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. 2010 Nov;83(1 Suppl):2.

7/31/2010

PMCID: PMC3399411
Biol Reprod. 2010 Nov;83(1 Suppl):2.

Anita Payne: Scientist, Teacher, Mentor, Advisor, Friend, and So Much More.

Phyllis M Wise 1

For many of us, our career success turns on whether we had a supportive mentor during our graduate or postdoctoral years. I was fortunate to have the very best. Anita not only taught me how to do research, but also was the quintessential lifelong advisor on all professional and personal issues. She kept up with all of us, tirelessly supported each of us and our families, and she took great pride in the success of her students. I look forward to sharing with you some of my experiences with Anita and how I benefitted immensely from having her by my side during my entire career.

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Biol Reprod. 2010 Nov;83(1 Suppl):2.

Anita Payne: The Inner Voice that Guides My Science.

Dale Buchanan Hales 1

The postdoctoral studies I conducted under Anita Payne's guidance set the stage for my career, and her pragmatic, conscientious, and thoughtful advice still guides my science today. I had the great fortune of working in Anita's lab soon after the primary Leydig cell culture system had been developed, which enabled me to pursue my interest in understanding the mechanisms that regulate the steroidogenic P450 enzymes. When I started my own laboratory at the UIC, I modeled the way I ran my lab after Anita's, and, with her blessing, continued to work with Leydig cell steroidogenesis. My talk will summarize the high points of this body of work and will describe the continuing importance of Anita's influence on my scientific pursuits.

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Biol Reprod. 2010 Nov;83(1 Suppl):2.

Nuclear and Cytoplasmic Maturation of the Mammalian Oocyte: New Insight from a Genome Wide Analysis of Maternal RNA Translation.

Marco Conti 1

Although working in a field not directly related to mine, Anita has had a preeminent role in shaping my professional and personal life. A model for good citizenship and tireless supporter of junior scientists, her positive attitude and her unquenchable enthusiasm have left a mark on the most important decisions for my career and my family. The research that I am going to present started more than four years ago when Anita was still with us and follows her example of not shying away from new intellectual pursuits. These data are dedicated to her.

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Biol Reprod. 2010 Nov;83(1 Suppl):2.

Cistromes Approach to Hormones Action.

Myles Brown 1

Abstract not available.

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Benefits of Germinal Vesicle Transfer by Intracytoplasmic Injection Versus Membrane Fusion on Homoplasmy and Maturational Success of Reconstructed Cat Oocytes.

Pierre Comizzoli, Budhan S Pukazhenthi, David E Wildt 1

Cytoplasmic defects in immature oocytes can be circumvented by transferring the germinal vesicle (GV) into a previously enucleated, good quality oocyte. However, the karyoplast containing the large donor GV must be fused with the recipient's cytoplast membrane, which results in a reconstructed oocyte with problematic heteroplasmy and impaired capacity to achieve nuclear maturation. Using the ability to artificially compact the GV in the cat model, the objective was to assess the impact of GV transfer by fusion of a smaller donor GV-karyoplast or by intracytoplasmic GV injection on (1) maintenance of homoplasmy and (2) nuclear maturation in the reconstructed oocyte. In Experiment 1 (3 replicates), immature cat oocytes were prepared for micromanipulation (cumulus removal, centrifugation and Cytochalasin B exposure). In each replicate, donor oocyte mitochondria were labeled (400 nM fluorescent Mitotracker Green; 15 min) before transferring the GV into a recipient cytoplast by: 1) membrane fusion (induced by Sendai virus); 2) membrane fusion after GV compaction with 1 mM resveratrol (1 h); or 3) intra-cytoplasmic injection (using a 15 μm diameter Piezo-actuated pipette) after GV compaction, aspiration in the pipette and extensive washing (n = 12 transfers/treatment). Diameters of donor GVs and resulting GV-karyoplasts were measured during each transfer. After membrane fusion or injection, reconstructed oocytes were fixed to assess the presence of labeled mitochondria. Diameters of donor GVs or GV-karyoplasts were smaller (P<0.05; ANOVA) after compaction (17.1 ± 2.0 μm and 21.6 ± 2.4 μm, respectively; mean ± SD) compared to without (41.5 ± 3.0 μm and 48.4 ± 5.4 μm, respectively). No fluorescence was detected after GV injection, whereas oocytes reconstructed by fusion always contained labeled mitochondria indicative of heteroplasmy regardless of volume of the donor GV-karyoplast. In Experiment 2 (4 replicates), oocytes were prepared for micromanipulation as above. Each replicate included GV transfers by fusion, fusion after compaction or injection after compaction (n = 20 transfers/method) as well as control oocytes that were either left intact, GV compacted, sham injected or GV compacted plus sham injected (n = 20/control group). After in vitro maturation (28 h), all oocytes were fixed to evaluate the efficiency of the GV transfer method as well as nuclear stage and metaphase II spindle integrity. GV injection resulted in more successful reconstructions (95.7 ± 3.1%; P<0.05) compared to fusions with large (74.4 ± 5.3%) or smaller (73.2 ± 3.3%) donor GV-karyoplasts. Proportions of reconstructed oocytes at the metaphase II stage were higher (P<0.05) after GV injection (71.7 ± 3.4%) than after fusion with large (59.6 ± 4.5%) or smaller (57.1 ± 6.0%) donor GV-karyoplasts, but were lower (P<0.05) than in all controls (range, 77.8 to 82.0%). Regardless of treatment (including controls), percentages of normal metaphase II spindles were similar (range, 83 to 87%; P>0.05). Results demonstrated that the cat GV retains meiotic competence after compaction and injection into a recipient cytoplast. Compared to membrane fusion, even with a reduced-volume GV-karyoplast, intracytoplasmic GV injection prevents heteroplasmy and allows a higher incidence of oocyte reconstruction and nuclear maturation. This approach offers new opportunities for addressing infertility and preserving the maternal genome.

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Biol Reprod. 2010 Nov;83(1 Suppl):2.

Regulation of Organelle Redistribution by PADI6 and the Cytoplasmic Lattices During Mouse Oocyte Maturation.

Rui Kan, Piraye Yurttas, Scott Alexander Coonrod 1

During oocyte maturation, endoplasmic reticulum (ER) and mitochondria undergo a dramatic re-localization that is important for calcium oscillations, calcium homeostasis, egg activation at fertilization, and oocyte and embryo competence. It has been suggested that the reorganization of ER is a multiple-step process controlled by the cytoskeleton and cytoplasmic dynein in mouse oocytes. The mechanism, however, is not well established. We have previously shown in the mouse that oocyte-restricted peptidylarginine deiminase 6 (PADI6) is essential for formation of the cytoplasmic lattices (CPLs) during oogenesis and for development beyond the two-cell stage. We have also found that the PADI6/CPLs complex contains a subset of insoluble tubulin and is involved in oocyte microtubule (MT) dynamics. Here we show that the PADI6/CPL superstructure is important for the MT-mediated accumulation of ER and mitochondria around the developing meiotic spindle at the time of germinal vesicle break down (GVBD) and for the migration towards metaphase II spindle and cortex during oocyte maturation. In PADI6-null oocytes, which lack CPLs, the ER and mitochondria migrate to the sub-cortical region and are redistributed in a non-polarized fashion throughout the cytoplasm from metaphase I (MI) to metaphase II (MII). Further, inhibition of dynein and kinesin, MT motor proteins driving organelle transport on trafficking, has no obvious effect on the organelle localization in PADI6-null oocytes. Therefore, a functional PADI6/CPL complex appears mandatory for normal organelle repositioning. This knowledge will aid in the identification of competent human oocytes and the optimization of the assisted reproductive technology. Research supported by NICHD grant RO1 38353 to SAC.

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Biol Reprod. 2010 Nov;83(1 Suppl):2.

Regulation of the Onset of Meiotic Maturation of Zebrafish Oocytes by Endogenous Estrogens Through the G Protein-Coupled Estrogen Receptor, GPER.

Peter Thomas, Yefei Pang 1

Estrogen inhibition of oocyte meiotic maturation (OM) and the role of GPER (formerly known as GPR30) were investigated in zebrafish. Estradiol-17beta (E2) and G-1, a GPER-selective agonist, inhibited OM and bound to zebrafish oocyte membranes suggesting the presence of GPER which was confirmed by immunocytochemistry using a specific GPER antibody. Incubation of follicle-enclosed oocytes with an aromatase inhibitor, ATD, and enzymatic and manual removal of the ovarian follicle cell layers significantly increased spontaneous OM which was partially reversed by co-treatment with either 100 nM E2 or G-1. Incubation of denuded oocytes with the GPER antibody blocked the inhibitory effects of E2 and G-1 on OM, whereas microinjection of estrogen receptor alpha (ERalpha) antisense oligonucleotides into the oocytes was ineffective. Moreover, OM was not inhibited with ERalpha- and ERbeta-selective agonists, which suggests this estrogen action is ER-independent. A cell-impermeable estrogen conjugated to a peptide mimicked the inhibitory effects of E2 on OM, indicating a cell-surface site of estrogen action. The results suggest that endogenous estrogens produced by the follicle cells inhibit or delay spontaneous maturation of zebrafish oocytes and that this estrogen action is mediated through GPER. Treatment with E2 and G-1 also attenuated the stimulatory effect of the teleost maturation-inducing steroid, 17,20beta-dihyroxy-4-pregnen-3-one (DHP), on OM. Moreover, E2 down-regulated the expression of membrane progestin receptor alpha (mPRalpha), the intermediary in DHP induction of OM. Conversely DHP treatment caused a > 50% decline in GPER mRNA levels. The results suggest that estrogens and GPER are critical components of the endocrine system controlling the onset of OM in zebrafish. A model is proposed for the dual control of the onset of oocyte maturation in teleosts by estrogens and progestins acting through GPER and mPRalpha, respectively, at different stages of oocyte development.

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Biol Reprod. 2010 Nov;83(1 Suppl):2.

Weakened Centromere Cohesion Is the Primary Cause of Age-Related Aneuploidy in Oocytes.

Teresa Chiang, Francesca E Duncan, Karen Schindler, Richard M Schultz, Michael A Lampson 1

Aneuploidy arising early in development is the leading genetic cause of birth defects and developmental disabilities in humans. It is now well established that the probability of a trisomic pregnancy (i.e., an embryo with an extra copy of a chromosome) increases dramatically with age: from only 2% for a woman in her 20s to about 35% by age 40. Most trisomies and all monosomies are inviable, and the few that are viable result in severe developmental disabilities (e.g., Down syndrome). Aneuploidies in the embryo usually originate from the egg instead of sperm, and more specifically, from chromosome segregation errors in Meiosis I of oocyte maturation. The frequency of these errors increases with maternal age, which is likely related to the long meiotic arrest that occurs during female meiosis in mammals. Primary oocytes enter meiosis during fetal development and remain arrested until ovulation in the adult, which in humans can be as long as 40 years. Although the phenomenon of age-related aneuploidy is well known, the molecular basis for the chromosome segregation errors in female meiosis is unclear. Here we show that weakened chromosome cohesion is the primary cause of aneuploidy in oocytes in a natural aging mouse model. In oocytes from old mice, we find that sister kinetochores are farther apart at both Metaphase I and II, indicating reduced centromere cohesion. Moreover, levels of the meiotic cohesin protein REC8 are severely reduced on chromosomes in these oocytes. In mice at different ages, REC8 levels gradually decrease while inter-kinetochore distances increase with age (from 3 to 15 month-old mice); yet despite this gradual loss of cohesion, chromosome segregation errors remain low until 12 months and then increase sharply between 12 and 15 months. These results suggest that cohesins are initially loaded in large excess, and errors arise only after cohesin levels drop below ~10% of the starting amount. To test whether cohesion defects lead to the observed aneuploidies, we monitored chromosome segregation dynamics at Anaphase I in live oocytes and assessed ploidy in the resulting Metaphase II eggs. We found that about 90% of age-related aneuploidies are best explained by weakened centromere cohesion. Together, these results demonstrate that the maternal age-associated increase in aneuploidy is primarily due to a failure to effectively replace cohesin proteins that are lost from chromosomes during aging. Because of the prolonged time interval in female meiosis, this may create a selective pressure to load an excess of cohesins in pre-meiosis S-phase to protect oocytes against impending age-related meiotic chromosome segregation errors, and thus enhance a mother's reproductive fitness by extending her period of fertility.

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Biol Reprod. 2010 Nov;83(1 Suppl):2.

Aneuploid Human Oocytes and Embryos Possess Significantly Reduced Quantities of Telomere DNA.

Nathan R Treff, Jing Su, Deanne Taylor, Richard T Scott 1

Aneuploidy represents the most prevalent form of genetic instability found in human embryos and is the leading cause of miscarriage and developmental delay in newborns. Although maternal age is a well known risk factor, the underlying molecular etiology of human aneuploidy is unknown. Since telomere DNA content is associated with genomic instability and is naturally reset during mammalian preimplantation embryonic development, telomere DNA quantity may play a role in the development of aneuploidy commonly found in human embryos. To begin to characterize the potential association of telomere biology with development of human aneuploidy, we have characterized relative telomere DNA quantity at various stages of human preimplantation embryogenesis. First, we developed a method capable of accurate quantitation of telomere DNA from single cells. Whole genome amplification (WGA) of picogram amounts of DNA from cells with previously established telomere DNA quantities provided results equivalent to those obtained using large quantities of purified total DNA (Pearson correlation coefficient=0.97). This technology was then applied to human oocytes and embryos. Excess WGA DNA was available for telomere DNA analysis of oocyte and embryo biopsies as a result of previously performing SNP microarray based 24 chromosome aneuploidy screening on human in vitro fertilization derived samples. Paired analysis of aneuploid and euploid polar bodies and embryonic cells from within the same patients' oocytes and embryos and within the same treatment cycle provided the greatest control over critical variables including maternal age. Embryo fragmentation rates, which were previously shown to correlate with telomere DNA length, weren't significantly different among aneuploid (9.2%) and euploid (9.7%) samples (P=0.75). Results demonstrated, for the first time, that aneuploid human polar bodies possess significantly less telomere DNA than euploid polar bodies (-3.07 fold, P=0.016). Since the first and second polar bodies are extruded upon completion of meiosis I and II, respectively, and reflect the chromosomal complement of the remaining oocyte, abnormalities observed in polar bodies are indicative of development of aneuploidy during meiosis of the oocyte. Therefore, the present results indicate that oocytes with reduced telomere DNA quantity are prone to development of aneuploidy during meiosis. We also show that aneuploid embryonic cells possess significantly less telomere DNA than euploid embryonic cells at the cleavage stage (-2.60 fold, P=0.002) but not at the blastocyst stage (-1.18 fold, P=0.340). These results indicate that reduced telomere length is associated with aneuploidy prior to embryonic genome activation between the cleavage and blastocyst stages of development. This further suggests that aneuploidy is not associated with failed resetting of telomere DNA length but is instead associated with failed telomere maintenance in the maternal genome during oogenesis and early embryo development.

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Biol Reprod. 2010 Nov;83(1 Suppl):2.

NOT PRESENTED.

NOT PRESENTED.

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Cofilin Localization in the Epithelium of Human Endometrium Changes During the Menstrual Cycle and in Endometriosis.

Kirsten Morris, Ivanna Ihnatovych, Emily Ionetz, Zuzana Strakova 1

The human uterine epithelium displays a distinct polarized organization within apical, lateral, and basal plasma membrane domains. Regulation of the actin cytoskeleton plays a role in establishment of epithelial cell polarity and protein trafficking. Actin dynamics, referring to the dynamics between actin polymerization and depolymerization, are regulated by actin-binding proteins such as cofilin. Cofilin binds to both monomeric (G) and filamentous (F) actin; its major known function is depolymerization of F-actin. This allows branching of actin filaments and cytoskeleton reorganization. In its inactive form, cofilin binds to monomeric G-actin. We have previously shown that cofilin participates in actin dynamics regulation during decidualization of human stromal fibroblasts. To investigate cofilin in human endometrium during the menstrual cycle, we localized cofilin in paraffin embedded fixed sections through immunohistochemical staining. During the proliferative phase of the menstrual cycle, cofilin is localized both in the apical and basolateral sides of luminal epithelial cells. Dual staining of cofilin and monomeric G-actin (represented by staining for DNAse I) indicated cofilin-G-actin colocalization in both locations. Interestingly, during the secretory phase of the menstrual cycle this colocalization was only present on the basolateral side of luminal epithelium cells; cofilin was not detected on the apical part. This suggests that inactive G-actin-bound cofilin translocates from the apical to the basolateral side of luminal epithelium. These findings imply that the different signals coming to the endometrium during preparation for blastocyst implantation in the secretory phase of the menstrual cycle have an effect on cofilin location thus regulating its participation in actin/cytoskeleton rearrangement in epithelial cells. Endometriosis is a chronic disease causing pain and infertility in more than 10% of reproductive age women. To investigate whether there is a different pattern of actin remodeling in endometriosis, we investigated cofilin localization in a baboon (Papio anubis) endometriosis model. The staining pattern of cofilin in baboon uterine endometrium of healthy animals confirmed our previous observations in human epithelial tissue. Proteins involved in regulation of cofilin activity (LIM kinase, slingshot, and binding protein 14-3-3) are localized along with cofilin. A very different staining pattern was detected in baboons with severe spontaneous endometriosis. In the secretory phase of the menstrual cycle, this tissue had a very similar pattern to the proliferative phase in a normal animal; distinct cofilin staining was observed in the apical parts of the luminal and glandular epithelium. This indicates that this severe dyssynchrony in menstrual cycle phases in an animal with endometriosis is connected with improper cytoskeleton rearrangements. This suggests that luminal epithelial cells of the endometrium are not properly prepared for blastocyst arrival, since these cells allow attachment of trophoblast cells to their apical pole during embryo implantation. In summary, we suggest that cofilin-mediated actin reorganization of uterine epithelial cells during the secretory phase might be important in preparation for implantation; dysregulation of this reorganization may lead to decreased fertility in women suffering from endometriosis (ARRA NIH HD044713 grant to ZS).

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Biol Reprod. 2010 Nov;83(1 Suppl):2.

Endogenous EMMPRIN Expression by Human Uterine Fibroblast Cells Regulates Metalloproteinase Production, Proliferation, and Decidualization.

Andrea Braundmeier, JiaJia Bi, Pavni Mehrotra, Romana Nowak 1

The uterine endometrium undergoes extensive proliferation and remodeling in preparation for embryo implantation. This tissue remodeling occurs, in part, through upregulation of matrix metalloproteinases (MMPs) in uterine stromal fibroblasts. Extracellular matrix metalloproteinase inducer (EMMPRIN) stimulates MMP production in a number of carcinoma cell lines and also in human uterine fibroblast (HUF) cells. We have shown in previous studies that human uterine epithelial cells (HES) express the cell surface form of EMMPRIN and secrete full length EMMPRIN via microvesicle shedding. This soluble form of EMMPRIN acts in a paracrine manner on underlying stromal fibroblast cells (HUF) to stimulate MMPs. The goals of the present study were to determine 1) whether soluble EMMPRIN is the major secretory product of HES cells that stimulates MMP production by HUF cells; 2) whether expression of EMMPRIN by HUF cells is necessary for these cells to be able to respond to HES secreted EMMPRIN; and 3) whether EMMPRIN expression in HUF cells is important for cell proliferation and decidualization. We harvested conditioned medium from HES cells and confirmed that this contains the full length form of EMMPRIN via immunoblotting. Treatment of HUF cells with either unconcentrated or 50-fold concentrated HES-cell conditioned medium (p<0.05) increased MMP-1, -2, and -3 mRNA levels without altering endogenous EMMPRIN mRNA levels. Removal of the soluble form of EMMPRIN from HES-cell conditioned medium by immunodepletion using a specific anti-human EMMPRIN antibody caused a reduction in MMP stimulation (7.7 fold for MMP-1, 4.2 fold for MMP-2 and 12.4 fold for MMP-3 (p<0.05)). Our results also showed that endogenous expression of EMMPRIN by HUF cells was important for MMP stimulation by HES-cell conditioned medium. Transfection of HUF cells with EMMPRIN siRNAs resulted in a dramatic reduction in the stimulatory effect of HES-cell conditioned medium on MMP mRNA levels (57 fold MMP-1, 69 fold MMP-2) but a marked increase in MMP-3 mRNA levels (36.7 fold, p<0.05). Treatment of uterine fibroblasts with EMMPRIN siRNAs also decreased the proliferative response of these cells to serum by 40% as measured by DNA synthesis assays. Effects of EMMPRIN siRNA on decidualization of stromal cells was determined by treating cells for 10 days with estradiol, progesterone and cAMP in the presence or absence of EMMPRIN siRNAs. Immunoblotting showed that EMMPRIN protein levels were significantly reduced by day 4 of treatment and nondetectable on days 6-10. Levels of prolactin mRNA, a marker of decidualization, increased 150-200 fold in control cells by days 6-8 but were increased less than 50 fold in EMMPRIN siRNA transfected cells. MMP-7 mRNA levels were increased 20-30 fold in control cells but only 5-7 fold in EMMPRIN siRNA transfected cells. These data indicate that EMMPRIN expression by uterine fibroblast cells is important not only for regulation of MMP production but also appears to play a role in regulating proliferation and decidualization of these cells. Future studies will focus on the role of EMMPRIN in cell cycle regulation in uterine stromal cells. (Supported by SCCPRR U54HD20093).

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Biol Reprod. 2010 Nov;83(1 Suppl):2.

Regulatory Factors in Secretory Phase Uterine Washings Are Derived from Endometrial Epithelial Cells and Can Be Selectively Altered by hCG, Providing a Means to Enhance Receptivity.

Natalie J Hannan 1, Premila Paiva 2, Cassandra Hincks 1, Katie Meehan 1, Enid Pruysers 1, Evdokia Dimitriadis 1, Lois A Salamonsen 1

Successful blastocyst implantation requires a synchronized dialogue between a receptive endometrium and a functional blastocyst. This is mediated in part by endometrial cytokines/growth factors secreted by glandular epithelium into the uterine cavity, which may contribute to endometrial receptivity and also support the blastocyst during implantation. Recent evidence suggests that blastocyst-derived hCG may further influence the endometrial milieu in conception cycles to enhance receptivity. The inability to achieve endometrial receptivity results in infertility in women and contributes to the failure of reproductive technologies. However, there is currently no robust test for endometrial receptivity, which can inform clinical decisions. This study aimed to investigate the profiles of a selected cohort of 42 cytokines/growth factors in human uterine fluid and secreted by primary human endometrial epithelial cells (hEECs). These factors included those with known and unknown roles in receptivity and implantation. The association between these factors and endometrial receptivity and infertility and the effect of hCG on their production by primary hEEC was also investigated. Uterine fluid (collected by flushing the uterine cavity with 5 ml saline) was obtained from fertile women during the receptive (mid-secretory phase; n=7) and non-receptive (mid-proliferative n=7) phases of the cycle, and from women with unexplained infertility during the receptive phase (n=9). hEECs isolated from endometrial biopsies collected from fertile cycling women (n=15), were treated under serum-free conditions ± recombinant hCG (0.2-20 IU/ml) for 48 hr. Uterine fluid and conditioned media from primary hEEC were analyzed using quantitative Milliplex Luminex cytokine assays. Both uterine fluid and primary hEEC conditioned media exhibited a remarkably similar cytokine profile. Over 30 cytokines/growth factors were identified, quantified and grouped according to their concentration ranges (from<1 to >1000 pg/ml). 8 factors including PDGFA, TNFB, sIL2RA, FLT3L, sCD40L, IL7 and IFNA2 were previously unknown in the endometrium. Comparison of uterine fluid from fertile non-receptive / receptive phase and infertile receptive phase cohorts revealed VEGF as significantly reduced (84%) in infertile compared to fertile cohorts (p<0.05). hCG (2 IU/ml) significantly increased the secretion of 6 factors including those with roles in receptivity and trophoblast function (IL11; 32±12%), blastocyst/trophoblast migration and adhesion (CXCL10; 31±11%), blastocyst development (GMCSF; 45±12%) and one with previously unknown roles in receptivity and implantation (FGF2; 52±23%)(p<0.05). Upregulation of known hCG-regulated proteins, VEGF and LIF, validated the study. Functional studies revealed that FGF2 (100ng/ml) stimulated phosphorylation of ERK1/2 in hEEC and both FGF2 and VEGF (100 ng/ml) stimulated hEEC adhesion to fibronectin (362±21%; 409±10%) and collagen type IV (391±64%; 513±15%) characteristic of blastocyst/trophectoderm-ECM (p<0.001; n=3). These findings indicate that soluble mediators in uterine secretions are derived from epithelial cells and support roles for hCG, VEGF and FGF2 in the molecular cross-talk important for endometrial receptivity and implantation. Identifying soluble mediators in uterine secretions may provide potential biomarkers of endometrial receptivity and insight into the unique microenvironment essential for the establishment of pregnancy.

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Biol Reprod. 2010 Nov;83(1 Suppl):2.

ICAM-1 Is Involved in Uterine Receptivity in Rats.

Laura Lecce, Margot Day, Christopher R Murphy 1

The uterine epithelium undergoes extensive molecular changes during early pregnancy to become receptive to an implanting embryo, and this is tightly regulated by ovarian hormones. Many adhesion molecules are known to reorganize during early pregnancy in order to facilitate embryo adhesion to the apical membrane of uterine epithelial cells and play an important role in uterine receptivity. This study examined the changes in distribution, expression and hormonal control of ICAM-1 in the rat uterus during early pregnancy, in ovariectomised rats, pseudo pregnant rats and cultured preimplantation embryos. The use of ICAM-1 specific vivo-morpholino antisense oligonucleotides was employed to discover the role of ICAM-1 in embryo development and implantation. ICAM-1 was visualized in the rat uterus using indirect immunofluorescence microscopy and was absent in the uterine epithelium on day 1 (pre-receptive) and day 3 of pregnancy. By day 6 (receptive) and day 7 of pregnancy at the time of implantation in the rat, ICAM-1 is present in the uterine epithelium and is localized to the apical region. ICAM-1 then disappears in uterine epithelial cells by day 9 (post receptive) of pregnancy. ICAM-1 thus is likely an important molecule in the adhesion of an implanting embryo to the apical membrane of uterine epithelial cells in rats. ICAM-1 is absent in the uterine epithelium of ovariectomised rats treated with oestrogen alone (non-receptive) and apical in the uterine epithelium of rats treated with progesterone alone, or combined with estrogen (receptive); and was apical in day 6 pseudo pregnant animals. Apical ICAM-1 staining in the uterine epithelium, on day 6 of pregnancy and in ovariectomised rats treated with progesterone and in pseudo pregnant rats always coincides with an increase in ICAM-1 monomeric expression on western blots. Dimeric expression of ICAM-1 did not alter from day 1 to day 6 of pregnancy and corresponds to the ICAM-1 protein found in endothelium. ICAM-1 was however present in endothelial cells of blood vessels found in the endometrial stroma at all stages of pregnancy. This indicates that ICAM-1 in uterine epithelial cells is under progesterone control and that endothelial ICAM-1 is not at all influenced by ovarian hormones. Previous studies have suggested that ICAM-1 might be up regulated by cytokines released by implanting embryos; however the use of pseudo pregnant and ovariectomised rats has shown that this is not the case. ICAM-1 is also present in pre-implantation embryos and may be necessary in facilitating embryo adhesion. Moreover, early day 3 embryos exposed to ICAM-1 vivo-morpholino antisense oligonucleotides fail to develop into blastocysts. In summary ICAM-1 is likely an important molecule in the receptivity of uterine epithelial cells and is under progesterone control. ICAM-1 is also a necessary molecule in embryo development.

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Biol Reprod. 2010 Nov;83(1 Suppl):2.

Steroid Receptor Coregulator-2 Is Required for Uterine Stromal Decidualization in Both Mouse and Human.

John P Lydon, Ramakrishna Kommagani, Jaewook Jeong, Atish Mukherjee, Selma Soyal, Steve Wu, Sophia Tsai, Ming Tsai, Francesco J DeMayo 1

Steroid receptor coregulator-2 (SRC-2) is a member of the SRC family of coregulators, which includes SRC-1 and SRC-3. Sharing strong sequence homology, all three coregulators have been shown to selectively modulate the transactivational potency of nuclear receptor family members, including estrogen receptor-α and the progesterone receptor. Knockout studies in mice have demonstrated that these coregulators exert diverse regulatory effects in vivo, ranging from determining mammary tumor susceptibility, modulating inflammation, to controlling metabolic homeostasis. Using innovative genetic approaches which either ablate or increase gene expression specifically in murine cell-lineages that express the progesterone receptor, we reveal SRC-2 as an indispensable coregulator for progesterone-dependent uterine decidualization. Although uterine luminal cells devoid of SRC-2 expression enable embryo apposition and attachment to their apical surface, these cells are incapable of facilitating further progression of the implantation program that ultimately leads to local decidualization of the underlying stroma. This specific block in uterine decidualization is reflected at the molecular level by a selective attenuation in the induction of progesterone target genes that are critical for the uterine decidual response as disclosed by microarray methods. Importantly, this uterine decidual response defect is not compensated by other coregulators, underscoring the unique coregulator importance of SRC-2 during this early stage of implantation biology. Moreover, clinical studies have shown that marked elevations in SRC-2 expression levels in the endometrium are associated with a number of uterine pathologies that lead to infertility, suggesting that perturbation of SRC-2 expression levels may be causal for abnormal uterine function. To address this proposal, we developed an innovative cre-loxP engineering strategy in the mouse to significantly increase SRC-2 expression levels selectively in uterine cells that express the progesterone receptor. Overexpression of uterine SRC-2 resulted not only in a compromised fertility state but also resulted in persistent hyperproliferation of the luminal and glandular epithelia of the endometrium. These findings support the supposition that SRC-2 levels require tight control to ensure normal progesterone responsiveness in the uterus and that deregulation of this coregulator is causal for unscheduled uterine epithelial proliferation which can undermine the normal functionality of this tissue. Finally, our observation that the same uterine cell-types in human and mouse express SRC-2 and that knockdown of SRC-2 expression in primary and immortalized human endometrial stromal cells blocks the ability of these cells to decidualize in vitro, lends strong translational support for a pivotal coregulator role for SRC-2 in the physiology and pathophysiology of the human endometrium. This research was funded by National Institutes of Health grants (U54 HD-07495) and (CA-077530).

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Biol Reprod. 2010 Nov;83(1 Suppl):2.

Demonstrating the Critical Role of Uterine Erbb Signaling in Fertility.

Michael J Large 1, Sean M Hartig 1, Heather L Franco 1, Ertug Kovanci 1, Michael A Mancini 1, David W Threadgill 2, John P Lydon 1, Jae-Wook Jeong 1, Francesco J DeMayo 1

The establishment of the appropriate embryo-uterine interactions required for successful implantation and pregnancy require the coordinate actions of growth factors and their receptors. The spatiotemporal expression of Erbb ligands and receptors in the periimplantation uterus and embryo suggests that they play a role in reproduction. Numerous Erbb ligands have overlapping expression patterns in the uterus during pregnancy, and gene ablations of individual Erbb ligands do not show a major uterine phenotype due to compensation by other family members. We have identified EGFR (ERBB1) as being induced during the implantation period and hypothesize that EGFR signaling regulates the ability of the endometrium to support embryo implantation. This hypothesis is being tested in vivo using mouse models and in vitro using primary human endometrial stromal (HES) cell culture and a novel high-throughput microscopy assay. In order to circumvent the embryonic lethality of Egfr-/- mice, conditional ablation of Egfr was achieved by crossing an Egfr floxed (Egfr f/f) mouse with a progesterone receptor cre (PRcre) mouse, resulting in Egfr deletion in all PR positive cells (PRcre/+ Egfr f/f; Egfr d/d). Egfr d/d mice are severely subfertile and exhibit reduced implantation and abnormal embryos. Mutant mice demonstrate an ablation of artificial decidualization including defects in morphology, proliferation, apoptosis, differentiation and gene expression. EGFR is induced during in vitro HES cell decidualization and that the use of EGFR knockdown impedes decidualization. Furthermore, these results have been confirmed using a novel, automated image-based microscopy assay. These results strongly indicate that Erbb signaling plays a critical role in female reproduction. Continuation of ongoing studies determining the mechanism of EGFR action and the role of Erbb2 will provide valuable insight and allow further elucidation of complex pathways and the orchestrated molecular interactions vital to reproductive health. This research was supported by NIH Grant RO1CA077530 (to J.P.L.), by NIH R01HD057873 (to JW.J), and by the Eunice Kennedy Shriver NICHD/NIH through cooperative agreement U54 HD07495 as part of the Specialized Cooperative Centers Program in Reproduction and Infertility Research.

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Biol Reprod. 2010 Nov;83(1 Suppl):2.

An ENU Mutagenesis Screen Reveals Known and Novel Genes That Cause Hypogonadism.

Rebecca Harris, Jeffrey Weiss, Lisa Hurley, Tim Barrett, Lisa Fisher, Donna Emge, Courtney Finlayson, J Larry Jameson 1

A genome-wide mutagenesis screen employing ENU in mice identified five mutant lines with male urogenital defects. Whole genome analysis using a highly parallel SNP assay mapped each mutation to a 20-90 Mb region. Candidate genes in these regions were identified and sequenced, allowing identification of mutations responsible for hypogonadism (Prdm9), cryptorchidism (Rxfp2) and germ cell loss (Plk4) phenotypes. Two of the mouse lines for which the causative mutations were identified (Rxfp2 and Prdm9) have known roles in male reproduction. Prdm9, a histone methyltransferase, is involved in the progression of meiotic prophase through trimethylation of lysine 4 of histone H3. Recently, PRDM9 was implicated as a key player in meiotic recombination hotspots and speciation. Rxfp2/RXFP2, a GPCR, is one of two known genes that cause cryptorchidism in both mice and humans. The mutation in mouse RXFP2 resulted in an amino acid alteration from aspartic acid to glycine at residue 294 (D294G). The D294G mutation impaired testicular descent and resulted in decreased testis weight in homozygous mice as compared to heterozygous and wild-type mice. Testicular histology of the homozygous mice revealed spermatogenic defects ranging from germ cell loss to tubules with Sertoli-cell only features. Genetic complementation analysis using a Rxfp2 loss of function allele confirmed causality of the D294G mutation. The functional implication of the D294G mutation was analyzed by measuring total and cell-surface expression of mouse RXFP2 protein in transfected cells. Total expression of the mutant (D294G) protein was only slightly reduced compared to wild-type, but cell-surface expression was markedly decreased. The D294G mutation underscores the role of the RXFP2 mediated pathway in testicular descent and expands the repertoire of mutations that affect receptor trafficking and function. Another identified gene, Plk4, encodes a serine/threonine kinase, which is highly expressed in the testis. PLK4 is involved in centriole formation and cell cycle progression, however the specific role of PLK4 in spermatogenesis is unknown. The mutation in PLK4 resulted in an amino acid alteration from isoleucine to asparagine at residue 224 (I242N). Homozygous mice are embryonic lethal, but heterozygous mice are hypogonadal and have germ cell loss. This loss is not due to a secondary defect in the hypothalamic-pituitary axis, as LH, FSH and testosterone are mainly unaffected. The germ cell loss appears to result from increased apoptosis. Genetic complementation analysis using a gene-trap allele confirmed causality of the I242N mutation. Further investigation into the underlying etiology of the defect, the mechanism of PLK4 action in the testis and potential downstream effector molecules is ongoing. Fine mapping of the two remaining hypogonadal lines was completed using a custom SNP array, reducing the regions of interest to 2 Mb. High-throughput sequencing of the regions is ongoing and it is anticipated that the mutated genes in the two remaining lines will provide novel insights, as the mapped intervals do not contain genes with known roles in the testis. Funding: U01 HD043425-01

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Biol Reprod. 2010 Nov;83(1 Suppl):2.

Hormonal Regulation of Testicular Descent.

Zaohua Huang 1, Elena Kaftanovskaya 1, Shu Feng 2, Rhea Pereira 1, Sukhjinder Kaur 1, Agusin Barbara 1, Anne Truong 2, Ivan Gorlov 3, Alexander Agoulnik 1

Cryptorchidism or testicular maldescent is the most common congenital birth defect in newborn boys. During male development testis moves from high intraabdominal position into the scrotum. Gubernaculum, a ligament connecting testis via epididymis with scrotal area of abdomen is believed to play a critical role in this process. Gonadal descent and gubernacular differentiation is controlled by two hormones produced in testicular Leydig cells, INSL3 and testosterone. INSL3, insulin-like3 peptide, signals through its G protein-coupled receptor RXFP2. Deletion of Insl3 or Rxfp2 in mice causes high intrabdominal cryptorchidism, indicating that this hormone governs first transabdominal stage of testis descent, whereas abnormalities of HPG axis or androgen signaling affect the second ingunascrotal stage. Both RXFP2 and androgen receptor (AR) are expressed in gubernaculum, however no local signaling pathways mediating the response to these hormones in gubernacular cells are known. It is also not clear what are their biological effects on different cellular components of gubernaculum and the interaction if any of these hormones during gubernacular differentiation. To address these questions we produced and analyzed various mutant mice with genetic alterations affecting INSL3 and androgen signaling. We have created shRNA transgenic mice targeting RXFP2 expression. The expression of the transgene is regulated by cre/loxP mechanism allowing modulation of RXFP2 activity in specific organs and at different developmental stages. We have shown that the transgene is able to knock-down gene expression and thus behaves as a hypomorphic allele of Rxfp2. Variable degree of uni-and bilateral cryptorchidism was detected in males with activated shRNA transgene on Rxfp2+/- background. Testis maldescent was also detected in mice with RXFP2 gene knock-down specifically in gubernaculum, demonstrating its importance in testis descent. We next performed gene expression analysis of mutant cremaster sac using Illumina microarrays. The expression of significant number of genes in Wnt/beta-catenin, Notch, AR signaling pathways in Rxfp2-deficient gubernacula was altered, suggesting their regulation by INSL3. We have demonstrated profound changes in expression pattern of beta-catenin, desmin, AR, Notch1, and other target genes in Rxfp2-/- male embryos, indicating INSL3 role in proliferation, differentiation, and survival of specific cellular components of gubernaculum. We have shown that INSL3/RXFP2 signaling is essential for myogenic differentiation and maintenance of AR-positive cells in gubernaculum. We then analyzed gubernacular development and testis descent in mice with conditional gubernacular-specific inactivation of beta-catenin, Notch1, Ar, and Wt1 genes; as well as in mice with conditional expression of active form of Notch1. We have demonstrated an abnormal gubernacular development and cryptorchidism in males deficient for beta-catenin, AR, and WT1. In summary, we have identified several crucial pathways controlling gubernacular development in response to INSL3 and androgens.

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Biol Reprod. 2010 Nov;83(1 Suppl):2.

Activin A Balances Sertoli and Germ Cell Proliferation in the Fetal Mouse Testis.

Kate L Loveland 1, Sirisha Mendis 1, Sarah J Meachem 2, Mai A Sarraj 2, Martin M Matzuk 3

Activin is important for male reproduction, and its role in the postnatal mouse testis has been extensively studied. This study defines the inverse relationship between activin actions on Sertoli cells and gonocytes in the fetal mouse testis, demonstrating the mechanisms by which physiological levels of activin A mediate normal testis growth. We began by investigating the normal activin signalling machinery that is present between the time of sex determination (E12.5) and birth (0 dpp). The mRNA encoding activin A (Inhba) is selectively upregulated in the testis from E12.5, followed closely by increased inhibin alpha (Inha) mRNA by E14.5. Quantitative PCR analyses revealed changes in activin receptor transcript levels: Acvr1 was significantly higher, while Acvr2b was lower in the 0 dpp testis compared with E13.5 and E15.5 timepoints. Transcript levels of Smads1, 3 and 4 were higher compared with E13.5 and E15.5, while Smads2, 5 and 7 were lower. Immunohistochemistry demonstrated phosphorylated SMAD2/3 (P-SMAD 2/3) in nearly all cell nuclei in the testis at E13.5, E15.5 and 0 dpp, indicating the widespread presence of active signalling by TGFbeta and/or activin ligands. Activin knockout (inhba-/-) mice have significantly smaller testes at birth relative to wild type littermates. This was attributed to decreased Sertoli cell proliferation from E13.5 using PCNA immunohistochemistry, and results in a 50% reduction in Sertoli cell number at birth. In contrast, the inhba-/- testis contained double the wild type number of gonocytes at birth, with some germ cells appearing to bypass quiescence prior to birth. The presence of P-SMAD 2/3 in nearly all cells in inhba-/- samples indicates the contribution of other TGFbeta superfamily ligands to fetal testis growth. Real time PCR interrogation of inhba+/+, inhba+/- and inhba-/- mouse testes at E13.5, E15.5 and at birth identified changes in activin signalling machinery and specific cell cycle regulators in relationship to inhba gene dosage that can be directly correlated with changes in the number of germ cells and Sertoli cells. This demonstrates the contribution of activin to establishing the balance between Sertoli and germ cell numbers that is required for male fertility in adulthood.

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Biol Reprod. 2010 Nov;83(1 Suppl):2.

shRNA-Mediated Knock-Down of Dickoppf Homolog 3 in Transgenic Mice Disrupts Regulation of Wnt/beta-Catenin Signaling in Sertoli Cells and Their Ability to Support Spermatogenesis.

Deepika Sharma, Neerja Wadhwa, Neetu Kunj, Subeer S Majumdar 1

Testis plays an important role in the development of male reproductive system, providing the hormones for sexual maturation as well as the site of spermatogenesis. Spermatogonia B does not appear in testis until onset of puberty in mice and monkeys, although circulating levels of LH, FSH and Testosterone (T) during infancy are as high as that found during puberty. This implies that mere presence of hormones is not enough, but processes downstream to their action in testicular Sertoli cells (Sc) are also important in controlling germ-cell (Gc) division and differentiation. Lack of noticeable spermatogenesis during infancy in the phase of high hormones is a situation similar to that found in certain categories of male infertility. Therefore, we undertook present study to analyse genes differentially expressed by Sc during infancy and puberty. To this end, Sc were cultured from infant and pubertal monkeys and maintained in-vitro. On 4thday of culture, Sc were treated with recombinant monkey FSH and T for 24 hours. Fluorescent differential display was performed using RNA from such treated Sc. Several differentially expressed genes were identified with the help of Bioinformatic tools. Dickoppf homolog 3 (Dkk3) was one of the genes which was over expressed by pubertal Sc. Since Dkk3 is a known candidate involved in induction of cell differentiation, a process akin to testis during spermatogenesis, we chose this gene to pursue our study. To knock down Dkk3, we generated transgenic mice expressing shRNA against Dkk3. In-vivo testicular electroporation technique developed by our laboratory was used to make such transgenic mice. Interestingly qRT-PCR analyses of testicular mRNA revealed an inhibition, usually between 50% to 90%, of Dkk3 expression in such transgenic mice. Testicular weights of adult transgenic mice were reduced as compared to age matched controls. Histological analysis of atrophic testis revealed a compromised differentiation of germ cells along with multinucleated giant cells and vacuoles. Inhibition of Dkk3 expression had lead to sloughing off of germ cells. TUNEL assay showed a higher incidence of Gc apoptosis which led to tubular degeneration. Severe reduction in sperm counts was also observed. Litter size of such mice was significantly reduced (p<0.5). Azoospermia was observed in one of the mice whose Dkk3 expression level was reduced by 96%. Since Dkk3 is a member of Dkk family of Wnt antagonists, interference with Dkk3 expression led to an increase in the expression of beta-catenin. beta-catenin stabilization is known to restrict Sc maturation. In conformity with this, we found elevated testicular expression of Mullerian inhibiting substance (MIS) and Glial cell-derived neurotrophic factor (GDNF), the known markers of Sc immaturity, in adult Dkk3 knock down mice. Lack of Sc maturity may underlie defective testicular spermatogenesis and Gc apoptosis in these transgenic mice. We have shown that knocking down of Dkk3 leads to constitutive activation of Wnt/beta-catenin signaling which inhibits differentiation and maturation of Sc as demonstrated by expression of GDNF and MIS in adult mice. Depending on the degree of shRNA mediated inhibition of Dkk3, the status of spermatogenesis was affected in such transgenic mice. Our findings indicated that Dkk3 is a major regulator of spermatogenesis in mice. This work was supported by the Department of Biotechnology, India.

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Biol Reprod. 2010 Nov;83(1 Suppl):2.

Identification and Function of Putative Stem Leydig Cells in the Adult Rat Testis.

Haolin Chen, Barry R Zirkin 1

Stem cells have the capacity to self-renew and to generate differentiated cells. Such cells are found in adult as well as in embryonic tissue. Previous studies have shown that a single injection of adult rats with ethane dimethanesulfonate (EDS) results in the elimination of the adult Leydig cells. Subsequently, a new generation of Leydig cells is restored to the testes, indicating that the elimination of the adult Leydig cells results in the induction of precursor cells to differentiate into functional Leydig cells. We developed an organ culture system with which to test this hypothesis. Interstitial and seminiferous tubule fractions of rat testes were separated from each other and isolated four days after the rats received an EDS injection. The two fractions were cultured in vitro with LH for four weeks. Immediately after their isolation, the tubules did not produce testosterone in response to LH. However, after two weeks in culture, testosterone was detected in the medium of the tubule fraction. Testosterone production by the tubules increased at least through four weeks. Western blots showed that the steroidogenic proteins, P450scc and StAR, which were undetectable immediately after the tubules were isolated, became detectable. Immunhistochemical staining showed that 3β-HSD positive cells were located around the tubules. In contrast to the tubules, the interstitial fraction failed to produce testosterone. The effects of hormones and/or growth factors, including IGF-1, PDGFAA, LIF, T3, TGF-a and PDGFBB, and of Notch inhibitor, on Leydig cell differentiation were tested on cultured tubules. These factors were selected based on previous studies suggesting that they may play roles in Leydig cell differentiation during normal prepubertal development. Among the factors examined, IGF-1, PDGFAA, LIF and Notch inhibitor were found to stimulate testosterone production, while T3, TGF-a and PDGFBB had no effect on Leydig cell differentiation. hese results suggest that Leydig cell differentiation in the adult testis after EDS treatment is similar to Leydig cell differentiation during normal prepubertal development. We suggest that the precursor cells that give rise to the adult cells might be stem Leydig cells. The results support the peritubular localization of these putative stem cells. The organ culture system used for these studies was particularly useful for identifying the factors (hormones and growth factors) involved in the regulation of the differentiation of the putative stem cells. Supported by grants R37 AG021092, RO1 HD050570 and RO1 AG030598.

