70 or d90 ovaries. Functionally qRT-PCR analysis demonstrated a significant decrease in ROBO1 expression (P<0.05) in these d70 fetally treated ovaries with a parallel, albeit not quite significant, decrease in both SLIT2 and SLIT3. These changes were not seen after indirect maternal administration of androgen. However maternal treatment was associated with an up-regulation of the inhibitor of differentiation 1 protein (Id1) in the d90 fetal ovary. These findings suggest that although androgenization in utero has no clear morphological or structural effects on the mid-gestation fetal ovary, early exposure to sex steroids may lead to functional changes that impact on ovarian development. These changes may differ after direct fetal androgen exposure giving further evidence that some of the fetal effects of maternal androgen exposure are mediated by products of placental metabolism such as estrogen. In conclusion, fetal ovarian development is vulnerable to alterations in the local environment and this might directly impact on post-natal ovarian function and the ovarian phenotype.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Equine Ovarian Aging and Differential Control of Gene Expression.

Juliano C Silveira, Elaine M Carnevale, Quinton A Winger, Gerrit J Bouma 1

Reproductive aging in mares and women coincides with a decrease in follicle numbers, generating cycle irregularities and decreased oocyte quality. The horse is a good model to study reproductive aging and oocyte quality, as follicular waves and hormone profiles are very similar between mares and women. Oocyte competence is dependent on synchronized events, with communication between the oocyte and somatic cells contributing to development and competence of the oocyte. Furthermore, follicular fluid (FF) provides an important environment of oocyte development and serves as a reservoir for products from surrounding cells. Our overall goal is to identify factors associated with oocyte quality using the aging mare as a model. MicroRNAs (miRNAs) are non-coding small RNAs that regulate gene expression and function, and play a role in female reproductive function and fertility. Cathepsin beta (CTSB), a lysosomal cysteine proteinase involved in a variety of cellular processes including apoptosis, has recently been correlated with low oocyte quality and competence in bovine. CTSB mRNA is a predicted target of miRNA 186 (mir-186). We postulated that mir-186 and CTSB expression could correlate with low oocyte quality in mares. To test this hypothesis, CTSB expression was determined in cumulus cells (CC) of young (good oocyte quality) and old (poor oocyte quality) mares. In addition, expression of miRNAs (including mir-186) was examined in FF and CC. Ovarian follicles from 22 young (4-12 years old), and 18 old (≥20 years old) mares were aspirated at three different time points (deviation (23-25mm), mid-estrus (30-33mm prior to des/hCG), and pre-ovulation (35mm 32-34hs after des/hCG). CCs were collected and processed for RNA and miRNA isolation. FF was collected in a separated tube, centrifuged and RNA was isolated using TRI-Reagent BD. Quantifiable cDNA templates for real time PCR were generated using qScript cDNA Synthesis Kit for mRNA and QuantiMir miRNA cDNA kit for miRNA. Real time PCR analysis of CTSB in CC demonstrated a significant (P<0.05) increase in expression in CC from old mares. A miRNA expression profiling screen in pre-ovulatory FF identified 38 miRNAs with differential expression (2 fold or higher) between young and old mares. Twelve miRNAs had a fold change ≥4 between the two groups, and 4 miRNAs were significantly different (p≤0.05) between young and old. Expression of mir-186 in CC and FF from young and old mares was not significantly different (P=0.09, and P=0.07 respectively). These results: 1) Indentify CTSB as being significantly higher expressed in CC from old mares, suggesting it plays a role in decreased oocyte quality observed in old mares. 2) Demonstrate the presence of differentially expressed miRNAs in FF, which could serve as novel diagnostic tool to assess oocyte quality. Further studies are needed to establish a direct correlation between miRNA function and oocyte quality. Supported by benefactors for Preservation of Equine Genome (PEG) and the Cecil and Irene Hylton Family Foundation.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Adenosine 5'-Monophosphate-Activated Protein Kinase Regulates 5 alpha-Reductase mRNA Expression in Ovarian Granulosa Cells.