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Biol Reprod. 2010 Nov;83(1 Suppl):2.

Sertoli Cells Survive as Allografts by Inhibiting Cell-Mediated Apoptosis.

Gurvinder Kaur 1, Saroja M Putrevu 1, Timothy J Doyle 2, Kwan Hee Kim 2, Jannette M Dufour 1

Immune-privileged Sertoli cells (SC) have the unique ability to provide protection to co-transplanted cells and survive long-term when transplanted allogeneically or xenogeneically. On the other hand, MSC-1 cells (a mouse Sertoli cell line) lack some of the immunoprotective abilities associated with primary SC, as they are unable to protect co-transplanted cells. The objective of this study was to compare the cell survival rate and gene expression profiles of primary SC and MSC-1 cells, to further understand the mechanism for SC immune-privilege. Aggregated primary SC or aggregated MSC-1 cells were transplanted as allografts into the renal subcapsular space of naive BALB/c mice and cell survival was analyzed by immunohistochemistry (IHC). Additionally, gene expression differences between these cells were investigated by microarray and pathway analyses. Primary SC grafts survived throughout the study and were not rejected, whereas, very few MSC-1 cells were detected by day 11 and MSC-1 cells were completely rejected within 20 days. Microarray analysis identified 3198 genes that were differentially expressed with a ± 4 fold or greater level in primary SC. Aggregated primary SC, as compared to aggregated MSC-1 cells, expressed immune-related modulators, such as immunosuppressive cytokines and complement inhibitors, regulators of apoptosis and lipid mediators for controlling inflammation. As complement and apoptosis were identified by pathway analysis and play an important role in allograft rejection, we hypothesized that SC survive as allografts by inhibiting antibody-mediated (complement) and/or cell-mediated (apoptosis) death. Primary SC or MSC-1 cell grafts collected at days 1-20 were analyzed for antibody deposition and complement activation by IHC for IgG, IgM, complement factor 4 (C4), complement factor 3 (C3) and membrane attack complex (MAC). Antibody deposition was not observed until day 14 post-transplantation whereas no complement deposition was observed throughout the study. In contrast, when grafts were analyzed for cell-mediated death, significant apoptosis, as measured by TUNEL assay, was observed in MSC-1 cell grafts as compared to primary SC grafts. This led to the conclusion that cell-mediated death plays an important role in allograft rejection of MSC-1 cells, and primary SC by inhibiting cell-mediated pathway enjoy long-term survival. Studies are ongoing to analyze MSC-1 and primary SC grafts for cellular infiltrate, cytokines and immune regulating molecules, which may further increase our understanding of the mechanism by which SC establish immune-privilege and thus improve transplantation success.

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Biol Reprod. 2010 Nov;83(1 Suppl):2.

BDADs - Progressing Towards an Oral Male Contraceptive.

Cathryn Hogarth 1, Ryan Evanoff 1, Travis Kent 1, Elizabeth Snyder 1, Debra Mitchell 1, Chris Small 1, John Amory 2, Michael Griswold 2

BDADs (Bis-(dichloroacetyl)-diamines) are a group of compounds that inhibit the conversion of vitamin A (retinol, (ROL)) to its active metabolite, retinoic acid (RA), which is essential for mammalian sperm production. This study investigates the feasibility of using BDADs to inhibit spermatogenesis leading to their potential use as male contraceptives. A novel testis organ culture technique and the expression of a marker of RA activity, Stra8, were utilized to demonstrate that a specific BDAD, WIN 18,446, can inhibit the metabolism of ROL to RA in the neonatal and adult murine testis, and the embryonic murine gonad. After culturing either whole testes or germ cells isolated from 2 days post partum (dpp) mice with BDAD or BDAD plus ROL, Stra8 expression was suppressed, demonstrating that BDAD inhibited the conversion of ROL to RA in both systems. We also utilized a transgenic mouse containing an RA-activated LacZ reporter gene to demonstrate that there was limited RA activation of LacZ in 2 dpp testes cultured with BDAD or BDAD plus ROL. The expression of Stra8 was also downregulated in adult mouse testis tubules cultured with BDAD when compared to tubules cultured with the vehicle control. BDAD also inhibited the conversion of ROL to RA in embryonic ovaries and testes cultured for 48 hours. These murine results, combined with the significant reduction in sperm count also observed in rabbits treated with WIN 18,446, provide critical insights into how the BDADs can inhibit spermatogenesis through blocking the ability of vitamin A to drive germ cell development. This work was supported by the NIH Grants HD 10808 to MDG and U01 HD060408 to JA.

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Biol Reprod. 2010 Nov;83(1 Suppl):2.

Maternal Passive Transfer of GnRH Antibodies Does Not Change Reproductive Development in Elk Calves.

Jenny G Powers, Dan L Baker, Jason E Bruemmer, Melissa G Ackerman, Terry M Nett 1

Fertility control is one alternative for managing overabundant wildlife populations; however, current technology is limited by treatment efficacy, duration and unacceptable side-effects. We previously investigated the effects of a single immunization with gonadotropin-releasing hormone (GnRH) vaccine on female elk (Cervus elaphus nelsoni) during mid-gestation. Vaccination did not affect existing pregnancy, calving rates, or neonatal growth rates during the year it was applied; however, it significantly decreased pregnancy rates for three breeding seasons following immunization. Strong immune and inflammatory responses, including robust GnRH antibody concentrations and injection site abscesses, were associated with vaccination. Calves nursing from GnRH vaccinated dams developed high serum GnRH antibody concentrations via colostral antibody transfer. Antibodies waned by 6 months of age. The effects of exposure to passively acquired GnRH antibodies during the neonatal period, on long-term reproductive development and function, are unknown. This study was designed to test the hypothesis that hypothalmo-pituitary-gonadal (HPG) axis development and function would be altered due to functional lack of GnRH during the neonatal period. The onset of puberty in male calves was estimated by measuring serum testosterone and secondary sex characteristics such as antler development, neck girth, scrotal size, and semen characteristics. Additionally, we measured serum luteinizing hormone (LH) and testosterone concentrations in response to stimulation with a potent GnRH agonist. Similarly, serum progesterone was measured in female calves at 10 day intervals to estimate pubertal onset. They were challenged with GnRH agonist to evaluate pituitary gonadotrope function before the second breeding season and exposed to fertile bulls to determine fertility. Gross, histologic, and endocrinologic examination of the HPG axis was performed on all calves post-mortem. There were no differences between groups with or without antibodies in pubertal onset for either males or females. Similarly, antibody status did not affect response to GnRH agonist. All males developed grossly normal antlers and had at least one high quality semen sample during their first breeding season. All females became pregnant during the second breeding season. There were no gross differences in reproductive tracts between groups and pituitary LH and follicle stimulating hormone as well as hypothalamic GnRH concentrations were similar. We failed to reject the null hypothesis that GnRH antibodies do not affect reproductive development. These results strongly suggest that neonatal passive transfer of maternal GnRH antibodies does not affect long-term reproductive potential of elk and supports the notion that precocial species such as elk do not require functional GnRH stimulation during the neonatal period for complete reproductive development. Research was supported by the Morris Animal Foundation D07ZO-054 (TN).

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Biol Reprod. 2010 Nov;83(1 Suppl):2.

Domestic Cat Contraception Using a Single-Dose, Micro-Encapsulated LHRH Vaccine.

Valeria Conforti 1, Jerry Reeves 2, David de Avila 2, Robert Bogden 3, Keith Stormo 3, Jackie Newsom 1, Helen Bateman 1, William Swanson 1

In the U.S., 30 to 100 million cats are estimated to survive in a feral environment, representing a major health, environmental and welfare issue. A single-dose contraceptive vaccine has several potential applications in felids, most notably for control of feral cat populations. In this study, we evaluated the safety and effectiveness of a single-dose luteinizing hormone-releasing hormone (LHRH) vaccine for contraception in cats. The vaccine was comprised of a recombinant ovalbumin-LHRH (ova-LHRH) fusion protein as the antigen mixed with adjuvant containing cytosine guanine oligodeoxynucleotide (CpG ODN). The single-dose vaccine contained both non-encapsulated and encapsulated forms of ova-LHRH; the latter presented the antigen in agarose microbeads for slow release. Fifteen young adult, intact female cats were randomly assigned to one of the following treatment groups (n=5 cats/group): 1) two injections of non-encapsulated ova-LHRH (350 μg each), 8 weeks apart (positive control); 2) one injection of a mixture of encapsulated (440 μg) and non-encapsulated (350 μg) ova-LHRH; and 3) one injection of saline (negative control). Injection sites were monitored for inflammatory responses. Blood samples were collected bi-weekly for one year, and LHRH antibody production assessed using a validated radioimmunoassay. Progestin metabolite profiles were monitored via a validated enzyme immunoassay in fecal samples collected every 2 to 3 days for one year. Fertility was assessed by housing a proven breeder male with females for four months beginning six months after initial injection. In the saline group, percent binding of 125I-LHRH in serum was zero throughout the study. At the end of the study (weeks 38-44 post-injection), antibody binding activity was higher (P < 0.05) in the two-dose group (63.4% ± 9.8; mean ± SEM) compared to the single-dose group (27.0% ± 7.9). Prior to immunization, all 15 females exhibited estrual behavior indicative of ovarian cyclicity, and most females (13/15) appeared to be spontaneous ovulators based on elevated fecal progestin levels lasting 5 weeks or longer. In the saline group, all five females continued to ovulate spontaneously throughout the study until pregnancy. In the single-dose group, four of five females were no longer ovulating by the beginning of the fertility trial; however, one of these four resumed ovulatory activity towards the end of the trial and became pregnant. The remaining female in this group ovulated regularly until pregnancy. In the two-dose group, three of five cats were spontaneous ovulators initially; all three ceased ovulating by the time of the fertility trial. One of these cats showed elevated progestin levels towards the end of this time period, but did not become pregnant. All females (5/5) in the saline group became pregnant (kittens/litter, 3.6; litter size, 1-5). No pregnancy occurred in the two-dose group. In the single-dose group, two of five females became pregnant (litter size, 3-4). Subcutaneous granulomas were observed at the injection sites in all immunized females, with the most severe granulomas occurring in the two-dose group including two with ulceration. Granulomas reduced in size spontaneously but were still palpable one year after injection. In conclusion, the single-dose LHRH vaccine provided some fertility control but adjustment of dose and/or delivery system may be needed for more consistent, long-lasting contraception. (Morris Animal Foundation grant # D08FE-029).

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Biol Reprod. 2010 Nov;83(1 Suppl):2.

Follicle Size and Gonadotropin Concentrations Influence In Vitro Growth of Preantral Dog Follicles.

Jennifer Nagashima, David E Wildt, Nucharin Songsasen 1

We previously reported that both follicle stimulating hormone (FSH) and luteinizing hormone (LH) may play an essential role in dog follicle growth in vitro. In the present study, we determined the influence of (1) follicle size and (2) the gonadotropin microenvironment on in vitro growth of dog preantral follicles. In Study 1, preantral follicles were enzymatically-isolated by incubating ovarian cortices at 38.5°C for 75 min in 0.7 IU/ml Liberase and divided into 3 groups based on diameter: a) < 200 μm (n = 16); b) > 200 to < 300 μm (n = 60); and c) > 300 to 400 μm (n = 17). All follicles were individually encapsulated in 0.5% alginate and cultured in 200 μl of α-MEM + 10% fetuin and 2.5 μg/ml insulin-transferrin-selenium (Basal Medium) supplemented with 100 μg/ml FSH in 5% CO2 for 240 h. In Study 2, follicles (180 to 300 μm in diameter) were individually encapsulated in 0.5% alginate and cultured in the Basal Medium with 0, 1, 10 or 100 μg/ml FSH and 0, 1, or 10 ng/ml LH for 480 h (10-14 follicles/group). In both studies, follicle diameter and survival (based on morphology) were determined every 48 h with growth rate assessed by ANOVA followed by the Duncan's Multiple Comparison Method and survival by Chi-square testing. In Study 1, follicle size influenced in vitro follicle growth, but had no effect on survival. Follicles of < 200 μm and < 300 μm increased (P < 0.05) in size 70% and 50%, respectively, by the end of culture period compared to 13% of > 300 μm counterparts. In Study 2, FSH and LH concentration and the interaction of these gonadotropins influenced in vitro follicle growth and survival. Specifically, all follicles cultured in medium supplemented with the highest FSH and LH concentrations became atresia within 288 h, whereas 10 to 50% of those cultured in lower gonadotropin concentrations remained viable for up to 480 h of culture. Follicles cultured in the absence or in 1 μg/ml of FSH grew slower (P < 0.05) than those exposed to higher dosages. The influence of LH on follicle growth was FSH concentration-dependent. When 10 μg/ml FSH was added, follicles cultured in 1 ng/ml LH grew faster and increased in size more than those cultured in other LH dosages (100% vs. 60%, respectively; P < 0.05). However, LH did not influence follicle growth (P < 0.05) in the presence of 100 μg/ml FSH. Our findings indicate that the stage of preantral development influences the culture environment required to promote dog follicle development in vitro. It is possible to demonstrate a dose-dependency for FSH in culture, illustrating the critical importance of this gonadotropin in dog folliculogenesis. Furthermore, results confirm a role for LH in dog follicle growth, especially in a microenvironment where FSH is available only at a modest concentration. These findings are relevant for two opposing challenges in canids, exploring: 1) methods for rescuing the maternal genome from immature follicles; and 2) potential targets for suppressing ovarian function for inducing sterilization.

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Biol Reprod. 2010 Nov;83(1 Suppl):2.

Birth of Sand Cat (Felis margarita) Kittens Following In Vitro Fertilization and Embryo Transfer.

Jason R Herrick 1, Farshid Mehrdadfar 2, Mark Campbell 3, Greg Levens 3, Kevin Leiske 4, William F Swanson 3

The sand cat (Felis margarita) is a small (2-3 kg BW) felid from Northern Africa and the Middle East that is classified as near threatened by the International Union for Conservation of Nature. Captive breeding programs have been developed for sand cats in the U.S., Europe, and Middle East, but the long term maintenance of genetic diversity in captivity will require periodic genetic exchange between these populations that historically have been managed separately. Since in vitro fertilization (IVF) and embryo transfer (ET) could reduce or eliminate the need to transport living animals between regions, these technologies could facilitate the integration of disjunct populations and allow for the development of global, rather than regional, management programs. Similarly, IVF and ET would allow offspring to be produced in zoos using spermatozoa collected from wild males, effectively integrating captive and wild populations without removing animals from the wild. However, production of viable offspring in sand cats has never been demonstrated using these technologies. Our objectives were to: 1) Determine the effectiveness of eCG (100 I.U.) and porcine luteinizing hormone (pLH; 1000 I.U., 84 h after eCG) for stimulating follicular development and ovulation in sand cats; and 2) Assess the in vivo viability of fresh and frozen-thawed IVF embryos and the suitability of the eCG/pLH-induced maternal environment following ET into sand cat recipients. Cumulus-oocyte complexes were collected from gonadotropin-treated (150 I.U. eCG, 100 I.U. hCG) females (n=7) by laparoscopic follicle aspiration (~24 h post-hCG) and cultured (~20-22 h) with fresh or frozen-thawed spermatozoa in Feline Optimized Culture Medium (Biol Reprod 82:552-562 2010). A total of 102 high-quality oocytes (14.6 ± 3.2/female; mean ± SEM) were inseminated, resulting in 76 embryos (76.9 ± 8.6% cleavage) that were transferred or cryopreserved at the 2-8 cell stage. Females (n=14) treated with eCG/pLH were examined by laparoscopy 50-55 h after pLH to determine the proportion of females ovulating (11/ 14; 78.6%), as well as the average number of corpora lutea (9.4 ± 2.5) and mature (≥2 mm) follicles (5.6 ± 1.3) present on the ovaries. Fresh (n=21) or frozen-thawed (n=26) embryos were laparoscopically transferred into the oviducts of 9 ovulating females (4 to 7 embryos per transfer). Following transfer of fresh embryos, 1 of 4 females (25%) became pregnant and delivered two healthy, female kittens (from 7 transferred embryos) after a 66 day gestation. At the time of abstract submission, 2 of 5 females (40%) receiving frozen-thawed embryos (5 embryos per female) had elevated (≥10x above baseline) concentrations of fecal progestagen metabolites at 53 days post-ET and are assumed to be pregnant. Our results suggest that the eCG/pLH regimen is effective for inducing both follicular activity and ovulation for embryo transfer synchronization in sand cats. Although pregnancy and embryo survival percentages were relatively low in this initial embryo transfer study, the successful production of kittens using IVF and ET demonstrates the value of these reproductive technologies for global management of sand cat populations. (Funded by IMLS Grant #LG-26-08-0155-08 and the Al Ain Wildlife Park & Resort).

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Biol Reprod. 2010 Nov;83(1 Suppl):2.

Development of Interspecies Somatic Cell Nuclear Transfer Embryos Reconstructed from Bactrian Camel (Camelus bactrianus) Fibroblasts and Dromedary Camel (Camelus dromedarius) Ooplasm.

Nisar Ahmad Wani 1

Interspecies somatic cell nucleus transfer could not only be a useful bioassay system for assessing the ability of mammalian somatic cells to develop into embryos but can also be used as a possible approach to conserve endangered species. It could provide a useful technique to preserve the wild Bactrian camels, which are threatened with extinction. In the present study embryos were reconstructed by using skin fibroblast cells from a Bactrian camel (Camelus bactrianus) as donor karyoplasts and dromedary camel (Camelus dromedarius) oocytes as recipient cytoplasts for investigating the developmental potential of the these embryos. Mature oocytes were collected from super-stimulated dromedary camels by ultrasound guided transvaginal ovum pick-up, 26 h after GnRH administration. Serum-starved adult Bactrian camel skin fibroblast cells were injected in to the perivitalline space of enucleated dromedary oocytes. The fibroblast cells and recipient cytoplasm were fused by two DC pulses of 100 V/cm for 15 μs each using an Eppendorf electroporator at room temperature. Reconstructs were activated 1 h postfusion with 5μM ionomycine followed by exposure to 6-dimethylaminopurine for 4 h. The activated oocytes were then transferred to 500 μL of embryo culture medium I (modified potassium simplex optimization medium with essential and non-essential amino acids [KSOMaa] supplemented with 1% BSA) and cultured at 38.5°C in an atmosphere of 5% CO2, 5% O2, and 90% N2 in air. On Day 3 (Day 0 = day of activation) the cleaved embryos were transferred into 500μL of embryo culture medium II (modified KSOMaa supplemented with 10% FCS) and cultured under the same conditions until Day 7. The proportion of oocytes that cleaved was recorded on Day 3, and those that reached morula and blastocyst stages were recorded on Day 7 of culture. The embryos reconstructed from dromedary camel (Camelus dromedarius) fibroblasts and oocytes were treated in similar manner and worked as control. Out of 58 reconstructed embryos, in 3 replicates, 77.4 ± 2.1 couplets fused successfully and were cultured after activation. The embryos that cleaved (59.1 ± 5.7) and those developed to the blastocyst stage (34.4 ± 3.9) were not different from the control group. This study demonstrated, for the first time, that somatic cells from Bactrian camel can be reprogrammed in the dromedary camel ooplasm and such interspecies nuclear transfer embryos can develop to the blastocyst stage. Further studies are needed to see the feasibility of transferring these embryos into dromedary camel recipients and the possibility of producing Bactrian camel calves from dromedary recipient camels.

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Biol Reprod. 2010 Nov;83(1 Suppl):2.

Alterations of the Fetal Primate Epigenome due to Maternal High Fat Diet Exposure Are Reversed in Subsequent Offspring with Cessation of High Fat Diet.

Melissa Suter 1, Philip Bocock 1, Min Hu 1, Lori Showalter 1, Cynthia Shope 1, Kevin Grove 2, Kjersti Aagaard-Tillery 1

The most recent report from the CDC shows that more than one third of American adult women are obese. The Developmental Origins Hypothesis postulates that potentially adverse conditions in utero, such as exposure to a maternal high fat diet and maternal obesity, are associated with fetal alterations, rendering susceptibility to adult metabolic disorders. We have previously reported in a non-human primate model that exposure to a maternal high fat diet results in both pathological and epigenetic changes to the fetal liver. We have shown that high fat diet exposure results in non-alcoholic fatty liver disease, concomitant with covalent modifications to fetal hepatic chromatin structure, specifically increasing histone H3 lysine 14 acetylation (H3K14ac) levels. We now show that cessation of the high fat diet during pregnancy, even in the face of persistent maternal obesity, returns acetylation levels to those of control diet animals (n=6/ group). We also show that this increased acetylation correlates with the reprogramming of several metabolic and circadian regulatory genes. The expression of Npas2, a peripheral circadian transcription factor, is disrupted in fetal hepatic tissue from animals exposed to a maternal high fat diet, returning to those of control diet upon high fat diet cessation. We also report the disruption of other circadian genes regulated by Npas2, such as Per1 and Reverb-alpha in the high fat diet exposed animals. Extensive analysis of the Npas2 promoter region shows that DNA methylation levels do not differ due to maternal diet. However, using chromatin immunoprecipitation of an important regulatory region of the Npas2 promoter, we find enrichment of H3K14ac in animals exposed to a high fat diet compared with those on a control diet. This enrichment is lost from the diet reverted group. We conclude that H3K14ac levels are perturbed by maternal high fat diet exposure, which correlates with altered gene expression. We find that reversion to a control diet, even with persistent maternal obesity, helps to attenuate some epigenomic and circadian perturbations.

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Biol Reprod. 2010 Nov;83(1 Suppl):2.

Xenoestrogen-Induced Sex Reversal of Chicken Embryonic Testes Involves Specific CpG Dinucleotide Demethylation at a DNA Hypomethylation Dip in CYP19A1/Aromatase Promoter.

Toshi Shioda, Keiko Shioda, Haley Ellis, Noel L Rosenthal, Kathryn R Coser 1

Evidence is accumulating that exposure of mammalian fetuses to environmental endocrine disruptors (EEDs) affects epigenetic regulation of gene expression in germline cells. Such epigenetic changes may be heritable, conveying EED effects trans-generationally. However, mechanistic bases of such EED-induced epigenetic reprogramming largely remain unexplored. Chicken has a long history as a favored homothermic vertebrate model animal for studies in developmental biology due to its excellent accessibility to live embryos at early stages of development. In contrast to mammals whose sex is strictly determined by the XY sex chromosomes, avian phenotypic sex determined by the ZW sex chromosome system [males have homologous (ZZ) and females have heterologous (ZW) sex chromosomes] is readily reversed by fetal exposure to exogenous sex steroids or inhibitors of sex steroid synthesis during early development. Taking advantage of the exogenous estrogen-inducible gonadal feminization of genetically male embryos, in the present study we attempted to establish an in vivo model of EED-induced changes in DNA methylation in germline and somatic cells in gonads. White Leghorn ZZ-male chicken embryos were exposed to 17alpha-ethynylestradiol (EE2) during early stages of development (Day 3-11), and their gonads were collected shortly before hatching (Day 19) for morphological characterization, mRNA expression profiling by microarray and qPCR, and pyrosequencing determination of promoter DNA methylation. Exposure of ZZ-male embryos to EE2 before completion of the morphological testicular differentiation (Day 7.0) induced strong morphological gonadal feminization. This sex reversal was associated with strongly enhanced mRNA expression for CYP19A1/aromatase, which is required for gonadal ovarian differentiation, and FOXL2, a transcription factor that activates CYP19A1 promoter. Expression of ESR1 mRNA encoding estrogen receptor alpha was not affected EE2 or normal sex differentiation of gonads. A CpG dinucleotide in a methylation-dip sequence of CYP19A1 promoter (869 nucleotides upstream of the transcription initiation site) was significantly and specifically hypo-methylated in the feminized ZZ-male gonads (30-35% methylation, n=18) as well as vehicle-exposed ZW-ovaries (20-25% methylation, n=24) compared to vehicle-exposed ZZ-testes (50-55% methylation, n=20). In contrast, neither EE2-induced feminization nor normal sex differentiation affected methylation of this CpG site in the heart, which was relatively hyper-methylated (40-45%, n=8). CpG methylation of ESR1 promoter in gonads was not affected by EE2 or normal sex differentiation. Whereas ESR1 promoter was differentially methylated between MACS-purified gonadal somatic cells and SSEA1-positive germline cells of ZZ-testes (>90% purity), CYP19A1 promoter methylation was nearly between these two cell types. EE2-induced ZZ-gonad feminization resulted in strong hypomethylation of the CYP19A1 promoter CpG(-869) site in both germline and somatic cells. Concomitantly, CYP19A1 mRNA expression was strongly enhanced in both gonadal somatic stromal cells and germline cells. The highly reproducible and sequence-specific DNA hypomethylation of chicken embryonic CYP19A1 promoter induced by exposure to EE2 during early in ovo development may provide unique opportunities to study mechanisms of the epigenetic and trans-generational effects of endocrine disruptors.

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Biol Reprod. 2010 Nov;83(1 Suppl):2.

Establishment of Cell Lineage-Specific DNA Methylation Profile Follows the Morphological Differentiation of Trophoblast and Embryonic Cell Lineages.

Momo Nakanishi, Koji Hayakawa, Kunio Shiota, Satoshi Tanaka 1

There are numerous tissue-dependent and differentially methylated regions (T-DMRs) in the mammalian genome. Cell type-specific DNA methylation status of T-DMRs constitutes DNA methylation profile of the cell, which ought to be reconstructed as cells differentiate. The first cell differentiation in the mammalian development separates the trophoblast and embryonic cell lineages resulting in the formation of trophectoderm (TE) and inner cell mass (ICM) in blastocyst. Although lower level of global DNA methylation in TE genome compared to ICM has been suggested, dynamics of DNA methylation profile during the TE/ICM differentiation has not been studied. In this study, we aimed to identify T-DMRs between trophoblast and embryonic cell lineages and analyzed their DNA methylation status in pre- and post-implantation embryos. Oct4 and Nanog are differentially methylated between trophoblast stem (TS) and embryonic stem (ES) cell lines (TS-ES T-DMRs). These two loci are hyper-methylated in TS cells and hypo-methylated in ES cells. Elf5 is another known TS-ES T-DMR but exhibits the opposite methylation pattern. A number of other TS-ES T-DMRs have been found so far in our laboratory. First, we addressed whether these TS-ES T-DMRs are indeed differentially methylated between trophoblast and embryonic cell lineages in vivo. Bisulfite sequencing analysis of 11 TS-ES T-DMRs revealed that the DNA methylation status of them in E6.5 extraembryonic ectoderm and epiblast coincide with those in TS and ES cells, respectively, showing that the TS-ES T-DMRs are surely cell lineage-based T-DMRs in vivo. We then collected TE and ICM cells separately from E3.5 blastocysts and subjected to bisulfite sequencing. Interestingly, genomes of TE and ICM exhibited no difference in the methylation status of all T-DMRs examined. They were in extremely hypomethylated status. Here we identified valuable T-DMRs for delineation of trophoblast/embryonic cell lineages. DNA methylation analysis of these T-DMRs suggested that the DNA methylation profiles specific to each cell lineage are established after the morphological delineation of trophoblast and embryonic cell lineages. DNA methylation may thus contribute to stabilize or fix the cell fate and the differentiation capacity. Supported by Grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (20062003).

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Biol Reprod. 2010 Nov;83(1 Suppl):2.

BRG1 Is Required for CDX2-Mediated Repression of Oct4 Expression in Mouse Blastocysts.

Kai Wang, Satyaki Sengupta, Luca Magnani, Catherine A Wilson, R William Henry, Jason G Knott 1

During mouse blastocyst formation the segregation of the inner cell mass (ICM) and trophoblast is governed by the mutually antagonistic effects of the transcription factors OCT4 and CDX2. Evidence indicates that suppression of the Oct4 gene in the trophoblast is mediated by CDX2. Nonetheless, the underlying epigenetic modifiers required for CDX2-dependent repression of Oct4 are largely unknown. Here we show that CDX2-mediated repression of Oct4 expression in mouse blastocysts requires the chromatin remodeling protein BRG1. Using a combination of RNA interference and gene expression analysis we found that BRG1 regulates Oct4 transcription specifically at the blastocyst stage. We found that in BRG1 deficient blastocysts Cdx2 expression is normal, yet there is widespread expression of OCT4 in the trophoblast cells. Using confocal microscopy we observed an overlap of BRG1 and CDX2 positive foci in the trophoblast of blastocysts. Moreover, using trophoblast stem (TS) cells as a model system for the blastocyst trophoblast we found that CDX2 interacts with BRG1 via co-immunoprecipitation. To examine the biological significance of these interactions CDX2 and/or BRG1 were depleted in preimplantation embryos. Microinjection of Brg1 siRNA or Cdx2 siRNA alone resulted in an increase in Oct4 mRNA compared to control blastocysts, respectively. Remarkably, in BRG1/CDX2 double depleted blastocysts we observed a significant increase in Oct4 mRNA compared to depletion of BRG1 or CDX2 alone. Such results suggest that BRG1 cooperates with CDX2 to repress Oct4 expression in the trophoblast lineage. Lastly, to elucidate the underlying epigenetic mechanisms by which BRG1 induces Oct4 repression, DNA methylation was examined at the Oct4 gene. We found that depletion of BRG1 had no effect on DNA methylation. These results suggest that at the blastocyst stage, BRG1 induces Oct4 repression via mechanisms independent of DNA methylation. In toto, these findings point to a novel role for BRG1 and CDX2 in transcriptional repression of the Oct4 gene in the developing trophoblast. This work was supported by the Michigan Agricultural Experimental Station (MAES) and an Intramural Research Grants Program (IRGP) grant at Michigan State University to J.G.K.

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Biol Reprod. 2010 Nov;83(1 Suppl):2.

Metastasis Tumor Antigen 2 (MTA2) Is Involved in Genomic Imprinting During Mouse Preimplantation Embryogenesis.

Pengpeng Ma, Shu Lin, Marisa S Bartolomei, Richard M Schultz 1

The epigenetic mechanisms involved in establishing and maintaining genomic imprinting are steadily being unmasked. The nucleosome remodeling and histone deacetylation (NuRD) complex is implicated in regulating DNA methylation and expression of the maternally-expressed H19 gene in preimplantation mouse embryos. The NuRD complex in mammalian cells is composed of at least 7 polypeptides. Four of the components, HDAC1 and HDAC2 and two histone-binding proteins (RBBP4/RbAp46, RBBP7/RbAp48), form a deacetylase complex that is also found in the Sin3A histone deacetylase complex. The other three components, CHD4 (Mi-2beta), MBD3, and MTA, however, appear to be unique to the NuRD complex. CHD4 is an ATP-dependent nucleosome-remodeling enzyme. Although mammalian MBD3 does not bind to methylated DNA, when NuRD binds MBD2, the resulting NuRD complex becomes an integral component of the MeCP1 complex that can bind to methylated DNA, thereby leading to histone deacetylation of DNA methylated nucleosomes. MTA proteins, which are the last characterized proteins of the NuRD complex, are encoded by three genes, and It appears that only one MTA family member is found within a given NuRD complex. To dissect further the function of the NuRD complex in genomic imprinting we employed an RNAi strategy to deplete the NuRD complex components Metastasis Tumor Antigen (MTA) 1, 2, and 3). We find that Mta2 is the only zygotically expressed Mta gene prior to the blastocyst stage and that RNAi-mediated knockdown of Mta2 transcript leads to biallelic H19 expression and loss of DNA methylation in the imprinting control region (ICR) in blastocysts. In addition, biallelic expression of the paternally-expressed Peg3 gene, but not Snrpn, is also observed in blastocysts following Mta2 knockdown. Loss of MTA2 protein does not result in a decrease in abundance of other NuRD components, including MBD2 and 3, HDAC1 and 2, and CHD4. No reduction in the amount of MTA1 or MTA3 was observed following targeting of their mRNAs, precluding any insight into the role of these two genes in genomic imprinting. Taken together, our results support a role of MTA2 within the NuRD complex in genomic imprinting. This research was supported by a grant from the NIH (HD042026 to M.S.B. and R.M.S., and HD022681 to R.M.S.)

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Biol Reprod. 2010 Nov;83(1 Suppl):2.

MiR-31 Is a Tumor Suppressor MicroRNA That Functions in Ovarian Cancer.

Zhifeng Yu, Chad Creighton, Michael D Fountain, Ankur K Nagaraja, Huifeng Zhu, Mahjabeen F Khan, Derek Y Han, Emuejevoke Olokpa, Shannon M Hawkins, Preethi Gunaratne, Matthew L Anderson, Martin M Matzuk 1

MicroRNAs (miRNAs) are short non-coding RNAs that could have large-scale biological effects by directing gene regulation through translational repression and degradation of multiple complementary target mRNAs. Like other regulatory molecules, altered miRNA expression has been implicated in the formation of cancers. To identify putative tumor suppressor microRNAs in ovarian cancer, we used Illumina next generation sequencing technology to comprehensively profile microRNAs in human ovarian cancers, cancer cell lines, and normal ovarian surface epithelium (NOSE). We found that miR-31 was the most down-regulated miRNA, decreased an average of 35-fold (range: 12-460-fold) in human serous ovarian cancers (n=8) compared to NOSE (n=4). Likewise, miR-31 levels were decreased 34-fold (range: 14-590-fold) in ovarian cancer cell lines compared to NOSE. We next profiled the mRNA expression levels in the cancers, cell lines, and NOSE cells. Using an established bioinformatic algorithms, we identified hundreds of potential miR-31:mRNA associations potentially underlying cancer. We followed up these findings by validating in vitro the interrelationship of the predicted upregulated target genes with the downregulation of the miR-31 microRNA. Overexpression of miR-31 in the serous ovarian cancer cell line, OVCAR8, resulted in inhibition of proliferation and induction of apoptosis. Similar responses to miR-31 were observed in a number of ovarian cancer cell lines with a dysfunctional p53 pathway (i.e., OVCAR8, OVCA433, SKOV3 and TOV112D); however, in other lines (i.e., HEY and OVSAYO) with functional p53, miR-31 had no effect. Moreover, the osteosarcoma cell lines U2OS and SAOS2 (p14ARF-deficient and p53-deficient, respectively) were also sensitive to miR-31. Studies on the colon cancer cell lines HCT116 (p53+/+) and HCT116 (p53-/-) further indicated that miR-31 function is associated with defects in the p53 pathway. Molecular analysis using QPCR showed that predicted miR-31 targets including E2F2 were downregulated in OVCAR8 cells overexpressing miR-31, while a pro-apoptotic gene BAK1 was upregulated. Interestingly, in HEY cells, E2F2 and BAK1 were not influenced by introducing miR-31, indicating that miR-31 may function through a direct or indirect regulation on E2F2 and BAK1. Our results reveal a tumor suppressive effect of miR-31 in ovarian cancer. Further in vivo studies to explore the impact of altered miR-31 expression are highly warranted and are being performed.

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Biol Reprod. 2010 Nov;83(1 Suppl):2.

Regulation of p21 Is Necessary to Control Pituitary Progenitor Cell Expansion.

Pamela Monahan, Ashley Himes, Lori T Raetzman 1

The pituitary gland is the master endocrine regulator responsible for controlling such physiologic functions as fertility. To understand the cellular processes that contribute to hormonal fluctuations and feedback signaling that regulate fertility we must first understand the development of the pituitary. During embryonic development, the pituitary is induced from the oral ectoderm, forming Rathke's pouch (RP), a primordial structure containing proliferating progenitors. As development progresses, progenitor cells encounter a cell fate decision: exit the cell cycle or remain proliferative. Recent evidence shows that critical mediators of this decision are cyclin dependent kinase inhibitors (CDKI). Increased CDKI expression is associated with a quiescent state and allows for differentiation. Disruption of this process can lead to abnormalities in pituitary development forming either a hypomorphic or hypermorphic organ, which can affect reproductive physiology. Loss of the CDKI, p21 leads to spontaneous tumor development in mice, indicating its role as a tumor suppressor. Recent evidence from our lab has shown that mice mutant for p21 have increased numbers of cells in mitosis in embryonic development. Furthermore, loss of p21 results in incomplete RP separation from the underlying ectoderm. With these observations, we hypothesize that loss of p21 disrupts pituitary progenitor cell proliferation and can affect pituitary development. To test this hypothesis, we examined wildtype and p21 mutant pituitaries throughout embryonic life and evaluated proliferation profiles, organ morphology as well as which molecules and pathways collaborate with p21. RP closure, in wildtype mice, occurs between e10.5 and e11.5. By utilizing somite staging, we now show that RP closure is delayed in the absence of p21. 3 out of 3 wildtype pituitaries at the 17-somite stage and beyond are closed, while some p21 mutant pituitaries have incomplete closure detected at 17-19 somite stages. Recent work from our lab shows that the transcriptional repressor, Hes1, of the Notch signaling pathway is able to influence p21 expression in the developing pituitary. Loss of Hes1 results in increased cell death, at e10.5 and e11.5, in an area coincident with ectopic p21 expression. Interestingly, loss of p21 in Hes1 mutant pituitaries recovers the ectopic cell death. Taken together these data indicate a potential regulatory mechanism controlling p21 expression as well as linking p21 to cell death regulation. Further experiments will clarify which cell death pathway is induced by p21. Recently our lab has revealed a synergistic role for p21 with another CDKI, p27, of the same protein family. Loss of p27, alone, results in pituitary hyperplasia. We now show that loss of both p21 and p27 results in hyperplastic pituitary growth as young as 21 days. This early hyperplastic phenotype may indicate that loss of CDKI action during embryonic development may contribute to adult phenotypes. Further experiments will clarify at which stage of development increased proliferation occurs. With these studies we show that the cell cycle inhibitor p21 alone and synergistically with other molecules is necessary to maintain a balance between progenitor expansion and cellular differentiation. Supported by NIH Grant R01 DK076647A.

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Biol Reprod. 2010 Nov;83(1 Suppl):2.

A Truncated, Activin-Induced Smad3 Protein Acts as a Transcriptional Repressor of FSHβ Expression in Mouse Pituitary.

So-youn Kim, Jie Zhu, Teresa K Woodruff 1

Activin signals are transduced by cell-surface receptors, which recruit and phosphorylate intracellular Smad2/3. Complexes of Smad2/3 (~52 kDa) and Smad4 translocate to the nucleus to act as transcriptional co-activators of activin-responsive genes. Within the pituitary gonadotrope, Smad2/3 degradation and turnover is believed to be a cellular mechanism for limiting the duration of activin-stimulated FSHβ expression. Immunoblots of pituitary gonadotrope cell (LβT2) lysates revealed a smaller than expected 30-kDa Smad3 protein, which was phosphorylated in response to activin treatment. Full-length (52 kDa) Smad3 was induced after 30 minutes of activin treatment, whereas the smaller Smad3 species accumulated slightly later, between 30 minutes and 1 hour, and then persisted beyond 2 hours. Activin-induced phosphorylation of both large and small phospho-Smad3 proteins was blocked by follistatin treatment or transfection of siRNA directed against Smad3. TGFβ failed to induce the small phospho-Smad3 protein in LβT2 cells. We determined that the small Smad3 protein was neither a proteolytic- nor a proteasome-processed form of Smad3, but a transcription product limited to the gonadotrope. 5' RACE performed with LβT2 RNA and a Smad3 MH2 region-specific primer confirmed the existence of two forms of Smad3 in LβT2 cells. To test the biological function of the small Smad3 protein, we constructed a constitutively active C-terminal fragment of Smad3 protein. This protein antagonized activin-stimulated FSHβ expression in LβT2 cells in a dose-dependent manner. These data explain prior observations in the pituitary cell, where activin-mediated FSHβ-expression is extinguished relatively rapidly compared with other TGFβ-regulated genes. Thus, we speculate that the pituitary gonadotrope may contain an ultra-short feedback loop by which activin activates both an agonist and an intracrine antagonist to limit FSHβ production during the normal reproductive cycle. This work is supported by NIH/NICHD grant R01 HD37096 "Inhibin Actions on Reproductive Target Tissues."

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Biol Reprod. 2010 Nov;83(1 Suppl):2.
Jeffrey V May, Vladimir Y Butnev, George R Bousfield 1

We have recently demonstrated that hFSH exists as two glycoform variants, a tetra-glycosylated form (hFSH-tetra), where both α and β subunits exhibit N-linked glycosylation at two sites, and a di-glycosylated form (hFSH-di), which exhibits dual N-linked glycosylation on only the α subunit. These glycoforms are distinct from isoforms, which reflect poorly-defined properties related to the amount, type, and charge of carbohydrate, not the number of protein sites glycosylated. Assaying a number of human pituitaries, we have demonstrated that in younger, reproductively active women, hFSH-di is present at higher levels than hFSH-tetra. In peri-menopausal women, the levels of the FSH glycoforms are roughly equal. In clearly menopausal women, the hFSH-tetra form is present in higher levels than hFSH-di. Owing to the apparent age-related shift from hFSH-di dominance to hFSH-tetra dominance, we set out to compare their relative bio-potencies in a series of in vitro assays. hFSH-di was isolated and purified via anti-hFSHβ immunoaffinity chromatography followed by Superdex 75 gel filtration. Human FSH-tetra was prepared by combining the HPLC-purified, glycosylated, 24 kDa hFSHβ subunit with hCGα and separating the resulting heterodimer from unassociated subunit by gel filtration. hFSH-di exhibited a 10- to 11-fold greater receptor binding affinity than hFSH-tetra in cultured porcine granulosa cells (pGC) and a 25-fold greater binding affinity in rat testis homogenate receptor assays utilizing both hFSH-tetra glycoform preparations as tracers. Purified hFSH-di exhibited an approximately 26-fold greater binding affinity towards the human FSH receptor transfected into CHO cells than purified hFSH-tetra utilizing [125I]iodo-hFSH-di tracer. In these cells, purified hFSH-di was of similar potency to that of equine FSH, which exists mostly as a tri-glycosylated hormone. In contrast, purified hFSH-tetra exhibited similar binding properties to a reference preparation (hFSH AFP7298B), which was 80% the tetra form. At equal concentrations, hFSH-di exhibited twice the potency of hFSH-tetra in stimulating E2β secretion in porcine intact hemi-follicles maintained in explant culture and five times the potency in stimulating P4 secretion in cultured pGC. In rat granulosa cell cultures, purified hFSH-di was 4-fold more potent than hFSH-tetra with respect to P4 secretion. In short, in every functional ovarian assay we have examined to date, hFSH-di is consistently more potent than hFSH-tetra (range = 2- to 26-fold). Our results suggest that as women age, they produce a less potent form of FSH. Moreover, we have determined that commercially available hFSH preparations utilized for controlled ovarian hyperstimulation (COH), regardless of whether they are recombinant or menopausal preparations, consist almost entirely of hFSH-tetra. The above in vitro results suggest that COH is being performed with a less potent FSH glycoform. Whether the increased activity of hFSH-di relative to hFSH-tetra observed in vitro extends to similar activity in vivo represents a potentially important therapeutic question that is currently being investigated. (Support: NIH Grant P01 AG029531 to GRB).

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Biol Reprod. 2010 Nov;83(1 Suppl):2.

Different Critical Periods Exist During Gestation for Differentiation of the External Genitalia and the Sexually Dimorphic Nucleus of the Hypothalamus-Preoptic Area in Sheep.

Charles E Roselli 1, Charles T Estill 2, Mary A Meaker 2, Fredrick Stormshak 2

In sheep, prenatal exposure to testosterone during a critical period for differentiation can masculinize both the external genitalia of fetuses and the ovine sexually dimorphic nucleus (oSDN) of the hypothalamus-preoptic area of fetuses. The present study investigated the possible independent control of these two anatomical end points, as revealed by their developmental history. Specifically, we tested the hypothesis that separate critical periods exist for androgenization of the genitalia and the brain. Pregnant ewes were treated weekly with 200 mg testosterone propionate (TP) in corn oil either from Day 30 to 60 of gestation (Early TP group) or 60 to 90 (Late TP group). As a control (C) pregnant ewes were treated with oil during the same gestational periods. At 135 ± 0.2 days of gestation, the fetuses were delivered by Caesarean section. Fetuses were weighed and their crown-rump length and ano-genital distance measured. Their brains were then immersion fixed for subsequent volumetric analysis. The oSDN was identified by its characteristic expression of aromatase mRNA using in situ hybridization and by thionin staining. Female fetuses from the Early TP group possessed a penis, scrotum devoid of testes, uterus and ovaries, whereas the external and internal genitalia of females from the Late TP and Control groups were normal. Neither period of exposure to TP grossly affected the genitalia of male fetuses. The volumes of the oSDN between early and late control fetuses were not different within sexes and thus were combined for comparison with the TP-treated groups. Despite the masculinized genitalia in Early TP females, the volume of the oSDN was normal (Early TP females, 0.32 ± 0.06 vs. C females, 0.24 ± 0.02 mm3), but was masculinized in the Late TP females (0.49 ± 0.04 mm3) when their genitalia appeared normal. In contrast, the oSDN from Late TP males (0.51 ± 0.02 mm3) were not different from C males (0.51 ± 0.04 mm3), but were significantly smaller in the Early TP males (0.35 ± 0.04 mm3). These results demonstrate that the critical period for sexual differentiation of the oSDN occurs between Days 60 and 90 of gestation indicating that masculinization of the oSDN can be separated from that of the external genitalia, which occurs between Days 30 and 60 of gestation. The observation that exposure of male fetuses to TP from Day 30 to 60 of gestation blocked masculinization of the oSDN suggests that there may be de-masculinizing effects of early exposure to exogenous androgen in the male fetal brain. Supported by NIH Grant R01RR014270.

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Biol Reprod. 2010 Nov;83(1 Suppl):2.

The Effect of Acute Estrogen Treatment on Hormone Release (Oxytocin, Vasopressin, Dopamine, Prolactin) from Hypothalamic Explants Isolated from Male Common Marmoset Monkeys, Callithrix jacchus with and Without Parental Experience.