Pradeep P Kayampilly, KMJ Menon 1

Ovarian follicular growth and functions are regulated by FSH, insulin and other paracrine factors through an array of signaling molecules. Pathophysiological conditions such as hyperandrogenism and hyperinsulinemia adversely affect the normal progression of follicular growth resulting in anovulation. Increased levels of 5 alpha reduced metabolites of androgen seen under these conditions further exacerbate the impairment of follicular growth. In the present study we examined the molecular mechanisms involved in the regulation of 5 alpha reductase, the enzyme that converts androgens to their 5 alpha reduced metabolite in granulosa cells. Insulin increased 5 alpha reductase activity in human granulosa-like tumor cell line (KGN) and primary cultures of rat granulosa cells in a dose and time dependent manner. Since insulin has been shown to regulate AMP Kinase activity, the possible role of this enzyme on insulin-mediated activation of 5 alpha reductase was examined. KGN Cells were grown in DMEM-F12 media with 10% FBS until 80% confluency and prior to the addition of test substances, they were maintained for 12 h in serum free media. The cells were then treated with the AMPK inhibitor (compound C, 20 µM) for different time intervals up to 6 hours, total RNA was isolated and expression of 5 alpha reductase mRNA was measured using real time PCR. The results showed that inhibition of AMPK significantly (p<0.05) increased the 5 alpha reductase mRNA expression in a time dependent manner. Since reduction of AMPK activity resulted in an increase in 5 alpha reductase mRNA expression, the effect of AMPK activation by the pharmacological agent AICAR on the expression was examined using KGN cells after 4 hours of treatment. Real time PCR analysis of 5 alpha reductase showed that AMPK activation significantly (p<0.05) reduced the 5 alpha reductase mRNA expression. Inhibiting the ERK mediated signaling pathway using specific inhibitor (U0126, 10 µM) significantly reduced the compound C-mediated increase in 5 alpha reductase mRNA expression whereas mTOR inhibition with Rapamycin (100 nM) did not have any effect. These results were then confirmed using primary cultures of rat granulosa cells. Cells were treated with AMPK activator (AICAR) or inhibitor (compound C) for 4 hours and mRNA expression of 5 alpha reductase was analyzed. As in the case of KGN cells, activation of AMPK reduced the 5 alpha reductase mRNA expression whereas AMPK inhibition produced stimulatory effect. Taken together, these results suggest that under hyperinsulinemic conditions, AMPK mediates the action of insulin on 5 alpha reductase mRNA expression in granulosa cells (supported by NIH Grant HD-38424)

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Ultrastructure of Human Prostate Cancer Shows Two Types of Stem Cells: Morphological Features Similar to Motile and Non-Motile Cells.

Sinha Akhouri 1

Metastasis is responsible for about 90% of human deaths from cancer, including prostate cancer (PCa). PCa stem cell (CSC)/progenitor (putative) stem cells have localized CD 133/CD44 in fresh frozen and/or formalin-fixed, paraffin-embedded radical prostatectomy tissue sections at light microscopic levels. Our objective was to establish ultrastructural features of stem cells by transmission electron microscopy (TEM). We used paraformaldehyde and glutaraldehyde for fixation and Epon-embeding prior to making thin sections for TEM as detailed before. Electron micrographs showed two types of CSC/PS cells located near the basal portion of prostatic acini in addition to basal cells adjacent to the basal lamina. At the EM level, one type of stem cell showed numerous plasma membrane foldings at the leading edge, but not at the trailing edge, whereas the other type did not show similar modifications. For this abstract, we have designated CSC/PS cells with membrane foldings as motile-type stem cell and cells without membrane foldings as non-motile-type stem cell. In addition, motile-type stem cells had relatively elongated nuclei, peripheral and elongated mitochondria, relatively few strands of endoplasmic reticulum and some ribosomes, but essentially no secretory granules and vesicles. Non-motile-type stem cells had relatively oval nuclei, relatively few mitochondria, ribosomes and occasional granules. Both types of cells appeared as undifferentiated cells. Basal cells had ‘spindle’-shaped nuclei which were parallel to the basal lamina and cytoplasmic organelles which were similar to the non-motile-type stem cell. Acinar cell nuclei were elongated along the baso-luminal axis and the cytoplasm contained numerous secretory vesicles and granules. These cells usually reached the acinar lumen. We suggest that motile-type stem cells probably migrated from acini to prostatic stroma and beyond whereas the non-motile-type cells remained in acini as did the basal cells. Localization of CD133 by immunefluorescence and immunoperoxidase did not show two types of stem cell. This study was conducted on Institutional Review Board approved protocol and supported by the Minneapolis Veterans Affairs Medical Center. There is no conflict of interest.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Stem Cells Prevent Radiation Exposue-Induced Ovarian Follicular Depletion.