Michael J Woller 1, Patrick Keelty 2, Jason E Moore 1, Shelly Prudom 3, Toni E Ziegler 3

The objective of this study is to determine if there are neuroendocrine correlates associated with male parental behavior. Besides humans, the common marmoset is one of only a few species of primates to show extensive paternal care. We designed the study to evaluate hormone release over time from hypothalamic explants collected from male marmosets in two groups: experienced fathers and inexperienced adults. Ten male marmosets were used in this study. Five of the males were experienced fathers who had at least two sets of offspring prior to the study and were between eight and eleven years of age. The other five males had not fathered offspring, were between the ages of four to six years, and lived with a female who was not fertile due to treatment with the prostaglandin F2alpha analogue, cloprostenol sodium. Hypothalamic explants were collected at autopsy, bisected down midline, and hemihypothalami were exposed to continuous flow of media (pH=7.40; 37°C) at a flow rate of 1ml/20min for a period of five hours using an Accusyst-S perifusion system. Samples were fractioned at 20-min intervals and assayed for hormone content using high pressure liquid chromatography with electrochemical detection (dopamine), radioimmunoassay (prolactin), and solid-phase extraction followed by enzyme-linked immunosorbent assay (oxytocin, vasopressin). During perifusion of the hemihypothalamic explants, one half from each animal was exposed to two hours of media followed by three hours of media containing 1µg/ml estrogen (cyclodextrin-encapsulated 17β-estradiol to solubilize it in media) while the corresponding hemihypothalamus from each monkey was exposed to two hours of media followed by 1µg/ml cholesterol as a control. Our results show that release of prolactin was higher from hypothalami isolated from experienced fathers compared to release from inexperienced males, and that estrogen treatment reduced prolactin secretion in both experienced and inexperienced males. Dopamine release was significantly lower in experienced compared to levels in inexperienced males. Oxytocin release tended to be lower (not significant) from inexperienced compared to release from experienced males both prior to estrogen treatment and afterward. Vasopressin release was not influenced by either parental status or estrogen treatment. Our results demonstrate that estrogen has an effect on the release of some of the hormones thought to influence parental behavior in primates (prolactin, oxytocin) when measured in vitro from hypothalamic explants. More interesting might be the effect of previous paternal experience by the marmosets prior to the in vitro measurement of release of prolactin, dopamine and oxytocin, suggesting a role for hypothalamic control of some aspects of paternal behavior in this biparental species. This project was supported by NIH Grants #HD057684 to TEZ MJW, ARRA summer supplement to TZ and P51 RR000167 to the Wisconsin National Primate Research Center.

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Biol Reprod. 2010 Nov;83(1 Suppl):2.

The Notch Inhibitor Numb Is Present in Gonadotropes and May Be Necessary for LH Expression and Function.

Leah Goldberg, Tyler Moran, Katherine Brannick, Lori Raetzman 1

The synthesis and release of the gonadotropins LH and FSH from the anterior pituitary are essential for fertility in males and females. Many cases of infertility that present with altered gonadotropin levels are idiopathic, making the identification of genes controlling gonadotrope development and function critically important. Notch signaling may be necessary for early gonadotrope lineage determination, however, it must be suppressed for timely hormone production in future gonadotropes. A candidate for suppressing Notch activity is the protein Numb, which causes degradation of the Notch receptor through polyubiquitination via interactions with the ubiquitin ligase Itch. Although the most characterized role of Numb is in suppressing Notch during development, it can also play a role in adult homeostasis. In fact, loss of Numb is correlated with ER, PR-negative breast cancer and Numb has been shown to play a critical role in regulating p53 stability. In the present study, we show by immunostaining that Numb is present in the rostral tip thyrotropes beginning at e13.5 and is detectable in αGSU expressing cells in the anterior lobe beginning at e15.5. Numb is preferentially localized in LH and FSH containing cells in the anterior lobe of the adult mouse pituitary. To determine the function of Numb in the gonadotropes, Cre recombinase driven by the αGSU promoter was used to delete Numb and its homolog Numblike in gonadotropes and thyrotropes of the pituitary. During the period of gonadotrope specification, from e15.5 to e16.5, loss of Numb results in higher levels of LH in gonadotropes, while TSH levels appear to be unaffected. The increase in LH levels appears to be ameliorated by birth. Preliminary results indicate loss of Numb may affect both female and male fertility. Conditional Numb knockout females display precocious puberty, with vaginal opening four to five days earlier than wildtype littermates. This observation is consistent with the elevated levels of LH seen in the pituitary. Four of five Numb conditional knockout females used for breeding have produced litters. However, the average litter size is reduced in conditional knockouts, with an average of 3.8 pups per litter as compared to an average of 9 pups per litter in wildtypes. The remaining conditional knockout female has not produced any litters, nor has shown evidence of a copulatory plug. Four of four male conditional Numb knockouts were fertile, however two males not used for breeding had reduced testis size, abnormal testis histology, and low LH mRNA levels. This study suggests Numb may have a previously unidentified critical role in gonadotrope synthesis and/or secretion, impacting reproductive fitness. Future studies will be critical in order to elucidate the mechanism by which Numb affects gonadotrope function.

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Biol Reprod. 2010 Nov;83(1 Suppl):2.

'Omics Gone Wild: The Genome Sciences and Sexual Reproduction.

Huntington Willard 1

Both the fundamental rationale for and outcome of sexual reproduction depend on the existence and perpetuation of two different sexes. To say that males and females are different is the understatement of the last 300 or so million years. Differentiation of the two sex chromosomes, evolution of the chromosomal basis for sex determination, differential expression of X- and Y-linked genes in males and females, and emergence of distinctive phenotypes in biology and medicine have focused attention on the two human genomes - male and female - that characterize our species. This presentation will explore the view that many aspects of reproductive biology can be explored via genomic, epigenomic, and transcriptomic approaches to understand the evolutionary and biological basis of reproduction, including studies of chromosome behavior, imprinting, allele-specific gene expression, transcription factor binding, and epigenetic regulation of the genome through differential chromatin states. Such 'omic studies are providing insights into the biological basis for sex differences and their impact on reproductive biology.

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Biol Reprod. 2010 Nov;83(1 Suppl):2.

Advances in Genomic and Genetic Technology That Enhance Detection of Fetal Abnormalities.

David H Ledbetter 1

Abstract not available.

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Stem Cells and Germ Cell Differentiation.

Robert E Braun 1

Abstract not available.

Biol Reprod. 2010 Nov;83(1 Suppl):2.

How the National Postdoctoral Association Can Help You to Maximize Your Postdoc Experience.

Cathee Johnson-Phillips, Philip S Clifford 1

The “postdoc” is a period of advanced mentored training that was originally designed to prepare scholars for independent research in the context of an academic tenure-track position. Today only 20 to 30 percent of postdoctoral scholars, or “postdocs,” obtain such a position. It is important for graduate students and postdocs to consider all of their career options in order to obtain a satisfying “permanent” position in the United States research enterprise. The National Postdoctoral Association supports postdocs in many ways, including the establishment of six Core Competencies designed to provide guidance for self-assessment and mentoring activities. This interactive presentation will provide an overview of the postdoctoral community today and discuss the Core Competencies and other NPA resources that provide postdocs and their mentors' guidance in successfully navigating the changing face of the U.S. research enterprise.

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Biol Reprod. 2010 Nov;83(1 Suppl):2.

Effect of Omega-3 Fatty Acids on Prostaglandin F2alpha-Induced Cyclooxygenase-2 (Cox-2) Gene Expression in Bovine Luteal Cells In Vitro.

Nicole R White 1, Patrick D Burns 1, Jirapat Charumilinda 1, Andrew D Bryant 1, Zane T Prosser 1, Jason E Bruemmer 2, Terry E Engle 2

The omega-3 fatty acids eicosapentaenoate (EPA) and docosahexaenoate (DHA) have been shown to attenuate agonist-induced Cox-2 gene expression and prostaglandin synthesis in several tissues. The objective of this study was to determine the effects of omega-3 fatty acids on PGF2alpha-induced Cox-2 gene expression in bovine luteal cells in vitro. Seven 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 = 4 cows) or corn gluten meal at 6% dry matter intake (controls; n = 3 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 dissected from ovarian stroma and digested with collagenase. Cell concentration was determined using a hemocytometer and viability using propidium iodide exclusion assay. Six-well culture dishes were seeded with 1x106 viable cells and treated in triplicate with 0, 0.1, 1, 10, 100, or 1000 nM PGF2alpha analog (cloprostanol). Cells were culture in a humidified atmosphere of 95% air and 5% CO2 at 37°C for 0, 6, 12 or 24 hr. Following incubations, total RNA was extracted and stored at -80°C. First strand cDNA was generated from mRNA and then subjected to RT-PCR. Cyclooxygenase-2 (target gene) and beta-actin (reference gene) was amplified using bovine specific primers and the comparative threshold cycle (CT) method was used for quantification of Cox-2 gene expression. There was an effect of treatment, time, dose of PGF2alpha, and treatment x time interaction on Cox-2 gene expression (P < 0.05). There was no effect of dose of PGF2alpha or treatment on Cox-2 gene expression at 0 h (P > 0.10). Prostaglandin F2alpha stimulated Cox-2 gene expression in a quadratic dose manner at 6 h and 12 h of incubation (P < 0.01), but not a 24 h (P > 0.10). This response dose was attenuated at 6 h (P < 0.01) in tissue obtained from fish meal supplement cows, but not at 12 h (P > 0.10). In conclusion, PGF2alpha induced expression of Cox-2 mRNA in bovine luteal cells and this induction was suppressed by omega-3 fatty acids at 6 h of incubation. 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, INC.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Effects of Glucocorticoid on Cell Death and mRNA Expression of Tumor Necrosis Factor Receptor Type I in Bovine Luteal Endothelial Cells.

Takuo Hojo, Akihiro Oda, Seung-Hyung Lee, Tomas J Acosta, Kiyoshi Okuda 1

The corpus luteum (CL) is mainly composed of luteal steroidogenic cells (LSCs) and luteal endothelial cells (LECs). Structural luteolysis is thought to occur due to death of LSCs and LECs. Therefore, it is important to understand the mechanisms regulating cell death in both types of luteal cells. Tumor necrosis factor alpha (TNF) and interferon gamma (IFNG) are known to induce cell death in LSC and LEC. The action of TNF is mediated by TNF receptor type I (TNFRI). We have reported that LEC expresses TNFRI, suggesting that cell death induced by TNF is mediated by TNFRI. Our recent study demonstrated that LSCs express glucocorticoid (GC) receptors (GR), and that cortisol (an active GC) suppressed cell death in LSCs. However, the presence of GR and the effects of GC on cell death in LECs remain unclear. We hypothesized that cortisol suppresses cytokines-induced cell death in LECs, and that this action of cortisol is mediated by regulating TNFRI expression. To test the above hypotheses, we investigated the effects of cortisol on cell death and TNFRI expression. LECs obtained from the mid-stage CL were used. In the preliminary study, we optimized the condition to induce cell death in LECs, by treating cultured LECs for 24 h with TNF (0.05, 0.5, 1.0, 2.5 nM) and/or IFNG (0.05, 0.5, 1.0, 2.5 nM). The viability of LEC was reduced by separated treatment with TNF or IFN dose-dependently (P < 0.05). Furthermore, the cell viability was further decreased by combination of TNF (0.5 nM) and IFN (0.5 nM) treatment compared with control (P < 0.001). Based on the above results, a dose of 0.5 nM was chosen for the treatment of LECs with TNF and IFN in the following experiments. In experiment 1, mRNA expression of GRalpha was detected by semi-quantitative RT-PCR in LECs. In experiment 2, to determine the effects of cortisol on cytokines-induced cell death, LECs were exposed to cortisol (10, 100, 1000 nM) alone or in combination with TNF and IFNG for 24 h. Cortisol (1000 nM) inhibited the cytokines-induced cell death (P < 0.05). In experiment 3, we investigated the changes of TNFRI mRNA expression in LECs by real-time PCR after treatment with cortisol (1000 nM), TNF, IFNG, TNF+IFN or TNF+IFN+cortisol. The expression of TNFRI mRNA significantly increased by treatment with IFNG alone or in combination with TNF compared with control (P < 0.05). However, there was no significant difference in the expression of TNFRI mRNA in LECs treated with cortisol in combination with the cytokines compared with that of control. In summary, the treatment with TNF and IFN increased the cell death, whereas cortisol inhibited the cytokine-induced cell death. The up-regulation of TNFRI mRNA expression by IFN and down-regulation by cortisol suggest that these factors play critical roles in cell death of not only LSCs but also LECs during luteolysis.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Involvement of Bone Marrow-Derived Endothelial Progenitor Cells in Neovascularization During the Corpus Luteum Formation.

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

Neovascularization is necessary for the corpus luteum (CL) formation. After ovulatory luteinizing hormone surge, vascular endothelial cells of the theca cell layer invade the avascular granulosa cell layer, which is the first step of the CL formation. Thereafter, blood vessels are rapidly formed in the CL, and the CL becomes one of the most highly vascularized organs in the body. Neovascularization includes vasculogenesis and angiogenesis. Vasculogenesis refers to blood vessel formation by endothelial progenitor cells that are derived from bone marrow. Angiogenesis refers to the sprouting of microvessels from the preexisting blood vessels. Vasculogenesis is essential for the development of systemic vascular network in the fetus. Interestingly, vasculogenesis also plays a role in physiological and pathological neovascularization in the adult body. It has been well accepted that angiogenesis contributes to the formation of vascular network in the CL. However, it is unclear whether vasculogenesis plays a role in neovascularization in the CL. Therefore, the present study was undertaken to investigate whether bone marrow-derived endothelial progenitor cells contribute to neovascularization during the CL formation. Bone marrow transplantation (BMT) was performed using C57BL/6-Tg (CAG-EGFP) mice (green mice) that ubiquitously express enhanced green fluorescent protein. Mononuclear cells (1 x ~107 cells) obtained from the bone marrow of green mice were injected intravenously to the C57BL/6 mice (recipients) that had received irradiation (9.0 Gy). To validate whether BMT was succeeded, the percentage of the cells expressing GFP was checked in the bone marrow of the recipient by flowcytometry 4 weeks after BMT. Since more than 90% of the cells of the recipient were from green mice, this animal model was used as BMT mice in this study. We examined the presence of bone marrow-derived endothelial cells in the ovary during the CL formation using BMT mice. BMT mice were pretreated with GnRH agonist (s.c., 50 g). They received 10 IU PMSG after GnRH agonist injection, followed by 10 IU hCG 48 h later. They were autopsied under light ether anesthesia just before hCG injection, or 12 h, and 24 h after hCG injection, and the ovary was removed. Immunohistochemistry on the ovaries in each time were performed using antibodies for anti-CD34 to identify vascular endothelial cells, anti-f4/80 to identify macrophages, and anti-GFP to identify bone marrow-derived cells. This study was approved by the committee for Ethics on Animal Experiments in Yamaguchi University. GFP-positive cells, CD34-positive cells, and f4/80-positive cells were present in the theca cell layer before hCG injection, and invaded into the granulosa cell layer after hCG injection in a same fashion. They increased in number in the CL after hCG injection accompanied by CL formation. By fluorescence microscopy, a few GFP-positive cells were identical to CD34-positive cells in the CL, suggesting that a few bone marrow-derived endothelial progenitor cells were incorporated into neovascularization in the CL. There were no GFP- and CD34-positive cells in the theca cell layer in the follicle before hCG injection. Many of GFP-expressing cells were f4/80-positive macrophages in the follicle and CL. In conclusion, the present study suggests that bone marrow-derived endothelial progenitor cells play a role, at least, in part, in neovascularization during the CL formation.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Inositol 1,4,5-Trisphosphate Receptor Isoform Expression During Development of the Bovine Corpus Luteum.

Marietta F Wright, Erica McDermott, EK Inskeep, Jorge A Flores 1

The developing bovine corpus luteum (CL) (days 1-5) is resistant to the luteolytic actions of prostaglandin (PG) F2alpha that are induced in the CL in mid- (day 5-10) and late-phases (days 11-17). Although incompletely understood, the intracellular signaling pathways responsible for this developmental difference are known to involve accumulation of inositol trisphosphate in both large and small steroidogenic cells. Although it is known that PGF2alpha stimulates an increase in the cytosolic calcium concentration in bovine luteal steroidogenic cells and that the magnitude of the increase in cytosolic calcium is affected by luteal development, the underlying mechanisms controlling this process are not entirely known. This study tested the hypothesis that developmental differences in expression of inositol 1,4,5-trisphosphate receptor (IP3R) calcium channels contribute, at least in part, to this developmental difference in PGF2alpha-induced regulation of intracellular calcium. Tests of the hypothesis included determining luteal expression of the three isoforms of inositol 1,4,5-trisphosphate receptor (IP3R) calcium channels, IP3R-1, IP3R-2, and IP3R-3 at three luteal developmental stages. Corpora lutea were collected from cycling beef cows on days 4 (n=4), 10 (n=4) and 15 (n=3). Total RNA was isolated and messenger RNA expression of each of these calcium channels was determined by real time RT-PCR. The results indicated that although all IP3R were expressed in the bovine CL, only IP3R-3 appeared to be up-regulated as the CL aged. Furthermore, IP3R-3 appeared to be the receptor that had the highest expression throughout the luteal life span. These data indicate that IP3R-3 expression may contribute, at least in part, to the developmental difference in PGF2alpha-induced regulation of intracellular calcium. Intracellular calcium homeostasis is crucial in regulating progesterone production and apoptosis. This finding therefore contributes to our understanding of the mechanisms that render the early CL resistant to luteolytic actions of PGF2alpha.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Direct Effects of Interferon Tau in the Corpus Luteum: The Possible Additive Signal in the Ovary for Maternal Recognition in the Cow.

Haruka Matsumoto 1, Koumei Shirasuna 1, Kaya Nagai 1, Akane Nitta 1, Shingo Haneda 1, Motozumi Matsui 1, Heinrich Bollwein 2, Akio Miyamoto 1

It is well established that the conceptus secretes interferon tau (IFN) as a maternal recognition factor in ruminants. IFN acts in the uterus and prevents luteolysis by inhibiting PGF2alpha release, resulting in the maintenance of the corpus luteum (CL) function toward pregnancy. Recently, it has been shown that IFN is released from the pregnant uterus to uterine vein and the index of IFN stimulus such as interferon-stimulated genes (ISG) 15 and 2',5'-oligoadenylate synthease (OAS)-1 are up-regulated not only in endometrium and blood cells but also in the CL at maternal recognition period (MRP) in pregnant ewes. In addition, IFN mRNA and protein are first detected in the conceptus on Days 7-8 of pregnancy. These findings suggest that IFN acts directly on CL and this action begins before Day 16 that is equivalent to MRP in the cow. On the other hand, the proteins of lymphangiogenic factors are expressed in the CL during the estrous cycle as well as early pregnancy in the cow, suggesting that lymph vessels have an essential role for cyclic and pregnant CL. Therefore, the present study aimed to determine the difference in expressions of IFN stimulus and lymph endothelial cell (LEC) makers between cyclic and pregnant CL, together with peripheral blood leukocyte (PBL) in vivo. CLs were collected by ovariectomy on Day 16 of the estrous cycle (C16) and Day 16 of pregnancy (P16) (n=3-4 for each luteal phase, ovulation=Day 1). In addition, blood samples were collected on Day 0 and Day 16 from pregnant and non-pregnant cows (n=10 for each group). The mRNA expressions of ISG15 and OAS-1 tended to be higher in the CL on P16 compared with C16 (P < 0.1). In markers of LEC, LYVE1 mRNA expression was significantly higher (P < 0.05) and podoplanin mRNA expression also tended to be higher in the CL on P16 compared with C16 (P < 0.1). Furthermore, ISG15 mRNA was significantly higher (P < 0.05) in PBL on P16 compared with C16. These data led us to focus the relationships between these phenomena in the pregnant CL and IFN, thus to investigate the direct effect of IFN on the CL depending on the luteal phases in vitro. CLs were collected at a local slaughterhouse and divided into 3 groups: Days 4-5, 7-8 and 10-12 of the estrous cycle (n=3 for each luteal phase). Recombinant bovine IFN (0.1, 1, 10, 100 ng/ml) were added to CL slices incubation and ISG15 mRNA expression was analyzed in each time of the incubation (1, 2, 4 h). In the CL of Days 10-12, ISG15 mRNA expression did not change at 1 h after IFN treatment. However, IFN at 10 and 100 ng/ml significantly stimulated ISG15 at 2 and 4 h. Additionally, IFN at 1 ng/ml significantly stimulated ISG15 at 4 h. IFN at 1 and 10 ng/ml also stimulated ISG15 on Days 4-5 and Days 7-8 at 4 h. The levels of ISG15 stimulation by IFN were higher by two times in the CL of Days 7-8 and 10-12 (about 1800% in 1 ng/ml IFN-treated group) compared with those in CL of Days 4-5 (890%). These results indicate that ISG15, OAS-1, LYVE1 and podoplanin mRNAs were up-regulated in the CL on P16 as well as ISG15 mRNA in PBL. IFN stimulated ISG15 mRNA expression in the CL depending on the dose and luteal phase in vitro. It is suggested that the CL can receive a stimulus of IFN from the conceptus and that IFN may have a potential to change the luteal microenvironment including lymph vessels during MRP in the cow. Supported by Global COE and JSPS programs.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Alpha beta+ T Cells Alter Gamma Delta+ T Cell Responsiveness to Luteal Cells.

Edyta Brzezicka, Joy L Pate 1

Recent evidence indicates that lymphocytes residing within the corpus luteum are a dynamic population with changes elicited by the functional state of the CL. γδ+ T cells (TC) are more responsive than αβ+ TC to luteal cells (LC) in vitro, and their response to LC is modified by interactions among subsets of T cells. In previous work it has been shown that when αβ+ TC were in contact with luteal cells, the proliferation of the physically separated γδ+ TC was significantly reduced. The ability of αβ+ TC to inhibit γδ+ TC proliferation may be due simply to competition among lymphocytes or to direct action of αβ+ TC on LC. Thus, it was of interest to determine if αβ+ TC or their secretory products can alter luteal cell function such that they elicit less response in γδ+ TC. The first objective was to determine if CD4+ can suppress LC-induced stimulation of γδ+ TC proliferation. To investigate the mechanisms responsible for regulating γδ+ TC proliferation, we developed a model using separated populations of lymphocytes in coculture with luteal cells. Corpora lutea were collected from cyclic cattle on days 10 of the estrous cycle, dissociated and placed in culture. αβ+ and γδ+ TC were isolated from the blood of the same cows immunomagnetically and αβ+ TC were placed in coculture in contact with luteal cells. The purity of the T cells fractions was 94%. After 48 hours, αβ+ TC were removed from wells by serial washes and γδ+ TC were added to the cultures either in contact with luteal cells or separated from LC by a 4 micron culture insert. In the absence of CD4+ cells, LC elicited a proliferative response in gamma delta TC, and the response was not dependent on cell contact. In contrast, after exposure to CD4+ TC, LC-induced proliferation of γδ+ TC required cell-cell contact. The second objective was to determine if CD4+ TC -secreted products alter the responsiveness of γδ+ TC to luteal cells. We used the same culture model as in objective one, except αβ+ TC were separated from LC by a culture insert. After 48 hours, αβ+ TC were removed and were replaced by γδ+ TC either in contact or without contact with LC. The response of γδ+ TC to LC precultured with CD4+ TC that were separated by an insert was similar to what was observed in the first experiment when the CD4+ TC contacted LC. γδ+ TC proliferation was greater when TC were placed in contact with LC (70%) and reduced to 10% when T cells were separated from LC. It is concluded that αβ+ TC secrete a paracrine factor that modifies LC. After exposure to the CD4+ TC secreted factor, LC-stimulated proliferation of γδ+ TC requires cell-cell contact. 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):2.

Interferon-tau Has Endocrine Action on the Ovine Corpus Luteum During Early Pregnancy That Is Independent of Its Paracrine Effect on Endometrial ESR1 and OXTR.

Alfredo Q Antoniazzi 1, Ryan L Ashley 1, Joao F Oliveira 2, Fuller W Bazer 3, Thomas E Spencer 3, Thomas R Hansen 1

The ovine conceptus secretes interferon-tau (IFNT) from Days 10 to 21-25 with greatest release on Days 14-16 of pregnancy. IFNT acts in a paracrine manner to silence transcription of endometrial estrogen receptor alpha (ESR1) and, indirectly, the oxytocin receptor (OXTR), thus preventing oxytocin-induced release of luteolytic pulses of prostaglandin F2 alpha (PGF). Endocrine release of IFNT into the uterine vein also occurs on Day 15 of pregnancy, which induces IFN-stimulated genes (ISGs) in extra-uterine tissues, e.g., the corpus luteum (CL). We also reported extension of estrous cycles beyond 32 days using osmotic pumps and endocrine delivery of recombinant ovine (ro)IFNT (2 X 107 antiviral units per day) into the uterine vein for seven days starting on Day 10 of the estrous cycle. Our working hypothesis is that endocrine release of IFNT into the uterine vein occurs as early as Day 13 of pregnancy in concentrations that activate the Type I IFN receptor (IFNAR1 and IFNAR2) in uterine vein, CL and liver. We also hypothesized that three-day infusion of roIFNT starting on Day 10 of the estrous cycle induces ISGs in the CL, liver and endometrium. Semi-quantitative RT-PCR was used to examine IFNAR1 and IFNAR2 in the CL; ISG15 in the CL, liver, uterine vein and endometrium; and ESR1 and OXTR in the CL and endometrium. Tissues were collected on Days 12-15 of the estrous cycle (NP) and pregnancy (P; n = 3-6 ewes per day and pregnancy status). Tissues also were collected three days following osmotic infusion of BSA or roIFNT (n = 5 ewes per group) into the uterine vein starting on Day 10 of the estrous cycle. All differences described are significant at P < 0.05. Concentrations of progesterone in serum, determined by RIA, were similar on Days 12 and 13 and then declined to less than 1 ng/ml in NP ewes between Days 14 and 15. Endometrial ISG15 mRNA increased in P versus NP ewes by Day 13 and remained greater through Day 15. Endometrial ESR1 and OXTR mRNAs were up-regulated in NP compared to P ewes by Day 14, and remained up-regulated on Day 15. Uterine vein ISG15 mRNA was not affected by pregnancy status. IFNAR1 and IFNAR2 mRNA were present in the CL, but did not change due to pregnancy status. ISG15 mRNA in CL and liver increased by Day 14 and remained up-regulated on Day 15 in P compared to NP ewes. Concentrations of progesterone in serum did not differ in roIFNT- compared to BSA-infused ewes. IFNAR1 and IFNAR2 mRNA in CL were not affected by infusion of roIFNT. ISG15 mRNA was induced in the CL, liver and endometrium in roIFNT- compared with BSA-infused ewes. Although endometrial ESR1 mRNA was lower in response to roIFNT infusion, OXTR mRNA was unaffected. In summary, endometrial ISG15 mRNA was up-regulated by Day 13 followed by abrogation of up-regulation of ESR1 and OXTR mRNA on Day 14 of pregnancy. Endocrine action of IFNT occurred through up-regulation of ISG15 mRNA in CL and liver by Day 14 of pregnancy. Three-day infusion of roIFNT also caused up-regulation of ISG15 mRNA in CL, liver and endometrium; and abrogation of up-regulation of ESR1 mRNA, but not OXTR mRNA in the endometrium. It is concluded that IFNT has endocrine effects on the CL between Days 13 and 14 of pregnancy and may protect the CL through mechanisms that are complementary, yet independent to its paracrine effects on the OXTR endometrial pathway. Supported by USDA-NIFA-NRI grant 2006-35203-17258.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Effects of Early Pregnancy and Estrous Cycle on Survival and Apoptotic Pathways Lead to Endurance or Death of Corpus Luteum in Sheep.

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

In ruminants, prostaglandin F2 alpha (PGF2a) is the luteolytic hormone and PGE2 is considered as luteal protective mediator. In sheep, days 14-16 are the critical period for regression or rescue of corpus luteum (CL) during which CL is exposed to variety of cell survival or death signaling. During luteolysis, pulsatile release of PGF2a by the endometrium is locally transported to CL and stimulates intraluteal PGF2a biosynthesis and results in luteal regression. If a healthy conceptus is present in the uterus, the conceptus/endometria derived factor would be locally transported to CL and alter intra luteal cell signaling pathways, and rescue the CL from regression, in addition to inhibition of PGF2a pulses. The objective of the present study was to unravel cell signaling pathways that lead to survival or death of CL, and to determine how the estrous cycle and pregnancy regulate these pathways. METHODS: Suffolk cross-bred ewes were ovariohysterectomized on days 3, 6, 10, 12, 14 and 16 of the estrous cycle (ECY) or 10, 12, 14, 16 and 18 of pregnancy (PNY), the CLs were collected, and temporal expression of cell survival and apoptosis pathway proteins were determiend by western blot. Blood samples were collected from the ovarian artery (OA) and ovarian vein (OV) and transport of PGF2a and PGE2 were determiend. RESULTS: Activation of cell survival pathway proteins p-ERK12, p-AKT, p-P38, p-PJNK, p-c-Raf, p-Ras, p-Src, p-beta-arrestin 1, p-p90RSK, p-70S6K, active-beta catenin, and p-GSK were temporally decreased on days 14-16 of the ECY compared to that of PNY. Expression levels of these cell survival proteins did not differ on day 10 of ECY compared to that of PNY. Expression of antiapoptotic proteins Bcl-Xl and Bcl2 were increased on days 14-16 of PNY compared to that of ECY. Proapoptotic proteins Bax and Bad were expressed at very low level in ECY and PNY. Expression of inhibitor of apoptosis (XIAP) protein was decreased; by contrast, apoptosis inducing factor (AIF) protein was increased on days 14-16 of ECY compared to that of PNY. Caspase-3 and PARP proteins were cleaved at steady-state levels on days 14-16 of ECY and PNY. The concentration of PGE2 in the OA on days 14-16 of the ECY was 50-250pg/ml and PNY was 4,800-5300pg/ml. During days 14-16 of PNY, the CL secreted less amounts of PGF2a (400-500 pg/ml and 1500-2000 pg/ml, PNY vs. ECY) and large amounts of PGE2 (and 3000-3500 pg/ml and 1000-1250 pg/ml PNY vs. ECY) into OV. DISCUSSION: These results together suggest that multiple cell survival signaling pathways are suppressed and intrinsic caspase-3 dependent and independent apoptotic pathways are activated which in turn promote luteal regression during luteolysis. Whereas, at the time of establishment of pregnancy activation of these cell survival pathways are sustained and induction of apoptotic pathways are suppressed by conceptus/uterine derived factor PGE2, which is the only other candidate besides PGF2a, can be locally transported from the uterus to the CL through the UOP. PGE2 has the ability to transactivate ERK1/2, AKT, and b-catenin survival pathways and suppress apoptotic pathways. CONCLUSION: Our data indicate that PGE2 produced by the endometria and/or conceptus is transported to the ovary through the UOP and activate several cell survival pathways and inhibit apoptotic pathways and thus protect the CL from apoptosis/death at establishment of pregnancy. Supported by USDA award 2008-35203-19101 to JAA.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

The Predominant Infiltration of Neutrophils at the Early Stage of Development into the Cyclic Bovine Corpus Luteum.

Sineenard Jiemtaweeboon 1, Koumei Shirasuna 1, Sybille Raddatz 2, Hans-Joachin Schuberth 2, Akio Miyamoto 1

In the ovary, ovulation induces a kind of inflammation with infiltration of a large number of macrophages and granulocytes and these leukocytes secrete inflammatory cytokines within the ovulatory/ovulated follicle. After ovulation, the corpus luteum (CL) rapidly develops within one week together with active angiogenesis and progesterone synthesis in the cow. In the life span of CL, the immune cell population presents throughout the estrous cycle. Especially in the regressing CL, T-lymphocytes and macrophages significantly increase, and the luteolytic function of these leukocytes is well illustrated. In the early CL, several types of immune cells including macrophages and eosinophils infiltrate and the primary function of these immune cells is considered to be phagocytosis, but they may also have other critical functions for the CL development. Therefore, we first characterized the distribution of mRNA expression in leukocytes especially for angiogenic factors such as vascular endothelial growth factor (VEGF), nitric oxide (NO) and tumor necrosis factor alpha (TNF). Blood samples were collected from cyclic cows and lymphocytes and granulocytes were purified using Ficoll solution. The mRNA expressions of chemokine receptor CXCR1 and CXCR2 were higher in granulocytes than lymphocytes. The mRNAs of VEGF, VEGF receptor (VEGFR) 1, interleukin-8 (IL8) and TNF were expressed in both lymphocytes and granulocytes, but VEGFR2 and inducible NO synthase mRNAs were expressed only in granulocytes. These results suggest that bovine leukocytes have a potential to act on angiogenesis. In the next, we focused on granulocytes in the early developing CL and investigated the distribution of neutrophils and eosinophils by periodic acid-schiff staining and luna staining, respectively, with related mRNA expressions. CLs were collected at a local slaughterhouse and divided into 4 groups: Days 1-2, 3-4, 5-6 and 7-8 of the estrous cycle (n=5-8 for each luteal phase). The number of neutrophils and eosinophils in the CL were significantly higher on Days 1-2 than Days 5-6 and 7-8. The mRNA expression of IL8, chemoattractant of neutrophils, was highest on Days 1-2 as well as the number of neutrophils in the CL, although mRNA expression of CCL5, major chemoattractant of eosinophils, was not high on Days 1-2. To investigate the function of IL8 as the possible luteal chemotactic mediator, we tested the migration of neutrophils using transmigration assay with the bovine IL8 and supernatants of the CL culture. In transmigration assay, the CL supernatants as well as IL8 (10-100 ng/ml) stimulated neutrophil migration. Although we tested the capacity of migration in neutrophils collected from circulating blood from Day 2, 5 and 10 of the estrous cycle (n=5 for each group), the levels of neutrophil migration were constant in all groups, suggesting that the migration of neutrophils rather depends on the level of IL8 in the CL but not on the stage of estrous cycle or the capacity of neutropils. In conclusion, the bovine granulocytes and lymphocytes express mRNAs for angiogenic factors, and neutrophils and eosinophils exist at the largest number in the early stage of development CL, besides those in regressing CL. Also, the bovine CL is able to active a migration of neutrophils possibly by chemoattractant such as IL8. Further study should be addressed on the possible interactions between the CL and immune cells to enhance luteal development in addition to phagocytosis in the cow.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Role of a Histone H3K27-Specific Demethylase (JMJD3) During Bovine Preimplantation Development.

Sebastian Canovas 1, Jose Cibelli 2, Pablo J Ross 3

Histone lysine methylation is an epigenetic mark involved in gene transcriptional repression or activation depending on the specific lysine residue and methylation level. JMJD3 is a histone H3 lysine 27 (H3K27)-specific demethylase with activity at di- and tri-methylated residues, both epigenetic marks associated with gene repression. JMJD3 has been involved in the temporal expression of different genes during posterior embryo development, neural commitment, inflammatory process and mammalian epidermal differentiation. Since we have shown that H3K27 trimethylation levels decrease during preimplantation development, it is possible that JMJD3 is active during early bovine development. We hypothesize that JMJD3 plays an important role in gene expression regulation during early embryo development. The aim of this study was to identify the expression pattern of JMJD3 in bovine oocytes and preimplantation embryos and to determine if depletion of JMJD3 by siRNA injection into oocytes or zygotes has any effect on preimplantation embryonic development. Embryos were produced by parthenogenetic activation or in vitro fertilization (IVF) and total RNA was collected at GV, MII, PN, 2-, 4-, 8-, 16-cell, morula, and blastocyst stages. Total and polyadenylated JMJD3 mRNA levels were determined by quantitative real-time PCR (qRT-PCR). The presence of JMJD3 protein was analyzed using western blot (WB) in 2- and 8- to 16-cell stage embryos. Two different siRNA species were designed and produced using the Silencer siRNA construction kit (Ambion). MII oocytes were injected with ~6pl of 25-µM JMJD3-1, JMJD3-2, or control (non-specific) siRNA, followed by parthenogenetic activation. Embryos were in vitro-cultured for 7 days. Four-cell and blastocyst stage embryos were collected to determine the levels of JMJD3, GADPH, B-Actin, 18s rRNA and RPL15 mRNA by qRT-PCR. Fertilized embryos were injected 17 hour postfertilization as described for MII oocytes and cultured for 7 days. Cleavage rate, blastocysts rate and blastocyst cell number were analyzed in all groups. Relative JMJD3 mRNA levels were highest at the GV stage, decreasing from MII to morula stage and increasing again at the blastocyst stage. No differences were observed between total and poly-A mRNA levels. WB analysis of embryos showed a ~190kDa specific band, which disappeared when the antibody was pre-incubated with JMJD3 peptide. MII injected oocytes with both JMJD3 siRNA species showed a significant reduction in JMJD3 mRNA levels at 4-cell (~90% decrease) and blastocyst stages (~50% decrease), compared to siRNA control injected embryos. Meanwhile, other genes tested were not affected. Also, blastocyst rate was significantly reduced in the JMJD3 siRNA treated groups (~50% reduction, P < 0.05). Furthermore, the cell number in parthenogenetic blastocysts tended to be lower in JMJD3 siRNA injected groups than in control groups (~20% reduction). Fertilized embryos, injected with JMJD3 siRNA, developed to blastocyst stage at similar rates than control groups, although the cell number in treated embryos was significantly reduced (~30% reduction, P < 0.05). The differential effects observed in the parthenogenetic and IVF studies could result from the different nature of the embryos used or the timing of siRNA injection. Nevertheless, these studies provide the first experimental evidence demostrating that JMJD3 plays an important role in preimplantation embryonic development.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

NOT PRESENTED.

NOT PRESENTED.

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Sendai Virus-Mediated Fusion Improves the Early Development of the Cloned Bovine Embryos Through Precise Nuclear Remodeling.

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

The success of somatic cell nuclear transfer (SCNT) is influenced by the several procedures, such as nucleus removal, fusion, and activation. Especially, fusion between donor nucleus and ooplasm is generally achieved by electric stimulus, but the electrofusion often leads to the developmental defects of the SCNT embryos. Thus, the current study was carried out to improve the development rates of bovine SCNT embryos using the inactivated Sendai virus envelop (HVJ-E)-mediated fusion and to define the underlying mechanism. SCNT embryos reconstructed using ear skin fibroblasts and in vitro matured oocytes were fused with electric stimulus or HVJ-E, subsequently cultured in CR1-aa medium for 7 days at 38.5°C, and subjected to the analyses for nuclear remodeling, fusion and development rates, blastocyst quality, and apoptosis. There were no significant differences in fusion and cleavage rates between electrofusion and HVJ-E-mediated fusion. However, the development rate and total cell number of blastocysts were higher in the embryos fused using HVJ-E than in electrofusion group (51.4 ± 7.1% vs. 34.7 ± 4.3%, P<0.05; 119.0 ± 31.7 vs. 87.6 ± 33.4, P<0.05; respectively). Furthermore, the number of trophectoderm in blastocysts was also increased by HVJ-E-mediated fusion compared to electrofusion (82.2 ± 24.1 vs. 58.0 ± 26.1, P<0.05), whereas apoptotic blastomeres were significantly decreased in HVJ-E treating group (1.8 ± 1.0 vs. 4.6 ± 2.7, P<0.05), indicating the improvement of blastocyst quality by the use of HVJ-E. Interestingly, the nuclei of embryos fused using HVJ-E were normally developed to the premature chromosome condensation state (84.0 ± 8.7% vs. 33.7 ± 5.6%, P<0.05), whereas the retarded or abnormal nucleus remodeling were frequently found in electrofusion method. Taken together these results suggest that the inactivated Sendai virus envelop is a useful fusion material for the improvement of SCNT technology.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Small Size Hyluronan Promotes Bovine Oocyte Maturation and Embryo Development In Vitro.

Ali A Fouladi-Nashta 1, Waleed Fawzy Marei 1, Fataneh Ghafari 2

The ability of the oocyte to develop to the blastocyst stage following in vitro fertilisation and subsequently a good quality embryo is affected by the microenvironment in which it is cultured. Hyaluronic acid (HA) is an important component of the extracellular matrix which is important for cumulus cells expansion and is present in the follicular oviductal and uterine fluids and is expressed in bovine oocytes and embryos. Hyaluronidase (Hyal) is an enzyme present in the oviduct and uterus and cleaves the HA into biologically active small fragments, facilitating its uptake or degradation. Bovine oocytes or cleaved embryos were matured in vitro in the presence or absence of HA, Hyal-2 or 4-Methylumbelliferone (4-MU), a HA synthesis inhibitor. The rates of oocyte nuclear maturation to the metaphase II stage and development of embryos to blastocyst stage and blastocyst quality were recorded. Large size HA and also Hyal-2 inhibited cumulus cells expansion without affecting oocyte maturation. 4-Mu at 1mM reduced cumulus cells expansion and oocytes maturation rate. Supplementation of embryo culture media with 300 Units/ml Hyal-2 significantly improved the blastocysts yield, and quality by increasing the total cell number and number of cells in the Inner Cell Mass (ICM). Fewer blastocysts were produced when 4-MU was added to the culture medium (P < 0.01). This data suggests that intrinsic HA production is critical for oocyte maturation and blastocyst development and also has an effect on embryo quality. This study therefore provides evidence for critical role of small HA fragments on improving embryo quality and development.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Sonic Hedgehog Is Important for Adrenal Development Whereas Dispensable for Gonadal Development in Mice.

Chen-Che Jeff Huang, Humphrey H-C Yao 1

The Hedgehog (Hh) family of proteins is one of the highly conserved paracrine/autocrine morphogens. In mammals, three Hh orthologs have been identified: Desert hedgehog (Dhh), Indian hedgehog (Ihh), and Sonic hedgehog (Shh). Although these three Hh genes have diverse expression patterns and functions, they are thought to induce a common signal transduction pathway in the target cells. In the developing adrenal and gonads, evidence of a paracrine crosstalk via the Hh pathway is emerging. The Hh ligands are produced by the earliest differentiated Steroidogenic factor 1 (SF1)-positive cells such as adrenocortical cells in the adrenal, Sertoli cells in the testis, and granulosa cells in the ovary. These tissue-specific ligands then act upon Hh-responding cells located mainly in the mesenchyme, including capsular cell in the fetal adrenal, fetal Leydig cells in the testis, and theca cells in the adult ovary. We investigated the function of Shh in adrenals and gonads by producing a conditional knockout model where Shh is inactivated in the SF1-positive cells in adrenals and gonads as early as at embryonic day 11.5. Inactivation of Shh in the SF1-positive cells affected fetal adrenal growth but yielded no ovarian/testicular phenotypes in fetal as well as in adult life. It was reported that other Hh ligands are present in gonads: Dhh in the testis and Ihh and Dhh in the ovary. Our results suggest that loss of Shh could be compensated by other Hh ligands in the gonads. (This study was supported by Billie Field Graduate Fellowship and NIH-HD059961.)

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Role of Peptidylarginine Deiminase 4 in Mammalian Preimplantation Embryonic Development.

Manjula Brahmajosyula 1, Teruhiko Wakayama 2, Masashi Miyake 1

Peptidylarginine deiminases are a family of calcium dependent, highly substrate specific enzymes that convert peptide bound arginine residues into non conventional amino acid citrulline in a post translational modification known as citrullination. Among the known 5 isomers, peptidylarginine deiminase 4 (PAD4) having nuclear localizing sequence, is localized in the nucleus and is known to target histone and nucleophosmin. It has been shown that modifications mediated by PAD4 are involved in regulating chromatin structure and gene expression in a variety of cell types. Recent studies have confirmed regulation of p53 target gene expression by peptidylarginine deiminase 4. Here we study the localization and expression of PAD4 in pig sus scrofa oocytes and electrically activated preimplantation embryos and analyze its role in the preimplantation development. 4-6 mm sized follicles were extracted from the ovaries that were collected from the local slaughter house and cultured for maturation in vitro. Oocytes were collected at different stages of maturation for analysis. Fully matured metaphase oocytes with extruded 1st polar body collected at 42-46 hrs post maturation culture were electrically stimulated. Thus activated embryos were collected at different developmental stages for the investigation. Indirect immunofluorescence assays and western blot analyses, showed that PAD4 is localized in the cytosol in all the stages of oocytes and embryos. However, in germinal vesicle oocyte, it was also found to have translocated into the nucleus before germinal vesicle break down and then onwards it was detected to be colocalized with the metaphase spindle. The association of PAD4 with chromatin was observed until the completion of 2nd meiosis. PAD4 substrate specificity was dissected using modified citrulline detection kit and it was discovered that histone was the target of peptidylarginine deiminase 4 in oocytes and embryos. It was noticed that the amount of histone citrullination was different in different stages of development. There was a dip in the citrullination signal at 2 cell stage which increased from 4 cell stage onwards, to be highest at the blastocyst stage. When PAD4 expression was knocked down via RNA interference, it was observed that there was delay in the development which lead to retarded growth. The delay was noticed at each developmental stage. There was significant decrease in the blastocyst cell number. When PAD4 expression and localization was also checked in mouse oocytes and parthenogenetic embryos, metaphase oocytes showed similar PAD4 localization pattern and the PAD4 expression was seen in all the stages of embryos too. To check whether oocyte associated somatic cells such as cumulus cells also contain PAD4 and are involved in the oocyte development, cumulus cells collected during denudation of metaphase oocytes were examined for the presence of PAD4 as well as citrullinated proteins in the nucleus by both indirect immunofluorescence and western blotting analyses. It was revealed that PAD4 was only cytoplasmic and histone 3 was the target. Thus our study confirms the presence of PAD4 in the pig oocytes, embryos and cumulus cells; PAD4 nuclear translocation in the later stages of oocyte development and during fertilization; and leads to the conclusion that PAD4 is involved in the development of pig preimplantation embryos.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

HSP90 Inhibitor, 17-AAG, Affects Meiotic Maturation and Preimplantation Embryos Development in Pigs.

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

Heat Shock Protein 90 (HSP90) is ATPase-directed molecular chaperon and affects survival of cancer cell. Inhibitory effect of HSP90 by inducing cell cycle arrest and apoptosis in the cancer cell was found. However, its role during oocyte maturation and early embryo development is very insufficient. Therefore, in the present study, we examined the roles of a HSP90 inhibitor (17-AAG) on meiotic maturation and early embryonic development in pigs, as well on the expression of HSP90-, cell cycle-, and apoptosis-related genes. Porcine oocytes were cultured in the NCSU-23 medium with or without 17-allylamino-17-demethoxygeldanamycin (17-AAG) for 44 hr and then cultured in fresh maturation medium without hormones for an additional 22 hr. In vitro fertilization was performed in the modified Tris-buffered medium with fresh ejaculated spermatozoa, and fertilized embryos were cultured in PZM3 medium. Meiotic maturation in porcine oocytes was examined in the presence of concentrations of 17-AAG (2 µM). After IVM, a lower proportion of 17-AAG treated compared with untreated oocytes was in MII (71.0 vs 27.9%, P < 0.05). Expression patterns of apoptosis related genes (Bax and Bcl-xl), HSP90 complex genes (HSP90, HSP70 and p23) and cell cycle genes (cdc2 and cdc25c) were significantly changed by using RT-PCR analysis. In addition, the frequency of penetration decreased in 17-AAG treated oocytes (35.6%) compared with control (68.2%), furthermore the rate of blastocyst formation decreased (P<0.05). Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) showed that the number of containing fragmented DNA at the blastocyst stage increased in the 17-AAG-treated group compared with control (8.8 vs. 6.6, respectively). In conclusion, HSP90 appears to play a direct role in porcine oocyte maturation by enhancing blastocyst development and survival.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

The Effect of Duration of the Preovulatory Period on the Expression of IFNtau and CSH-1 mRNA by Embryos Collected on Day 17.5 of Pregnancy in Beef Cattle.