Shiow-Chwen Tsai 1, Chien-Chen Lu 2

Ovarian failure is commonly caused by aging, autoimmune disease, menopause, and cancer therapy. We used a radiation exposure model in the ovary to test the hypothesis that stem cells are helpful for ovarian regeneration after injury. Prematured female Sprague Dawley rats were divided into three main groups, such as sham-operated control, radiation plus PBS, and radiation plus immortalized human bone marrow stromal cells (stem cells) groups. Radiation (4 Gy) exposure were performed on 10 AM, the Hoechst 33342 (H33342, 1 μg/ml Hosch) prestained stem cells or PBS were injected into rat ovaries immediately. Then, in the afternoon of the same day, and 7, 14, 21, and 28 days after radiation exposure, equine chorionic gonadotropin (eCG) was given intra-peritoneally to initiate folliculogenesis. Sixty hours after eCG injection, animals were then killed. The ovary gland was weighed, and ovarian folliculogenesis, and vascular neogenesis were evaluated by hematoxylin-eosin staining (H&E staining) and immunofluorescence staining. Fluorescence in situ hybridization (FISH) was employed to detect human-specific X chromosomes and evaluate the differentiation of stem cells in the ovary. In order to study the associated mechanisms of improved folliculogenesis after radiation exposure, the mRNA expression of luteinizing hormone receptor (LHR), steroidogenic acute regulatory protein (StAR), and cytochrome P450 side chain cleavage (P450scc) were measured by Northern blot. The expression ofStAR specifically were evaluated by Western blot. Results of H&E staining indicated that ovarian size was smaller and that the rate of folliculogenesis was lower in radiation exposure animals, but both recovered after stem cell treatment. Based on FISH data, the stem cells migrated into the ovary and differentiated into the theca cells, granulosa cells, corona radiata cells, and vascular endothelial cells. The stem cells increased von Willebrand factor (vWF) expression in the ovary compared with the sham-operated or radiation exposure plus PBS groups. The colocalization of stem cells and vWF or CD31 expression meant stem cells injected into the ovary and became vascular endothelial cells. The mRNA levels of LHR, StAR, P450scc were significantly decreased in the radiation exposure plus PBS group, but treatment with stem cells restored mRNA levels of LHR, StAR, and P450scc as compared to the sham-operated group. The StAR protein expression was also recovered by stem cells treatment in the radiation exposure ovaries. These results suggest that stem cells might be helpful for ovarian regeneration after injuries by promoting vascular neogenesis and steroidogenesis.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Basic Fibroblast Growth Factor Promotes Proliferation of Chicken Primordial Germ Cells via MEK/ERK Signaling Pathway.