Lucas A Souto 1, Leandro H Cruppe 1, Martin Maquivar 1, Martin L Mussard 1, Vagner J Guerreiro 1, Dave E Grum 1, Troy L Ott 2, Joy L Pate 2, Michael L Day 1

The objective of the present experiment was to determine the influence of manipulation of preovulatory estradiol concentrations through altering the duration of the preovulatory period on amounts of mRNA for interferon tau (IFNtau) and chorionic somatomammotropin hormone - 1 (CSH-1) in day 17.5 bovine embryos. Non-lactating beef cows were pre-synchronized to a common day of estrus and ultrasonographic-guided follicle aspiration was performed 6.5 ± 0.3 days after estrus (day of aspiration = day -7 of experiment). The experiment was performed in two replications. Cows were assigned to receive 25 mg of prostaglandin F2alpha (Lutalyse) on either day -3 (high estradiol treatment; Hi-E), or on day -2 (low estradiol treatment; Lo-E). All cows received 100 µg of GnRH (Cystorelin) on day 0 (hr 0), resulting in a preovulatory period of either 3 (Hi-E) or 2 (Lo-E) days. Ovarian ultrasonography was performed on days -7, -3, 0, 2 and 6 to monitor emergence and diameter at ovulation of the preovulatory follicle, and formation of a new corpus luteum after GnRH. Blood samples collected at 12 h intervals from hr -72 to hr -12 and at hr -6 and 0 were used to determine the preovulatory concentrations of estradiol. Blood samples for progesterone analysis were collected on day -3, every other day from day -2 to 6 and day 9 to 13, and daily until slaughter on day 17.5. Embryos were collected from donor cows on day 7 of gestation following standard MOET procedures and implanted into experimental cows immediately after collection on day 7 of the experiment (7 days after GnRH). Cows were slaughtered on day 17.5, and intact filamentous embryos were collected from 4 cows in the Hi-E treatment and 8 cows in the Lo-E treatment. Statistical analyses were conducted using mixed procedures of SAS with a model that included treatment and replication for gene expression. For analyses of progesterone and estradiol concentrations over time, the appropriate repeated measure of hour or day was included in the statistical model. Gene expression was determined using RT-qPCR for IFNtau, CSH-1 and RPL19. Diameter of the ovulatory follicle on day 0 for the Hi-E (15.0 ± 1.0 mm) and the Lo-E (14.6 ± 1.1 mm) treatment did not differ. Concentrations of estradiol during the preovulatory period tended (P = 0.06) to be greater in the Hi-E than Lo-E treatment. A treatment x day interaction for progesterone concentrations was detected. Concentrations of progesterone did not differ between the Hi-E and Lo-E treatments on days 0, 2, 14, 15, and 17 but were greater (P < 0.05) in the Hi-E than Lo-E treatment on days 4, 6, 9, 11, 13, and 16. Relative amount of mRNA did not differ between the Hi-E and Lo-E treatments for IFNtau (109 ± 23.5 and 181 ± 25.1 units, respectively) or CSH-1 (3.6 ± 1.7 and 15.7 ± 6.7 units, respectively) between the Hi-E and Lo-E treatments. In conclusion, increasing the length of the preovulatory period resulted in increased preovulatory concentration of estradiol and concentrations of progesterone during the ensuing luteal phase. However, these alterations in systemic concentrations of estradiol and progesterone did not influence expression of IFNtau and CSH-1 genes by day 17.5 bovine embryos.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Ultra-Structure of Bovine IVP Embryos Cultured in Two Different Serum Concentrations after Chemical Lipolysis with Forskolin.

Daniela Martins Paschoal 1, Mateus Jose Sudano 1, Tatiana S Rascado 1, Luis Carlos Ona Magalhaes 1, Leticia F Crocomo 1, Joao Ferreira Lima Neto 1, Alicio Martins 2, Fernanda da Cruz Landim-Alvarenga 1

Almost all media developed to support in vitro production (IVP) of bovine embryo contain fetal calf serum (FCS) and/or bovine albumin (BSA). Nevertheless, serum inhibits cleavage and development of early embryos and induces a decrease in embryo tolerance since its presence is responsible for the greater cytoplasmic lipid accumulation observed in IVP embryos. The manipulation of culture media and the use of drugs to modify embryo metabolism have been used to produce embryos with greater resistance to cryopreservation. Among these, the use of forskolin has been reported to induce an increase in pig embryo lipolysis. Therefore, the present experiment studied the ultra structure of bovine embryo produced in the presence (2.5%) or absence (0%) of FCS and forskolin. Cumulus oocyte complexes (COCs) were matured in TCM 199 (Gibco, Grand Island, NY, USA) supplemented with 10% FCS, sodium pyruvate, FSH (Pluset, Hertape Calier Saude Animal, Juatuba, Brazil), LH (Lutropin, Bioniche, Canada), penicillin and streptomycin, in a humidified atmosphere with 5% CO2 in air at 38.5°C for 24 hours. Frozen bull (Bos taurus indicus) semen from a single batch was selected through a Percoll gradient and IVF was performed in HTF (Human Tubal Fluid, Irvine Scientific, Santa Ana, California, USA) supplemented with caffeine, heparin, penicillamine, hypotaurine and in a humidified atmosphere with 5% CO2 in air at 38.5°C for 20 to 22 hours. Presumptive zygotes were cultured in SOFaa (Synthetic Oviductal Fluid) with 1% non-essential amino acids (Gibco, Grand Island, NY, USA) and 1% essential amino acids] supplemented with BSA in the presence of 2.5% FCS or in the absence of FCS. Embryos were kept in a humidified atmosphere with 5% CO2, 5% O2 and 90% N2 at 38.5°C. On day 6, embryos from each group were divided into 2 groups with or without the addition of 10 µM Forskolin. Therefore, the 4 treatments used in this study were: 2.5% FCS without forskolin, 0% FCS without forskolin, 2.5% FCS plus forskolin (2.5%+F) and 0% FCS plus forskolin (0%+F). Embryo cleavage was recorded on day three (day zero = fertilization), and blastocyst production on day 7. Transmission electron microscopy (TEM) was performed in 6 embryos from each group trough standard protocol. For statistical analysis, % cleaved and % blastocyst were transformed using the arcsine transformation (√y/100) and analyzed using ANOVA followed by Tukey test. The level of significance adopted was 5%. No differences (P>0.05) on cleavage rates were observed between groups. However a higher (P<0,05) blastocyst formation rate was obtained on groups that used FCS (2.5% FCS--46.8±13.28a and 2.5% + forskolin--46.31±11.39a) in comparison with the groups without FCS (0%FCS--21.85±8.38b and 0% + forskolin--25.71±11.58b). The ultra structural analysis showed similar features in embryos from all studied groups. However, embryos cultured in absence of FCS presented lesser and smaller lipid droplets. Moreover, embryos cultured without FCS presented more cellular debris in the perivitelinic space and in the blastocoele, indicating loss of blastomeres. The use of forskolin was not able to reduce lipid droplets in serum produced embryos. The results indicate that the removal of FCS induces a reduction in IVP embryo lipid content. In spite of this, the removal of FCS reduced embryo quality demonstrated by the increase in cellular debris and by the lower blastocyst formation rates.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Temporal and Spatial Characterization of Hyaluronic Acid and Associated Transcriptional Activity During Early Porcine Embryo Development.

J Alex Pasternak, Francois Paradis, Susan Novak, Richard RE Uwiera, George R Foxcroft, Michael K Dyck 1

Hyaluronic acid (HA), a member of the glycoaminoglycan family, has been shown to have positive effects on in vitro embryo development in mice, cows, and pigs. Furthermore, its addition to culture media has been shown to increase embryo development following transfer in mice and improve pregnancy rates resulting from embryo transfer in human populations with multiple previous failures. However, the mechanisms by which HA affects early embryo development is yet unknown, a situation further complicated by changing effects in response to both the concentration and molecular weight of the HA used. The objective of this experiment was to determine the normal status of HA within the porcine reproductive tract and resolve any temporal and spatial variations within the system which may affect embryo viability up to the blastocyst stage. Primiparous sows (n=21), on their second estrus following weaning, were randomly designated for sample collection at seven periods corresponding to the germinal vesicle (GV), metaphase II (MII), 2cell, 4cell, early morula, morula, and blastocyst stages of embryonic development. Timing of collection for each the post-ovulatory stages was based on detection of ovulation by real time transcutaneous ovarian ultrasound at 4 hour intervals; while the staging of the GV group was based on detection of standing estrus. A terminal hysterectomy was performed, and flushing of the oviduct and lower, middle, and upper thirds of the uterine horns relative to cervix carried out. Tissue samples from each of the uterine sections along with the ampula, isthmus and ampulary-ismic junction were collected and flash frozen in liquid nitrogen, while embryos were isolated from the flushings, imaged and frozen in pools of five embryos at like stages. HA content in each segment was determined through quantitative ELISA and concentrations adjusted to account for flush volume. Total RNA was extracted from tissue samples and embryos, DNase treated, and analyzed using real time PCR for determination of relative gene expression of HAS1, 2 and 3 and CD44 using 18S as an endogenous control. Expression was analysed using the ΔCT method for endometrial samples, and the residuals of 18S as a covariate for embryos. ELISA data was log transformed for normality and all data was analysed for temporal and spatial variation using the general linear model of SAS. Minimal spatial variation was found in the post-ovulatory stages, however temporal differences in HA concentration and gene expression were identified. ELISA results show elevated concentrations of HA at the GV stage that decrease rapidly at the 2cell stage and then increase toward the blastocyst stage. HAS1 and HAS3 were identified as the predominantly transcribed synthase forms in both the reproductive tract and embryos, while low level expression of HAS2 was detected in the reproductive tract irrespective of embryonic stage. Synthase gene expression appears to be reciprocal between the uterus and embryos, with highest expression peaking at the GV and 4 cell stages respectively. These results suggest HA does not play a significant role in the development of spatial asynchrony in the reproductive tract, but may play a significant role in temporal asynchrony. Furthermore, the complex interplay between embryonic and uterine expression profiles suggests an extracellular signaling role for HA during early development.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Expression of Human Erythropoietin Gene in Bovine Somatic Cell Nuclear Transfer Embryos.

JY Lee 1, EJ Jung 1, SH Jeong 1, CJ Yang 1, YK Kim 1, EM Jung 2, YW Jeong 1, SH Hyun 1, T Shin 1, EB Jeung 2, WS Hwang 1

Erythropoietin (EPO) is a glycoprotein hormone that controls erythropoiesis. As current methods of EPO production cannot satisfy the demand, cloned animals to be used as bioreactors can help patients with chronic anemia caused by kidney dysfunction. This study was performed to produce human EPO-transgenic cloned cows. Somatic cell nuclear transfer was performed with a vector that was created to induce production of hEPO in cow's milk. AlphaS1-casein was selected as the promoter to be used in this study through the specific promoter activity test, and enhanced green fluorescent protein(EGFP) gene was attached to the CMV promoter to allow observation of the donor cell during the experiment. Two cell lines(calf and adult fibroblast cells) were transfected with lipofectamine. After G418 selection, transfection efficiency (EGFP expression) was 27.3% and 50.0% for calf and adult cells, respectively. The transfected cells were injected to the enucleated oocytes, and injected embryos were accomplished by cell to cell fusion. These embryos were then activated with calcium ionomycin and 6-dimethylaminopurine. Comparing the efficiency of calf and adult fibroblast cells as nuclear transfer donor cells, there were no significant differences in the fusion rate(73.0±10.0% vs. 71.6±12.1%), but in the cleavage rate(63.1±11.7% vs. 56.3±17.1%) and blastocyst rate(16.4±9.0% vs. 14.6±8.0%), the calf cell showed better effects on the embryos(P < 0.05). GFP was expressed in 92.3±2.8% of the blastocysts formed(Calf: 90.4%±3.2 vs Adult: 95.3±2.2%) and the total cell number of blastocysts except the ones used for embryo transfer and DNA sampling was 102.4±22.3(Calf: 99.8±21.4 vs. Adult: 104.6±23.2). We confirmed the successful integration of hEPO genes and EGFP in bovine embryos through polymerase chain reaction (PCR). As the calf fibroblast cell was more efficient than the adult fibroblast cell in nuclear transfer embryo development, we used the calf fibroblast cell as a donor cell for embryo transfer. A total of 58 blastocysts were transferred to 29 surrogate mothers and 4 recipients were found to be pregnant. After a calf is born, microsatellite analysis for determining transgenicity and functional analysis for measuring hEPO in the milk are scheduled.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

NOT PRESENTED.

NOT PRESENTED.

Biol Reprod. 2010 Nov;83(1 Suppl):2.

The Potential of the Oocyte In Vitro Maturation System for the Reproductive Toxicology.

Anna R Beker van Woudenberg, Mariska Mulderij, Andre Wolterbeek, Rob Stierum 1

There is a rising concern about the effects of environmental contaminants (EC), many of them estrogen-like substances, on the reproductive function of animals and humans. Although standard screening assays for EC are available, they might not reveal the full physiological relevance of the potential harm of those compounds with respect to oocyte maturation and fertilization. Moreover, to screen on reproductive and developmental toxicity, a substantial amount of experimental animals has being used. The immature oocyte (in germinal vesicle stage) is a very susceptible cell, considering that it is still needs to complete the final stages of nuclear and cytoplasmic maturation before it can properly support fertilization and the early stages of embryo development. Previously, we demonstrated by using the in vitro oocyte maturation system (IVM), that even physiological concentrations of 17beta estradiol can be deleterious for the final maturation of oocytes (malformation of the meiotic spindle) if those oocytes were exposed in an wrong window of exposure. Therefore, it can be expected that, a chronic exposure of woman to environmental estrogens may affect spindle formation and chromosome segregation in vivo as well. Our hypothesis is that the in vitro oocyte maturation system could contribute for an integrated testing strategy (ITS) for screening chemicals that affects female fertility. Furthermore, by using ovaries from slaughterhouse waste, it could also contribute to reduce the number of experimental animals being used for those studies. We have selected compounds with a known mechanism of action (i.e. cycloheximide, estradiol, DES, cadmium chloride) to be tested in this system. As the first step to investigate this proof of principle, we cultured bovine oocytes, harvested from slaughterhouse ovaries, in a M199 media at 39°C during 22 h , under 5%CO2 and in the presence of the following concentrations of cycloheximide (CHX),a known protein synthesis inhibitor: 0.75; 0.60; 0.45; 0.30; 0.15 and 0 µM. The toxicological endpoint was the ability of the oocyte to reach the metaphase II stage (i.e. the final stage of maturation). In addition, we investigated the general cytotoxicity of CHX by using MTT-test. Our results show no general cytotoxicity for CHX in all of the concentration used. The meiosis was inhibited in a dose-response manner and the EC50 for CHX was 0.55µM, much lower than the concentration used to block protein synthesis in somatic cells,indicating the high sensivity of the oocyte to express toxicity. The following step is to extend this investigation to the other selected compounds and to combine functional parameters with the use of proteomics and transcriptomics to obtain mechanistic insights in oocyte maturation and the effect of chemicals hereon. It is expected that markers derived, along with functional parameters, can form a powerful assay for the in vitro prediction of reproductive toxicity. In conclusion, we do believe that the use of the IVM assay can grant a powerful and physiological model in an ITS of effects of chemicals on female fertility. Research supported by TNO Research Program on Hazardous Substances and Occupational Safety; and The Netherlands Toxicogenomics Center.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Evidence That Mammary Gland Infection/Injury During Pregnancy in Dairy Cows May Have a Negative Impact on Size of the Ovarian Reserve in Their Daughters.

James J Ireland 1, Danielle M Scheetz 1, Fermin Jimenez-Krassel 1, Joseph K Folger 1, George W Smith 1, Francesca Mossa 2, Alexander CO Evans 2

The effect of disease on maternal environment, which has a crucial role in embryo development and subsequent health of offspring, has not been examined extensively in cattle. Therefore, the purpose of this study was to determine if a persistent mammary gland infection such as mastitis and correspondingly a chronically high (> 200,000 units) somatic cell count (SCC) in milk of pregnant dairy cows has a potential negative impact on size of the ovarian reserve (total number of healthy follicles and oocytes in ovaries) and potentially fertility of their offspring. This study was conducted at Green Meadow Farms Inc. in Elsie, MI and used 192, 12-mo-old Holstein heifers. Each animal was injected with 2 injections of prostaglandin F2 alpha (PG) spaced 11 d apart. To assess relative size of the ovarian reserve in each heifer, a single blood sample was removed 4 d after the 2nd PG injection and assayed for concentration of anti-Müllerian hormone (AMH), and ovarian ultrasonography was used to determine number of follicles ≥ 3 mm in diameter and ovary size. Results confirm previous observations that AMH concentrations were highly variable among animals but positively correlated (P < 0.001) with both ovary size and number of antral follicles. To determine if a chronic mammary gland infection during pregnancy of dairy cows was associated with alterations in AMH concentrations and correspondingly size of the ovarian reserve in their offspring, we next tested whether number of SCC measurements > 200,000 for each heifer's dam was associated with alterations in the serum AMH concentration in each heifer. Number of SCC measurements > 200,000 per individual cow is an accurate index of recurrent udder infections, injury and/or inflammation. A total of 5 to 7 measurements of SCC were made for each dairy cow beginning 2 mo before pregnancy and continuing throughout pregnancy. Number of SCC measurements > 200,000 ranged from 0 to 5 per individual cows, thus cows were separated into groups based on their number of SCC measurements > 200,000. Cows with 4 or 5 SCC measurements > 200,000 were ~2 years older (P < 0.01, 6.1 vs 4.4 yrs old) and tended (P < 0.09) to produce less milk compared with cows that had 0 to 3 SCC measurements > 200,000. Results showed that the daughters of cows with 4 and 5 SCC measurements > 200,000 also had markedly lower (P < 0.02) AMH concentrations as young adults compared with daughters of the cows with 0 to 3 SCC measurements > 200,000. Although mechanisms are unclear, our results imply that a chronic mammary infection or injury during pregnancy of older cows (as predicted by a high number of SCC measurements > 200,000) is associated not only with low milk production, but also with diminished size of the ovarian reserve and perhaps reduced reproductive performance (fertility and reproductive lifespan) of their daughters. Research supported by USDA-NRI 2004-01697, 2007-01289 to JJI.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Mono-(2-ethylhexyl) Phthalate Inhibits Expression of Antioxidant Enzymes in Mouse Antral Follicles.

Jeffrey M Singh, Rupesh K Gupta, Jodi A Flaws 1

At birth, the ovaries contain a finite number of primordial follicles. Only a limited number of these primordial follicles will mature into antral follicles. Antral follicles are essential for the proper functioning of the ovary because they synthesize and secrete sex steroid hormones important for the maintenance and regulation of estrous cyclicity and fertility. Di-(2-ethylhexyl) phthalate (DEHP) is a plasticizer found in many commercially used products such as plastic bottles, IV tubes, toys, fragrances and cosmetics. DEHP, a toxic parent compound, can be metabolized by esterases in the body to form mono-(2-ethylhexyl) phthalate (MEHP). MEHP is known to restrict growth, reduce estradiol levels and cause death of antral follicles via apoptosis. Many pathways can lead to cell death via apoptosis, but oxidative stress is a pathway specifically known to cause apoptosis in both non-reproductive and reproductive tissues. Balance between reactive oxygen species production and detoxification by antioxidant enzymes such as Cu/Zn superoxide dismutase (SOD1), glutathione peroxidase (GPX), and catalase (CAT) keep the cell in a healthy and reduced state. Any environmental toxicant exposure that disrupts this balance results in oxidative stress in the cell. Since it was not known if MEHP induces oxidative stress in antral follicles, the goal of this study was to test the hypothesis that MEHP induces the oxidative stress pathway by suppressing the expression of SOD1, GPX and CAT in antral follicles. To test this hypothesis, antral follicles were mechanically isolated from adult cycling CD-1 mice and cultured for 96 hours in alpha-minimal essential media containing either MEHP (0.1-10 µg/ml) or the vehicle control, dimethylsulfoxide (DMSO). Following culture, follicles were subjected to quantitative real time PCR to measure mRNA levels of the Sod1, Gpx, and Cat genes. The results indicate that MEHP significantly decreased mRNA expression of Sod1 compared to controls (DMSO=67.33±8.51 genomic equivalents (GE), MEHP 0.1 µg/ml=24.03±7.73 GE, MEHP 1 µg/ml=19.03 ±4.25 GE, MEHP 10µg/ml=18.73 ±9.41 GE; n=3; p≤0.05). In addition, MEHP significantly decreased mRNA levels of the Gpx gene compared to controls (DMSO=29.16±3.99 GE, MEHP 0.1 µg/ml=3.69±0.45 GE, MEHP 1 µg/ml=4.90±2.04 GE, MEHP 10 µg/ml=2.47±0.60 GE; n=3; p≤0.05). However, the expression levels of Cat were not significantly affected by MEHP exposure compared to controls (DMSO=9.78±0.81 GE, MEHP 0.1 µg/ml=3.85±2.13 GE, MEHP 1 µg/ml=3.47±1.21 GE, MEHP 10µg/ml=4.47±1.77 GE; n=3). Therefore, it is possible that MEHP represses the expression of specific antioxidant enzymes, allowing apoptosis of antral follicles via the oxidative stress pathway. Support: R01ES012893; T32ES07326.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Bisphenol A Alters Steroidogenic Acute Regulatory Protein and Progesterone Receptor Gene Expression in the Mouse Ovarian Antral Follicle.

Jackye Peretz, Isabel Hernandez-Ochoa, Jodi A Flaws 1

Bisphenol A (BPA) is a chemical which is used as the backbone for plastics and epoxy resins, especially those used in various food and beverage containers, including water bottles, frozen entrees, and the lining of food and beverage cans. In a government funded human study (NHANES), BPA, with a half-life of 4-6 hours, was present in 95% of random urine samples, indicating that humans are constantly exposed to this chemical. BPA has also been detected in ovarian follicular fluid of adult women. Few studies have investigated the effects of BPA on antral follicles, the main producers of sex steroid hormones in the female and the only follicles capable of ovulation. Previous studies in our lab have shown that exposure to BPA inhibits antral follicle growth, induces atresia, and decreases sex steroid hormone production in antral follicles. The current study was designed to expand these previous findings by testing the hypothesis that BPA alters the mRNA expression level of sex steroid hormone receptors in antral follicles. In addition, this study tested whether BPA alters expression of enzymes required for sex steroid biosynthesis in antral follicles. To test these hypotheses, antral follicles were mechanically isolated from adult cycling FVB mice, individually cultured for 120h in supplemented α-minimum essential medium, and treated with either vehicle control (DMSO) or various concentrations of BPA (0.4 µM, 4.4 µM, or 44.4 µM). After 120h, follicles were collected and RNA was extracted, transcribed into cDNA, and subjected to real-time PCR for analysis of estrogen receptor alpha (Esr1), estrogen receptor beta (Esr2), androgen receptor (Ar), progesterone receptor (Pgr), steroidogenic acute regulatory protein (Star), 3beta-hydroxysteroid dehydrogenase (3βHsd), and cytochrome P450 17alpha (Cyp17alpha). Exposure to BPA (44 µM) significantly increased the expression levels of Pgr mRNA in antral follicles compared to DMSO controls (DMSO: 0.66 ±0.14 ge; BPA 44.4µM: 10.0 ± 3.3 ge; genomic equivalent (ge); n=3; p =0.019). In contrast, BPA exposure (44 µM) significantly decreased the expression levels of Star mRNA in antral follicles compared to DMSO controls (DMSO: 0.39 ± 0.08 ge; BPA 44.4 µM: 0.09 ± 0.03 ge; n=3; p =0.014). BPA exposure did not affect the other selected sex steroid hormone receptors or enzymes in antral follicles. Collectively, these data indicate that BPA may alter some enzymes and receptors important for sex steroid biosynthesis and responsiveness. It is possible that BPA-induced changes in gene expression may mediate the ability of BPA to inhibit follicle growth, decrease sex steroid hormone production, or induce atresia in the ovarian antral follicle. Supported by NIH 1P20ES018163.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Acute and Chronic Exposure to Bisphenol A Disrupts Ovarian Follicle Maturation in Sprague-Dawley Rats.

Kaitlin L Knackstedt, Jennifer M Reinhart, Monika G Baldridge 1

Bisphenol A (BPA) is a ubiquitous man-made chemical capable of endocrine disruption. Globally more than 6.4 billion pounds of BPA are manufactured yearly, making BPA one of the most widespread and persistent industrially produced compounds capable of altering endocrine homeostasis. Several studies have shown that unregulated disposal of products containing BPA has resulted in global environmental contamination; therefore, making BPA responsible for the majority of the estrogenic activity that leaches from landfills. BPA is a monomer in polycarbonate plastics such as PVC pipes, dental sealants and fillings, and the resins used to line food and drink containers (including baby bottles). Chemical Safety Reports claim that BPA is safe in plastic food containers if they are used as intended. Yet, several studies have documented that exposure to even low doses of BPA causes multiple health problems including irregular reproductive tissue development. The deleterious impact of BPA on the mature male and female reproductive tracts have been established and well documented; however, the data remain especially limited regarding reproductive effects in offspring. This study was designed to determine (1) whether an in-utero, lactational and chronic exposure to a low or high dose of BPA (LBPA, HBPA) has deleterious effects on ovarian follicle maturation in the Sprague-Dawley rat; (2) if circulating estrogen and progesterone concentrations are altered following acute or chronic exposure; and (3) if estrous cycles are modulated during chronic exposure. Animals receiving an acute exposure were treated in utero and lactationally from gestational day 7 through postnatal day 28 (weaning). Animals receiving a chronic exposure were treated directly for an additional 65 days. Acute- and chronic-exposure groups were treated daily via oral gavage with LBPA (10mg/kg/day) or HBPA (100mg/kg/day). Acutely exposed pups were sacrificed on postnatal day 28 (chosen in order to follow the pups' exposure through lactation). Chronically exposed rats were sacrificed on postnatal day 93. Following sacrifice; the ovaries were excised, fixed for histology, and analyzed. The analysis included a count, and measurement and classification of preantral and antral follicles in the greatest cross-sectional area of the ovary. Trunk blood collected at the time of sacrifice was analyzed for estrogen and progesterone concentrations via radioimmunoassay. No statistical change in estrogen (p=0.747) or progesterone (p=0.672) concentrations was noted among acute-treatment groups. Radioimmunoassay results for the chronically exposed groups indicated a significant difference in estrogen (p=0.034) and progesterone (p<0.001) concentrations between the treatment and control groups. Increased atresia levels and a decreased number of follicles were observed in the LBPA and HBPA treatment groups (p<0.05). Modulation of estrous cycles was noted in the HBPA treatment group. These data support the hypothesis that BPA reduces the number of follicles of certain size classes in rats exposed during the critical period of development and during chronic exposure after birth. This study begins to decipher the acute and chronic effects of BPA on reproductive parameters in the female Sprague-Dawley rat.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

In Vivo and In Vitro Studies on Nickel Induced Reproductive Toxicity and the Protective Role of Melatonin.

Niraj R Joshi 1, Sudip Banerjee 1, Raktim Mukherjee 1, Shail Kumar Chaube 2, TG Srivastava 3, AV Ramachandran 1

Male reproductive toxicity of Nickel and putative protective role of melatonin have been assessed in 180 days old wistar rats. Ni was administered through drinking water and, melatonin (18:00 hrs) intraperitoneally for 60 days. On 61st day animals were sacrificed and testis was taken for estimation of tissue metal load by ICP-AES and biochemical estimation of antioxidants (both enzymatic- CAT, SOD, GPx, GR and non-enzymatic GSC, Ascorbic acid) together with level of lipid peroxidation. Serum titres of steroids and melatonin and activity of testicular steroidogenic enzymes (3β and 17β HSDH) were measured along with epididymal sperm parameters(Sperm count, motility and abnormality). Further, studies were extended to assess the effect of Ni on in vitro testosterone production by isolated rat Leydig cells under basal and stimulated conditions. Results of the present study reveal increased tissue metal content following Ni treatment. Ni induced oxidative stress in testis is marked by increased lipid peroxidation and depletion and inhibition of endogenous non-ezymatic and enzymatic antioxidants respectively. Marked inhibition of testicular steroidogenic enzymes was reflected in the form of altered serum titres of testosterone and estradiol in Ni administered animals. Disruption of spermatogenesis is marked by loss of sperms, decreased number of motile sperms and increased abnormal spermatozoa in the Ni treated group. Isolated Leyding cells incubated with Ni showed decreased testosterone secretion under both basal and hCG stimulated conditions. In conclusion, the present study has shown Ni induced oxidative stress and deleterious effects on steroidogenesis and adverse quantitative and qualitative effects on sperm. Melatonin has been found to be effective in preventing these effects to a greater extent though not completely.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Exposure to Bisphenol A Modulates Hormone Concentrations in Gammarus pseudolimnaeus.

Susan E Lewis, Angela M Yokofich, Rebecca Giuliani, Cassandra Kurth, Megan Mohr, Monika G Baldridge 1

Bisphenol A (BPA) is a pervasive anthropogenic chemical used to construct polycarbonate plastics. Worldwide more than 2.2 million metric tons of BPA are produced each year and 100 tons is released into the atmosphere. Several studies have shown that unregulated disposal of products containing BPA has resulted in global environmental contamination, making BPA responsible for the majority of the estrogenic activity that leaches from landfills into the ecosystem. The effluents from sewage treatment plants introduce BPA into rivers and streams where gammarid amphipods are often the most prevalent invertebrates. Gammarus pseudolimnaeus can provide a powerful invertebrate model system to investigate the endocrine-disruptive capabilities of BPA. However, techniques to assay hormone concentrations in amphipods have not been well developed. This study was designed to (1) quantify estrogen concentrations in juveniles and in adult female amphipods; (2) determine testosterone concentrations in juveniles and in adult male amphipods; and (3) delineate the effects of BPA on estrogen and testosterone concentrations following exposure. Amphipods were collected from a local stream that has high water quality and categorized as juvenile or adult based on size. Adults were subsequently sexed based on external morphology. Animals were assigned to one of four treatment groups: control (0 ug/L BPA), 10 ug/L BPA, 50 ug/L BPA and 100 ug/L BPA. Animals were housed in groups of 5 (adults) or 10 (juveniles) in BPA-free containers, and BPA in solution was added to each experimental container every 3 days over a 9 day treatment period. At the conclusion of the trials, amphipods were sacrificed, homogenized with protosol, and frozen at -80°C. Tissue extracts from homogenized amphipod samples were analyzed for estrogen or testosterone concentrations via radioimmunoassay. Hormone concentrations were quantified for each reproductive class. BPA was found to modulate hormone concentrations across treatment levels. The results of this study begin to elucidate the effects of BPA on reproductive hormones in freshwater amphipods, a new model system. Information gleaned from this and future studies may have profound implications for invertebrates and vertebrates since amphipods are central to many aquatic food webs.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Exposure of Pregnant Mice to Cadmium Oxide (CdO) Nanoparticles (NP) Poses a Risk to the Developing Offspring.

Jason L Blum, Carol Hoffman, Judy Q Xiong, Judith T Zelikoff 1

There has been and will continue to be exponential growth of the nanotechnology industry; by 2012, the industry is projected to reach $4.5 billion and exceed $1 trillion by 2015. Nanosized particles of cadmium oxide (CdO) serve as the starting point in the manufacture of quantum dots. Applications for CdO nanocrystals also include flame retardant coatings, plastics, fiber and textiles, and in certain alloy and catalyst applications. Further research is being pursued to determine their potential use in electrical, magnetic, optical, and other bioscience applications. Despite the fact that there is continuing interest by regulatory agencies and ~5 million women in the United States are employed in the manufacturing sector (of which 75% are of reproductive age), virtually nothing is currently known about the potential deleterious effects of nanomaterials, particularly for female reproductive health and fetal development. Thus, a study was carried out using pregnant CD-1 mice. The overall goal of which was to determine whether CdO nanoparticles (NP; ~11 nm in diameter), inhaled (nose-only) at a concentration of 100 μg/m3 every other day (2.5 hours per day) for a total of 18 days, could reach the placenta, ultimately, leading to obstetric consequences such as low birth weight and/or restricted development in utero. Analysis of maternal and fetal tissue (by atomic absorption spectroscopy) revealed that pulmonary and blood Cd levels in exposed dams were 9.6 µg Cd/mg per mg of tissue and 211.2 ng Cd/ml, respectively, while uteri and placentas averaged 34.3 and 43.3 ng Cd/g of tissue, respectively. In contrast, the uteri of virgin mice exposed via the same inhalation route to CdO had no measurable Cd levels. In all cases, Cd levels from control mice were below detectable limits. Inhalation exposure of CdO NP at a higher concentration (550 µg/m3) more than doubled the incidence of resorbed/resorbing fetuses and litter sizes at gestational day 17.5 were decreased compared to pregnant mice receiving only filtered air. These data show, for the first time, that inhaled nanoparticles of any type can reach the placenta and lead to an environment less favorable for the developing fetus. Supported by NYU NIEHS Center Pilot Project and NIEHS RO1 ES017427-01.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

In Vitro Risk Assessment of a Nanoparticulate Formulation of Arsenic Trioxide in Isolated Ovarian Follicles Is Representative of Effects In Vivo.

Susan L Barrett 1, Richard W Ahn 2, Meera R Raja 2, Jennifer K Jozefik 1, Lidia Spaho 1, Haimei Chen 2, Marcel Bally 3, Michael J Avram 1, Leo I Gordon 1, Lonnie D Shea 1, Thomas V O'Halloran 2, Teresa K Woodruff 1

We report here the first methods to assess the fertotoxicity of chemotherapeutic drugs in both in vitro and in vivo systems. Using a novel nanoparticulate formulation of arsenic trioxide (ATO), [NB(Ni,As)] encapsulated in liposomal vesicles, that has preclinical efficacy in both lymphoid and breast tumors, ovarian follicles were assessed for uptake of free arsenic and was shown to have lower arsenic content than using ATO alone. Arsenic induced toxicity to the female reproductive tract was examined using isolated, ovarian, secondary follicles embedded in a degradable fibrin-alginate matrix and found that healthier follicles degraded fibrin at an advanced rate compared to unhealthy follicles seen after ATO treatment. The serum and ovarian pharmacokinetics were measured for NB(Ni,As) and ATO in mice and while the serum levels (AUC) are drastically increased for NB(Ni,As) compared with ATO, the ovarian uptake is reduced. Extended dosage studies revealed that the NB(Ni,As) was not detrimental on the ovary unlike ATO, which resulted in constant diestrus. Ovarian tissue displayed normal histology from NB(Ni,As) treated mice however ATO treatment revealed numerous hemorrhagic ovarian cysts. In short, we have validated a novel series of assays to determine the fertotoxicity of chemotherapetuic agents, and demonstrated that the NB(Ni,As) has reduced toxicity to the female reproductive tract in mice compared with free drug. This is the first known test for chemotherapeutic fertotoxicity and could be adapted for use in human to aid in the development of fertoprotective chemotherapeutics.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

DNA Methylation and Histone Acetylation Status in the Promoter Region of Insulin-Like Growth Factor-Binding Protein-1 (IGFBP-1) and Prolactin (PRL) in Human Endometrial Stromal Cells.

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

A number of genes are up-regulated or down-regulated in human endometrial stromal cells (ESC) undergoing decidualization. Epigenetic mechanisms including DNA methylation and histone modification play key roles in the gene transcription. DNA methylation at the CpG site in the promoter region and histone acetylation of the promoter are associated with inactivation and activation of transcription, respectively. Epigenetic regulation is also involved in cell-/tissue-specific gene expression. It is interesting to note that insulin-like growth factor binding protein-1 (IGFBP-1) and prolactin (PRL) are preferentially expressed in ESC undergoing decidualization, and are recognized as specific markers of decidualization. The present study investigated the involvement of epigenetic mechanisms in the regulation of IGFBP-1 and PRL gene expressions in ESC undergoing decidualization. This study was approved by IRB of Yamaguchi University and informed consents were obtained by all patients. ESC isolated from the proliferative endometrial tissue were incubated with dibutyryl-cAMP (cAMP, 0.5 mM) for 4 days. IGFBP-1 and RPL mRNA expressions were induced by cAMP accompanied by decidualization. We first examined whether IGFBP-1 and PRL expressions are induced by cAMP in the cells that are not derived from the endometrium. For this purpose, skin fibroblast cells (FBC), which are derived from human skin and do not express IGFBP-1 and PRL, were cultured with cAMP for 4 days. PRL mRNA expression, but not IGFBP-1 was induced cAMP in FBC. Histone acetylation and DNA methylation status of the IGFBP-1 and PRL promoters were analyzed in both cells by ChIP assay and sodium bisulfite genomic sequencing method, respectively. Histone acetylation status of the IGFBP-1 promoter was lower in FBC compared with ESC. Both cells showed similar hypomethylation status of the IGFBP-1 promoter. There were no differences in histone acetylation and DNA methylation status of the PRL promoter between both cells. To investigate the functional role of histone acetylation of the IGFBP-1 promoter in the regulation of IGFBP-1 mRNA expression, FBC were cultured with cAMP in the presence or absence of a histone deacetylase (HDAC) inhibitor that induces histone acetylation. Treatment with cAMP in the presence of an HDAC inhibitor induced IGFBP-1 mRNA expression with the increased histone acetylation level in FBC. These results suggest that histone acetylation status of the IGFBP-1 promoter is a critical factor for IGFBP-1 mRNA expression in the presence of transcriptional factors. ESC and decidualized ESC induced by cAMP treatment showed similar DNA methylation status and histone acetylation status of the IGFBP-1 and PRL promoters. DNA methylation status of both gene promoters in the endometrium was hypomethylated DNA status in the proliferative phase, secretory phase, and early pregnancy (decidua). These results suggest that DNA methylation status and histone acetylation status of both gene promoters are not affected by decidualization in ESC. In conclusion, the promoter regions of IGFBP-1 and PRL showed hypomethylated DNA status and high histone acetylation status in human ESC, suggesting the loose chromatin structure of the promoter regions, which allows transcription factors to bind the response element of the promoter when decidualization-related transcriptional factors are recruited.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

The Histone Methyltransferase EZH2 Adopts a Nuclear Localization During Cleavage in Porcine Embryos Fertilized In Vitro.

Celeste E Morrison, K Park, X Wang, R A Cabot 1

Histone methyltransferases play a vital role in early mammalian embryo development by regulating chromatin structure and gene expression. Methylation of the lysine 27 residue of histone H3 (H3K27) is closely linked with repression of transcription. The histone methyltransferase known to be responsible for methylating H3K27 is Enhancer of Zeste (EZH2). Our lab has previously shown that the global patterns of H3K27 methyation undergo reorganization during cleavage development in the porcine embryo. Specifically, H3K27 trimethylation is present on chromatin in mature porcine oocytes, pronuclear and 2-cell stage embryos, with H3K27 trimethylation decreasing at the 4-cell stage and H3K27 trimethylation not detectable in blastocyst stage embryos. The purpose of this study was to determine the intracellular localization of the EZH2 protein in pronuclear, 2-cell and 4-cell stage porcine embryos. Because H3K27 methylation undergoes global changes during cleavage development, we hypothesized that EZH2 protein would adopt a nuclear localization during cleavage development. To test this hypothesis, the porcine version of EZH2 was subcloned into an expression vector to generate an N-terminal GFP fusion protein; mRNA was synthesized in vitro and injected into porcine oocytes that were fertilized in vitro. Immature porcine oocytes were harvested from prepubertal gilt ovaries and matured in a chemically defined medium (TCM-199 supplemented with 0.1% PVA, 0.069 mg mL-1 cysteine, 10ng mL-1 EGF, 0.5 IU mL-1 LH and FSH) for 42-44 hours at 39°C in 5% CO2). Following removal of cumulus cells, oocytes were placed in a Tris-buffered medium containing 1mg/ml BSA and 1mM caffeine and co-incubated with boar sperm (5 x 105 sperm per ml) for 5 hours. Fertilized oocytes were then injected with GFP-EZH2 mRNA (concentration of 1 µg µL-1) using a FemtoJet microinjector (Eppendorf, Hamburg, Germany); successfully injected oocytes were cultured in PZM medium supplemented with 3mg mL-1 BSA. Embryos were removed from culture at the following times following gamete mixing to determine the intracellular localization of ectopically expressed GFP-EZH2: 12, 24 and 48 hours for pronuclear, 2-cell and 4-cell stages, respectively. Upon removal from embryo culture medium, embryos were stained with Hoeschst33342 (1 µg µL-1 for 15 mintues) in order to visualize nuclear DNA. The location of the fusion protein was determined by either direct epifluorescent microscopy on live cells (2-cell and 4-cell stage embryos), or immunocytochemically in cells fixed in 3.7% paraformaldehyde and probed with an antibody directed against GFP (pronuclear stage embryos). Our results revealed that GFP-EZH2 was localized in the nuclei of the majority of pronuclear stage embryos (n=29/38), the remaining embryos showed ubiquitous distribution of GFP-EZH2 throughout the entire embryo (GFP detected in equal amounts in both nuclear and cytoplasmic compartments). GFP-EZH2 was also demonstrated a predominant nuclear localization in 2-cell (n=2/2) and 4-cell (n=8/13) stage embryos; the remaining 4-cell stage embryos showed ubiquitous distribution of GFP-EZH2 throughout the entire embryo (GFP detected in equal amounts in both nuclear and cytoplasmic compartments). These results demonstrate that EZH2 has the ability to localize to the nucleus and in cleavage stage embryos and therefore likely serves a role in modifying histone proteins at these developmental time points.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Effects of Embryo Transfer and Methyl-Enriched Maternal Diet on Fetal Outcomes in Viable Yellow (Avy/a) and a/a C57Bl6J Mice.

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

In viable yellow (Avy/a) mice, which have an intracisternal A particle (IAP) embedded in the Agouti locus, exposure throughout gestation to a maternal diet enriched in methyl donors results in methylation of IAP site and increased number of pseudoagouti coat color offspring. Yellow coat color offspring with a demethylated IAP site develop diabetes and obesity but their C57 genetically identical siblings with a darker coat color remain healthy. No previous study has examined DNA methylation patterns in offspring exposed to the methyl supplements just after fertilization, i.e. at a time analogous to when most pregnant women might begin to consume high amounts of folic acid supplement, and whether in utero exposure to methyl components can alter global DNA methylation patterns in a/a (black) mice. We bred a/a females (maintained on NIH-31 control diet) to Avy/a males. On morning of coital plug, 1-cell embryos were collected from donors and transferred into the oviducts of surrogate dams maintained on the methyl-supplemented diet, 3MS ZM, or NIH-31 control diet (Harlan-Teklad, Madison, WI). Surrogate dams were "bred" to vasectomized males and examined morning of the transfer for a coital plug. Recipient females were permitted to give birth, and the coat colors, body weights, and serum glucose concentrations of the resulting offspring were analyzed. Tail DNA was isolated from a/a black females, restriction digested with Mse I to yield DNA fragments with intact CpG islands, and immunoprecipitated with anti- 5-methyl cytidine antibody. Labeled and total input DNA samples were co-hybridized to Nimblegen Methylation, Mouse, 2.1M whole-genome tiling arrays (Roche-Nimblegen, Madison WI). Our preliminary results suggest that post-fertilization exposure to methyl-enriched diet is insufficient to alter coat color in Avy/a offspring. Post-fertilization exposure to the methyl-supplemented diet did not affect overall weight of Avy/a and a/a female and male offspring compared to respective controls. From weaning to 9 weeks of age, Avy/a daughters in the methyl and control groups consistently weighed more than a/a daughters in both groups (P<0.05). Surprisingly, a/a and Avy/a male offspring, who were exposed to the methyl-enriched diet in utero, had increased average serum glucose concentrations from 4 to 9 weeks of age compared to a/a and Avy/a counterparts exposed to control diet (Methyl Diet: a/a-186.6 ± 10.6, Avy/a- 212.0 ± 8.5; Control Diet: a/a-144.1 ± 10.0; Avy/a- 181.8 ± 10.0 mg/dl, P<0.05). Conditional heat maps based on tail DNA methylation patterns for each chromosome revealed a remarkably consistent pattern across the genome, namely that the daughters from three surrogate dams on the methyl diet formed one distinct cluster and two of three daughters from three control dams formed a separate cluster. Substantially more genes were methylated in daughters born to surrogate dams on methyl diet compared to those from dams on control diet (3,483 hyper-methylated genes for methyl daughters vs. 139 hyper-methylated genes for control daughters, P<0.05). Examples of genes that were hypermethylated in a/a methyl daughters include Ar, Xist, Tsix, and Dnmt3b. In utero exposure of a/a and Avy/a offspring to a methyl-enriched diet can result in substantially increased DNA methylation that could, in turn, modulate the risk for later adult-onset diseases in the exposed offspring. Research supported by an American Institute for Cancer Research (AICR) grant.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Mice Deficient in a Drosophila Homeotic Selector Ortholog Exhibit Female Infertility and Reduced Male Fertility.

Sally A Camper, Michelle L Brinkmeier, Krista A Geister, Morgan Jones, Ivan Maillard 1

One in seven couples have problems conceiving, and genetic abnormalities may account for 15% of the cases. Quantitative and structural chromosomal aberrations such as Turner's syndrome and fragile X are the most common known genetic causes of female infertility, and much remains to be learned about the effects of individual genes and epigenetic regulation on reproduction in women. We report female infertility and reduced fertility in males homozygous for mutations in Ash1l, (absent small or homeotic)-like 1. Ash1l is an ortholog of the Drosophila gene ash1, which is a histone methyltransferase that regulates the expression of the Hox gene cluster. We generated Ash1l knockout mice to test the role of Ash1l during development. Homozygous mutants have a pleiotropic phenotype that includes poor viability and involvement of the hematopoietic and immune systems. Ash1l mutant females have hypoplastic uteri and very few stromal glands. All stages of the estrous cycle can be identified in Ash1l mutant females, and fertilized eggs from mutant females can survive to birth in pseudopregnant wild type females, suggesting that the uterine defect as the source of infertility. Hoxa11a mutant mice have similar uterine defects, supporting the hypothesis that ASH1L regulates Hoxa11 in intact animals. Ash1l mutant males exhibit reduced fertility yet display normal testis morphology as well as normal sperm numbers and motility. Mutant males exhibit malformations in the formation of the vas deferens similar to the homeotic transformations seen in both Hoxa10b and Hoxa11c mutant males. These studies demonstrate a role for ASH1L in the development of the female and male reproductive tracts and suggest a role for ASH1L in the regulation of Hox gene expression in vivo. Research supported by NIH grant R37-HD30428.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Establishment of a Phenotypical Model of Adverse Outcomes Associated with Assisted Reproductive Technologies.

Katherine M Robbins 1, Kevin D Wells 1, Tom Geary 2, Chad O'Gorman 1, Michael D MacNeil 2, Michael F Smith 1, Ky Pohler 1, Emma Jinks 1, Rocio Melissa Rivera 1

Genomic imprinting is an epigenetic modification that directs parent-specific gene expression. Imprinted genes are involved in regulating growth and development of the conceptus (fetus and placenta). Beckwith-Wiedemann Syndrome (BWS) is an overgrowth condition that is associated with loss-of-imprinting in humans. BWS has been shown to have an increased incidence in children conceived by the use of assisted reproductive technologies (ART). Currently, there are no animal models that recapitulate the ART-induced human overgrowth phenotype. As in humans, ART can induce an overgrowth syndrome in ruminants which is phenotypically similar to BWS. In ruminants this syndrome is called large offspring syndrome (LOS). We hypothesize that BWS and LOS share epigenetic origins. The goal of our research is to determine the baseline methylation and allele-specific expression in bovids of imprinted loci known to be misregulated in BWS as a result of minimal ART procedures. First, DNA was isolated from Bos taurus taurus (Angus, Hereford, Holstein breeds) and Bos taurus indicus (Nelore breed) tissues to determine DNA sequence polymorphisms between the two breeds at differentially methylated regions (DMRs) as well as within the transcriptional unit (i.e. exons) of BWS-associate loci. For DNA methylation analyses, bisulfite-specific PCR primers were designed to amplify over the polymorphic regions at putative DMRs. For gene expression analyses, PCR primers which encompassed the polymorphic regions were developed and were used to determine parent-specific gene expression. We produced three day 65 Bos taurus taurus x Bos taurus indicus F1 concepti by artificially inseminating three Bos taurus taurus heifers with Nelore semen to establish baseline DMR methylation and imprinted gene expression. The following tissues were collected from the day 65 concepti; chorioallantois, amnion, brain, tongue, lung, heart, liver, kidney, intestines, and reproductive tract. Results show that, as expected, the BWS-associated genes KCNQ1OT1 and CDKN1C are monoallelicaly expressed in the chorioallantois of the F1 concepti. Methylation analyses for the DMRs that regulate these genes is underway. In order to determine if ART causes misregulation of imprinted genes in bovine embryos, cumulus-oocytes complexes from Bos taurus taurus cows were matured in vitro and inseminated with Nelore semen. Putative zygotes were stripped of their cumulus cells after fertilization and allowed to develop for 8 days. Half of the embryos received 10% fetal calf serum supplementation on day 5. The addition of serum is known to cause LOS in ruminants and therefore the blastocysts exposed to serum are expected to have biallelic expression of KCNQ1OT1 and loss of expression in CDKN1C. Blastocysts were collected and RNA and DNA isolated from individual embryos for expression and methylation analyses, respectively. Preliminary data suggest that serum supplementation results in silencing of the maternally-expressed cell cycle regulator CDKN1C. Studies in mice demonstrate that silencing of Cdkn1c can result in placental overgrowth. In vivo-produced F1 blastocyst will be generated by inseminating Bos taurus taurus heifers with Nelore semen. Embryos will be flushed from the uterus on day 8 gestation to use as controls for the in vitro-produced F1 blastocysts. These data will serve to determine the appropriateness of using LOS as a epigenetic model for BWS.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Progesterone Receptor Membrane Component-1 Is Localized with Aurora Kinase B During Oocyte Meiosis.

Alberto Maria Luciano 1, Valentina Lodde 1, Federica Franciosi 1, John J Peluso 2

Although bovine, rat and human oocytes express progesterone receptor membrane component-1 (PGRMC1), its role in oocyte function has not yet been conclusively determined. We have previously demonstrated that during oocyte maturation PGRMC1 localizes on Metaphase I (MI) and Metaphase II (MII) chromosomes. During the anaphase/telophase stages of meiosis, the majority of PGRMC1 dissociates from the chromosomes and concentrates between the separating chromosomal plates. Moreover, an injection of a PGRMC1 antibody disrupts the MI to MII transition and results in the disorganization of the chromosomal plate. For these reasons, we hypothesize that PGRMC1 is involved in the machinery by which chromosomes segregate during meiosis. To test this hypothesis, we conducted an immunocytochemical study to co-localize PGRMC1 with Aurora-B protein kinase (AURKB), a component of the chromosomal passenger complex that regulates several meiotic events including chromosome condensation, spindle dynamics, kinetochore-microtubule interactions, chromosome orientation and establishment of the metaphase plate. The present immunocytochemical and confocal imaging study demonstrated that in germinal vesicle (GV) stage oocytes, PGRMC1 and AURKB were present throughout the GV and co-localized. However, neither protein associated with the chromatin. With the resumption of meiosis, the localization of PGRMC1 and AURKB changed such they concentrated at the centromeric region of the MI chromosomes. During the anaphase/telophase stages, the localization of PGRMC1 and AURKB dissociated from the chromosomes and concentrated in the midzone and the midbody of the separating chromosomal plates. Finally at the MII stage, both PGRMC1 and AURKB again became associated with the centromeric region of the chromosomes. In a second experiment, the relationship between the AURKB and the phosphorylated (active) form of AURKB was assessed in order to determine if the localization of PGRMC1 was correlated with the presence of the activated form of AURKB. This study revealed that AURKB was not phosphorylated until prometaphase I. At MI AURKB became phosphorylated on Thr-232 and remained Thr-232 phosphorylated up to the metaphase II stage. Importantly our data indicate that, after GV breakdown, PGRMC1 is always concentrated to those subcellular compartments where the active, phosphorylated form of AURKB is present and exerts its regulatory functions. Based on these immunocytochemical observations, our data indicates that PGRMC1 co-localizes with AURKB during oocyte meiotic division and this association supports an involvement of PGRMC1 in the process by which chromosomes align and segregate. Future studies must now be conducted in order to determine whether PGRMC1 promotes AURKB phosphorylation or whether PGRMC1 only co-localizes with AURKB that is phosphorylated at Thr-232. Work supported by PUR2009 UniMI (AML).

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

MicroRNA-21 and PDCD4 Expression During In Vitro Maturation of Porcine Oocytes.

Elane C Wright, Cai-Xia Yang, Robyn Scanlon, Ben Selman, Jason Ross 1

MicroRNAs (miRNAs) are short (18-24 nt) non-coding RNAs that are endogenously expressed and have the ability to influence both mRNA stability and protein translation efficiency. Following germinal vesicle breakdown (GVBD) during oocyte maturation, transcription is limited and changes in mRNA and protein abundances within the oocyte may occur through post transcriptional gene regulation mediated by miRNAs. Our preliminary data suggest that miR-21 expression increases during in vitro porcine oocyte maturation. In numerous oncogenic cell lines, miRNA-21 has been demonstrated to have a post transcriptional regulatory impact on programmed cell death 4 (PDCD4; also known as nuclear antigen H731) mRNA and protein abundance. The objective of this study was to identify the temporal relationship between miRNA-21 and PDCD4 expression during in vitro oocyte maturation in the pig. Sow ovaries were obtained from a local abattoir and aspirated to collect cumulus oocyte complexes (COC) from follicles of 3-5 mm in diameter. Germinal vesicle stage oocytes (GV) were collected immediately from COCs following removal of several compact layers of cumulus cells using 1% hyaluaronidase in TL-HEPES. Remaining COCs containing several compact layers of cumulus cells were washed and placed in maturation media for 42-44 hr, stripped of expanded cumulus cells, and metaphase II (MII) arrested oocytes, as indicated by the presence of an extruded polar body, were collected. GV and MII oocytes (n = 4 per stage) were pooled in groups of 25 for quantitative real time PCR (QT-PCR). Pools of 50 GV and MII oocytes (n=4 per stage) were utilized for western blot analysis using a monoclonal antibody against PDCD4. QT-PCR mRNA abundance demonstrated that miR-21 was up-regulated approximately 5-fold during progression from GV to MII (P = 0.001). In the same samples PDCD4 mRNA abundance was not statistically different (P = 0.34). Western blot images were quantitatively evaluated using image analysis software (ImageJ 1.42) and demonstrated PDCD4 protein abundance was significantly reduced during the transition from GV to MII (P = 0.02). These data suggest a temporal relationship between the up-regulation of miR-21 and post-transcriptional regulation of PDCD4 during oocyte maturation. Developing a better understanding regarding the roles miRNAs and their ability to impact the mRNA and protein inventories during oocyte maturation may contribute to the development of strategies to positively impact in vivo oocyte quality. This work was supported in part by the USDA National Institute of Food and Agriculture (2008-35205-05309).

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Microtubule Integrity Influences the Activity and Localization of AMP-Activated Protein Kinase During Mouse Oocyte Maturation.

Ru Ya, Stephen M Downs 1

Our lab has previously shown that AMP-activated protein kinase (PRKA) is not only involved in the resumption of mouse oocyte maturation, but also plays an important role in completion of maturation. Blocking PRKA activity inhibits germinal vesicle breakdown (GVB) as well as polar body formation. Immunoflurescent staining demonstrated that the active form of PRKA is initially associated with condensed chromosomes and later with the meiotic spindle apparatus. We have further examined this relationship in the present study. Primed immature mice were injected with human chorionic gonadotropin and at appropriate times thereafter oocytes were isolated, fixed and triple-stained for DNA (DAPI), active PRKA (anti-PT172 Ab) and tubulin (anti-alpha-tubulin Ab). Active PRKA colocalized with condensed chromatin and was localized at the spindle poles at MI and MII and the midzone during anaphase. The localization patterns were similar in isolated oocytes induced to mature in vitro. We next examined the interaction of active PRKA with microtubules (MTs) during maturation in vitro. The microtubule destabilizing agent, nocodazole, dose-dependently suppressed both FSH-induced maturation in cumulus-enclosed oocytes (CEO) and AICAR (PRKA activator)-induced maturation in denuded oocytes (DO) when each was maintained in meiotic arrest with dbcAMP. A second destabilizing agent, vinblastine, also completely suppressed meiotic induction in DO, but was less effective in CEO. Coincident with suppression of meiotic induction, nocodazole also prevented the increase in GV staining of active PRKA in FSH- and AICAR-stimulated oocytes prior to meiotic resumption. To examine the effect of MT perturbation at later stages, CEO were cultured for 17 h to the MII stage, then either nocodazole or the microtubule-stabilizing agent, paclitaxel, was added to the culture medium and oocytes were fixed 1h later and processed for triple staining. Nocodazole treatment and immunofluorescent staining showed that proper localization of active PRKA was disrupted upon spindle depolymerization. However, the effect of nocodazole on active PRKA localization was reversible; when nocodazole was washed out and oocytes were cultured for 1 h, the spindle reformed and active PRKA was localized back to the spindle poles. If oocytes cultured 17 h were instead treated with paclitaxel, MTs were excessively polymerized, with exaggerated spindles and numerous asters. Interestingly, active PRKA localized to the spindle poles and all asters. Taken together, these data suggest that the meiosis-inducing action and localization of active PRKA is dependent on microtubule/spindle integrity.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Role for MATER in Cytoplasmic Lattice Formation in Murine Oocytes.

Boram Kim 1, Rui Kan 1, Lynne Anguish 1, Lawrence M Nelson 2, Scott A Coonrod 1

Mater and Padi6 are maternal effect genes that are first expressed during oocyte growth and are required for embryonic development beyond the two-cell stage in the mouse. We have recently found that PADI6 localizes to, and is required for the formation of, abundant fibrillar Triton X-100 insoluble structures termed the oocyte cytoplasmic lattices (CPLs). Given that Mater and Padi6 have similar expression profiles and mutant mouse phenotypes, we have been testing the hypothesis that MATER may also play a role in CPL formation and/or function. Here we show that PADI6 and MATER co-localize throughout the oocyte cytoplasm and cortex and that this co-localization is retained following Triton extraction, suggesting that MATER co-localizes with PADI6 at the CPLs. Additionally, while indirect immunofluorescence confocal microscopy and western blotting data showed that PADI6 levels remained similar in non-extracted Mater+/+ and Matertm/tm oocytes, the solubility of PADI6 was dramatically increased in Matertm/tm oocytes following Triton extraction, suggesting that the CPL structures are altered in the mutant oocytes. This prediction is supported by transmission electron microscopic analysis of Mater+/+ and Matertm/tm germinal vesicle-stage oocytes which found that volume fractions of CPLs were reduced by approximately 90% in the Matertm/tm oocytes compared to Mater+/+ oocytes. Taken together, these results suggest that, in addition to PADI6, MATER is also required for CPL formation. Given that PADI6 and MATER are essential for female fertility, these results also support the hypothesis that the lattices play an important role in mediating events during the egg-to-embryo transition. Research supported by NICHD grant RO1 38353 to SAC and the Intramural Research Program on Reproductive and Adult Endocrinology to LMN.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

The Expression of ENPP2 in Rat Uterus During the Estrous Cycle.

Hyo-Jin Ahn, Eui-Bae Jeung 1

Ectonucleotide pyrophosphatase/phosphodiestrase 2 (ENPP2), also known as autotoxin is originally isolated from human melanoma cells. The ENPP2 gene is a recently discovered phospholipase D that transforms lysophophatidylcholine (LPC) into lysophosphatidic acid (LPA). Lysophosphatidic acid (LPA) is becoming a new player in regulation of the reproductive processes. We studied the relation that expression of ENPP2, an LPA-generating enzyme, and secretion of sex stroid hormones estrogen and progestrone in the uterus during the estrous cycle in rat. We identified mRNA expression of ENPP2 during the estrous cycle in rat using RT-PCR. The result, uterine expression of ENPP2 mRNA was decreased at estrus as compared to proestrus and diestrus. To demonstrate performed sex stroid hormone of effect in the uterus, immature rat were treated with E2 and P4 or E2 plus P4 for 3days. The level of ENPP2 mRNA in the uterus of rat was significantly reduced by E2 treatment. ENPP2 protein level was observed during estrous cycle in rat uterus by western blotting. The protein level of ENPP2 was decreased at esturs compare to proestrus and diestrus. Using Immunohistochemistry with a specific antibody, we demonstrated the expression of ENPP2 in rat of estrous cycle. The result, ENPP2 was observed mainly in the diestrus stage and the glandular epithelia cell in the uterus endometrium. Taken together, these result showed that mRNA level of ENPP2 down-regulation during Estrous cycle by estrogen in rat. Thus uterine of ENPP2 might be involved in reproductive fuction during the estrous cycle in female rat.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Mouse Primary Uterine Cell Coculture System Revisited: Ovarian Hormones Mimic the Aspects of In Vivo Uterine Cell Proliferation.

Daesuk Chung, Sanjoy Das 1

Despite several attempts, previous studies using the mouse uterine epithelial-stromal co-culture system had limited success to mimic in vivo situation in respect of uterine cell responsiveness to ovarian hormones. Here we provide a comprehensive analysis in respect of hormonal regulation of uterine cell proliferation, in conjunction with the status of hormone receptors by confocal immunofluorescence studies. Uterine epithelial and stromal cells were isolated from day 4 pseudopregnant mice, by enzymatic digestion method. Individual cell type with maximum purity (>99%) as judged by the staining for cell-specific markers (cytokeratin for epithelial cells and desmin for stromal cells) were used in the present study. In our initial studies, we observed that when both cell types were placed in direct contact, they were unable to exhibit any proliferation after applications either by estradiol-17beta (E2, 1-25 nM) itself or in a combination of E2 (1-25 nM) and progesterone (1-50 µM), suggesting cell-cell contact is not supportive of hormone-dependent cell proliferation. In contrast, when both cell types were co-cultured separately using cell culture inserts coated with Matrigel (basal membrane extract), the epithelial cell layers, as opposed to the stromal bed, showed significant level of increase in BrdU labeling with different concentrations of E2 (1-25 nM), suggesting our primary co-culture system is capable of inducing estrogen-directed epithelial cell proliferation through an effective interactions by the stromal cells. Moreover, the combination treatment by E2 (10 nM) and progesterone (1, 10, or 50 µM) exhibited significant level of increase in proliferation exclusively in the stromal bed, while the progesterone (1-50 µM) alone had minimal effects. Overall, these results strongly suggest that our primary co-culture system effectively mimics in vivo regulation of estrogen and/or progesterone dependent cell proliferation as observed in adult mice. Further analysis of estrogen and progesterone receptors revealed that both the ERα and PR were primarily detected in the nucleus of both cell types, while the expression of estrogen receptor β (ERβ) was below the detection limit. Current studies are underway, using the specific agonists and antagonists, to examine the involvement of ER or PR during the regulation of hormone dependent cell proliferation and gene expression. In conclusion, our primary mouse uterine co-culture system is an alternative model to explore molecular aspects of uterine biology, especially hormonal regulation of cell proliferation via cell-cell communication between the epithelial and stromal compartments in mice. (Supported by NIH grants ES007814 and HD056044).

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Bovine Retained Placenta: Expression of Pro- and Anti-Apoptotic Factors in Fetal and Maternal Placenta.

Kaori Wakamiya 1, Yasuyuki Kamemori 2, Shintaro Ohtani 3, Tomas J Acosta 1, Kiyoshi Okuda 1

Retention of fetal membranes (RFM) is normally defined as the condition in which the fetal membranes are not expelled from the uterus within a period of 12 h after expulsion of the fetus. RFM is economically one of the most important disturbances during the postpartum period in cattle, because it affects herd health, milk production and reproductive efficiency. Recent studies demonstrated that apoptosis induced decidual regression and separation from the endometrium in human and rodents. In previous study, we showed that the levels of CASP3 mRNA expression were higher in maternal placental tissue (caruncula) of RFM cows than those of non-RFM cows (P < 0.05). The above findings suggest that apoptosis of fetal membranes are crucial for the normal separation and expulsion of the placenta after parturition. The present study examined the expressions of pro-apoptotic factors (BAX, FAS, CASP8) and anti-apoptotic factors (BCL2, CFLAR) in cows with and without RFM. The placentomes were manually collected from the uterus via intra vaginal immediately after parturition (0 h). Samples were classified retrospectively into two groups (RFM (n=5) and non-RFM (n=5)) according to whether placental membranes were expelled or not within 12 h after delivery. Fetal and maternal membranes of placenta were manually separated. Real time RT-PCR analysis of fetal and maternal placenta revealed that FAS mRNA expression were lower in maternal placental tissue (caruncula) of RFM cows than those of non-RFM cows (P < 0.05). The levels of CFLAR mRNA expression, anti-apoptotic factor, were lower in both maternal and fetal placental tissue of RFM cows than those of non-RFM cows (P < 0.05). However, the protein expressions of FAS and CFLAR examined by western blotting was not significantly different between RFM and non-RFM groups. The expression of other apoptotic factors analyzed in fetal and maternal placenta were not significantly different between RFM and non-RFM cows. The overall results suggest that apoptosis are necessary for normal placental separation in cows and that RFM occurs at least in part due to a dysfunctional apoptotic process in the maternal placenta tissue. The different expressions of mRNA and protein may be due to the fact that placental tissues were collected immediately after parturition. Further temporal studies are in progress to clarify the expression of pro- and anti-apoptotic factors in placentomes.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Infertility and Ovarian Pathology in Knock-In Mice with a Constitutive Active Luteinizing Hormone Receptor.

Stacey McGee, Prema Narayan 1

The activation of the luteinizing hormone receptor (LHR) in the gonads by luteinizing hormone (LH) is critical for steroidogenesis and gametogenesis. Several activating mutations in human LHR have been identified in boys with sporadic or familial male limited precocious puberty (FMPP) or testotoxicosis. The majority of the activating mutations are in transmembrane helix 6 of the 7 transmembrane G protein-coupled receptor, the most common being a mutation of aspartic acid 578 to glycine. Boys with this disorder present with elevated levels of testosterone in the context of prepubertal LH levels and the onset of puberty by age four or earlier. Surprisingly, females harboring these mutations are normal. We have created a knock-in mouse (KiLHR) with an activating point mutation, caused by substitution of glycine for aspartic acid at position 582 (D582G) in mouse LHR, corresponding to the common D578G mutation in human LHR. The constitutive activity of D582G mLHR was verified by transient transfection in HEK 293 cells. Competitive binding and signaling studies showed that D582G mutant binds hCG with slightly higher affinity than WT LHR and exhibits a 14-fold elevation in basal cAMP levels compared to WT LHR. RT-PCR analysis of RNA from the gonads of heterozygous KiLHR mice demonstrated expression of the wild-type (WT) and mutant alleles. The WT and mutant alleles were distinguished by the introduction of a unique Hinf1 restriction enzyme site in the mutant sequence. Female KiLHR mice exhibited precocious puberty as evidenced by a premature vaginal opening. These mice have irregular estrus cycles and are infertile. The ovaries of the KiLHR mice were significantly larger than that of WT mice and histological analysis revealed the presence of multiple large hemorrhagic cysts as early as 5 weeks of age. Only a few follicles were present at the periphery, but none had progressed beyond the preantral stage. The uteri of KiLHR mice were larger with a dilated lumen, disorganized luminal epithelial and myometrial layers and reduced stromal layer. The female phenotype of the KiLHR mice is consistent with elevated androgens resulting from the constitutively active LHR and is different from that observed in human females. Supported by NIH HD044119.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Effects of Tumor Necrosis Factor Alpha on Luteinizing Hormone-Induced Oocyte Maturation in Trout.

Josep V Planas, Diego Crespo 1

In fish, like in other vertebrates, luteinizing hormone (LH) is an essential hormone for the completion of oocyte maturation. In salmonid fish (i.e., salmon and trout), oocyte maturation is induced by LH through its stimulation of the production of the maturation-inducing steroid (17α,20β-dihydroxy-4-pregnen-3-one or MIS). Specifically, LH stimulates the production of MIS through the production of its steroidal precursor (17α-hydroxyprogesterone or 17-OHP) as well as the expression and activity of 20β-hydroxysteroid dehydrogenase (20β-HSD), the enzyme responsible for the conversion of 17-OHP to MIS. In mammals, several factors have been reported to modulate the effect of LH on oocyte maturation. For example, a strong body of evidence suggests that tumor necrosis factor α (TNFα) could play multiple physiological roles in the control of ovarian function. In the present study, we have investigated the possible involvement of TNFα in the regulation of oocyte maturation in the brown trout (Salmo trutta). Our results show that in vitro treatment of brown trout preovulatory follicles with purified coho salmon LH (sLH) significantly increased oocyte maturation, as assessed by germinal vesicle breakdown (GVBD), and this effect was blocked by TAPI-1 (an inhibitor of TNFα converting enzyme or TACE/ADAM17), indicating a possible involvement of TNFα on LH induction of GVBD. Furthermore, treatment of trout preovulatory follicles with sLH resulted in a significant increase in the expression of TNFα. Interestingly, recombinant trout TNFα (rtTNFα) caused a significant increase in GVBD. In view of these results, we explored the possibility that TNFα could be involved in the production of MIS. sLH significantly stimulated the production of 17-OHP, and this effect was blocked by TAPI-1. Furthermore, although we have not yet measured the production of MIS, rtTNFα significantly stimulated the expression of 20β-HSD. Interestingly, rtTNFα also increased the ovarian expression of the LH receptor. These results strongly suggest that TNFα may contribute to the regulation of oocyte maturation by LH in trout.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Anti-Oxidative and Anti-Inflammatory Activities of Adrenomedullin (ADM) in the Rat Uterus.

Lowell Hei Man Kong, Wai-sum O, Fai Tang 1

Adrenomedullin (ADM) is a multifunctional peptide that signals through a G-protein -coupled receptor. Previous studies in our laboratory showed that in rats, the genes encoding for ADM peptide, Adm and its receptor omponents are expressed in male accessory sex glands (ASGs), including the prostate, the coagulating gland and the seminal vesicle while ADM is found in both the tissues and the secretions of these ASGs. The high level of ADM found in post coital uterine flushing suggests that this hormone may play a possible role in reproductive physiology, especially in the female reproductive tract. The aim of this study is to investigate the functional roles of ADM in the female reproductive tract. After pretreatment with 100nM ADM for 30 min, the reactive oxygen species (ROS) level generated by 1µM hydrogen peroxide in isolated endometrial epithelial cells were measured by the fluorescence intensity method using 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA). We found that incubating with ADM significantly decreased the hydrogen peroxide-induced ROS level in endometrial epithelial cells, suggesting that ADM secreted by the ASGs may protect the sperm against ROS generated in uterus. A similar decrease in ROS in epithelial cell was also observed when treated with post coital uterine flushings, suggesting the ADM from seminal plasma can protect uterine epithelial damage from ROS. ADM has been shown to be an anti-inflammatory agent that acts through nitric oxide system. To find out the anti-inflammatory effect of ADM on the rat uterus, uterine tissue was cultured with ADM for 4 h and the levels of tumor necrosis factor-alpha\(TNF-alpha\) in the culture media were measured using enzyme-linked immunosorbent assays (ELISA).Treatment with ADM significantly decreased TNF-alpha\ production suggesting that ADM may play an anti-inflammatory role in the uterus. The present study provides evidence for a role of ADM in the seminal plasma in ROS production and the maternal immune response in the uterus. This study is supported by a grant from the Research Grant Council of Hong Kong SAR (GRF775908).

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Expression of Toll-like Receptors (TLRs) Is Regulated by Early Pregnancy in the Mare Endometrium.

Mehmet Osman Atli 1, Ercan Kurar 2, Seyit A Kayis 2, Selim Aslan 3, Ahmet Semacan 2, Sefa Celik 4, Aydin Guzeloglu 2

Intrauterine immune regulation is involved in defense mechanism against pathogen microorganisms and is critical in mediating tolerance for embryonic/fetal development inside the uterus. TLRs are known to be an important part of the innate immune system. To our best knowledge, there is no data regarding the expression of TLR genes in mares during early pregnancy. Our objective was to determine the effect of early pregnancy on the expression profiles of TLRs in the mare endometrium. Endometrial biopsy samples were obtained from mares on days 14 (P14, n=4) and 18 (P18, n=4) of pregnancy. Biopsy samples were also collected from cyclic mares during the comparable stages of the cycle with P14 and P18 of early pregnancy, that is, during late diestrus (LD, n=4) and after luteolysis in estrus phase (AL, n=4) of the cycle. Stages of estrous cycle were defined by daily measurement of plasma progesterone concentrations and ultrasonography. Pregnancy was confirmed by ultrasonography. Total RNA was extracted and converted to cDNA. PCR was used to detect expression of genes studied and then relative expression levels were quantified using real-time PCR analysis in duplicate. A mixed model was fitted on the normalized data and least significant difference test was employed to determine significantly different groups. Ten TLR genes (TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, and TLR10) were detected to be present in the endometrium in early pregnancy and all stages of estrous cycle days. When LD and P14 were compared, expressions of TLR1, 2, and 10 did not differ. Pregnancy at P14 significantly suppressed the expressions of TLR3, 4, 7, and 8, but induced TLR5, 6, and 9 compared to LD. Expressions of TLR2, 3, 5, 6, 8, 9, and 10 were unchanged between AL and P18. However, while TLR4 was increased on P18, the steady state levels of TLR1 and 7 were decreased on P18 compared to AL. In conclusion, the results indicate that mRNA expressions of Toll-like receptors are differentially regulated in the mare endometrium by factors associated with pregnancy. It can be suggested that regulation of TLR genes in the endometrium may be playing a role in immune tolerance mechanism against embryo during early pregnancy in mares. (Funded by TUBITAK).

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Expressions of Insulin-Like Growth Factor-Binding Proteins (IGFBP) in Mare Endometrium during Estrous Cycle and Early Pregnancy.

Ercan Kurar 1, Mehmet O Atli 2, Seyit A Kayis 1, Selim Arslan 3, Ahmet Semacan 1, Sefa Celik 4, Aydin Guzeloglu 1

The insulin-like growth factors (IGFs) are low molecular weight single chain peptides which are associated with female reproduction and regulation of various endometrial functions. IGFs with mitogenetic action involve in endometrial differentiation and provides uterine environment for preimplantation of the mammalian embryo. IGFBPs are known to play important roles in IGF system and mediate the activities of IGF peptides in target tissues. The objective of this study was to investigate temporal expression profiles of IGFBPs in equine endometrium during the estrous cycle and early pregnancy. Biopsy samples were obtained from mares on day of ovulation (d0, n=4), late diestrus (LD, n=4, high progesterone [P4]), and after luteolysis in the estrus phase (AL, n=4, <1 ng/ml P4) of the cycle. Biopsies were also taken on days 14 (P14, n=4), 18 (P18, n=4), 22 (P22; n=4), and 60 (P60, n=2) of pregnancy. Total RNA was isolated and subjected to first strand cDNA synthesis. Relative mRNA expression levels of the genes encoding IGFBPs were quantified using real-time RT-PCR. The data was normalized and then analyzed via one-way ANOVA. Least significant difference (LSD) test was employed to determine significantly different groups. All of the IGFBPs (1, 2, 3, 4, 5 and 6) were found to be expressed in mare endometrium. IGFBP-1 expression was unchanged during the estrous cycle and early pregnancy up to P22, but was greatly induced at P60. IGFBP-2 level remained constant during the cycle, suppressed during the early pregnancy, however increased at P60 to the levels observed at d0. IGFBP-3 expression decreased at LD and then tended to increase at AL. Pregnancy attenuated the expression of IGFBP-3 throughout the early pregnancy. IGFBP-4 levels were stable during the cycle. Interestingly, its level was decreased by pregnancy up to P22 and was high again at P60. Expression of IGFBP-5 was similar between d0 and LD, and then was decreased at AL. Pregnancy suppressed IGFBP-5 expression during the early pregnancy. Endometrial expression of IGFBP-6 was upregulated only at LD and downregulated on the other days of the cycle and during pregnancy. It appears that early pregnancy is usually suppressive on the expression of IGFBPs in the mare endometrium. However, expression of IGFBP-1, -2, and -4 were significantly upregulated later in pregnancy at P60. These findings suggest that endometrial expression of IGFBPs of the IGF system appears to be regulated in the equine by both cyclic changes and early pregnancy. Study was funded by TUBITAK 107O035.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Activation of G-Protein-Coupled Receptor, GPR30, Opposes Estrogenic Growth Regulation via Inhibition of ERK1/2 Activation Signaling in the Mouse Uterus.

Fei Gao, Xinghong Ma, Alicia Blackwell, Sanjoy K Das 1

Early- and late-phase estrogenic signaling in the uterus have been well recognized, yet molecular mechanisms involved in their relationships remain poorly understood. General consensus is that the early signals, occurring within the first 6 h, undergo appropriate molecular cross-talk to prepare the uterus for later (18-30 h) growth regulation. In this regard, GPR30 has been identified as a candidate of extra-nuclear receptor for estrogen and implicated in various early non-genomic signaling in both normal and cancer tissue biology. However, the genetic deletion of GPR30 in mice exhibited no apparent defects for estrogen-dependent uterine growth regulation. In contrast, here we show novel evidence to suggest that the uterine epithelial activation of GPR30 can elicit antagonistic signaling to the stroma to effectively modulate early signaling events, including the ERK1/2 activation for estrogen-dependent growth regulation. In our initial studies, we examined whether GPR30 activation can have any impacts to the uterus of ovariectomized mice. Consistent to the literature, we observed that an injection of GPR30 selective agonist, G-1 (at doses: 0.5, 2, 10, 50 or 100 µg/mouse) did not induce any significant alteration in uterine wet weights at 6 and 24 h, while an injection of estradiol-17β (E2, at doses 1, 5, 15, 25 or 100 ng/mouse) caused significant increase in uterine weights for both phases by doses ≥15-100 ng/mouse, suggesting G-1, as opposed to E2, lacks estrogenicity. In contrast, an injection of G-1 (at doses ≥25-100 µg/mouse) given 30 min prior to E2 (25 or 100 ng/ mouse) exhibited significant inhibition of E2-dependent uterine late response, while the early weight response remained unaffected. These results were further consistent with the analysis of E2-dependent uterine epithelial cell proliferation. To evaluate the mechanism involved in GPR30 mediated inhibitory role, we first examined the status of ERα and GPR30 expression under above conditions. Both mRNA and protein analyses indicate that E2 caused significant reduction of GPR30 levels, but not the ERα, during the early phase, while G-1 was unable to alter these proteins. Double immunofluorescence studies also show that GPR30 primarily co-expressed with ERα in uterine epithelial cells, while stroma primarily detected ERα. Further analysis of well-recognized early and late E2-responsive genes (viz., Bip, SFRP-2, c-fos, c-myc, cdkn1a and Ltf) revealed that expressional effects were either compromised or potentiated by G-1, suggesting G-1 modulates E2-regulated gene responses in the uterus. Additionally, analysis of several early non-genomic signaling events in respect of phosphorylation of ERK1/2, Akt (pThr308 and pSer473), CREB (pSer133) and ERα (pSer118) demonstrated that E2-dependent activation of ERK and ERα was predominantly suppressed by prior G-1 treatment, while Akt phosphorylation remained unaffected. In contrast, CREB phosphorylation was enhanced by such treatment. Most importantly, further studies using a selective ERK inhibitor (SL327) caused abrogation of E2-dependent uterine growth response, suggesting ERK activation critically controls E2-depedent uterine growth regulation. Collectively, our studies revealed that GPR30 acts as an upstream negative regulator for ERK activation that effectively controls uterine growth regulation under the direction of E2. (Supported by NIH grants ES007814 and HD056044).

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Chemoprevention in the Hen Model of Ovarian Cancer.

Lindsey S Trevino, Patricia A Johnson 1

Ovarian cancer is the leading cause of reproductive cancer death in U.S. women. This high mortality rate is due to the lack of early detection methods and ineffectiveness of therapy for advanced disease. Until more effective screening methods and therapies are developed, chemoprevention strategies are warranted. Oral contraceptive (OC) use significantly reduces the risk of ovarian cancer in women; however, the mechanisms of OC action are still not clear. The hen has a high spontaneous incidence of ovarian cancer and is a unique model for studying ovarian cancer chemoprevention. Our objective was to determine the effect of progestin (P) alone, estrogen (E) alone, or progestin and estrogen (P+E) in combination (as found in OCs) on ovarian cancer incidence in the hen. A total of 231 one year-old hens were divided into 4 treatment groups: 1) control (n=59; vehicle injection and sham implant); 2) P (n=59; 50 mg medroxyprogesterone acetate (MPA) injection and sham implant); 3) E (n=57; vehicle injection and 25 mg estradiol implant); and 4) P+E (n=56; 50 mg MPA injection and 25 mg estradiol implant). Injections were administered every 4 weeks and implants were replaced after 180 days. Egg production was monitored daily for each individual bird. Hens were approximately 2.5 years-old at the end of the 16 month treatment period. Necropsies were performed to determine presence of nodules on the ovary, oviduct and/or other organs, as well as presence of ascites, and stage of cancer if present. Diagnosis was confirmed by histology. Ovarian cancer incidences for control, P, E, and P+E treatment groups were 14, 2, 19, and 4%, respectively. Treatment with P alone (P < 0.05) or the combination of P+E (P < 0.08) decreased ovarian cancer compared to the control treatment. A significant risk reduction of 88% was observed in the group treated with P alone (risk ratio 0.125: 95% confidence interval 0.016-0.999). Treatment with E alone had no effect on cancer incidence compared to the control treatment. Average egg production throughout the study period was 65, 31, 57 and 35% for control, P, E, and P+E treatments, respectively. Administration of P alone or P+E significantly decreased egg production compared to control (p<0.01). E alone also decreased egg production compared to control; however, egg production in this group was greater than the P alone and P+E groups which were associated with decreased cancer incidence. Our results suggest that ovulation is related to incidence of ovarian cancer in hens because hens injected with P exhibited decreased egg production as well as decreased incidence of ovarian cancer. However, other factors, such as hormones, more than likely modify the risk because hens treated with P+E exhibited decreased egg production but only a trend toward a decreased incidence of ovarian cancer. P may be important for the prevention of ovarian cancer, although this could be due to its effect of inhibition of ovulation or through its ability to regulate cellular processes such as apoptosis and proliferation. Further studies are underway to determine the mechanism of action of these hormones in ovarian cancer development in the hen. Our study supports the use of oral contraceptives to prevent ovarian cancer and further validates the hen as a suitable animal model to test chemoprevention strategies. This research was supported in part by NIGMS F31GM078742 to LST.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Dominant Activation of Hedgehog Signaling Alters Development of the Female Reproductive Tract.

Fernando F Migone, Yi Ren, Rebecca M Harman, Robert G Cowan, Susan M Quirk 1

The hedgehog (HH) signaling pathway regulates the development of multiple organs in the embryo as well as remodeling in adult tissues. Signaling occurs through binding of one of three secreted HH ligands (Indian, desert and sonic) to the membrane receptor patched (PTCH); this relieves inhibition of the signal transducer smoothened (SMO), and downstream signaling occurs through GLI transcription factors. The objective of this study was to determine the role of HH signaling in development of the female reproductive tract. Mice were created in which expression of a dominant active allele of Smo, known as SmoM2, and a fusion protein, yellow fluorescent protein (YFP), are induced in the Mullerian duct by CRE-mediated recombination using the Amhr2cre/+ allele. The Amhr2cre/+ allele is known to be expressed during embryonic development of the Mullerian duct and the ovary. In Amhr2cre/+SmoM2 mutant mice, YFP was detected in the reproductive tract on the day of birth, confirming that expression of the SmoM2/Yfp fusion gene had been induced. At 24 days of age, expression of genes that are known transcriptional targets of HH signaling, Gli1, Ptch1 and Hhip, were increased in uteri and oviducts of Amhr2cre/+SmoM2 mutant mice compared to genotype-matched Amhr2+/+SmoM2 controls. Thus, over-activation of HH signaling continued in prepubertal mutant mice. The reproductive tract of mutant mice fails to develop normally. Unlike the uterus in control mice, in which the luminal epithelium consists of a single layer of columnar epithelial cells, the luminal epithelium in mutant mice has stratified squamous cells, resembling those of a normal cervix. In addition, the uterus of mutant mice lacks glands, the myometrium is disorganized and the oviduct lacks coiling. These characteristics suggest partial homeotic transformation of the uterus in mutant mice to resemble the more posterior cervix and failure of the oviduct to develop normally. The anterior-posterior axis of the reproductive tract is known to be regulated by differential expression of genes in the Hox family. Wnt7a null mice have defects in patterning of the reproductive tract. Furthermore, neonatal treatment with DES was reported to alter development of the reproductive tract and the expression of Hox genes and Wnt7a. Therefore, expression of Hoxa9, Hoxa10, Hoxa13 and Wnt7a were determined in oviduct, uterus and vagina of 24 day old Amhr2cre/+SmoM2 mutant and control mice. In control mice, expression of Hoxa9, Hoxa10 and Hoxa13 were most prevalent in the oviduct, uterus and vagina, respectively. Expression of Hoxa9 and Hoxa10 in the oviduct and uterus of mutant mice was similar to that in control mice. In contrast, expression of Hoxa13 in uterus of mutants was 16-fold higher than in controls. Wnt7a expression was similar in the reproductive tract of mutants and controls. Mutant mice are infertile, in large part due to an ovarian defect that prevents ovulation. In addition, mating with either fertile or vasectomized wild-type males induced a severe inflammatory response in the reproductive tract by a mechanism that remains to be determined. In summary, over-activation of HH signaling during development of the reproductive tract results in a defect in anterior-posterior patterning that is associated with an altered pattern of expression of Hoxa13. These results show that development of the reproductive tract can be influenced by HH signaling. Supported by NICHD R03HD057648.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Estradiol-17beta Treatment Enhances the Sensitivity to Bradykinin-Induced Relaxation of Ovine Uterine Arteries.

Leslie A Lekatz, Praveen Shukla, Stephen T O'Rourke, Kimberly A Vonnahme 1

Bradykinin, a potent vasorelaxant, elicits vasodilation of blood vessels through the release of nitric oxide, prostacyclin, and/or endothelium-derived hyperpolarizing factor. Limited data exist on how estrogen may impact the vasoactivity of bradykinin via nitric oxide release in sheep uterine arteries. We hypothesized that estradiol-17beta (E2) treatment for 24 h would enhance the bradykinin-induced relaxation in sheep uterine arteries. Further, we hypothesize that bradykinin is acting through the release of nitric oxide, and incubation with a nitric oxide antagonist will prevent bradykinin-induced vasodilation. To examine the effects of E2on the bradykinin-induced relaxation of uterine arteries, 8 ovariectomized ewes were implanted with E2 for either 0 (n = 4) or 24 (n = 4) h before tissue collection. Ewes receiving E2 implants were implanted subcutaneously in the axillary region with 2 Silastic implants (3.35 mm i.d. x 4.65 o.d. x 15 mm length) each containing 50 mg of E2. At tissue collection the uterine arteries were collected for vasoreactivity studies. Arterial rings were suspended in organ chambers filled with 25 mL of physiological salt solution aerated with a mixture of 95% O2 and 5% CO2 and kept at 38.6°C. Optimal tension was found by progressively stretching the rings until the contractile response to KCl (20 mM) was maximal. All doses were previously determined to be optimal for arterial responses. The presence or absence of endothelium was verified by testing the ability of bradykinin (10-7 M) to produce endothelium-dependent relaxation during contraction evoked by norepinephrine (10-6 M). Rings were contracted with U46619 (10-8 M), and the dose response curve to bradykinin (10-10to 10-5 M) was obtained. Estradiol-17beta treatment caused a leftward shift in the bradykinin dose response curve. Incubation with bradykinin receptor 2 antagonist resulted in a rightward shift in the bradykinin dose response curve for both 0 and 24 h ewes, with E2 treatment causing a greater shift compared to the 0 h ewes. Incubation with L-nitroarginine, an inhibitor of nitric oxide synthase, also resulted in a rightward shift in the bradykinin dose response curve for both 0 and 24 h ewes, with E2 treatment causing a greater shift compared to the 0 h ewes. Therefore, we fail to reject our hypothesis as these results indicate that E2 treatment for 24 h enhances the sensitivity of the uterine artery to bradykinin-induced relaxation. It appears that E2 increases the sensitivity of the uterine artery to the bradykinin-induced vasodilation, resulting from bradykinin receptor 2 binding. It appears the majority of the bradykinin-induced vasodilation to be the consequence of nitric oxide release.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Gestational Exposure to Chromium (VI) Impairs Pregnancy Outcome and Fetal Development.

Sakhila K Banu 1, Jone A Stanley 1, JeHoon Lee 1, Thamizh K Nithy 1, Joe A Arosh 1, Patricia B Hoyer 2, Michael M Aruldhas 3, Robert C Burghardt 1

Hexavalent chromium (Cr VI) is a heavy metal environmental endocrine disruptor. Cr VI has been used in a wide range of applications in the chemical industry, graphics industry, artistic paints, anticorrosion paints, electroplating, steel alloys, stainless steel welding, and a multitude of other uses. The tanning and leather industries use Cr to a larger extent in developing countries. Increased use and improper disposal of Cr in the developing countries and in the U.S have increased Cr levels in the air, soil and drinking water. Significant contamination with Cr(VI) has been found in approximately 30% of the drinking water sources in California. The Blacksmith Institute has reported the "Top 10 world's worst polluted places" which include Bangladesh, China and India for their drinking water heavily contaminated with Cr(VI). Women working in Cr industries and living in Cr contaminated area experience infertility problems, and their children have various developmental defects. Cr is found in all human organs of adults and newborns. Soluble Cr(VI) is rapidly transported inside the cell by anion transport mechanisms, and it leads to multiple cellular dysfunctions that include DNA adduct formation, deregulated cell cycle, apoptosis and carcinogenesis. Vitamin C and antioxidants protect the cells from Cr toxicity. We hypothesize that pre-natal exposure to chromium (1) impairs follicle development and steroidogenesis in the F1 offspring; (2) deteriorates fetal development in F0 and pregnancy outcome in both F0 mother and F1 offspring. The objectives were to study the effects of gestational exposure to Cr(VI) on: (1) implantation, pregnancy outcome and fetal development in the mother rats (F0); (2) Serum hormonal profiles and pregnancy outcome in the F1 offspring, and (3) ovarian steroidogenesis and follicular atresia in F1 offspring. Study 1: Pregnant rats (n=75) were treated with different doses of Cr(VI) (potassium dichromate 5-200 ppm) through drinking water from gestational days (GD) 9 to 20. After birth female offspring (F1) were given regular drinking water and diet, and were killed on postnatal days (PND) 3, 7 and 18. Numbers of healthy and atretic follicles were counted and serum hormones were measured. Cr(VI) decreased follicle number, serum hormones and increased follicle atresia in a dose-dependent manner. Study 2: Pregnant rats were treated with Cr(VI) (50 ppm) through drinking water from GD 9 to 20. On GD 20 mother rats were killed and uteri were removed with fetuses. Implantation sites, number of pups, fetal resorption sites, number of corpora lutea (CL) were counted. Cr(VI) increased fetal resorption, decreased number of CL, and implantation sites. Weights of the uteri, placenta and fetuses were significantly decreased. Study 3: Pregnant rats were treated with different doses of Cr(VI) (potassium dichromate 5-200 ppm) through drinking water from GD 9 to 20. After attainments of puberty, the female rats (F1) were mated with normal male rats, pregnancy outcome was determined. Cr(VI) increased pre-term labor and decreased full-term labor. Study 4: Granulosa cells were isolated from prepubertal rats (23 days), treated with 10 µM CrVI for 24h, mRNA expression of steroidogenic enzymes and receptors were detected by RT-PCR. CrVI significantly decreased (p<0.05) steroidogenic enzymes and receptors. Thus, Cr(VI) caused two-generational toxicity in female rats. This study was supported by a grant from NIH (R03 ES016605-01A1).