Jae Y Han 1

Long-term maintenance of avian primordial germ cells (PGCs) in vitro is beneficial for understanding the biology of PGCs and their application to avian transgenesis. In the present study, we investigate the effects of various combinations of LIF, SCF, and bFGF on the survival and proliferation of PGCs under feeder-free conditions. Proliferation of PGCs was observed in medium containing bFGF, which increased cell numbers in a dose-dependent manner. Moreover, after withdrawal of bFGF for 24 h during culture, mitotic cell populations decreased, while numbers of apoptotic cells increased. Subsequent characterization confirmed that the cultured PGCs maintained expression PGC-specific markers, telomerase activity, and normal migrational activity. Subsequently, we also investigated the molecular mechanisms supporting PGC proliferation and found that bFGF activates mitogen-activated protein kinase kinase/extracellular-signal regulated kinase (MEK/ERK) signaling, a major pathway for cell survival. Also, microarray analysis showed that the expression of a total 133 transcripts was altered by bFGF withdrawal, these changes being reversed by subsequent administration of bFGF. Our results show that bFGF induces PGC proliferation in vitro by activating MEK/ERK signaling and regulating downstream genes that may be important in inducing PGC proliferation. This research was supported by a grant from Easybio System Co. and from the World Class University (WCU) program (R31-10056) through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Proteomic Analysis of Rabbit Embryonic Stem Cells Derived from Fertilized and Parthenogenetically Activated Embryos.

Jyh-Cherng Ju 1, Payungsuk Intawicha 1, Shih-Han Wang 1, Ya-Jeng Shie 1, Neng-Wen Lo 2, San-Yuan Huang 1

Rabbit embryonic stem (rES) cells have been established from different sources of embryos, however, understanding regarding their gene and protein expressions is still limited. In this study, we aimed to determine the proteomics profiles of the fertilized embryo-derived and parthenote-derived ES cells, designated as f-rES and p-rES cells, respectively. Protein analyses using 2-DE and LC-MS/MS on the differentiated fibroblasts and undifferentiated f-rES and p-rES cells were performed. Collectively, the expression levels of one hundred and seventy-nine spots differed significantly among these three cell types (P<0.05). Of those differentially expressed spots, 66% were identified as known sequences to GenBank and 34% were unknown. Of the known proteins identified, 11% are the nuclear proteins, 3% membranous proteins and the rest of 86% reside in cytoplasmic compartment as cytoskeletal, mitochondrial, endoplasmic reticulum, and cytosolic proteins (14%, 7%, 11% and 41%, respectively). These proteins were also categorized based on their biological functions involved in energy and metabolic pathways (25%), cell growth and/or maintenance (24%), protein metabolism (11%), protein folding (10%), signal transduction (6%), regulations on nucleobase, nucleoside, nucleotide and nucleic acid metabolisms (4%), and other processes (20%). As few as 10 proteins were observed expressing in both f-rES and p-rES cells. Among them, 5 proteins (cytoplasmic ezrin, cofilin-1, α-4a, α-actin and nuclear lamin-B1) were found to express exclusively in f-rES, 2 (vimentin and β-actin) in fibroblast, and 12 in p-rES cells. Our results demonstrated a differential proteomic profiling and revealed some novel key proteins in specific rabbit cell types. This analysis provides insights into rES cell biology and would invite more in-depth studies toward rES cell applications.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Telomere Length Did Not Differ Between Blastocysts Produced by SCNT or IVF in Bovine, Ovine, and Mouse.

Pablo Bermejo-Alvarez 1, Chih-Jen Lin 2, Inchul Choi 3, X Cindy Tian 2, Keith Campbell 3, Alfonso Gutierrez-Adan 1