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Tissue-Specific Expression of Calcium Transporter Genes, TRPV5, TRPV6, NCX1, PMCA1b, in Horse (Equus caballus).

In-Ho Hwang, Eui-Bae Jeung 1

Calcium transporter genes such as transient receptor potential cations channel subfamily V member 5/6 (TRPV5/6), Na+/Ca2+ exchanger (NCX1), plasma membrane calcium-transporting ATPase 1b (PMCA1b) are essential to maintain homeostasis and to metabolize Ca2+ ions. TRPV5/6 is a protein that plays an important role in the absorption of Ca2+. NCX1 and PMCA1b are proteins which play a critical role in intracellular calcium homeostasis. In this study, in order to understand physiological roles of these calcium transporter genes, we investigated the tissue-specific mRNA and protein expressions of calcium transporter genes in the duodenum, kidney and heart of horse (Equus caballus) tissues using reverse transcriptase-polymerase chain reaction (RT-PCR) and western blot, and localization of exhibited calcium transporter genes in various horse tissues by immunohistochemistry. As our results, TRPV5 was highly expressed in the kidney, while scarcely expressed in the duodenum and heart. TRPV6 expression was similarly expressed in the duodenum, kidney and heart. In addition, NCX1 and PMCA1b expression was high in the heart, while there was no difference in NCX1 and PMCA1b expression in the duodenum and kidney. The expression patterns of these proteins by western blotting were similar with gene expressions by RT-PCR. As the result of this study, the aspect of calcium transporter genes expression in horse is almost similar with other vertebrates, suggesting that the calcium transporter genes may have an essential role such as maintaining calcium homeostasis in horse.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Duodenal and Renal Transient Receptor Potential Vanilloid 6 Appear to be Distinctly Regulated by Sex Steroid Hormones, Estrogen, and Progesterone, in Immature Female Rats.

Ji-Hae Kim, Eui-Bae Jeung 1

Calcium-related proteins include transient receptor potential vanilloid (TRPV) 5 and 6, plasma membrane calcium-ATPase 1b (PMCA1b), and calbindin-D9k and -D28k. The TRPV6 is a major calcium channel located in the apical and basolateral membranes of cell and distributed widely in many other organs, especially in the exocrine tissues such as intestine and uterus. TRPV6s are generally regulated by vitamin D, a dietary calcium ion and hormone. In particular, uterine TRPV6 appears to be affected by sex steroid hormones, which are altered according to estrous cycle and pregnancy. In order to discover the effect of sex steroid hormones on the regulation of TRPV6, we examined the expression of TRPV6 mRNA by using RT-PCR and real-time PCR, and protein expression of TRPV6 by immunohistochemistry (IHC) in the uterus, duodenum, and kidney. To evaluate the effect(s) of sex steroid hormones on its uterine, duodenal, and renal regulation, 17beta-estradiol [E2; 40 µg/kg of body weight (bw)] and/or progesterone (P4; 4 mg/kg of bw) or vehicle (n = 6/each group) were subcutaneously injected into Sprague-Dawley immature female rats (14 days old, n = 24 in total) for 3 days. As a result, the treatments of immature rats with E2 or P4 increased TRPV6 mRNA for calcium function or regulation in the uterus of immature rats. To confirm the specificity of E2 or P4 through their receptors, we treated the immature rats (extra n = 24 in total) with an estrogen receptor-antagonist, ICI 182,780 (ICI; 30 µg/kg of bw), and/or progesterone receptor antagonist, RU 486 (10 mg/kg of bw), at 3 days prior to E2 or P4 injection. Consequently, an increase in TRPV6 mRNA was observed in the following 2 treatments; ICI plus E2/P4 and E2/P4 alone. In IHC, we further observed that the expression of duodenal TRPV6 was increased by E2 or P4 and E2 or P4 plus ICI, while no difference was observed in renal TRPV6 by the treatments of sex steroid hormones. In conclusion, these results indicate that the expressions of uterine and duodenal TRPV6 may be induced by E2 and P4, but its renal expression may not be controlled by these steroids.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

A New Tool for Reproductive Biology: Exploring Recombinases for Conditional Mutagenesis.

Lois J Maltais, Randy Babiuk, Constance M Smith, Steve Murray, Michael Sasner, Kim Forthofer, Pete Frost, James Kadin, Martin Ringwald, Janan T Eppig 1

Conditional mutagenesis in mouse is a powerful tool for examining effects of stage-specific gene regulation and for studying gene function in cases where critical early developmental expression causes gene knockouts to be lethal. Conditional genotypes are "knocked-out" at the site of interest in a spatial and temporal manner, determined by the driver/promoter element of the recombinase containing transgene or knock-in allele used. Many new conditional-ready targeted mutations are being made through the efforts of the International Knockout Mouse Consortium (www.knockoutmouse.org). To maximize the utility of these new mutations, a companion set of specific cre driver mice is needed ("cre" is used here to stand-in for all variations of recombinase constructs). Fundamental to choosing cre-bearing strains for mating with these mutants and to interpreting phenotypes from conditional genotypes is the need to understand the temporal and spatial distribution of cre recombinase expression and specificity. Thus, for example, it is important to know that a conditional knockout is gonad-specific or has wider tissue specificity that could secondarily affect reproductive traits. We have built a Recombinase (cre) Portal as a central access point to cre expression and specificity data (www.creportal.org) to aid researchers using conditional mutagenesis. For cre-containing transgenes or knock-in alleles, key data include the molecular description of the cre construct, the driver/promoter element, whether the construct is inducible or not, publications, and availability of the cre-bearing mice through a public repository. Of particular importance are the curated annotations (with images, when available) of the tissues, specific structures, and ages assayed that define the specificity of the cre transgene or knock-in allele. Recombinase transgenes or knock-ins can be searched by specificity in a particular anatomical system or by specific promoter/driver (http://www.creportal.org). Nomenclature searches can be done by transgene or knock-in symbol or synonym. Downloads and views of complete recombinase data summaries are also provided. As of March 1, 2010, there are over 1,160 unique recombinase-containing transgenes and knock-in alleles cataloged in the Cre Recombinase Portal. We welcome comments on www.creportal.org and user contributions on their experiences with specific cre constructs. The Recombinase (cre) Portal project is funded by NIH grants HG000330 and HD033745 and EU grant HEALTH-F4-2009-223487.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

The Polycomb Group Protein EED Is Able to Localize to the Nucleus During Cleavage Development in the Porcine Embryo.

Kallie Blaine Foust, Kieun Park, Ryan Cabot 1

Chromatin-modifying complexes, including the polycomb group proteins, serve essential functions during mammalian embryonic development. Polycomb group proteins EED, SUZ12, and EzH2 form the Polycomb-Repressive Complex 2 (PRC2); this complex has been shown to mediate methylation of the lysine 27 residue of histone protein H3 (H3K27), an epigenetic mark that is linked with transcriptional repression. Our lab has previously shown that the global patterns of H3K27 methylation undergo reorganization during cleavage development in the porcine embryo. Specifically, H3K27 trimethylation is present on chromatin in mature porcine oocytes, pronuclear and 2-cell stage embryos, with H3K27 trimethylation decreasing at the 4-cell stage and not detectable in blastocyst stage embryos. In addition, we have shown that the transcript levels of EED, SUZ12 and EzH2 are present in differing amounts at discrete stages of cleavage development. The goal of this study was to determine the intracellular localization of ectopically expressed EED in 2-cell, 4-cell, morula and blastocyst stage porcine embryos produced by in vitro fertilization. We hypothesized that the global changes in H3K27 trimethylation were due to restricted nuclear access of PCR2 complex proteins at discrete stages of porcine cleavage stage development. To test this hypothesis, mRNA encoding a GFP fusion protein (N-terminal GFP tag) of porcine EED was synthesized in vitro and injected into mature porcine oocytes. Briefly, porcine oocytes harvested from prepubertal gilt ovaries were matured in a chemically defined medium (TCM-199 supplemented with 0.1% PVA, 0.069 mg mL-1 cysteine, 10 ng mL-1 EGF, 0.5 IU mL-1 LH and FSH) for 42-44 hours at 39°C in 5% CO2. Following removal of cumulus cells and in vitro fertilization, oocytes were injected with GFP-EED mRNA (concentration of 1 μg μL-1); successfully injected oocytes were cultured in PZM medium supplemented with 3 mg mL-1 BSA. Embryos were removed from culture at the following times subsequent to gamete mixing to determine the intracellular localization of ectopically expressed GFP-EED: 24, 48, 96 and 144 hours for 2-cell, 4-cell, morula and blastocyst stages, respectively. Upon removal from embryo culture medium, embryos were stained with Hoechst 33342 (1 μg μL-1 for 15 minutes) in order to visualize nuclear DNA. Individual embryos were then examined using epifluorescence microscopy to visualize GFP-EED and DNA. Two to four biological replicates were performed for each stage of development. Our results revealed that GFP-EED possessed a strong nuclear localization in 2-cell (n=4/4), 4-cell (n=9/9), morula (n=5/5) and blastocyst (n=1/1) stage embryos. These results indicate that EED has the ability to interact with chromatin to participate in H3K27 methylation throughout porcine cleavage development. It is possible that restricted access of other PRC2 proteins may contribute to the global changes in H3K27 trimethylation or that activity of histone demethylases counteracts or overrides the histone methyltransferase activity of PRC2.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Seasonal Changes in FSH-Induced FSHR mRNA Splice Variant Expression During In Vitro Maturation of Cat Oocytes.

Rebecca J Hobbs 1, JoGayle Howard 1, Robert C Fleischer 1, David E Wildt 2, Pierre Comizzoli 1

Successful in vitro maturation (IVM) of cat oocytes can only be achieved during the non-breeding season (September through January) with a 10 times higher concentration of follicle-stimulating hormone (FSH; 10 μg/ml) compared to the breeding season (February through August). Despite the reduced sensitivity to FSH during this anestrual interval, we have observed an increase in expression of full-length FSH receptor (FL-FSHR) mRNA, but not the truncated version (Tr-FSHR), in immature cumulus-oocyte complexes (COCs). The objective of this study was to assess the impact of season on overall FSHR, FSHR splice variants (FL-FSHR, Tr-FSHR) and estrogen receptor 2 (ESR2; as estrogen is known to synergize with FSH during oocyte maturation) mRNA following IVM (using 1 μg/ml versus 10 μg/ml FSH). Immature Grade 1 COCs (dark oocyte cytoplasm and compacted cumulus cells) were collected from adult cat ovaries in the breeding (Br) and non-breeding (NBr) seasons. The COCs were stored immediately in RNAlater (Br, n = 45; NBr, n= 47; in 3 replicates per season) or after IVM with 1 µ g/ml (IVM1; Br, n = 59; NBr, n = 50) or 10 µg/ml (IVM10; Br, n = 54; NBr, n = 54) FSH for 26 hr. Total RNA was extracted using a Picopure RNA isolation kit and cDNA generated using random primers and Superscript III reverse transcriptase. cDNA samples were assessed by real-time qPCR to compare relative expression levels of overall FSHR, FL-FSHR, Tr-FSHR and ESR2 mRNAs (normalized to ACTB, RPS7 and YWHAZ mRNA expression). The significance of expression ratios was tested using the Pair-Wise Fixed Reallocation Randomization Test. Compared to immature COCs, overall FSHR mRNA expression was decreased (P < 0.01) after IVM1 and IVM10 in the breeding season; but remained similar (P > 0.05) after IVM10 during the non-breeding season. FL-FSHR mRNA expression was decreased (P < 0.01) after IVM1 and IVM10 compared to immature COCs in both seasons. Tr-FSHR mRNA expression was decreased (P < 0.05) after IVM1 and IVM10 compared to immature COCs in the non-breeding season, but was similar (P > 0.05) after IVM10 during the breeding season. ESR2 mRNA was decreased (P < 0.01) after IVM in all treatment groups except after IVM10 during the non-breeding season (P > 0.05). Results indicate that (1) FSH-regulated expression of overall FSHR, FL-FSHR and Tr-FSHR mRNAs differ seasonally, (2) increased overall FSHR associated with a decrease in Tr-FSHR mRNA expression may be necessary for optimal maturation of oocytes during the non-breeding season and (3) deficits in COC sensitivity to FSH during seasonal anestrus may be overcome by increased ESR2 mRNA expression. Findings suggest that differential regulation of FSHR mRNA splice variants (FL-FSHR, Tr-FSHR) and ESR2 mRNA appears important in FSH-induced oocyte maturation in vitro during the non-breeding season. (Supported by the Kelly Fellowship and Lalor Foundation).

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Karyopherin Alpha 1, 2, and 7 Adopt a Nuclear Localization in Porcine Oocytes and Cleavage Stage Embryos.

Xix Wang, Kieun Park, Kallie Yeoman, Ryan Cabot 1

The transport of cellular proteins into the nucleus is critical to many cellular functions. One of the best characterized nuclear trafficking pathways is nuclear import mediated by the importin alpha/beta heterodimer. Importin alpha, also called karyopherin alpha (KPNA), binds to cellular proteins that contain nuclear localization signals (NLS); upon cargo binding, importin alpha then binds with importin beta and the trimeric complex translocates through the nuclear pore complex and disassociates in the nucleus. Seven porcine KPNA molecules have been identified to date. The aim of the study was to determine the localization of KPNA1, KPNA2 and KPNA7 proteins in GV-stage oocytes and 2-cell and 4-cell stage embryos produced by in vitro fertilization. Transcripts for these importin alpha subtypes are detectable in porcine oocytes and cleavage stage embryos, we therefore hypothesized that these importin alpha subtypes participate in nuclear import at these stages of development and would adopt a nuclear localization in these cell types. To test the hypothesis, N-terminal GFP fusion proteins were constructed for porcine KPNA1, KPNA2 and KPNA7 and mRNA encoded by these constructs was injected into porcine oocytes. Briefly, porcine oocytes were collected from prepubertal gilt ovaries; cumulus oocyte complexes were placed in maturation medium (TCM-199 supplemented with 0.1% PVA, 0.069 mg mL-1 cysteine, 10 ng mL-1 EGF, 0.5 IU mL-1 LH and FSH) for 42 to 44 hours at 39°C, in an atmosphere of 5%CO2, 100% humidity. Following cumulus cell removal, mature oocytes were co-incubated with spermatozoa for 5 hours. For experiments involving germinal vesicle (GV) stage oocytes, cumulus cells were removed after oocyte aspiration and allocated to treatment groups. Denuded GV-stage oocytes and mature, fertilized oocytes were assigned to one of four treatment groups: GFP-KPNA1 mRNA injection, GFP-KPNA2 mRNA injection, GFP-KPNA7 mRNA injection and GFP mRNA injection. After microinjection, fertilized oocytes in each treatment group, as well as non-injected control oocytes, were cultured in Porcine Zygote Medium 3, containing 3 mg mL-1 BSA. Two-cell and 4-cell stage embryos were collected after 24 or 48 hours of culture, respectively. GV-stage oocytes were harvested 12 hours after microinjection. All oocytes and embryos were stained with Hoechst 33342 for 15 minutes and examined on an epifluorescent microscope to determine the localization of the nuclei and GFP fusion protein. We found that KPNA1, KPNA2, and KPNA7 proteins were localized in GV oocytes (n=74), 2-cell (n=15) and 4-cell (n=13) embryos, whereas cells injected with GFP did not show nuclear localization to the nucleus in all the stages tested (n=17). These results suggest that in GV-stage oocytes and 2-cell and 4-cell stage embryos, KPNA1, KPNA2, and KPNA7 participate in nuclear import.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Characterization of Ubiquitin Carboxyl-Terminal Hydrolase L1 (UCH-L1) in the Fate Determination of Spermatogonia.

Lin Tang 1, Marie-Claude C Hofmann 2, Ina Dobrinski 1

Spermatogenesis is a highly coordinated and complex process, which requires a fine balance between self-renewal and differentiation of Spermatogonial Stem Cells (SSCs) to support continuous sperm production. In the mammalian testis, SSCs represent a subset of undifferentiated Type A spermatogonia that are located at the basement membrane of the seminiferous tubules. The mechanisms that govern the fate decision of SSCs to maintain the stem cell identity versus to initiate differentiation remain largely unknown. Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is a deubiquitinating enzyme that is selectively expressed in the brain, testis and ovary. It has been implicated in neurological diseases as well as oncogenic processes. In the mouse and pig testis, UCH-L1 is first detected in gonocytes, the precursors of spermatogonia. Later on, the expression of UCH-L1 is restricted to undifferentiated spermatogonia (Asingle, Apair, Aalign) that are associated with the basement membrane of the seminiferous tubules. UCH-L1 is rapidly down-regulated in differentiating spermatogonia and completely undetectable in spermatocytes and spermatids. The objective of this study was to further characterize involvement of UCH-L1 in regulating the fate decision of spermatogonia. Expression of UCH-L1 was characterized by immunohistochemistry and RT-PCR in primary mouse SSCs derived from day 6 mouse testes and in the immortalized type A spermatogonial cell line C18-4. UCH-L1 was detected in mouse SSC clusters grown in vitro. Interestingly, we observed unequal segregation of UCH-L1 in daughter cells of undifferentiated Type A spermatogonia in vitro similar to what we reported previously in vivo. This suggests that undifferentiated spermatogonia are heterogeneous on the molecular level and that variation in the UCH-L1 level may serve as a potential means of regulating the fate decision of spermatogonia. In C18-4 cells, UCH-L1 was highly up-regulated in cells undergoing mitosis. These observations, combined with a previous report that over-expression of UCH-L1 in the mouse testis results in a block in meiosis and infertility, prompt us to hypothesize that a high level of UCH-L1 expression is associated with the maintenance of an undifferentiated status of spermatogonia and down-regulation of UCH-L1 is necessary for differentiation and entry into meiosis. The proposed role of UCH-L1in fate determination is potentially on both transcriptional and post-transcriptional levels. Future work will focus on identifying downstream targets regulated by UCH-L1 and manipulation of UCH-L1 to direct germ cell self-renewal vs. differentiation. Supported by 2RR17539-09, UCVM and CIHR Training Program in Genetics, Child Development and Health.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

GnRH Signals Through Calcineurin, NFAT and JUN to Regulate Expression of at Least Three Genes Required for Gonadotrope Function.

April K Binder, Jean C Grammer, Maria K Herndon, John H Nilson 1

Gonadotropes of the anterior pituitary synthesize and secrete the gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH). LH and FSH are essential for normal reproductive function and act on the ovary and testes to regulate steriodogenesis and gametogenesis. The genes that encode these two hormones are regulated by gonadotropin-releasing hormone (GnRH), which is secreted from the hypothalamus and binds to its receptor on gonadotropes. Upon binding its receptor, GnRH activates Gαq that in turn activates multiple members of the mitogen-activated protein kinase (MAPK) signaling family, including extracellular regulated kinase (ERK), JUN N-terminal kinase (JNK), and p38 MAPK, as well as increases intracellular Ca++ concentrations. Increased activity of the MAPK family members leads to mRNA accumulation of several key immediate early genes (IEGs) including Atf3, Jun and Egr1. These IEGs confer hormonal responsiveness to the gonadotrope specific genes Gnrhr, Cga, Fshb and Lhb. Calcium has been implicated in regulation of several of these genes, although the role for Ca++ within gonadotropes has been controversial depending on the cell line utilized for the study. In this study we test the hypothesis that GnRH regulates activity of the protein phosphatase calcineurin and consequently several immediate early genes through increased intracellular Ca++. Herein we report that pretreatment of murine LβT2 cells with either a Ca++ blocker, BAPTA-AM, or a calcineurin specific inhibitor, cyclosporin A, reduces the ability of GnRH to regulate the accumulation of Atf3 and Jun mRNA while Egr1 mRNA is not affected. Furthermore, pretreatment with thapsigargin, an intracellular Ca++ protein pump inhibitor, increases Ca++ and accumulation of Atf3 and Jun mRNA in the presence of both vehicle and GnRH. Calcineurin also regulates the transcriptional activity of NFAT proteins. Additional data indicate that LβT2 cells express NFAT and that GnRH increases the luciferase activity of an NFAT-dependent promoter reporter. We also show that GnRH regulates NFAT-dependent transcription through intracellular Ca++ and calcineurin activation. NFAT proteins have been shown to cooperate with proteins bound to AP1 response elements suggesting that AP1 responsive genes including Jun may also require NFAT. Together, these data suggest a functional link between GnRH, calcineurin, NFAT and a specific immediate early gene (Jun) that regulates expression of at least three genes required for gonadotrope function. These data also suggest that NFAT is a new member of the gonadotrope transcriptional network and may provide another input that confers hormonal responsiveness to several key genes required for gonadotropin synthesis and secretion. This work was supported by NIH R01 HD055776 to J.H.N.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Cloning of Candidate Genes for TMEM48 Binding Proteins, Which Could Be Involved in Gametogenesis.

Shimpei Kajita 1, Kouyou Akiyama 1, Michiko Hirose 2, Narumi Ogonuki 2, Atsuo Ogura 2, Takehito Tsuji 1, Tetsuo Kunieda 1

The skeletal fusions with sterility ( sks) is a mutation of mouse showing defects of gametogenesis and axial skeletal formation. Recently, we have identified a mutation of Tmem48 (Ndc1) gene in the sks mutant mouse. To confirm that the mutation is responsible for the phenotypes of the sks mutant mouse, we attempted to rescue the phenotypes of the sks mutant mouse by using bacterial artificial chromosomes (BAC) transgenic mouse, containing a normal Tmem48 gene. As a result, the BAC transgene completely rescued phenotypes of the sks mutant mouse including defective gametogenesis. Thus, Tmem48 is confirmed to be the causative gene for sks. TMEM48 is highly conserved in eukaryotes and known for a member of nuclear pore complexes (NPC). TMEM48 is predicted to be required for NPC assembly and binds to several NPC proteins in somatic cells. Since the sks mutant mouse show defects of gametogenesis, Tmem48 should have a essential role in gametogenesis in mouse. However, functions of Tmem48 and NPC in gametogenesis are still elusive. The purpose of this study is, therefore, to reveal the functions of Tmem48 in gametogenesis, and we attempted to identify proteins that bind to TMEM48 in the mouse testis. We employed yeast two hybrid system to screen TMEM48 binding proteins. For preparation of the yeast two hybrid screening, mouse Tmem48 cDNA and mouse testis cDNAs were cloned into bait and pray vectors, respectively. As a result of yeast two hybrid screening, we obtain 58 positive clones, and determined their nucleotide sequences. Consequently, 29 independent genes were identified as candidate genes for TMEM48 binding proteins. Some of these candidate genes encode proteins which could be involved in nucleocytoplasmic transport through NPC or intracellular membrane fusion. Next, we examined expression of these candidate genes and Tmem48 in various mouse tissues by RT-PCR. A significant part of these candidate genes were expressed strongly in testis, and some of them showed expression patterns similar to that of Tmem48. These findings suggested that Tmem48 is involved in germ cell specific nucleocytoplasmic transport.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

A Novel N-Linked Glycosylated G-CSF (Phe140Asn) Protects H9c2 Rat Cardiomyocytes from H2O2-Induced Apoptosis.

Hee Kyoung Chung, Eun-Mi Ko, Sung Woo Kim, Sung June Byun, Jae Gyu Yoo, Soo-Bong Park, Kyung-Woon Kim 1

Granulocyte-colony stimulating factor (G-CSF) is one of the hematopoietic growth factors that play an important role in stimulating proliferation, differentiation, and functional activation of blood cells. As a therapeutic agent, G-CSF is used to reduce the risks of serious infection in immunocompromised individuals undergoing chemotherapy and thus facilitates recovery. In addition, G-CSF mobilizes bone marrow stem cells (BMSCs) into the infarcted hearts and accelerates the differentiation in vascular cells and cardiac myocytes. The carbohydrate portions of glycoprotein molecules are reported to have many diverse functions, including the effects on the biosynthesis and secretion, immune protection, conformation, stability, solubility and biological activity of the molecules. While G-CSF has an O-linked glycosylation on Thr133, it has no N-linked glycosylation that plays a role in enhancing biological activity. In this study, we constructed six mutant human G-CSF expression vectors with modified glycosylation-sites. The expressions of these mutants are tested in CHO cells using ELISA and Western blotting. The stability and biological activity of these mutant proteins were determined using MTT assay in rat heart myoblasts, H9c2 cells. Cells were incubated with 100 ng/ml G-CSF wild type (WT) and mutants for 24 hrs. Among them, mut #6 (Phe140Asn) had the higher cell viability than other mutants in H9c2 cells. This mutant also had higher anti-apoptotic effects than WT upon H2O2 treatment. Cells were pretreated with 400 ng/ml G-CSF WT and mut #6 (Phe140Asn) for 90 min and then 1 mM H2O2 was added to the cells for 1 hr. The anti-apoptotic effects of G-CSF mut #6 (Phe140Asn) were identified by using FACS analysis (Annexin V and PI staining) and Western blotting for PARP cleavage. As a result, mut #6 (Phe140Asn) had about 10% more cell survival rate than WT in H9c2 cells under H2O2 treatment. And, cleaved PARP upon G-CSF mut #6 (Phe140Asn) had less expressed than WT. In addition, bcl-2 expression level upon G-CSF mut #6 (Phe140Asn) was higher than G-CSF WT in time dependent manner. Therefore, mut #6 (Phe140Asn) may be a potent therapeutic target for the cure of patients with heart failure. These results will contribute to the development of not only pharmaceutical industry but also medical research.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Homeorhetic Adaptation During the Pregnancy to Lactation Transition: A Comparative Transcriptome Analysis of Key Metabolic Tissues Using a Rat Model.

Osman V Patel 1, Karen Plaut 2

Homeorhetic adaptation during the pregnancy to lactation transition elicits adjustment in metabolism of several organs and augments shuttling of nutrients to the mammary gland. Therefore, the goal of this study was to characterize coordinated gene interaction among liver, mammary and adipose tissue mediating homeorhetic adaptation from pregnancy to lactation. Total RNA was isolated from mammary, liver and adipose tissues collected from rat dams on day 20 of pregnancy (P20) and day 1 of lactation (L1) and gene expression was measured using Rat 230 2.0 Affymetrix GeneChips. Parametric gene set enrichment analysis method was utilized to identify transcriptional differences between P20 and L1 tissues and Ingenuity Pathway Analysis (IPA) for uncovering the corresponding regulatory network structures. IPA revealed that pathways associated with development (ERK-MAPK, IGF, PI3K-AKT, PTEN, JAK-STAT), endocrine receptor-associated signaling (AHR, NR3C1, INSR, PRLR, ER) and metabolism (carbohydrate, amino acid, lipid, cholesterol, protein) were enriched (P<0.001) in the mammary gland during P20 to L1 transition. Genes engaged in conveying glucose (INSR, GLUT1, GLUT4, SGLT1 and SGLT2), bicarbonate (SLC4), sodium (SLC9), zinc (SLC30), copper (SLC31) and iron (SLC40) in tandem with rate-limiting lipogenic genes (ACACA, FASN, PRLR, SREBP2, THRSP) were specifically enriched in the mammary gland during the P20 to L1 evolution. Collectively, 50% of the genes associated with solute transport and anabolic process were upregulated in the mammary gland during P20 to L1 evolution compared to 10% in liver and 15% in adipose tissue. Our results show that the mammary gland becomes a factory for macromolecular biosynthesis at L1 through coordinated upregulation of genes associated with nutrient transfer and lipid biosynthesis paralleled with downregulation of the same genes in liver and adipose tissue. (Supported by NASA NNA05CP91A).

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Recombinant Sox2 Protein Rarely Adopts a Nuclear Localization in Immature Porcine Oocytes.

Yanfang Li, Kieun Park, Ryan Cabot 1

Nuclear import mediated by the importin alpha/beta heterodimer serves a key role in partitioning intracellular proteins between nuclear and cytoplasmic compartments during development and differentiation. In this system, importin alpha recognizes the nuclear localization signal (NLS) on intracellular proteins; upon cargo binding, importin alpha interacts with importin beta to transport the NLS-bearing cargo into the nucleus. Multiple importin alpha isoforms exist in mammals. While some intracellular proteins can interact with all known importin alpha subtypes, certain intracellular proteins appear to preferentially interact with only certain importin alpha subtypes. In addition, it is known that transcripts encoding the different importin alpha subtypes vary in abundance within porcine oocytes. We hypothesized that cargos transported by discrete importin alpha isoforms would localize to the nucleus within germinal vesicle (GV)-stage oocytes differently than cargoes that interact with multiple importin alpha isoforms. The goal of this study was to determine the intracellular localization adopted by recombinant Sox2, a protein previously shown to be preferentially imported by importin alpha 5 and recombinant protein bearing the NLS derived from the SV40T antigen, a classical NLS shown to interact with all known importin alpha subtypes. Briefly, glutathione-S-transferase (GST) fusion proteins, containing GST fused with either Sox2 (GST-Sox2), the NLS from SV-40 T antigen (GST-NLS), or the reversed sequence of the 8 amino acids compromising the SV40 T antigen (referred to as GST-SLN), were produced in bacteria. Following purification, both GST-NLS and GST-SLN were labeled with Alexa 594, while GST-Sox2 was labeled with fluorescein-5-maleimide. Immature porcine oocytes were harvested from prepubertal gilt ovaries, cumulus cells were removed from GV-stage oocytes by vortexing in 0.1% hyaluronidase in Hepes-buffered medium. Oocytes were then divided into two treatment groups and coinjected with either GST-NLS and GST-Sox2 or GST-SLN and GST-Sox2. Following microinjection, all oocytes were stained with Hoechst 33342 for 15 minutes and examined on an epifluorescent microscope to determine the intracellular localization of all injected proteins. Our results show that while GST-NLS localized to the nucleus in all oocytes (n=53/53) and GST-SLN was restricted to the cytoplasm in all oocytes (n=45/45), GST-Sox2 was excluded from the nucleus in the majority of oocytes (n=80/98). In the remaining oocytes, GST-Sox2 was detectable in the nucleus, although considerable fluorescent signal remained detectable in the cytoplasm. These results suggest that proteins preferentially imported by importin alpha 5 may be restricted in their ability to access the nucleus in GV-stage oocytes.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

NOT PRESENTED.

NOT PRESENTED.

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Blastocyst Dicer Expression and miRNA Biogenesis Related to Human Infertility.

Susanna McReynolds 1, Blair R McCallie 1, Jason C Parks 1, Ann Janesch 1, John Crocker 1, William B Schoolcraft 2, Mandy G Katz-Jaffe 1

MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate the abundance and translation of mRNAs. Pre-miRNAs are processed sequentially by a series of RNase III-related enzymes, such as Dicer, to yield miRNAs. Recent data indicate the importance of miRNAs in human development including our previous report of altered miRNA profiles in blastocysts derived from infertile patients. Our group showed that blastocysts derived from patients with polycystic ovaries (PCO) exhibited six significantly altered miRNA profiles compared to blastocysts derived from fertile controls including let-7a, miR-19a, miR-19b, miR-24, miR-92 and miR-93. Predicted miRNA target genes, including ARIDH2, KHSRP and NFAT5, were projected by TargetScan (www.targetscan.org) and demonstrated to have altered expression in blastocysts derived from PCO patients, consistent with their respective miRNA profiles (P<0.05). In an attempt to understand the mechanisms behind these altered blastocyst miRNA profiles and PCO infertility prognosis we studied the mRNA expression of 20 genes involved in the miRNA biogenesis pathway including DICER1 & DGCR8 (pre-miRNA) and MOV10 (mRNA cleavage). All human blastocysts were cryopreserved as surplus embryos and donated for research with informed patient consent and IRB study approval. Results showed no significant expression differences between blastocysts derived from patients with PCO (n= 24) and fertile controls (n= 24) for all 20 gene transcripts involved in the miRNA biogenesis pathway. Based on these findings, we further hypothesized that alterations in the miRNA biogenesis pathway could be occurring post-translation at the protein level, specifically a decrease in Dicer protein expression in blastocysts derived from PCO patients compared to fertile controls. To test the hypothesis we examined Dicer protein expression using fluorescent immunocytochemistry in blastocysts derived from patients with PCO (n=8) compared with blastocysts from fertile controls (n=12). Individual viable blastocysts were fixed and stained for the cytoplasmic protein Dicer. Positive and negative controls were performed and results revealed no differences in the Dicer protein levels between the two groups of blastocysts, consistent with the mRNA profile. In summary, these data suggest that PCO infertility prognosis does not affect the miRNA biogenesis pathway of human blastocysts. Rather another form of regulation may be responsible for the observed altered miRNA profiles, such as the very targets they control. Growing evidence indicates the importance of miRNAs during development, including an association between altered blastocyst miRNA profiles and infertility prognosis. Aberrant miRNA expression may be considered an additional contributing factor to reproductive failure with the potential for therapeutic intervention.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Sertoli and Granulosa Cell-Specific Vascular Endothelial Growth Factor (VEGF) Loss Using pDMRT-1 Cre Alters Gonadal Morphogenesis and Function.

Ningxia Lu 1, Debra T Clopton 1, William Pohlmeier 1, Vanessa Brauer 1, David Silversides 2, Napoleone Ferrara 3, Andrea S Cupp 1

Inhibition of VEGF disrupts both testis-specific vascular development and cord formation. In the female, VEGF isoforms regulate follicle development and may affect the initial primordial follicle pool. Therefore, the objective of the current study was to evaluate effects of Sertoli and Granulosa cell-specific production of VEGF isoforms on vascular development and gonadal morphogenesis. The pDmrt1 gene is expressed in the indifferent gonad at 10.5 dpc in precursor Sertoli and Granulosa cells. Therefore, we used a DMRT1-cre crossed to a floxed VEGF mouse to determine potential phenotypes in male Sertoli-cell (VEGF-DSert) and female Granulosa-cell (VEGF-DGran) specific knockouts. Mice from these crosses had litters and had approximately one less mouse/litter than controls. Reproductive organs were collected from adult mice, and weighed (male: control n=9, VEGF-DSert; n=8; female: control n=11, VEGF-DGran n=10). Testosterone and estrogen levels in blood were determined by ELISA. In VEGF-DSert , the weight of the body (28.52±0.48 VS 32.52±1.20 g); testes (0.1658±0.0064 VS 0.1924±0.0061 g) and epididymus (0.0627±0.0046 VS 0.0792±0.0042 g) were significantly smaller than those from the control (P<0.05). The prostate weight also tended to be different (P=0.0636) than controls. Testosterone was not different when averaged across treatment and controls groups; however, three VEGF-DSert males had 10 fold greater testosterone concentrations than controls (12.81 vs 1.18 ng/ml). For the females, the weight of ovaries from VEGF-DGran were significantly smaller than that of the control (0.0144±0.0014 VS 0.0183±0.0009 g, P<0.05). Two VEGF-DGran ovaries had abnormal morphology in structures that appeared to be CL. Furthermore, the estrogen concentrations in the VEGF-DGran mice were 56% lower than those in control mice (48.83±9.58 VS 91.53±14.75 pg/ml, P<0.05). Therefore, we can conclude from these data that lack of VEGF in Sertoli and Granulosa cells alters reproductive organ morphogenesis, reduces gonadal and male accessory gland organ weights, alters steroidogenic profiles and potentially causes subfertility in both male and female mice. This research was supported by NIH/NICHD HD051979.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Identification of Multiple Isoforms of the Ashwin Gene in Cattle.

Everardo Gonzalez-Rodriguez, Elier Soto-Orduno, Veronica Moreno-Brito, Ruth Lechuga-Valles, Eduviges Burrola-Barraza, Moises Barcelo-Fimbres, Ernesto Orozco-Lucero, Irene Leal-Berumen 1

The ashwin gene in Xenopus is expressed in the neural plate, and later in the embryonic brain, eyes, spinal cord, and regulates cell survival during gastrulation and anterioposterior patterning. ashwin is also maternally and zygotically expressed, suggesting its importance in embryo development. Here, the bovine ASHWIN gene homolog was isolated from an embryonic ovary cDNA library. The expression analysis of this gene by RT-PCR of bovine embryo and adult brain, heart, lung, muscle, ovary, and testis demonstrated the ubiquitous expression of its 699 bp transcript. Interestingly, its expression analysis in adult tissues showed the expression of two alternative variants of 345 and 543 bp, which are expressed in heart, spleen, and kidney, while the 615 and 478 bp alternative variants are present in ovary, lung, muscle, and brain, with a strong presence in testis. In conclusion, our results demonstrate the expression of alternative variants of ASHWIN in adult tissues, which suggests multiple functions of this gene in bovine. The high expression of two alternative variants in testis suggests the possible importance of ASHWIN in the cellular processes in the male gonad of this species, as spermatogenesis and morphogenesis.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

NOT PRESENTED.

NOT PRESENTED.

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Effect of GnRH Agonist (Buserelin) on Bovine Endometrial mRNA Expression of Growth Factors and Cytokines.

Thavaneetharajah Pretheeban, Ravinder Singh, Rajadurai Rajamahendran 1,2

Gonadotropin releasing hormone (GnRH) agonists are widely used in bovine reproductive management. Administration of GnRH agonist prior to breeding and during the luteal phase has given mixed results in terms of embryo survival and pregnancy rates. The demonstration of extra hypothalamic GnRH and GnRH-R system, in bovine reproductive tract, has led to the study of local modulatory role of this system in reproductive processes such as fertilization, early embryo development and implantation. In a recent study, we have demonstrated the presence of differential mRNA expression of luteal phase endometrial genes, associated with fertility, between dairy heifers and lactating dairy cows. In this study, we investigated the effect of a GnRH agonist (Buserelin) on the expression of FGF2, IGF1, IL1A, and TNF in the luteal phase endometrium. Endometrial explants (100mg) obtained from slaughter house reproductive tracts were cultured at 37°C in air with 5% CO2 in Dulbecco modified eagle medium containing 0.1% BSA, 100 IU/mL penicillin G and 100 µg/mL streptomycin for 20 h. Media was replaced and then treated with buserelin (0 and 200 ng/mL), GnRH antagonist- antide (500 ng/mL), and antide + buserelin (500+200 ng/mL) for further 6 h. Two µg of total RNA, extracted from treated explants, was reverse transcribed using gene-specific primers and the PCR products were electrophoresed and visualized under UV illumination. The relative mRNA levels were measured using GAPDH as the house keeping gene. GnRH agonist (buserelin) up regulated the mRNA expression of IGF1 and no effect was found on the expressions of FGF2, IL1, and TNF. GnRH antide + GnRH treatment reversed the effect of GnRH on IGF1 expression, suggesting a receptor mediated response. Further studies are warranted to confirm the functional significance of up regulation IGF1 mRNA expression on the regulation of endometrium and therefore bovine early embryo development.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Effects of Porcine Follicular Fluid on the Developmental Characteristics of the Alpha 1,3-Galactosyltransferase Knock-Out Cells for SCNT.

Seongsoo Hwang 1, Keon Bong Oh 1, Hwi-Cheul Lee 1, Yeoung-Gyu Ko 1, Byong-Chul Yang 1, Gi-Sun Im 1, Eung-Woo Park 1, Jin-Hoi Kim 2, Jae-Seok Woo 1, Soo-Bong Park 1

Follicular fluid contains growth factors, electrolytes, hormones, amino acids and unknown factors. Unlike eggs, donor cells are not exposed to reproductive hormones such as estrogen, progesterone or FSH, directly. This study was performed to investigate the effects of porcine follicular fluid (pFF) on the developmental characteristics of the donor cells knocked-out with alpha 1,3-Galactosyltransferase (GalT KO) during porcine SCNT procedure. The pFF was collected from ovarian follicles (3-6mm in diameters) and centrifuged for 20min (10,000 x g) at 4°C. The GalT KO donor cells were cultured and divided into three experimental groups: the cells were cultured in (1) DMEM containing 10% FBS (control); (2) DMEM supplemented with 0.5% FBS (serum starvation, SS); (3) control added with 10% pFF (pFF) under 5% CO2 in air at 38.5°C for 72 hrs. After the culture period, the donor cells were trypsinized and analyzed for cell proliferation (WST-1, TAKARA) and cell membrane alterations assay (Annexin-V-FLUOS Staining kit, Roche) and the concentration of 16 amino acids (Cys, Met, Asp, Thr, Ser, Glu, Gly, Val, I-Le, Leu, Tyr, Phe, Lys, His, Arg and Pro) in the conditioned medium was measured. The reconstructed embryos were fused with two electric pulses (DC) of 1.2 kV/cm for 30 µsec. The cloned embryos were transfer to a surrogate (Landrace). The cell proliferation rate was SS (0.47) and pFF (1.25) compared with the control (1.0), respectively. The percentage of Annexin V-positive (apoptotic and necrotic) cells was significantly higher under SS (32.5, 9.9) than that of control (1.0, 2.1) and pFF (1.9, 2.4) (p<0.05), respectively. The concentration of amino acids in the conditioned medium was similar among three treatment groups, except for higher concentration of Asp, Thr, Glu and Pro in pFF group (p<0.05). Using the donor cells, we produced three GalT KO TG cloned miniature pigs. Based on the results, the treatment of pFF was no harmful effects on the developmental characteristics in the transgenic donor cells and the production of the TG cloned pigs. This work received grant support from the Agenda Program (No. PJ006688), Rural Development Administration, Republic of Korea.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.
Kyle W Selcer, Laura L Vollmer 1

The importance of estrogen in bone regulation is exemplified by the rapid loss of bone density at the onset of menopause. Post-menopausal women have low levels of estrogens, but high levels of inactive sulfated steroids. These can be converted to active estrogens by the enzyme steroid sulfatase (EC 3.1.6.2), which removes the sulfate group. It is known that estrogens can stimulate the growth and maintenance of bone cells, particularly osteoblasts; however, it is not known if sulfated steroids can induce a similar response. The purpose of this study was to determine if sulfated steroids are able to induce proliferation of a human osteoblast-like cell line (MG-63), and to establish if steroid sulfatase inhibitors were capable of blocking any response. A growth assay was developed to assess proliferation of MG-63 cells in the presence of various steroids. A variety of different steroids were tested, and the results indicated that estradiol and estrone sulfate both increased proliferation of the osteoblast-like cells. A dose-response assay of MG-63 cell proliferation in response to estradiol and estrone sulfate revealed that estradiol was more potent at stimulating cell proliferation. The estrogen receptor antagonist ICI 182,780 inhibited both estrone sulfate- and estradiol-stimulated growth, indicating that the proliferation response is mediated by the estrogen receptor. Steroid sulfatase activity was then assessed in MG-63 microsomes and in whole cells in the presence of two steroid sulfatase inhibitors (EMATE and DU-14). Steroid sulfatase activity in the presence of either of the inhibitors was virtually eliminated. One of these inhibitors, DU-14, was able to block estrone sulfate-stimulated growth. EMATE, which is known to be estrogenic, stimulated cell growth in the presence of estrone sulfate. These data demonstrate that proliferation of the human MG-63 osteoblast-like cells is stimulated by estrogens and by sulfated estrogens, supporting the concept that sulfated steroids and steroid sulfatase may be important in the regulation of bone density.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Defining the Porcine Milk Proteome.

Alejandro J Silva 1, Anne A Wiley 1, Amy Lynn Frankshun 2, Carol A Bagnell 2, Frank F Bartol 1

The importance of colostrum (first milk) for immunological and nutritional support of newborn mammals is well known. Many bioactive peptides are present in milk at higher concentrations than in maternal circulation. Milk-borne, lactocrine-acting factors affect patterns of gene expression in neonatal somatic tissues, including the reproductive tract and heart. Thus, it is important to understand the biochemical nature of colostrum/milk. Data for relaxin, a prototypical lactocrine-acting factor in porcine colostrum, indicate that transmission of such factors is significant prior to gut closure in the neonate. Additionally, amino acid sequences encoding potentially bioactive peptides are encrypted within porcine milk proteins. Such factors could be released into the neonatal circulation through proteolytic cleavage after consumption. The array of proteins/peptides constituting the porcine colostral proteome has not been defined. Objectives of this study were to employ two-dimensional gel electrophoresis (2DE) and image analysis to: (1) define the porcine colostral proteome on lactation day (LD) 0; and (2) determine if and how this proteome changed from LD 0 to LD 6. Colostrum (LD 0) and milk (LD 6) samples were obtained from six lactating sows. Protein was extracted from individual samples and total protein concentrations were determined. Extracted proteins were subjected to both standard SDS-PAGE (10% total monomer) and 2DE. For 2DE (Criterion; Bio-Rad Laboratories), first dimension separations were carried out using pH 3-10 immobilized pH gradient strips followed by SDS-PAGE using gradient polyacrylamide gels (10-20% total monomer). Individual samples were run on duplicate 2DE gels and stained with Sypro RUBY (Bio-Rad). Digital images of individual gels were obtained and analyzed using a Typhoon 9400 digital scanner (General Electric) and PDQuest 2-D Analysis Software (Bio-Rad). Total protein concentrations for colostrum (LD 0) and milk (LD 6) were 8.5 mg/ml and 8.3 mg/ml. Standard SDS-PAGE analyses revealed distinct differences in the distribution of protein bands between LD 0 and LD 6. PDQuest analysis identified 304 spots on 2DE gels that defined the colostral/milk proteome. Of these, 34 were unique to LD 0 and 8 were unique to LD 6. Differences in relative spot intensity between groups were also identified. There were 143 spots common to LD 0 and LD 6 that changed quantitatively at least 2-fold between days. Of these 143 spots, 73 increased in abundance while 70 decreased in abundance from LD 0 to LD 6. Data indicate that the colostral proteome (LD 0) is distinct from that identified for milk (LD 6). Thus, newborn pigs that nurse obtain a complex mixture of proteins and peptides from birth which changes with time during a critical developmental period. This idea is supported by the fact that gilts deprived of colostrum for only two days from birth exhibit altered gene expression patterns associated with development of reproductive tract tissues and which persist when colostrum-deprived neonates are returned to milk feeding immediately thereafter. The presence of encrypted peptides in colostral/milk proteins increases the complexity of the porcine milk proteome. Future studies will focus on identifying colostral proteins and peptides and determining their role as mediators of lactocrine programming in the neonate. [Support: USDA-NRICGP 2007-35203-18098 and NSF-EPS-0447675].

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

NOT PRESENTED.

NOT PRESENTED.

Biol Reprod. 2010 Nov;83(1 Suppl):2.

A Novel Method of Monitoring Placenta-Specific Transgene Expression Throughout Pregnancy by Noninvasive Bioluminescence Imaging.

Xiujun Fan, Peigen Ren, Sabita Dhal, Stuart B Goodman, Sanjiv S Gambhir, Maurice L Druzin, Nihar R Nayak 1

Placental dysfunction underlies numerous complications during pregnancy affecting both maternal and fetal health. The recent development of placenta-specific gene activation and inactivation by lentiviral vector transduction of mouse blastocysts using the Cre/LoxP system is a significant step forward in understanding placenta-specific gene functions during pregnancy. However, in this system, there is considerable variability in gene expression among different placentas, and the extent of gene expression can only be determined after termination of pregnancy. Also, several genes are known to be expressed in different trophoblast lineages only at specific stages of development, not consistently throughout the pregnancy, underscoring the critical need for an efficient noninvasive method of monitoring gene expression at different stages of placental development during pregnancy. In this study, we have developed a method of repetitively tracking in vivo gene expression in the mouse placenta at multiple time points during pregnancy after placenta-specific lentiviral gene delivery using a double reporter gene expression system, and demonstrated the feasibility of preselecting blastocysts for uniform gene expression in all placentas of the same litter. We prepared a lentiviral vector containing both firefly luciferase and tomato fluorescent protein driven by constitutive ubiquitin C promoter (LV-Fluc/Tomato). After zona removal, the blastocysts were incubated with LV-Fluc/Tomato (2.5x109particles/ml) and transduced blastocysts were transplanted into day 3 pseudopregnant mice. In another group, LV-Fluc/Tomato transduced blastocysts were examined for levels of Fluc expression by bioluminescence imaging (BLI), and blastocysts with total photon flux (photons/sec) in the range of 2.0E+4-6.0E+4 were transferred. All animals were examined for establishment of pregnancy and Fluc expression by live BLI on days 4, 5, 6, 9, 12, 16 and 18 of pregnancy, and the placentas and fetuses were collected on day 18 for both BLI and examination of tomato expression in different cell types. Fluc expression was detectable in all surrogate dams by day 5 of pregnancy by live imaging, and the signal increased dramatically thereafter each day till day 12, reaching to a peak level at day 16 and maintained at the same level till day 18. All of the placentas, but none of the fetuses, analyzed on day 18 by BLI showed different degrees of Fluc expression. However, all the placentas of dams transferred with selected blastocysts showed uniform photon distribution with no significant variability of photon intensity among the placentas of the same litter. Tomato expression in the placentas was limited to all trophoblast cell lineages, with more intense expression in the spongiotrophoblasts, giant cells and interstitial glycogen trophoblast cells. Our results, for the first time, demonstrate the feasibility of selecting lentiviral transduced embryos for uniform gene expression in all placentas of the same litter, and early detection and tracking of trophoblast-specific gene expression throughout pregnancy by live imaging. This method will be highly useful for wide range of applications involving trophoblast-specific gene manipulations in utero.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Differential Microarray Analysis of Human Placental microRNAs Revealed Upregulation of Angiogenesis-Associated microRNAs (miR-15a, miR-126, and miR-210) in Preeclampsia.

Wen Wang 1, Wu-Xiang Liao 1, Jing Zheng 2, Dong-bao Chen 3

Impaired placental angiogenesis is a hallmark of preeclampsia (PE) that affects ~7% of all human pregnancies. MicroRNAs are a class of noncoding 21-25 nucleotide RNAs that negatively regulate gene expression primarily at the level of post-transcription. MicroRNAs are differentially expressed in preeclamptic vs. normal placentas and angiogenesis associated ones are increased by PE. Placentas were collected from severe PE and healthy term pregnancies (n=10/group). Total microRNA samples were isolated from placental tissues by using the mirVanaTM miRNA isolation kit. Differential microRNA expression profiling of PE vs. normal placentas were performed with human microRNA array containing 894 mature microRNAs from miRBase database. Angiogenesis-associated microRNAs were analyzed by searching for their potential targets using online target prediction software (i.e., TargetScan, picTar, and RegRNA). Differentially expressed microRNAs of interest in PE placenta were confirmed by real-time quantitative RT-PCR. Differential microarray analysis identified 44 highly expressed placental microRNAs that differ significantly between PE and normal controls; 74 miRNAs were also differentially expressed but with low expression levels in human placenta. Bioinformatic analysis revealed that these microRNAs are involved in the expression of genes with various biological functions and a subset was associated with angiogenesis. With real-time PCR analysis we confirmed that three highly expressed placental microRNAs, i.e., miR-15a, miR-126 and miR-210, were upregulated in PE placentas. Placental microRNAs are differentially expressed in preeclamptic and healthy term human placentas. Upregulation of angiogenesis-associated microRNAs implicates a potential mechanism for deregulation of angiogenic genes by PE (NIH RO1 HL74947 & HL70562).

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

NOT PRESENTED.

NOT PRESENTED.

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Expression of SPP1 (Osteopontin) and the Aggregation of Integrins into Focal Adhesions Is an Adaptation to Mechanical Forces at the Uterine-Placental Interface in Pigs.

James W Frank 1, Xilong Li 1, David W Erikson 1, Guoyao Wu 1, Fuller W Bazer 1, Kayla J Bayless 2, Robert C Burghardt 1, Greg A Johnson 1

In pigs, the luminal epithelium (LE) adjacent to the implanting conceptus (embryo/fetus and placental membranes) secretes SPP1, an ECM protein involved in cell-cell and cell-ECM communication. SPP1 binds the apical surfaces of trophectoderm (Tr) and LE, and is hypothesized to mediate uterine-conceptus interactions during implantation. Our laboratory demonstrated that the alpha v beta 6 integrin receptor in cultured porcine Tr cells, and the alpha v beta 3 integrin in LE cells bind SPP1 to increase Tr migration, and Tr and LE adhesion. These integrins were also shown to aggregate at the interface between Tr and LE on Day 20 of gestation, suggesting SPP1 binding and assembly of focal adhesions at implantation sites. Similar focal adhesions were detected at the interplacentomal uterine-conceptus interface of sheep that increased in area along the interface as pregnancy progressed, but not until well after the periimplantation period. Results from sheep support the idea that focal adhesions assemble at the uterine-conceptus interface to stabilize adhesion as an adaptation to increasing compression, tension and shear forces generated by the growing fetus and fluid compartments formed by extraembryonic membranes. Therefore, we hypothesized that SPP1, alpha v beta 6 and alpha v beta 3 form focal adhesions along the epitheliochorial uterine conceptus interface throughout pig pregnancy, and that focal adhesions increase with fetal/placental growth. To test this hypothesis we evaluated the expression of SPP1, and integrins alpha v (ITGAV), beta 3 (ItGB3), and beta 6 (ITGB6) at the uterine-conceptus interface of pigs on Days 25, 50, 70 and 90 of gestation. In situ hybridization and immunohistochemistry localized SPP1 mRNA and protein to uterine LE and GE. Interestingly, SPP1 was not expressed in LE facing placental areolae. Using immunofluorescence analyses, large immunoreactive aggregates of ITGAV were detected at the apical surfaces of both LE and Tr on Day 25, and ITGB3 aggregates were observed on the LE only. Unexpectedly, by Day 50 through Day 90, aggregates of ITGAV and ITGB3 were no longer detectable at the uterine-conceptus interface, but ITGB3 was localized specifically to the basolateral surfaces of chorionic epithelia in areolae. Antibodies that cross-reacted with the pig ITGB6 subunit were not available, therefore in situ hybridization was utilized to evaluate ITGB6 mRNA. ITGB6 mRNA was detected in elongating conceptuses and in the uterine LE by Day 12 of pregnancy, and expression was maintained through Day 90. The interesting differences in focal adhesion assembly during pregnancy between sheep and pigs suggest differences in the distribution of mechanical forces at the uterine-conceptus interface. In sheep, interplacentomal regions of endometrium lack the extensive interfolding that is characteristic of the pig uterine-conceptus interface, and therefore require expansion of focal adhesions to maintain continuous contact between uterus and placenta. In contrast, extensive endometrial folding and coordinate interdigitation with the chorioallantois at the uterine-conceptus interface of pigs serves to disperse forces caused by fetal growth, negating the need for focal adhesion assembly by Days 50 through 90. Further, the localization of SPP1 to LE in direct contact with chorion, and not to LE adjacent to placental areolae, suggests that SPP1 is involved in direct adhesion between LE and Tr. Supported by USDA CSREES NRI Grants 2006-35203-17199 and 2008-35203-19120.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Candidate Gene Expression in Bovine Binucleated Trophectoderm and Changes in Expression Profiles after Culture.

Kathleen A Pennington 1, Qien Yang 2, Alan D Ealy 2

A giant, hyperploidic, fusogenic cell termed the binucleate cell (BNC) comprises 15 to 20% of the trophectoderm in ruminant placentae. This cell differentiates from mononucleated trophoblast cells (MNCs) by unidentified mechanisms. In the bovine, BNCs migrate and form a feto-maternal syncytium made up of trinucleated feto-maternal hybrid cells. This laboratory previously described a fluorescence-activated cell sorting (FACS) procedure for obtaining BNCs from bovine placental homogenates. The primary objectives of this work were to describe the expression of specific trophectoderm genes in BNCs and determine if BNCs isolated from mid-gestation bovine placentae could be maintained in culture. In one study, several trophectoderm specific genes were examined in enriched BNC and MNC samples using qRT-PCR. Transcript abundance for the trophectoderm lineage specifier, CDX2, was not different between MNCs and BNCs. The relative abundance of CSH1 and PAG1 mRNA was greater (P<0.05) in BNCs than MNCs (13.4 ± 6.0 and 9.9 ± 4.0 fold induction, respectively). A second a study was completed to describe differences between MNCs and BNCs in mRNA concentrations for putative placental differentiation factors. Primary transcripts of interest were HAND1 (heart and neural crest derivative 1), an endoreduplication factor, and GCM1 (glial cells missing homolog-1), a fusogenic factor. HAND1 mRNA concentrations tended to be greater (P=0.07) in BNCs than in MNCs (3.5 ± 1.97 fold induction). GCM1 mRNA concentrations were greater (P<0.05) in BNCs than in MNCs (2.2 ± 0.9 fold induction). The abundance of these transcripts was also evaluated in one bovine and one ovine trophectoderm cell line (CT1 and oTR). In both cells, HAND1 mRNA was barely detectable and GCM1 expression was absent. In a final study, the maintenance of BNCs in culture was tested. FACS-enriched BNC samples (n=4 isolations) were cultured in serum containing DMEM on Matrigel or no coating (plastic) for 3.5 days at 38.5°C. A greater (P<0.01) number of BNCs and percentage of CSH1-immunoreactive cells were observed when plated on Matrigel (12.15 ± 2.2 BNCs/field; 91 ± 2.1% CSH1-positive) than when plated on plastic only (5.5 ± 1.2 BNCs; 51 ± 7.4% CSH1-positive). Transcripts for CDX2, CSH1, PAG1 and CYP19 were evident after culture, but the abundance of these transcripts decreased (P<0.05) when compared to BNCs examined immediately after collection. To summarize, FACS was effective at enriching BNCs from bovine placental homogenates, and these BNCs contained transcripts for several trophectoderm- and BNC-specific genes. Transcripts for two factors that may regulate BNC formation and function also were found in greater abundance in BNCs than MNCs, and these factors were absent in trophectoderm cell lines. FACS-enriched BNCs maintained their morphology after several days in culture. However, key BNC-specific genes were lost during culture, suggesting these cells rapidly lost their ability to produce trophectoderm- and BNC-specific factors. The loss of these BNC-specific features makes them a poor model for studying placental biology in cattle.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Decreased Lin28 Expression Is Required for Mouse Trophoblast Stem Cell Differentiation.

Quinton A Winger, Brittany A Fromme, Vanessa A Enriquez, Russell V Anthony, Gerrit J Bouma 1

Trophoblast cell proliferation and differentiation are necessary for embryo implantation and establishment of the placenta. In the mouse, trophoblast cells differentiate into trophoblast giant cells and enter a unique endocycle. Mouse trophoblast stem (TS) cells can be maintained in a proliferative state in culture by adding FGF4 to the media and can be forced to differentiate by FGF4 removal or by addition of the CDK1 inhibitor RO3306, even in the presence of FGF4. Inhibition of CDK1 mimics the action of p57 and results in TS cell differentiation and initiation of endocycles. We have previously demonstrated that mRNA levels of pluripotency factors Lin28, Sox2 and Nr0b1 decrease as TS cells differentiate into trophoblast giant cells by FGF4 removal. However, it is possible that the decrease in gene expression is due to the influence of FGF4 removal and not due to TS cell differentiation. In this study we compared relative mRNA amounts in TS cells differentiated by RO3306 treatment in the presence of FGF4 by real-time RT-PCR and found Lin28 was down regulated, however Sox2 and Nr0b1 were not significantly decreased. Lin28 generally is detected in pluripotent stem cells and decreases upon cell differentiation. One established role of Lin28 is interference with miRNA processing and blocking the accumulation of mature let-7 miRNA. In differentiated TS cells Lin28 expression decreases, resulting in increased amounts of mature let-7 miRNA. Tcfap2c mutant TS cells are an interesting cell line for studying TS cell differentiation because they do not show a morphologic change to giant cells, do not down regulate Lin28 mRNA or increase mature let-7 miRNA with FGF4 removal. Treating Tcfap2c mutant cells with RO3306 results in cell death. Therefore, the two methods of differentiating TS cells either results in cell death or no differentiation in Tcfap2c mutant cells. Chromatin immunoprecipitation (ChIP) experiments confirm that TCFAP2C binds to the Lin28 promoter region in TS cells. To determine the effect of Lin28 knockdown in Tcfap2c mutant TS cells we infected cells with lentivirus expressing shRNA targeting the Lin28 transcript. In control cells, 79% Lin28 knock-down efficiency was achieved compared to a lentivirus scrambled shRNA control. The control cells maintained a proliferative state after Lin28 knockdown. Lin28 knockdown efficiency was 88% in the Tcfap2c mutant cells and resulted in rescued ability to differentiate into trophoblast giant cells upon removal of FGF4. Therefore, our conclusion is that Tcfap2c mutant cells block TS cell differentiation due to an inability to decrease Lin28 expression that when removed by Lin28 shRNA knockdown rescued their ability to differentiate. This result suggests that TS cell differentiation is dependant on Lin28 down regulation. Lin28 does not appear to be required for TS cell proliferation and may only be necessary as a secondary control mechanism to prevent let-7 miRNA expression in proliferating TS cells.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.
Tiffany A Wilmoth, Caleb O Lemley, Jill M Koch, Juanita Engels, Kenneth P Blemings, Matthew E Wilson 1

Both pig and placental size vary greatly, not only between litters, but within individual litters. Nutrient availability and the efficiency of the placenta to deliver nutrients to the fetus can influence both fetal and placental growth and development. Placental efficiency, defined as fetal weight divided by placental weight, is influenced by specific placental factors such as size, which in turn may influence the size of the fetus. Amino acids are one of the major nutrients transported to the fetus, and are of particular interest because they not only contribute to the growth and development of the fetus in utero, but also lay the groundwork for potential growth and muscle development postnatally. Therefore, the objective of the current study was to determine the amino acid concentrations in the umbilical artery and vein of fetal pigs on day 90 and 110 of gestation, whether they were affected by day of gestation and to ascertain any relationships among placental efficiency, placental weight and fetal weight. Gilts (N=13) were observed for estrous behavior and bred by artificial insemination 12 and 24 hours following the onset of estrus of the second estrous cycle. At the time of breeding, gilts were randomly assigned to be ovariohysterectomized on day 90 or 110 of gestation. At the time of ovariohysterectomy, fetal weights, placental weights and placental efficiencies were determined for each conceptus and a blood sample from the umbilical vein and umbilical artery was collected for each conceptus. Plasma was harvested from these blood samples and used to determine amino acid concentrations by high performance liquid chromatography. Amino acid concentrations for venous aspartate increased (P < 0.05) from day 90 and 110 (222 ± 32 µM and 720 ± 237 µM, respectively). Venous alanine concentrations decreased (P < 0.05) between day 90 and 110 of gestation (600 ± 67 µM and 290 ± 44 µM, respectively). Venous glutamine decreased (P < 0.05) on days 90 and 110 of gestation (339 ± 38 µM and 213 ± 31 µM, respectively). Arterial glycine (1502 ± 193 µM and 968 ± 58 µM) and alanine (489 ± 54 µM and 240 ± 56 µM) decreased (P < 0.05) between days 90 and 110 of gestation, respectively. On day 90 of gestation, venous proline was negatively correlated with placental efficiency (r = -0.27, P < 0.01). On day 110 of gestation, eight venous amino acids were negatively correlated with placental efficiency, including aspartate (r = -0.29, P < 0.03), serine (r = -0.28, P < 0.04), glutamate (r = -0.30, P < 0.03), asparagine (r = -0.28, P < 0.03), glycine (r = -0.26, P < 0.05), arginine (r = -0.28, P < 0.04) and proline (r = -0.34, P < 0.009). The negative relationship between some venous amino acid concentrations and placental efficiency in the last third of gestation would be consistent with a more efficient placenta having an increased ability to extract nutrients from maternal blood.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Ginger Juice Activates Endothelial Cell ERK1/2 and eNOS and Induces Vasodilation.

Caifeng Dai 1, Lu Lin 2, Mingjiang Li 2, Ziming Yu 1, Dinesh Shah 1

Preeclampsia is pregnancy-specific hypertensive disorder unique to humans that is characterized by elevated blood pressure, proteinuria and edema. Causing substantial maternal and perinatal morbidity and mortality, preeclampisa represents a common and significant obstetric problem worldwide. Although numerous synthetic anti-hypertensive drugs have been developed over the last few decades, none can be safely used in preeclamptic women to benefit the mother but without any significant risk to harm the fetus. Therefore, exploration of novel, safe and effective therapeutic strategies for preeclampsia is highly desirable. Ginger is a natural dietary substance. As a commonly used remedy to alleviate symptoms of pregnancy-associated nausea and vomiting, ginger has proven pregnancy-safe. In the present study, we sought to investigate the possible anti-hypertensive effect of ginger and the underlying mechanism(s). To this end, juice was freshly prepared from ginger roots and its effects on vasoreactivity and on ERK1/2 and eNOS activation were evaluated respectively in dissected mouse mesenteric artery rings by wire myography and in primary human umbilical vein endothelial cells (HUVECs) by Western blotting. The results clearly demonstrated that ginger juice robustly dilated the mouse mesenteric artery pre-constricted by potassium or phenylephrine and significantly induced phosphorylation of ERK1/2 at Thr202/Tyr204 and eNOS at Ser1177 in a dose-dependent manner. Given that the ERK1/2-mediated eNOS activation and NO production in the vascular endothelium play an important role the regulation of vascular smooth muscle relaxation, we can conclude that ginger is capable of dilating arterial vessels through an endothelial ERK1/2 and eNOS-dependent mechanism. Our study strongly suggests that ginger juice possess a great potential to be developed as a novel, effective and non-toxic antihypertensive drug for preeclampsia.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Adherens Junction Proteins in the Hamster Uterus: Their Contributions to the Success of Implantation.

Bibhash C Paria, Tianbing Ding, Amanda Stinnett, Jeff Reese, Liming Luan 1

The adherens junction (AJ), which is an indispensible intercellular unit in the uterine luminal epithelial lining, is important for maintaining uterine structural integrity, composition of luminal environment, and initiation of implantation by virtue of its properties of cell-cell recognition, adhesion, and establishment of cell polarity and permeability barrier by developing tight junctions. In this study, we investigated the uterine changes of AJ components E-cadherin, beta-catenin and alpha-catenin at their protein and mRNA levels together with the cellular distribution of phospho-beta-catenin and active beta-catenin. Studies were performed in hamsters that show only ovarian progesterone-dependent uterine receptivity and implantation similar to humans. By immunofluorescence, in situ hybridization, immunoprecipitation and immuno blotting, we have demonstrated that uterine epithelial cells expressed three of these AJ proteins and their mRNAs prior to and during the initial phase of implantation. With advancement of the implantation process and loss of the epithelial lining surrounding the implanting blastocyst, three components of AJs were primarily expressed in cells of the secondary decidual zone (SDZ), but not in the primary decidual zone (PDZ). In contrast, we noted strong expression of beta-catenin and alpha-catenin proteins in cell of the PDZ, but not in the SDZ, of mice. We also report that uterine beta-catenin may be involved in mediating the Wnt-signaling pathway in the uterus during implantation and decidualization. Taken together, these results demonstrate that uterine AJ junctional proteins are important for cell-cell adhesion and signaling in continuation of pregnancy to ensure protection of the embryo by creating a favorable uterine environment. Research supported by NIH HD044741.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Pregnancy Increases Total S-nitrosylation, but Decreases S-Nitrosylation of Endothelial Nitric Oxide Synthesizing System in Sheep Uterine Artery In Vivo.

Honghai Zhang 1, Ronald R Magness 2, Dong-bao Chen 1

Pregnancy-associated uterine vasodilatation occurs via enhanced endothelial nitric oxide (NO) synthase (eNOS) expression and activation thereby increasing NO production locally at the level of the uterine artery (UA) endothelium. Indeed local inhibition of uterine vascular NO production with L-NAME in vivo dose dependently decreases ovine uteroplacental blood flow. However, it is unknown if increased NO production directly affects UA protein post-translational modifications and thus their functions. Formation of S-nitrothiols (S-nitrosylation) via addition of NO-derived nitrosyl groups to cysteines regulates the function of a plethora of proteins including eNOS. Hypotheses: total protein S-nitrosylation is upregulated, however S-nitrosylation of the NO synthesizing system is downregulated by pregnancy. Methods: UA segments were obtained from day 120-130 pregnant (P) and day 10-12 luteal phase nonpregnant (NP) ewes (n=3/group). UA endothelium (UAendo) and smooth muscle (UAsm) were mechanically separated. Total levels of nitrosylated proteins were labeled by a 3-step biotin switch technique and determined by immunoblotting with anti-biotin antibody. Biotinylated proteins were then pulled down by NeutrAvidinTM Protein coated beads for measuring levels of nitrosylated proteins of the NO synthesizing system (eNOS and HSP90) by immunoblotting with specific antibodies. Results: Levels of total UAsm nitrosylated proteins were significantly greater in P UAsm that of NP UAsm (p<0.01). By contrast levels of total UAendo nitrosylated proteins did not differ in P vs. NP. Total eNOS protein was upregulated whereas total HSP90 was downregulated in UAendo by P. As expected, eNOS protein was barely detected in UAsm and total HSP90 was downregulated in UAsm by P. Levels of nitrosylated eNOS were downregulated in UAendo by P, and levels of nitrosylated HSP90 were also downregulated in UAendo, but unchanged in UASM. Conclusion: Upregulation of UAsm protein nitrosylation implicates a possible pathway of NO-dependent vasorelaxation, whereas de-nitrosylation of UAendo eNOS and HSP90 implicates a mechanism for increasing UAendo NO production via eNOS activation during pregnancy associated with increased NO production. NIH HL98746, HL74947, HL70562, HL49210, HD38843, HL87144.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Proline-Rich Acidic Protein 1 (PRAP1) as a Marker for Established Uterine Receptivity.

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

The proline-rich acidic protein 1 (PRAP1) is mainly expressed in the epithelial cells and has been suggested to play an important role in maintaining normal growth homeostasis in epithelial cells. We have examined the expression and regulation of PRAP1 in mouse uterus. In situ hybridization indicates that PRAP1 mRNA is exclusively expressed in the uterine luminal epithelium (LE). In ovariectomized uterus, PRAP1 expression levels can be upregulated by 17β-estradiol treatment and downregulated by progesterone treatment. PRAP1 mRNA has unique temporal expression pattern in the early pregnant mouse uterus. In normal wild type uterus, PRAP1 mRNA is strongly detected in day 0.5 LE (mating night as day 0), disappears in pre-implantation day 3.5 LE, and then is intensively expressed in day 4.5 LE after implantation has initiated. These data indicate that the temporal expression of PRAP1 is associated with the establishment of uterine receptivity. This indication has been further demonstrated in experimentally-induced delayed implantation wild type mouse uterus and Lpar3(-/-) mouse uterus with delayed implantation. Indeed, PRAP1 mRNA is undetectable in the non-receptive uterus in both delayed implantation mouse models. These results suggest that PRAP1 may serve as a marker for the established uterine receptivity. However, the function of PRAP1 in the LE has not been established. (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):2.

Essential Roles of BMP Signaling in Uterine Biology.

Caterina Clementi 1, Mark A Edson 1, Qinglei Li 1, Ruihong Chen 1, Vesa Kaartinen 2, John P Lydon 1, Francesco J DeMayo 1, Martin M Matzuk 1

The TGFβ superfamily is the largest family of secreted proteins in mammals. The importance of the TGFβ pathways is demonstrated by the massive literature that has been produced in the past decades to investigate their involvement in a variety of physiological and pathological processes both in adults and during embryonic development. The bone morphogenetic proteins (BMPs) belong to this family and signal through heteromeric complexes of type 1 and type 2 serine/threonine kinase receptors. Significant data has been produced to support a central role of BMP2 in the uterus during implantation. The focus of our studies was to define the roles of the three BMP type 1 receptors [also known as activin like kinases (i.e., ALK2, ALK3, and ALK6)] in uterine biology. Since their sequences are highly conserved in mammals, we adopted a loss-of-function approach to evaluate the consequences of their absence on mouse fertility. ALK6 null female mice have already been shown to be infertile due to defects in cumulus cell expansion. Since both ALK2 and ALK3 are embryonic lethal, we created several cre-loxP conditional knockout (cKO) mouse models in which the silencing of the target gene (ALK2 or ALK3) in female somatic tissues is accomplished by a cre recombinase enzyme that is expressed under the control of 1) the progesterone receptor promoter (PR-cre), 2) the anti-Mullerian hormone receptor 2 promoter (AMHR2-cre), and/or 3) the cytochrome P450 family 19 promoter (CYP19-cre). The use of these distinct cKO models has allowed us to obtain and compare different phenotypes resulting from the unique spatiotemporal patterns of cre deletions of ALK2 or ALK3. In particular, we discovered that PR-cre mediated deletion of either ALK2 or ALK3 results in female infertility. Starting from these encouraging phenotypes, we have begun to perform experiments to pinpoint the specific function of these proteins. Our preliminary data indicates that these type 1 receptors have unique and non-redundant roles in the peri-implantation period. The importance of these findings can be appreciated by considering the impressive proportion of pregnancies that fail secondary to defects in implantation (75%) and by the high number of later complications due to implantation abnormalities. On the other hand, considering the broad involvement of the TGFβ superfamily members in many different processes, it is crucial to dissect these pathways further in uterine biology and to identify possible targets for therapeutic manipulation in the future. This research is supported by National Institutes of Health grant HD32067.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Mucins in Blastocyst Implantation: Opposing Roles for 13 and 15.

Connie E Poon, Margot Day, Christopher R Murphy 1

Receptivity of the uterine luminal epithelium to blastocyst penetration is an important determinant of pregnancy success and the glycocalyx of the uterine luminal epithelium forms the first point of contact between the uterus and blastocyst. A family of proteins called Mucins are a major component of the glycocalyx and a few (Mucins 1, 4 and 16) provide an anti-adhesive surface on the uterine luminal epithelial surface through the mechanism of "steric hindrance." The presence of one of them (Mucin 1) is proven to inhibit implantation. Given their important role, this study sought to elucidate a functional role for other members of the Mucin family, specifically Mucins 13 and 15. Immunofluorescence, western blotting and ovariectomy studies were utilised to examine the localisation and expression of Mucins 13 and 15 in the uterus during early pregnancy and the pre-implantation blastocyst and determine whether the proteins are hormonally regulated in the uterus. Immunofluorescence studies show that Mucin 13 is present apically in uterine luminal epithelial cells at time of implantation while Mucin 15 is lost from the cell surface at implantation. Both proteins are present in the blastocyst and localise to the trophoblast cells and inner cell mass. However, Mucin 15 is additionally present in the zona pellucida of the blastocyst, an anti-adhesive structure which prevents attachment of the blastocyst outside the uterine chamber. Western blotting results support these immunofluorescence findings and further demonstrate that these proteins may undergo changes to their glycosylation profiles which have implications for their functions. Ovariectomy studies demonstrate that Mucin 13 is upregulated to the apical cell surface by progesterone while Mucin 15 is hormonally upregulated by estrogen. Collectively, this study suggests that Mucins 13 and 15 are hormonally regulated in the uterus and play contrasting roles in the implantation event, with Mucin 13 contributing to an adhesive surface to facilitate attachment of the blastocyst to the uterine luminal epithelium. Conversely, Mucin 15 appears to form an anti-adhesive molecule and its absence may allow implantation to occur. These findings suggest that different Mucins have differing and opposing roles in the uterus and blastocyst.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

NOT PRESENTED.

NOT PRESENTED.

Biol Reprod. 2010 Nov;83(1 Suppl):2.

NOT PRESENTED.

NOT PRESENTED.

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Effects of Pregnancy Status on Organic Anion Transporters and Prostaglandin Receptors in the Equine Endometrium: Insights into Maternal Recognition of Pregnancy in the Mare.

Ellane R Cleys, Gerrit J Bouma, Jason E Bruemmer 1

The physiological mechanism of maternal recognition of pregnancy (MRP) in the mare has yet to be identified. However, it has been determined that the signal for MRP occurs between days 12 to 16 post ovulation, when the conceptus migrates throughout the uterus, preventing pulsatile luteolytic prostaglandin F2alpha release from the endometrium and rescuing the corpus luteum. With failure of MRP, endometrial prostaglandin F2alpha release will initiate luteolysis on day 18. Recent description of down-regulation of solute carrier organic anion transporter family member 2A1 (SLCO2A1), also known as the prostaglandin transporter (PGT), in pregnant verses non-pregnant ovine endometrium suggests that SLCO2A1 plays a role in embryonic signaling for MRP and endocrine release of luteolytic prostaglandin F2alpha. This provokes the hypothesis that SLCO2A1 was down-regulated in equine endometrium during MRP. Another anion transporter, multidrug resistant associated protein 4 (ABCC4), which also is a transporter of prostaglandin F2alpha, was hypothesized to be down-regulated in endometrium during MRP in mares. Also, prostaglandin F2alpha receptor (PTGFR) was expected to be down-regulated and prostaglandin E2 receptor subtypes 2 (PTGER2), 3 (PTGER3), and 4 (PTGER4) were expected to be up-regulated in endometrium in response to pregnancy. Endometrial biopsies were obtained from three mares in a cross-over study, with each mare serving as both a pregnant treatment and a non-pregnant control. For the first estrous cycle, mares were artificially inseminated every other day until ovulation. Pregnancy was diagnosed by transrectal ultrasound and confirmed by terminal uterine lavage on the corresponding day of biopsy. Mares were not inseminated (control) on the following cycle. Transcervical uterine biopsies were obtained on days 12, 14, 16, and 18 post ovulation for both pregnant and control cycles. Samples were snap frozen in liquid nitrogen for real time RTPCR and Western Blot analysis. RTPCR revealed that endometrial PTGER2 mRNA was up-regulated six-fold in non-pregnant mares on day 18 (P < 0.05). Endometrial PTGER2 (three-fold) and PTGER3 (two-fold) mRNAs were up-regulated in endometrium from non-pregnant mares on day 16 (P < 0.05). Endometrial PTGFR mRNA expression was not affected by pregnancy status or day of tissue collection. Interestingly, while two- to four-fold changes in expression were observed for SLCO2A1, ABCC4, and PTGER4 mRNA, no statistical significance was found across all time points of sample collection. This is likely due to the variability of expression observed between individual mare samples, suggesting that a larger sample size be used to clarify these responses in future studies. In conclusion, a viable embryo affects PTGER2 and PTGER3 mRNA expression in a time specific manner, possibly facilitating signals for MRP. Future experiments will focus on consequential changes in protein expression in response to pregnancy status. This study is supported by a Research Grant from the American Quarter Horse Foundation.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Structural and Mechanical Indices of Remodeling in Uterine Arteries During Ovine Gestation Manifest During Early Pregnancy.

Daniel A Henao 1, Brittney Hills 2, Jill Koch 1, Benjamin J Sprague 3, Ronald R Magness 1

Uterine vascular remodeling facilitates the dramatic increases in uterine blood flow required for normal fetal growth. Insufficient vascular remodeling during pathological pregnancies is associated with low birth weights and as described by the "Barker Hypothesis" Developmental Origins of Adult Onset Disease. Causes of insufficient vascular remodeling that occur with preeclampsia/growth restriction are unknown, but they are thought to initially be revealed during early pregnancy. In sheep the period of maternal recognition of pregnancy occurs between days 10-16 when the initial increases in uterine blood flow of gestation are seen. During this time the embryo elongates and signals very dramatic changes in the uterine luminal environment which may be an initial contributing factor in uterine artery (UA) remodeling. We hypothesized that UA structural and mechanical properties associated with remodeling will be observed during early gestation spanning the time of maternal recognition of pregnancy. In this study we apply bioengineering principles in order to provide numerical comparisons of compliance changes at distinct physiologically important times of gestation. Structural and mechanical functional properties were measured in UAs obtained from Nonpregnant multigravida (n=11: 8 Luteal and 3 Follicular) ewes, early pregnant sheep (n=8; day 10-16), and late pregnant sheep (n=10; day 120-130; term = 147d). The Nonpregnant sheep had lambed 7-12 months prior to study. UAs (1st & 2nd generation) were dissected from the mesometrium; outer diameters and axial lengths were measured digitally under passive conditions with increasing pressures (0-120 mmHg) in a stair-step manner of 10-mmHg increments. Vessels were then fixed (4% formalin) at physiologic pressures (85-95 mmHg), and inner and outer diameter, lengths and wall thickness measured. Pressure length and pressure diameter curves were constructed using the arctangent Langewouters model. With assumptions of "conservation of mass" and curve fitting, we calculated Circumferential Green Strain and Cauchy Stress as well as Circumferential Incremental Elastic Modulus. There were no differences (P>0.05) in structural or mechanical properties between UAs from Luteal or Follicular animals; these data were combined. Wall thickness was unaltered by pregnancy, however compared to Nonpregnant UAs Circumferential Cauchy Stress was partially reduced in Early Pregnancy, but was substantially elevated during Late gestation (P<0.01). UAs from Nonpregnant sheep exhibited less (P<0.05) Circumferential Green Strain when compared to either Early or Late Pregnant which were equal. These data collectively demonstrate the functional consequences of UA remodeling are seen during early pregnancy. Young's Elastic Modulus, a measure of vessel stiffness, was determined from the slope of the Stress-Strain curve in the physiologic pressure range. This measure of Elastic Modulus was reduced significantly (P<0.01) during early pregnancy, and remained low in Late Pregnant UAs. These results illustrate that major mechanical and functional changes occur in UAs during maternal recognition of pregnancy, when key critical embryonic signals substantially modify the uterine milieu. These changes in UA properties coincide with the initial early gestation rises in uterine blood and likely contribute to early embryonic survival. NIH HL49210, HD38843, HL87144.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Endometrial Profiling to Demarcate the Cellular Events Induced by Embryonic Factors.

Geetanjali Sachdeva 1, Shruti Nimbkar-Joshi 1, Rajendra Katkam 1, Dhananjay Manjramkar 1, Siddhant Metkari 1, Uddhav Chowdhury 1, Sanjiva Kholkute 1, Indira Hinduja 2, Chander Puri 1

A complete understanding of the endometrial events occuring during very early stages of pregnancy in humans has been elusive, owing to the ethical constraints. Nonetheless, this information is essential to devise the effective strategies for infertility management and also to identify the potential targets for preventing embryo-endometrial interactions. Towards this, studies have been undertaken in our laboratory to investigate the endometrial phenotype at the time of embryo attachment in bonnet monkeys (Macaca radiata) and validate some of the observations using human endometrial epithelial cell line (Ishikawa) and embryo conditioned media. Early pregnancy was confirmed in the mated animals by pre-implantation factor bioassay and also by in-situ localization of the embryo attachment site. 2D PAGE profiling of sera from pregnant and nonpregnant animals was also done. At structural level, gestational endometrium showed epithelial and stromal cell proliferation and angiogenesis. Interestingly, when stimulated with embryo conditioned media, ishikawa cells demonstrated significant proliferation, thereby, indicating that embryo secretes certain factors that induce proliferation. This prompted us to investigate the expression of estrogen receptor (ER) alpha and beta in the endometrium collected during embryo attachment stage. Immunohistochemical and QPCR studies indicated significant upregulation in the expression of ER alpha. Estrogen responsive genes such as c-MYC were also found upregulated in gestational endometrium. In vitro stimulation with embryo conditioned medium and subsequent immunoflorescence using confocal microscopy led us to infer that the in vivo increase in the expression of cell adhesion molecules like alpha v and beta 3 integrin, during embryo attachment, could be the result of stimulation by the embryonic factors of soluble nature. Further, transcriptome profiling of endometrial stromal cells (isolated by laser capture dissection microscopy), using 44 K agilent array revealed altered regulation of several pathways such as actin cytoskeleton pathway, cytokine-cytokine signaling pathway, cell adhesion pathway during embryo attachment. All these inferences point at the dynamic nature as well as complexity of the endometrial response to embryonic signals. These observations will help us to establish functional networks that become active during very early stages of pregnancy in humans and primates. Research Supported By Indian Council of Medical Research and Department of Science and Technology, Government of India.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Bovine Seminal Plasma (BSP) Proteins, which Enhance Bull Sperm Binding to Oviductal Epithelium, Respond Differently to Capacitating Conditions.

Pei-hsuan Hung, Susan S Suarez 1

When bull sperm enter the oviduct, most bind to the epithelium in the lower oviduct to form a sperm reservoir. Sperm become capacitated before they leave the reservoir. It was reported that Bovine Seminal Plasma proteins (BSPs) enhance binding of bull sperm to the oviductal epithelium. BSPs are heparin- and phospholipid-binding proteins secreted by bovine seminal vesicles and absorbed by epididymal sperm when they come in contact with vesicular secretions. There are three BSPs in bovine seminal plasma: PDC109 (BSPA1/A2), BSPA3, and BSP30K. It was shown that PDC109 is shed from sperm during capacitation, thereby releasing sperm from oviductal epithelium; however, it was not known whether BSPA3 or BSP30K are also shed during capacitation. Because the BSPs show great differences in distribution of surface charges, we hypothesized that they are shed at different rates during capacitation. This could serve to regulate movement of sperm out of the reservoir. To test for differential shedding of BSPs during capacitation, ejaculated bull sperm were incubated at 38.5°C for 5 h in TALP medium with or without heparin (10 µg/ml), 3-isobutyl-1-methylxanthine (IBMX) (100 µM) and dbcAMP (1 mM), or heparin plus IBMX and dbcAMP to enhance capacitation. Proteins were extracted from sperm and western blot assays were performed, using antibodies made against unique polypeptides from the N-terminal domain of each BSP. Sperm capacitation was confirmed by western blot analysis of tyrosine phosphorylation, using anti-beta-tubulin as a loading control. At 5 h, a small amount of PDC109 was shed from the sperm under all of the capacitating conditions; however, when capacitation was inhibited by glucose, shedding was not reduced. BSP30K was shed much more quickly than PDC109 and was even shed significantly in TALP medium alone, when little tyrosine phosphorylation was detected. On blots probed with anti-BSPA3 antibody, a band of lower molecular mass appeared during incubation, indicating that BSPA3 is modified by sperm during capacitation; however, the formation of the lower molecular mass was not reduced in the presence of glucose. We concluded that each BSP responds differently to various capacitating conditions. Surprisingly, the response is not always directly related to the extent of tyrosine phosphorylation. Altogether, the different responses of BSPs could modulate the release of sperm from the reservoir, perhaps causing a more gradual release or even guiding sperm toward the oviductal ampulla, where fertilization takes place. USDA CSREES NRICGP 2008-35203-19031.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Effects of Hypotaurine, Cysteamine, and Aminoacids Solution on Post-Thaw Microscopic and Oxidative Stress Parameters of Angora Goat Semen.

Kenan Coyan 1, Purhan B Tuncer 2, Serpil Sariozkan 2, Pinar A Ulutas 3, Mustafa Numan Bucak 2, Nuri Baspinar 1, Birol Ozkalp 1

This study was conducted to determine the effects of cysteamine, hypotaurine and aminoacids solution (BME) on standard semen parameters, lipid peroxidation and antioxidant activities of Angora goat semen after the freeze-thawing process. Ejaculates collected from four Angora goats were evaluated and pooled at 37°C. Semen samples, which were diluted with a Tris-based extender containing the antioxidants hypotaurine (5 mM) and cysteamine (5 mM), and an aminoacid solution (13%), and an extender containing no antioxidants (control), were cooled to 5°C and frozen in 0.25 ml French straws in liquid nitrogen. Frozen straws were thawed individually at 37°C for 20 s in a water bath for evaluation. Supplementation with cysteamine, hypotaurine and BME caused significant (P < 0.05) increases in sperm motility, and significant (P < 0.05) decreases in total abnormality rates in comparison to the control group. While all in vitro treatments did not affect the acrosomal abnormality rates, hypotaurine and BME but not cysteamine significantly (P < 0.05) increased the HOST results as compared to the control group. Supplementation with antioxidants and BME did not significantly affect MDA levels and CAT activity in comparison to the control group (P > 0.05). The antioxidants hypotaurine and cysteamine decreased SOD activity when compared to the BME and control groups (P < 0.001).

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

The Effect of Antioxidants on Post-Thawed Angora Goat (Capra hircus ancryrensis) Sperm Parameters, Lipid Peroxidation, and Antioxidant Activities.

Purhan Barbaros Tuncer 1, Mustafa Numan Bucak 1, Serpil Sariozkan 2, Fatih Sakin 3, Ahmet Atessahin 4, Recai Kulaksiz 5, Mesut Cevik 6

The aim of this study was to determine the effects of the antioxidants curcumin, inositol and carnitine on microscopic seminal parameters, lipid peroxidation (LPO) and the antioxidant activities of sperm, following the freeze-thawing of Angora goat semen. Ejaculates were collected via artificial vagina from three Angora goats and microscopically evaluated and pooled at 37°C. The pooled semen samples were diluted in a Tris-based extender, including curcumin (2.5, 5 or 10 mM), inositol (2.5, 5 or 10 mM), carnitine (2.5, 5 or 10mM) and no antioxidant (control). The diluted semen was slowly (at a rate of 0.2-0.3°C/min) cooled to 5 °C and then cryopreserved in 0.25mL French straws. Frozen straws were thawed individually at 37°C for 20 s in a water bath, for microscopic sperm evaluation. The freezing extender supplemented with 2.5mM curcumin led to higher percentage of computerassisted semen analyzer (CASA) sperm motility (65±3%), when compared to the control, inositol and the 10mM carnitine (P < 0.01) groups, following the freeze-thawing process. The addition of antioxidants did not provide any significant effect on the percentages of post-thaw subjective analyses and CASA progressive motilities, as well as sperm motility characteristics (VAP, VSL, LIN and ALH), compared to the controls. Freezing extenders with antioxidants at three different doses led to lower percentages of acrosome and total sperm abnormalities, when compared to the controls (P < 0.001). However, the addition of 5mM inositol did not induce any difference in total sperm abnormalities, when compared to the controls. The antioxidants also did not show any effectiveness in the elimination of malondialdehyde (MDA) formation and the maintenance of glutathione peroxidase (GSH-PX) activity, when compared to the controls. Superoxide dismutase (SOD) activity was found to be higher in the presence of curcumin at all three dose levels and carnitine at 5mM, compared to the other groups. Glutathione (GSH) concentration was demonstrated to be maintained at a higher level with the addition of inositol, compared to the other groups. However, these differences in SOD and GSH levels were not significant, compared to the controls. All the antioxidants at all three dose levels resulted in a better protection of the sperm morphology (except for 5mMinositol with respect to the total sperm abnormalities), compared to the control samples. According to CASA, the best post-thawing sperm motility rate was recorded when the freezing extender was supplemented with 2.5mM curcumin. Further studies are required to obtain more conclusive results regarding the characterization of microscopic and oxidative stress parameters in cryopreserved goat sperm, using the different antioxidants.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Microarray-Based Analysis of Differential Expression of DNA Repair Genes During Spermatogenesis in the Mouse.

Dipanwita Roy Choudhury 1, Eric de Waal 1, Chris Small 2, Michael D Griswold 2, Christi A Walter 3, John R McCarrey 1

Spermatogenesis is the process by which male germ cells develop and differentiate to give rise to mature spermatozoa. The "Disposable Soma Theory" predicts that the integrity of the germline genome will be selectively maintained so that a pristine set of genetic information will be passed on to the next generation. A variety of individual DNA repair genes have been reported to be up-regulated in male germ cells compared to somatic tissues. The aim of the present analysis was to mine available microarray data characterizing gene expression during spermatogenesis to characterize expression of entire DNA repair pathways in 1) premeiotic type A spermatogonia, 2) meiotic pachytene spermatocytes and 3) postmeiotic round spermatids. We detected expression of 269 genes related to DNA repair in one or more of these cell types. These include genes involved in 12 different DNA pathways or gene groups including direct repair (15 genes), nucleotide excision repair (36 genes), base excision repair (26 genes), mismatch repair (21 genes), non-homologous end-joining (10 genes), homologous repair (23 genes), Fanconi anemia proteins (9 genes), check point factors (14 genes), translesion synthesis (11 genes), modulation of nucleotide pools (4 genes), DNA damage response (24 genes) and other factors either associated with the above mentioned pathways or suspected to function as DNA repair genes (76 genes). Although a majority of these genes were expressed at some level in all three spermatogenic cell types, we observed significant differences in levels of individual transcripts between spermatogonia and spermatocytes, followed by a general down-regulation of DNA repair gene expression in spermatids. Non homologous end-joining repair and homologous repair pathways were found to be up-regulated in spermatocytes relative to spermatogonia, while mismatch repair pathway genes are were up-regulated in spermatogonia. Checkpoint factors and translesion synthesis genes were up-regulated in spermatids relative to spermatocytes. In general we found that DNA repair pathways up-regulated in mitotic spermatogonia are associated with DNA replication, while those up-regulated in spermatocytes are associated with homologous recombination repair and meiosis, and those up-regulated in spermatids are involved with repair functions in non-replicating cells. These results suggest the use of distinct DNA repair pathways is emphasized during the mitotic, meiotic and postmeiotic stages of spermatogenesis, respectively, reflecting the distinct functions ongoing in each of these cell types.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Roles of Sulfogalactosylglycerolipid (SGG) and Its Binding Proteins in Fertilization.