In somatic cell nuclear transfer, an overall epigenetic reprogramming must occur to abolish the somatic cell expression profile and to establish a new embryo-specific expression profile. The epigenetic reprogramming involves processes such as DNA methylation, histone modifications, X-chromosome inactivation and adjustment of telomere length. Telomere length is thought to be established during early preimplantation development, and thus, an incomplete telomere lengthening during this period may lead to long term effects, such as cellular premature ageing. Currently, there is no consensus about the possible effect of SCNT on telomere length. Discrepancies among studies may be caused by the analysis in fetal or adult tissues, as telomere length differs greatly among different adult cell types and within the same cell type in different stages of aging or maturation, as occurring for lymphocytes and epidermal cells. To avoid this skew, we analyzed by qPCR the telomere length at the blastocyst stage, when telomere length is thought to be established, in embryos produced by IVF or SCNT in three species: bovine, ovine and mouse (B6D2F1). SCNT was carried out with fetal fibroblasts in the bovine and ovine and with cumulus cells in the mouse. For each species, 10 blastocysts per group were snap frozen individually and stored at -80°C until analysis. The samples were digested with 8 µl of a 100 µg/ml proteinase K solution in water at 55°C overnight. After digestion, proteinase K was inactivated at 95°C for 10 min. Two µl of the digested sample were used in each qPCR, which was carried out with specially designed primer sequences to amplify the telomere region or with primers designed for 18S rRNA gene. To avoid dimmer interaction during the fluorescence acquisition in the qPCR, CXR fluorochrome (Promega) was used as double stranded DNA staining. Average telomere length was calculated according to the comparative CT method. The ΔCT value was determined by subtracting the CT value for the multicopy gene 18S, to correct the telomere copy number to relative telomere amount the total DNA present in the sample. Calculation of ΔΔCT involved using the highest sample ΔCT value (i.e. with the sorthest telomere) as a constant to subtract from all other ΔCT values. Fold-changes were determined using the equation 2-ΔΔCT. Statistical analysis was carried out by ANOVA. No significant differences were found between blastocysts produced by IVF or by SCNT within each specie (mean±SEM; Bovine: IVF:1±0.01, SCNT:1.12±0.14; Ovine: IVF:1±0.37, SCNT:1.13±0.52; Mouse IVF:1.23±0.24 SCNT:1±0.21). In conclusion, SCNT can efficiently restore the correct telomere length in bovine, ovine and mouse blastocysts.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

Mice Cloned from Induced Pluripotent Stem Cells.

Shaorong Gao, Zhaohui Kou, Lan Kang, Ye Yuan, Yu Tao, Jing He, Tong Wu, Yu Zhang 1

Differentiated somatic cells of various species could be reprogrammed into induced pluripotent stem cells (iPSCs) by ectopically expressing a combination of several transcription factors which are highly enriched in embryonic stem cells (ESCs). The generation of iPS cells in large animals has raised the possibility of producing genetically modified large animals through nuclear transplantation approach. However, it remains unknown whether iPSCs could be used for generating cloned animals through somatic cell nuclear transfer (SCNT). Here, we show the successful production of viable cloned mice from inducible iPSCs through SCNT approach, and the efficiency is similar to using ESCs derived via normal fertilization. Furthermore, the cloned mice are fertile and can produce second-generation offspring. These efforts strengthened the possibility of utilizing iPSCs to generate gene modified large animals for pharmaceutical purposes in the future.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):4.

CD9 Is a Surface Marker on Human Male Germline Stem Cells.

Khaled Zohni, Xiangfan Zhang, Seang Lin Tan, Peter Chan, Makoto C Nagano 1

Spermatogonial stem cells (SSCs) are responsible for the lifetime production of sperm, and are an important target cell for restoring male fertility after chemotherapy. In the mouse, we can immunologically enrich testis cells for SSCs, expand in culture, cryopreserve, and transplant them into the testis of chemically-induced infertile mice, resulting in fertility restoration. Although this scheme represents a promising clinical approach to male fertility preservation and restoration for cancer survivors, we have virtually no information about biological properties of human SSCs. As a first step to realize the above scheme for a clinical application of human SSCs, we evaluated the expression of molecules known as mouse SSCs markers in human spermatogonia. CD9 is a type IV glycoprotein involved in binding of cells to the extracellular matrix and was shown to be mouse SSCs marker. The intense signals were detected in the basal compartment of the human seminiferous tubules, which were also stained with a pan-spermatogonia marker, MAGE A4 (an oncofocalprotein). To test if CD9 can enrich for human spermatogonia, we used immunomagnetic cell sorting (MACS). Our results showed 5to 7 enrichment for human spermatogonia including SSC using CD9. To test if CD9 is expressed on human SSCs we used a functional transplantation assay. Our data show that CD9 is expressed on human SSCs and thus can be used as a marker for this rare population.

(poster)

Articles from Biology of Reproduction are provided here courtesy of Oxford University Press

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