Kessiri Kongmanas 1, Clarissa Sugeng 1, Puneet Souda 2, Julian Whitelegge 2, Kym Francis Faull 2, Nongnuj Tanphaichitr 3

SGG is an evolutionarily conserved sulfoglycolipid present at 10 mole% of total lipids on the surface of mammalian male germ cells. On mature sperm, SGG is localized exclusively on the sperm anterior head plasma membrane (APM), the site where sperm interact with zona pellucida (ZP). We have shown that SGG not only has direct ZP affinity, but also plays an important role in the formation of capacitated sperm lipid rafts, which possess ZP binding ability. Also, SGG and its analog, Sulfogalactosylceramide (SGC), possess affinity for a number of molecules, including heat shock protein 70 (Hsp70) and a ZP-binding protein, arylsulfatase A (ASA), both existing in lipid rafts. Since SGG is present abundantly in sperm APM, we hypothesize that it may recruit other sperm surface proteins important for fertilization to the APM region during capacitation. Synergistic action between SGG and these APM proteins, which may also have SGG affinity, would stabilize the binding of sperm to the ZP. Nitrogen cavitation at 650 psi selectively disrupts the APM. APM vesicles containing lipids and membranous proteins can be pelletted upon ultracentrifugation, whereas peripheral plasma membrane proteins of the APM would remain in the supernatant (SN). Notably, the levels of APM vesicles from capacitated sperm were higher than those from non-capacitated gametes, confirming the significance of APM vesicles in ZP binding. Our proteomic analysis further revealed a number of ZP binding proteins in these vesicles, including SED1, zonadhesin, angiotensin converting enzyme, Sp32, and glutathione S-transferase mu 2 (GST). Some of these proteins may have direct SGG affinity. In fact, our initial attempt to characterize SGG binding proteins using the "SN" fraction indicated that SP-10, Sp32, and GST, all with a similar molecular mass range (25-28 kDa), formed complexes with SGG liposomes, which could then be floated up in a sucrose gradient by ultracentrifugation. Similar experimental approaches are being carried out to identify SGG binding proteins in APM vesicles, with the expectation that some of them would have ZP affinity. Interestingly, chaperone proteins, Hsp70 and Hsp86, were also present in higher amounts in APM vesicles of capacitated sperm. Hsp70 is known to oligomerize upon its binding to SGC. We therefore propose that SGG may induce oligomerization of Hsp70 and perhaps also Hsp86 in the APM entity, especially in capacitated sperm. This would promote the clustering of ZP binding proteins and subsequently their synergistic action in ZP binding. Experiments are underway to validate this hypothesis. Our study should reveal whether SGG can recruit ZP binding proteins to the APM entity via its direct affinity to these proteins and/or indirect influence through their interaction with the chaperone proteins, Hsp70 and Hsp86. Funded by CIHR, NSERC, and DPST (The Development and Promotion of Science and Technology Talented Project Thailand scholarship).

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Comparison of Sperm Counts Between a Relocated Computer System and Human Technicians.

Bill Plummer, Andrew Schaffner, Carli Grimbleby, Jessica Einstein, Paola Wessinger, Emy Pace, Lindsay Kirscher 1

When evaluating sperm numbers two methods are generally used, either a technician with microscope and counting chamber or a computer assisted sperm analysis (CASA) system. CASA systems are usually set in place and all samples are brought to it. Microscopes and technicians are more mobile. When large numbers of males are to be reproductively evaluated at a distant site and in a relatively short period of time, taking the CASA system to the work site may be more appropriate for research. However, moving the system is a momentous task and is to be avoided if possible. The goals of this project were to compare the results of the two methods and to determine the value of moving the CASA versus using technicians and microscopes. One semen sample from each of 151 yearling bulls, on the 2009 Cal Poly Yearling Bull Test, was evaluated for sperm concentration and motility. Semen was collected via electro-ejaculator, diluted to 80 million sperm/ml and distributed to two technicians (Tech A and Tech B), using Makler Counting Chambers and inverted microscopes with 20X objectives, plus two technicians running the CASA system, SpermVision (Minitube of America, Verona, WI). Each Makler technician utilized two Maklers and the same microscope for the entire test. Of the two technicians teamed up to run the SpermVision, one performed all of the semen dilution and distribution to the Makler and SpermVision systems. Manufacturer's procedures for use of each system were utilized. A comparison was undertaken to determine a correlation between the two systems. Results were very surprising. For concentration, the two technicians, when compared with each other, were not significantly different (p > 0.999). However, the difference between the technicians and SpermVision was significant (p < 0.0001), for the SpermVision counted approximately 31% fewer sperm than the technicians. For motility, Tech A produced estimates that were significantly different (13% higher, 9% lower; p < 0.0001) than Tech B and SpermVision, respectively. Tech B's estimates were consistent with SpermVision. Investigating the concentration differences utilized samples of know sperm concentrations and resulted in data which suggests the Makler technicians over counted the number of sperm. In conclusion, further studies are necessary to decide on which system is best for field work. This research is supported in part by California Research Initiative grant for 2008.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Oxidative Stress on Canine Semen Before and after Oral Antioxidant Supplementation.

Bethania V Lopes 1, Gabriel A Monteiro 1, Viviane Chirinea 1, Ana ISB Villaverde 1, Heder Nunes 1, Sergio Rodrigues 1, F Arduino 2, Maria Denise Lopes 1

The spermatozoon, constantly faces the oxygen paradox: oxygen is required for life, but oxidative metabolism of biological molecules can be potentially toxic because of the formation of highly reactive oxygen species (ROS) that can modify cell viability, causing sub or infertility. The concentration of ROS in seminal plasma is inversely related to sperm quality. The imbalance between ROS antioxidant concentration cause oxidative stress on semen. Oxidative stress has been proposed to have a causal/concausal role in many cases of male infertility. So, many treatments with antioxidants has been proposal to decrease the problems cause by oxidative stress. In humans, oral administration of vitamin E improved sperm function and gave a significant dose-dependent suppression of ROS production from human spermatozoa. Ascorbate is most effective as an antioxidant in the aqueous phase, and its role might be to scavenge free radicals and regenerate Vitamin E. The aim of this work was to evaluate the oxidative stress on seminal plasma from subfertile dogs after oral supplementation with vitamin C and E. Six healthy and sexually mature dogs were used in this study: four Labrador Retrievers, one Rottweiller and one German Sheperd, ranging between 2 and 7 years of age. Those dogs had low sperm count (<20x106sptz/mL) and/or more than 30% of total sperm pathology. They were all from Military Police from Bauru-SP- Brazil and were fed with a commercial dog food (Maxi adulto for large breeds; Royal Canin,Brazil).The dogs had their diet supplemented with vitamin C and E (500 µg each)for 60 days. Semen was collected, from those dogs, by digital manipulation. The ejaculate was diluted with TRIS/egg-yolk extender and then centrifuged twice, in order to obtain seminal plasma without sperm cells and remove as much as possible the extender. The concentration of thiobarbituric acid-reactive substances (TBARS) were measured in seminal plasma before, after 30 and 60 days of supplementation. The TBARS concentration were used as index of oxidative stress. Results were analyzed using repeated measures ANOVA. No statistic difference were found on TBARS concentration, between the 3 moments evaluated. Based on these findings, it can be concluded that the oral supplementation for 60 days, with 500 µg of vitamin C and E does not decrease the oxidative stress on canine seminal plasma from subfertile dogs. Acknowledges: FAPESP, Royal Canin, Poilicia Militar de Bauru.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

A Functional Polymorphism c.-712C>T in the FGF9 Promoter Is Associated with Sertoli Cell-Only Syndrome.

Chia-Ling Chung 1, H Sunny Sun 2, Ching-Yi Lin 1, Yung-Ming Lin 1

Fibroblast growth factor 9 (FGF9) is an autocrine/paracrine growth factor considered to be important for a variety of biological processes, including proliferation, differentiation and cell survival activities in embryogenesis and adult. Recent studies from Fgf9 knockout mice emphasized the importance of FGF9 in male sex determination and differentiation. The reproductive system phenotypes of Fgf9-/- null mice range from testicular hypoplasia to male-to-female sex reversal and gonadal dysgenesis, suggesting the crucial role for FGF9 signaling in sex determination and testicular development. The RNA and protein expressions of FGF9 in the testes of Sertoli cell-only syndrome (SCOS) were significantly decreased, compared to controls. We further systematically examined the sequences of FGF9 gene in 29 SCOS infertile men and 79 unrelated healthy controls to test whether sequence variants of FGF9 gene play a pathological role in SCOS phenotype. A single nucleotide polymorphism and two polymorphic microsatellites were detected. The allelic and genotypic frequencies of the c.-712C>T in the promoter region of FGF9 were significantly different between SCOS patients and controls (P = 0.005 and 0.0008, respectively), while the microsatellites c.-124dupT(8_10) in the 5'UTR and c.*275_76TG(13_17) in the 3'UTR did not show any difference between these two groups. Reporter assay demonstrated that the polymorphism c.-712C>T dramatically attenuated promoter activity. By transcription element search system (TESS), E2F transcription factor 1 (E2F-1) was predicted to bound to -712C and this binding was confirmed by chromatin immuno-precipitation (ChIP) assay. Moreover, in electrophoretic mobility shift assay (EMSA), substitutional mutation of E2F-1 binding site (i.e. -712C>T) reduced DNA-protein complex formation. Collectively, our data suggest the functional polymorphism c.-712C>T within the promoter region of FGF9 gene, which results in decreased FGF9 expression and may contribute to one of the causes of SCOS.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Toxicity of Cadmium on Piglet Sertoli Cells.

Hui Yuan 1, Li-xin Wen 1, Zu-ping He 2, Ming Zhang 1, Jing Wu 1, Li-yun Yuan 1, Yin Lu 1, Cheng-zhi Guo 1, Li Zhu 1, Si-jun Deng 1

Although there are reports showing that cadmium has severe toxicology in male germ cells and affects male reproduction, very little information is known about the toxic effects of cadmium on somatic cells in testis. The objective of this study is to explore the toxicity of cadmium on piglet Sertoli cells. The Sertoli cells were isolated from piglet testes using a two-step enzyme digestion and followed by differential plating. Piglet Sertoli cells were identified by oil red O staining and Fas ligand (FasL) expression as assayed immunocytochemistry, and they were cultured in DMEM/F12 supplemented with 10% fetal calf serum. The cultured Sertoli cells were classified into five groups, i.e., group A, control without cadmium chloride; group B with 10 μM cadmium chloride; group C with 20 μM cadmium chloride; group D with 40 μM cadmium chloride; and group E with 80 µM cadmium chloride. We found that cadmium chloride inhibited the proliferation of Piglet Sertoli cells as shown by MTT assay, and it increased malondialdehyde (MDA) but reduced superoxide dismutase (SOD) and GSH-Px activity. Comet electrophoresis showed that cadmium chloride caused DNA damage of Piglet Sertoli cells and resulted in cell apoptosis as assayed flow cytometry. Abnormal and apoptotic ultra-structure was observed in Piglet Sertoli cells treated with cadmium chloride compared to the control under transmission electronic microscopy. In conclusion, cadmium has obvious adverse effects on the proliferation of Piglet Sertoli cells and causes their DNA damage, cell apoptosis, and aberrant morphology, which may lead to decrease in reproduction of male pigs.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Expression of Ets Transcription Factors Etv4, Etv1 in Mouse Testis.

Xueming Zhang, Lei Si, Zhanpeng Yue, Yang Liu, Bin Guo, Ziyi Li, Dexue Li 1

Spermatogonial stem cells (SSCs) are primordial spermatogonia being capable of self-renewal and differentiation throughout the life of the male. The recent blizzard of researches implied that SSCs self-renewal is modulated by the integration of SSCs intrinsic factors with their microenvironment niches. Signaling from Sertoli cells, the primary component of SSCs niches, determine the fate of SSCs by either supporting self-renewal or initiating differentiation leading to meiotic entry and production of spermatozoa. However, the detailed mechanisms of SSCs self-renewal and differentiation still remains unclear. Recently, Ets related molecule (ERM, Etv5) was reported as an essential molecule for the maintenance of SSCs niches in mouse testis. Since Etv5, Etv4 and Etv1 belong to the same subfamily of Ets transcription factors and they have highly conserved homologous domain, this research focused on whether Etv4 and Etv1 are expressed in mouse seminiferous epithelium and what roles they play on SSCs behavior. To this end, Specific primers for Etv4, Etv1, Etv5 and internal control beta-actin were designed and synthesized to investigate the mRNA expression level of the interested gene in mouse testicular tissue at different developmental stages by employing Real-time RT-PCR analysis. Four time points including 5-day, 7-week, 16-week and 10-month were selected as the representative developmental stages for puberty, sexual maturity, adulthood and old age, respectively. The ANOVA test of the data was carried out by using SPSS. The results showed that the expression of Etv5 mRNA was lower at 5-day, extremely increased at 7-week (P<0.001), then significantly decreased at 16-week and 10-month (P<0.01). The expression of Etv1 mRNA was at the same level with that of Etv5 at 5-day, increased significantly at 7-week (P<0.05), decreased slightly at 16-week and then increased evidently at 10-month. The expression of Etv4 was very lower at all four time points compared with that of Etv5 and Etv1. However, it also increased significantly at 7-week and decreased afterwards. The present results indicate that expression of Etv5 and Etv1 in testis reaches peak along with the sexual maturity, and then decreases and maintains at a relatively high level during the spermatogenesis, which putatively suggests Etv1 may has functions in mouse spermatognesis. However, the exact roles of Etv1 on SSCs self-renewal and differentiation still need to be addressed in next step. This research was supported by the National Natural Science Foundation of China (No. 30771555, No.30200195, No.30471246).

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Localization and Characterization of the Newly Elucidated Teneurin C-terminal Associated Peptide (TCAP) in the Mouse Testis.

Dhan Chand 1, Alexandra Kollara 1, Theodore J Brown 2, Dalia Barsyte-Lovejoy 1, David A Lovejoy 1

The teneurins are a phylogenetically conserved family consisting of four type-II transmembrane glycoproteins implicated as signaling molecules, receptors and transcription cofactors during tissue development in vertebrates. Teneurins possess a carboxy terminal sequence oriented in their extracellular domain that may be cleaved to generate a 40- or 41-amino acid modulatory bioactive peptide known as teneurin C-terminal associated peptide (TCAP). Synthetic TCAP-1 regulates cellular proliferation, increases protein synthesis and promotes cell survival under certain cellular stress situations. Based on these findings, we hypothesized that TCAP may be localized in areas of high metabolic activity, proliferation, and cellular differentiation such as the testes. The localization of TCAP expression was determined by immunohistochemistry in the mouse testis, epididymis and seminal vesicles using TCAP-1 specific antisera. Immunoreactive TCAP was detected in spermatogonia, primary and secondary spermatocytes, spermatids and on the flagella of developing spermatozoa. Confocal microscopy localized TCAP expression in the cytoplasm, with highest levels in spermatogonia. TCAP was not detected in Sertoli or Leydig cells, or in the epididymus or seminal vesicle suggesting a potential role in spermatogenesis. Testis slices were incubated with FITC-labeled TCAP-1 to localize TCAP binding sites, which showed specific binding to spermatids. To determine whether TCAP is expressed independently from the teneurins, the cellular localization of TCAP-1 and teneurin-1 in mouse F9 testicular carcinoma cells were examined by immunofluorescent microscopy. These studies localized teneurin-1 protein to the plasma membrane, whereas intense expression of TCAP-1 was observed in the cytoplasm. As expected, some co-localization was observed at the plasma membrane. Thus, TCAP-1 may be processed as part of the full-length teneurin-1 where it is then liberated by proteolytic cleavage by vesicle- or plasma membrane-bound peptidases or possibly synthesized as part of a smaller teneurin-TCAP transcript. Western blot analysis revealed a 50kDa and 70kDa TCAP-related protein in F9 cells and testis tissue respectively. High performance liquid chromatography and immunoblotting also suggests the existence of an extended 14.8kDa TCAP form, consistent with a cleavage site encoded within the last exon of the teneurin gene. Altogether, these data provide novel information indicating the expression of TCAP-1 in areas of the testes distinct from those known to express teneurin. Moreover, our findings suggest that TCAP may play a role in spermatogenesis. This research was supported by NSERC funding to DAL and TJB.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Hypoxia May Play a Role in Steroidogenesis in the Testes of Infertile Men.

Chia An Lin, Shaw-Jenq Tsai 1

Hypoxia-inducible factor-1 alpha (HIF-1alpha) is one of the most important factors that responses to the change of oxygen homeostasis. In human testis, two HIF-1alpha isoforms have been identified, HIF-1alpha and HIF-1alphaTe. HIF-1alphaTe is derived from an alternative promoter-first exon combination, but with a different genomic organization and a different nucleotide sequence. HIF-1alphaTe still forms heterodimeric complexes with HIF-1beta; however, HIF-1alphaTe is incapable of forming a DNA binding HIF-1 complex. Our previous result showed that a significant decrease in hCG-induced testosterone production in Leydig cells was also found after hypoxia treatment compared to normoxia treatment. HIF-1alpha is known to modulate transcriptions of a number of genes by binding to DNA via the hypoxia response element (HRE) consensus sequence RCGTG. A bioinformatics search (The Binding Element Searching Tool, http://thebest.binfo.ncku.edu.tw/thebest/) of upstream promoter sequences for testicular genes in human testes containing HRE binding sites were done in NCKU Bioinformatics center. A total of 15 testis-expressed genes were identified to possess the HRE in their promoter regions, one of these genes is StAR. The objective of this study is to unravel the expression profiling of HIF-1alpha, HIF-1alphaTe and StAR in the human testis, and to determine the relationships between the HIF-1alpha/HIF-1alphaTe expressions and StAR expression. IHC demonstrated that HIF-1alpha protein could be mainly detected in interstitial Leydig cells and post-meiotic germ cells in human testes. Real time RT-PCR showed an increasing tendency of HIF-1alpha transcript levels in patients with spermatogenic failure (P = 0.12), a significantly decreasing tendency of HIF-1alphaTe transcript levels in patients with spermatogenic failure (P = 0.004) and an increasing tendency of StAR transcript levels in patients with spermatogenic failure (P = 0.068). A significant positive correlation between HIF-1alpha transcript levels and StAR transcript levels was noted (r = 0.467; P = 0.025), whereas a negative correlation between HIF-1alphaTe transcript levels and StAR transcript levels was observed (r = -0.229; P = 0.293). Given that HIF-1alphaTe isoform has been recognized as a dominant-negative regulator of HIF-1, our results suggest the association of hypoxia with steroidogenesis defect in infertile men.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Interaction of VSIG1 with ZO-1 During Spermatogenesis.

Ekyune Kim 1, Youngjeon Lee 1, Yonggeun Hong 2, Kyu-Tae Chang 1

Mammalian spermatogenesis takes place in the seminiferous epithelium, which is composed of Sertoli cells and germ cells. The interaction between spermatogenic and Sertoli cells as well as elongated spermatids and Sertoli cells is tightly regulated by junctional adhesion molecules (JAMs). JAMs, which are cell adhesion molecules, are known to play roles in various biological processes such as fertilization, neurogenesis, cancer progression, and spermatogenesis. Members of the JAM family have a unique structure: they contain an N-terminal signal peptide domain, immunoglobulin (Ig)-like domains, transmembrane and cytoplasmic tail domains, each of which has distinct functions. The extracellular Ig-like domains interact in a homophilic or heterophilic manner, whereas cytoplasmic tail domain mediates the tight junction assembly. Although members of the JAM family are exclusively present in or restricted to the testis, their precise roles in spermatogenesis and fertilization have not yet been completely explored. The functional roles of Nectin-2, Nectin-3, JAM-C, cell adhesion molecule1 (CADM1), and coxsackie and adenovirus receptor (CAR) have been evaluated by analysis of null mutant mice. Unfortunately, CAR-deficient mice had an embryonic lethal phenotype; this demonstrates the importance of CAR in development, but its physiological role in spermatogenesis is not known. The loss of CADM1, Nectin-3 and JAM-C resulted in male infertility caused by loss of adhesion between germ and Sertoli cells. A variety of JAMs participate in the interaction between germ and Sertoli cells; the different physiological phenotypes of JAM- knockout mice indicate that these molecules not only play different roles but also cannot be functionally replaced by other cell adhesion molecules. Recently, the Scanlan research group characterized human VSIG1, which was originally known as A34, as a new member of the JAM family; VSIG1 is composed of two extracellular Ig-like domains, a transmembrane domain, and a cytoplasmic domain. However, this molecule has not been functionally characterized, so this was one of the aims of our present study. Therefore, in the present study, RT-PCR and immunoblot analyses were used to study VSIG1 expression, VSIG1 was specifically expressed in testicular germ cells but not in sperm. Pull-down assay with glutathione S-transferase (GST) or His-fused first Ig and second Ig domains of VSIG1 and SDS-PAGE under mild non-reducing conditions demonstrated that VSIG1 functions as an in vitro homophilic adhesion molecule. Furthermore, cells expressing a deletion of the C-terminus of VSIG1 failed to interact with ZO-1, the central structural protein of the tight junction. These findings suggest that mouse VSIG1 interacts with an unknown molecule in Sertoli cells via its extra-cellular domain, while its cytoplasmic domain is needed for binding to ZO-1. Thus, we suggest that mouse VSIG1 may play an important role(s) in spermatogenesis rather than fertilization by forming heterophilic complex with a molecule similar to JAM family in its first immunoglobulin-like domain.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Effect of Cryptorchidism on Human Testicular Transcriptome.

Marie Eve Bergeron, Christine Legare, Ezequiel Calvo, Marc Simard, Robert Sullivan 1

Cryptorchidism is the most common congenital disorder in boys. One major consequence of this anomaly is male infertility due to undescended testis to which an increased risk of testicular cancer is associated. The genetic cause of cryptorchidism remains to be elucidated. The objective of this study was to compare the transcriptome of human cryptorchid testis with normal tissues. Seminoma tissues were used as a positive control of genes known to be expressed in testicular cancer. Normal tissues were obtained with collaboration of our local organ transplantation program; the five donors were from 23 to 53 years of age. Cryptorchid testes were obtained by surgical orchidectomy performed on 4 patients from 29 to 43 years of age presenting with inguinal testis. Donors of seminoma tissues were from 22 to 34 years of age and were obtained through the pathological tissues bank of our institution. Tissues samples were kept on ice and snap freeze immediately upon arrival in the research lab. Total RNA was extracted from the tissues and used to probe Affymetrix GeneChip Human Gene 1.0 ST arrays which include 28,869 genes with 764,885 distinct probes. In normal and cryptorchid tissues 2278 and 589 genes were up-regulated, respectively. As expected, transcripts known to be associated with spermatogenesis characterized the normal tissues whereas, in cryptorchid testes, transcripts of Leydig and Sertoli cells were found in higher amount. For example, PRM1 and ADAM2 transcripts associated to germ cells were in higher quantity in normal than cryptorchid testes, but CYP17A1, LHR and HSD17B3 transcripts of Leydig cells as well as GATA4, INHA and INHBA transcripts of Sertoli cells were higher in cryptorchid than normal testes. Some transcripts known to be associated with testicular cancer, such as CALR3 and PIWIL1, were up-regulated in normal tissues whereas KITLG and CCND2 were more expressed in cryptorchid testes. In seminoma tissues, PIWIL1 transcript was more expressed in cryptorchid but less in normal testicular cancer; the CALR3 and KITLG were less expressed in seminoma than in other tissues whereas the CCND2 was much more expressed in cancer tissues. Markers for testicular descent were more expressed in cryptorchids than in normal and seminomas as expected. These transcript expression patterns from the microarray results were confirmed by quantitative real-time PCR. Differences in transcriptome due to cryptorchidism will give us a clue to find the genetic cause of this disease. This work was supported by Canadian Institute of Health Research grant to Robert Sullivan.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Characterization of Seminal Plasma Proteins from Dairy and Meat Goat Sires.

Desiree Rucker, Kristin Baker, Ursula Tress, Louis Nuti, Gary R Newton 1

Male sub-fertility is a serious problem in many farm species. Current clinical tests to assess fertility include analysis of sperm number, morphology, motility, chromatin quality, and acrosomal integrity. These endpoints may fluctuate due to factors that are unrelated to fertility. Our working hypothesis is caprine seminal plasma contains specific proteins that are crucial for fertility. The identification of these proteins may lead to the development of a reliable, non-invasive marker for male fertility. To test this hypothesis, semen was collected monthly for one year from Alpine (n=4) and Boar (n=4) bucks. Scrotal circumference and sperm concentration in the ejaculate was recorded. Scrotal circumference varied throughout the year (P<.05) and was greatest at the start of the breeding season for Alpine (29.9±0.98 cm) and Boer (32.7±0.98 cm) sires. Concentration of sperm in the ejaculate also varied throughout the year (P<0.01). Highest concentrations were recorded during periods of sexual quiescence (April) in both Alpine (7.24±0.74 x 109/ml) and Boer (6.41±0.74 x 109/ml) sires. Concentration of sperm was lowest during the breeding season in November/December for both Alpine (1.22±0.74 x 109/ml) and Boer (1.02±.074 x 109/ml) sires. Weight of the total ejaculate collected each month did not differ (P>0.1). Seminal plasma was separated from spermatozoa by centrifugation and protein concentrations in seminal plasma were determined. Concentrations of proteins in seminal plasma gradually increased from August (Alpine-43.10±16.96 mg/ml; Boer-39.15±16.96 mg/ml) until the months of October- December when concentrations peaked sharply (P<.01) for Alpine and Boer sires (194.85±16.96 and 216.30±23.98 mg/ml, respectively) and then declined. Isoelectric focusing and SDS-PAGE were used to characterize seminal plasma protein profiles in ejaculates. Analysis revealed a complex profile of seminal plasma proteins. Major proteins were identified with approximate molecular weights of 139.0, 132.0, 96.7, 75.2, 61.0, 55.5, 41.6, 33.0, 22.0, 17.7, 16.9, 15.7 and 14.8 kDa. Of particular interest were a complex of low molecular weight (<20 kDa) proteins with isoelectric points between pH 4-7. These abundant proteins were subjected to electrospray ionisation mass spectrometry. Mascot search results indicated the sequence of the 14.8 kDa protein was similar (Score = 127) to bodhesin-2 from Capra hircus. Bodhesin-2 belongs to a new family of animal lectins called spermadhesins and constitutes one of the main protein components of seminal plasma from pigs, cattle, horses, and sheep. Identification of key seminal plasma proteins may ultimately lead to tests that will predict, at an early age, the utility of sires used in animal production systems.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Immunophenotyping of Undifferentiated Spermatogonia in Human Testes.

Hanna Valli 1, Serena L Dovey 1, Marc C Hansel 2, Kyle E Orwig 1

Spermatogonial stem cells (SSCs) initiate and maintain spermatogenesis in the testis and may have application for treating male infertility that results from gonadotoxic cancer therapies. In this scenario, a patient could theoretically cryopreserve testicular tissue (including SSCs) prior to their oncologic treatment. Cryopreserved SSCs could then be reintroduced into the testes of cancer survivors to regenerate spermatogenesis. The proof in principle for this approach is already established in mice, rats, pigs, goats and dogs. Before this fertility preserving therapy is translated to the human clinic, preclinical safety and feasibility studies are required. Human testicular tissue harvested from a male cancer patient prior to oncologic treatment has the potential to be contaminated with tumor cells. This is especially problematic for systemic hematologic malignancies (e.g., leukemia). In the mouse model it has been demonstrated that malignant cells can be separated from a testicular cell suspension by fluorescence-activated cell sorting (FACS) and that SSCs in the remaining suspension can be safely transplanted to regenerate spermatogenesis in an infertile recipient (Fujita et al., 2005). To translate this approach to human testis cell preparations, it is necessary to gain insights about the cell surface characteristics of human SSCs. We hypothesize that human SSCs have unique cell surface characteristics that can be exploited for their enrichment and to separate them from contaminating malignant cells by FACS. Normal human testis tissues were obtained from organ donors via the University of Pittsburgh Health Sciences Tissue Bank. Human testis cell suspensions were stained for 22 cell surface markers, including several established markers of rodent SSCs (e.g., CD49f, CD29 and CD90). Our preliminary results indicate that CD49f (α6-integrin) is a conserved marker of human spermatogonia because all SALL4+ cells (marker of undifferentiated spermatogonia) were recovered in the CD49f+ fraction of human testis cells. Human to nude mouse xenotransplantation experiments will be used to confirm SSC activity in the CD49f+ fraction. The results for CD29 and CD90 were more equivocal and will require further investigation. MOLT-4 (T-cell leukemia) cells were phenotyped for the same 22 cell surface markers. MOLT-4 cells do not express CD49f, but do express CD49e (α5-integrin), which is not expressed by SALL4+ human testis cells. Thus, we hypothesize that CD49f and CD49e are two cell surface markers that can be exploited to separate SSCs from contaminating malignant cells in human testis cell suspensions. To test this hypothesis in future studies we will perform FACS on a mixed cell suspension containing both human testicular cells and MOLT-4 leukemia cells. Each sorted fraction will then be transplanted into infertile nude mice to assess SSC activity and malignant potential, respectively. This work was supported by NIH grant HD055475 and Magee-Womens Research Institute and Foundation.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

NOT PRESENTED.

NOT PRESENTED.

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Seasonal Variations in Physiological and Semen Parameters, Activity of Antioxidant Enzymes in the Seminal Plasma and Peripheral Concentrations of Steroids in Saanen (Capra hircus) Bucks Raised Under Tropical Conditions in Brazil.

Carlos EA Souza, Maria GF Salles, Italo CS Lima, Arlindo A Moura, Davide Rondina, Airton A Alencar 1

The purpose of this work is to evaluate the physiological responses of Saanen bucks to environmental changes throughout the year in a tropical environment. We used 9 adult Saanen bucks, a dairy goat breed characteristic of temperate regions, bred in the Northeast brazil, a tropical environment, characterized by two distinct seasons, rainy (February to may) and dry (August to November), based on environmental temperature and air humidity. We evaluated rectal and body skin temperatures, heart and respiratory rates. Semen samples were collected biweekly, to evaluate sperm parameters. After evaluations, samples were centrifuge to separate the seminal plasma (SP). SP samples were assayed for total protein, and catalase and superoxide dismutase activities. Also, blood samples were collected from the bucks to determine serum concentrations of testosterone and cortisol. Environmental temperature was significantly higher during dry season. Inversely, humidity reached maximum values during the rainy period. All physiological parameters measured were increased (p<0.05) in the dry, compared to the rainy season. Although massal motility and vigor did not vary (p>0.05) between seasons, ejaculates displayed higher values (p<0.01) for sperm motility (54.1±4.2% versus 29.9±4.3%) and concentration (2.41±0.1 versus 1.92±0.1 billion cells/mL of ejaculated semen) during the rainy period. Total protein concentration and activity of superoxide dismutase (per mg of protein) in SP were similar for both seasons, but catalase activity was 4.5-fold higher in the dry season (p<0.01). As described for bovine, reactive oxygen species production and catalase activity were less influenced by seasonal variations than catalase. Serum cortisol concentrations were significantly (p<0.05) increased during dry season (1.13±0.1 versus 1.83±0.3 ng/mL), opposed to the rainy period. Testosterone concentrations, however, did not differ between seasons. Thus, during the dry period, Saanen bucks raised in a tropical environment showed characteristic signs of heat stress during the dry season, including increased body temperature, heart and respiratory rates and serum cortisol, as well as decreased sperm motility and concentration. In such condition, males typically display a shift in the reproductive fluids protein profile, increasing expression of chaperonins and heat shock proteins. The next steps are preparation of protein maps of seminal plasma and sperm membrane samples, to compare the protein profiles between seasons, and to identify differentially expressed spots. This information will add-up to the knowledge of the molecular mechanisms by which heat stress affects reproductive biology and sperm function of Saanen goats raised in tropical environments. This study has been funded by grants from the Brazilian Research Councils (CNPq and CAPES).

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Sertoli Cell Number Is Similar Following Aromatase Inhibition and Estrogen Receptor Blockade.

Trish Berger, Barbara Nitta-Oda, Kim Katleba, Erin Legacki, Alan Conley 1

Sertoli cell proliferation is prolonged and numbers at sexual maturity increased when aromatase is inhibited with the aromatase inhibitor Letrozole prepuberally in male pigs. Since the number of Sertoli cells control adult testis size and sperm production capacity, increased Sertoli cell numbers lead to larger adult testes and increased sperm production capacity. Our previous work demonstrated that Letrozole reduced endogenous estrogens but had no detectable effects on gonadotropins. Reduced estrogen receptor activation due to reduced endogenous estrogen is hypothesized to increase the window of Sertoli cell proliferation but other mechanisms are possible. To test the involvement of estrogen receptors, littermate trios of intact male pigs were treated with canola oil vehicle, the aromatase inhibitor Letrozole, or with ICI 182,780, a blocker of alpha and beta estrogen receptors. Treatment was initiated at 1 week of age and tissues were recovered at 6 1/2 weeks of age. Testes were weighed, and a sample fixed for Sertoli cell enumeration using the optical dissector method with GATA-4 immunohistochemical labeling of Sertoli cells. Accessory sex glands were weighed. Either aromatase inhibition or estrogen receptor blockade increased the number of Sertoli cells at 6 1/2 wks of age by approximately 35% compared with the control littermates, P ≤ 0.05, 3 replicates. However, while Letrozole stimulated growth of seminal vesicles and bulbourethral glands (P < 0.05), there was no effect of estrogen receptor blockade on accessory sex gland growth. These data identify a primary role for nuclear estrogen receptors in constraining Sertoli cell proliferation prepuberally, hence limiting numbers in testes at maturity and thereby determining sperm production capacity. Further studies are required to determine if Sertoli cell proliferation is regulated similarly in other domestic species. (Supported in part by NRI grant 2008-35203-19082 from the USDA National Institute of Food and Agriculture.)

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Identification of RNA Targets of the Conserved Spermatogenesis Factor, Boule.

Michael JW VanGompel, Eugene Yujun Xu 1

Though many RNA-binding proteins have been identified in the testis, validated mRNA targets of these proteins remain scarce, and how such protein-RNA interactions mediate RNA storage and translation activation remains unclear. Boule is one such RNA-binding protein that is thought to activate the translation of associated mRNAs. Boule is the ancestral member of the DAZ (Deleted in Azoospermia) family, with orthologs in nearly all metazoans, and gave rise to homologs Dazl (DAZ-Like) and DAZ in vertebrates and higher primates, respectively. Boule mutations lead to a pachytene arrest in Drosophila males and C. elegans females, and human BOULE can rescue meiosis in the fly testis, suggesting a conservation of Boule meiotic function. In Drosophila, boule is thought to regulate the translation of the Cdc25 homolog twine, and thus control entry into meiotic metaphase. A direct interaction has not been demonstrated, however, and other Boule targets are not yet known. We have shown that Boule is not required for meiosis in mice, but instead is necessary for differentiation beyond the round spermatid stage. To further determine Boule function, we sought to identify RNA targets of Boule and any associated functions. Boule was immunoprecipitated from whole testes, and co-precipitating RNA was purified and analyzed. We detected interactions between Boule and mRNAs important for spermatid differentiation, as well as with Cdc25a mRNA, and followed up on both observations. Our data is the first evidence for a direct interaction between Boule and Cdc25 homologs, so we pursued the possibility that Dazl compensates for Boule in our mutants by Dazl and Boule mutant mice. Since male Dazl-/-i mice have an early germ cell phenotype, we removed only one copy of Dazl in a Boule null background. These mice completed meiosis normally, however, and showed a round spermatid arrest similar to Boule deletion alone. This shows that removing three of four copies of DAZ family genes in mice is not sufficient to disrupt meiosis, but is inconclusive as to any Boule function. To further examine how Boule regulates its spermatid RNA targets, we performed microarrays on control and knockout 24-day old testes, when round spermatids are the most advanced cells present and defects first appear in mutants, and also on control and Boule null purified adult round spermatids. Interestingly, we found that several mRNA targets that interact with Boule are absent specifically in mutant round spermatids. This suggests that Boule is involved in RNA stability, in addition to any role it may have in translation activation. Our results show that Boule can bind Cdc25a mRNA and therefore may control meiosis in mice, though how important this function is remains unclear. Additionally, we have identified a novel role for Boule in maintaining RNA stability, the first indication of a role for any DAZ family protein beyond translational activation. Further studies are needed to determine if DAZ and Dazl function similarly, but our results have widened the possibilities of how Boule and the DAZ family control fertility.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Assessment of Succinate Deydrogenase and Lactate Dehydrogense Enzyme in Idiopathic Aesthenozoospermia.

Rashmi Tomar, Arun Kumar Jain, Nayan Kumar Mohanty 1

Evaluation of the possible implication of the Succinate Dehydrogenase (SDH) in male infertility was performed on the basis that sperm concentration in the ejaculate has been previously correlated with nuclear-encoded mitochondrial enzyme activities (the four subunits of succinate dehydrogenase/complex II of the respiratory chain and citrate synthase). We performed an exhaustive analysis of the five genes for the presence of sequence variants that could be associated with impairment of sperm production. And the lactate dehydrogenase (LDH) Enzyme family members characteristically are distributed in tissue and cell-type specific patterns and serve as the terminal enzyme of glycolysis, catalyzing reversible oxidation-reduction between pyruvate and lactate. However, total LDH enzymatic activity was considerably lower in Ldhc-/- sperm, indicating that the LDHC homotetramer (LDH-C4) is responsible for most of the LDH activity in sperm. Although initially motile when isolated, there was a more rapid reduction in the level of ATP and in motility in Ldhc-/- sperm. Moreover, Ldhc-/- sperm did not acquire hyperactivated motility, were unable to penetrate the zona pellucida in vitro, and failed to undergo the phosphorylation events characteristic of capacitation. These studies showed that LDH plays an essential role in maintenance of the processes of glycolysis and ATP production in the flagellum that are required for male fertility and sperm function. Then, the correlation between the motility and concentration of sperm and the positive percentage of sperm mitochondrial SDH & LDH enzymes activities in infertile & fertile human spermatozoa were analyzed. We hypothesized that disruption of ATP production via this metabolic pathway would impair motility, concentration and fertilizing capacity of spermatozoa similar to that observed with male aesthenozoospermia. However, here we demonstrate that targeted disruption of LDH & SDH enzyme affects only male fertility. The results showed that strongly suggest that an SDH & LDH enzymes activities largely depends on the whole energy production originating in the mitochondrial enzymes. The present results not only satisfy proof of principle but also suggest that these Enzymes may be a useful target in developing a male contraceptive. The immunocytochemical assessment of mitochondrial Succinate dehydrogenase & Lactate dehydrogenase enzymes in sperm samples of idiopathic infertile individuals could help to identify cases of mitochondrial-based asthenozoospermia.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Development Testicular and Spermatogenesis in Nellore Bulls.

Carlos H Lobo, Erika SB Menezes, Claudia R de Andrade, Rodrigo V de Oliveira, Carlos EA Souza, Airton A Araujo, Raimundo M Filho, Arlindo AA Moura 1

Nellore and their crosses represent the largest group of beef herd in Brazil. Their reproductive performance still needs to be improved once conception rate is not consistently high at the end of breeding seasons. Part of this problem is related to the sires which were not selected considering their reproductive capacity. In this regard, the knowledge of testicular physiology and the main factors that affect sperm producing capacity is crucial for selection and management of sires. Thus, the present study was conducted to evaluate the relationships among quantitative aspects of the testis size, testosterone secretion and spermatogenesis in Nellore bulls raised in the Northeast of Brazil. For this purpose, bull scrotal circumference (SC), testis diameter and length were measured from 10 to 29 months (mo.) of age in 28 bulls. At 12, 13, 15, 17, 18, 19, 20 and 21 mo., plasma testosterone concentrations were measured before and after intramuscular injection of GnRH (0.05 mg/kg body mass). At 30 mo., all bulls were slaughtered and the testis samples taken for histological analysis. Correlations among all variables were determined by Pearson's method. We observed that several aspects of the seminiferous tubules, including the average number of AI spermatogonium and round spermatids in the tubule cross sections, number of round spermatids per Sertoli cells, Al spermatogonium and spermatocyte were correlated to concentrations of GnRH-stimulated testosterone. Animals with higher testicular mass had more tubules with mature spermatids as the most developed germ cell type (r=0.52; p<0.05). All correlations between quantitative aspects of spermatogenesis and testis diameter and length were similar to those found with testis weight. Testis and epididymal weight were closely related at the age of 30 months (r=0. 72; p<0.05). Scrotal circumference at 23, 25 and 26 months was related to the number of round spermatids per tubule per section (r=0.49 to 0.59; p<0.05) and SC from 18 to 29 months was associated with the population of Sertoli cells per testis (r=0.50 to 0.59; p<0.05). In conclusion, these results suggest that testes measure evaluated in pre and pubertal phase can be an important indicator of gonadal development in adult Nellore bulls. Confirming that animals with larger scrotal circumference have more Sertoli cells, which provide a specialized, protected environment within the seminiferous tubules of the testis for germ cell development. These results extend our knowledge in physiology of male reproductive tract mainly in spermatogenesis. This study was supported by grants from Brazilian Research Councils, CNPq and CAPES (to AAAM).

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

NOT PRESENTED.

NOT PRESENTED.

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Fer Tyrosine Kinase Is Required for GVBD and Metaphase-I Spindle Organization.

Lynda McGinnis, Xiaoman Hong, Lane K Christenson, William H Kinsey 1

The control of microtubule and actin mediated events that direct the physical arrangement and separation of chromosomes during meiosis is critical since failure to maintain chromosome organization can lead to germ cell aneuploidy. Mammalian oocytes express the tyrosine kinase Fer at high levels relative to other tissues and the objective of the present study was to determine the function of this kinase in the oocyte. Fer kinase was uniformly distributed in the ooplasm of small oocytes but became concentrated within the germinal vesicle during oocyte growth. After germinal vesicle breakdown, Fer associated with the MI and MII spindles. Suppression of Fer expression by siRNA knockdown in germinal vesicle stage oocytes did not prevent activation of CDK1 activity or chromosome condensation during in vitro maturation, but did arrest oocytes prior to germinal vesicle breakdown or during meiosis-I. The resultant phenotype displayed condensed chromosomes trapped in the germinal vesicle, or condensed chromatids at the cell cortex but with an underdeveloped or no spindle microtubule structure. This phenotype was different than that resulting from nocodazole treatment suggesting that Fer kinase suppression leads to disruption of events other than just spindle assembly. The results demonstrate that, in oocytes, Fer kinase plays a critical role in maturation through GVBD and coordination of metaphase spindle organization. This study was supported by NICHD 14846 to W.H.K. and P20RR024214 from the National Center for Research Resources.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Xenopus Dicalcin, an Intrinsic and Suppressive Mediator of Sperm-Egg Interaction and Subsequent Fertilization.

Naofumi Miwa 1, Motoyuki Ogawa 2, Yoshiki Hiraoka 3, Ken Takamatsu 1, Satoru Kawamura 4

In a currently favored model of Xenopus laevis fertilization, sperm bind to vitelline envelope (VE) that surrounds an egg, and undergo acrosome-reaction that enables sperm to penetrate the VE. Despite identification of several regulatory proteins and sugar residues, molecular details of these interactions remain elusive. To contribute to the study of sperm-egg interaction and subsequent fertilization, we have isolated and characterized Xenopus dicalcin in Xenopus eggs. Xenopus dicalcin is localized prominently in the VE and the cytoplasm of the cortex of both animal and vegetal hemispheres of Xenopus eggs. Our overlay assay revealed that dicalcin exhibits a Ca2+-dependent binding to two ZP glycoproteins, both of which constitute a polymeric framework of VE. Since these VE glycoproteins are considered to function as sperm-receptors, we tested the effect of dicalcin on sperm-VE binding, sperm-VE penetration, and fertilization in vitro. Preincubation of eggs with recombinant dicalcin reduced the number of sperm that bound to VE as well as the efficiency of fertilization. In contrast, inhibition of intrinsic dicalcin by preincubation with anti-dicalcin antibody increased sperm-binding to VE and the efficiency of fertilization. To assay the effect of dicalcin on penetration through the VE, we developed an in vitro penetration assay method using fenestrated-filter upon which VE protein layer was prepared. In this penetration assay, recombinant dicalcin inhibited sperm-VE penetration significantly. Accordingly, it seems likely that dicalcin is capable of controlling either favorable or unfavorable conditions for sperm-egg interaction in Xenopus laevis. To further characterize this observation, we examined molecular action of dicalcin on sugar distribution within the VE. Exogenously applied dicalcin increased in vivo lectin-reactivity of the VE, which was accompanied by an increase in the lectin-binding ability of electrophoretically isolated VE protein. Furthermore, preincubation wih dicalcin increased the extent of incorporation of exogenously applied fluorescent molecule into the VE, which suggests that dicalcin causes changes in the three-dimensional structure of the entire VE framework. In conclusion, these results strongly suggested that dicalcin binds to egg-coating glycoproteins and alters the pattern of sugar presentation within the VE. Correlating with this alteration, dicalcin regulates both sperm-binding and sperm-penetration processes and thereby exert a suppressive function in sperm-egg interaction during fertilization. We believe that our present study would provide insights to define highly coordinated molecular machinery of fertilization processes. This research was supported by the JSPS to N.M. and S.K.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

FAK and PYK2 Are Rapidly Activated Following Fertilization of the Zebrafish Oocyte.

William H Kinsey 1

Fertilization triggers rapid activation of protein kinases which play multiple roles in egg activation and zygote development. The present study investigated the closely related Focal Adhesion Kinase (FAK) and Proline Rich Tyrosine Kinase (PYK2) which were detected in plasma membrane fractions prepared from zebrafish oocytes. Western blot analysis using antibodies specific for the autophosphorylation sites of FAK and PYK2 revealed that these kinases underwent activation/autophosphorylation within 2.5 minutes post fertilization. Antibodies specific for additional phosphorylation sites known to be targets of Src-family PTKs revealed that FAK and PYK2 also were phosphorylated by Src-family PTKs within 2.5 minutes post fertilization. Immunofluorescence studies confirmed the findings made by western blot and demonstrated that PYK2 activation was initiated at the site of sperm-egg interaction and progressed to include the newly forming fertilization cone. FAK activation was first detected in the cortex adjacent to the fertilization cone and both PYK2 and FAK activation progressed laterally to involve the entire oocyte although the most intense activation included the animal hemisphere of the zygote. Functional studies using a dominant-negative fusion protein encoding then N-terminal band 4.1 homology domain of PYK2 to suppress PYK2 activation caused an irregular fertilization cone structure and failure of sperm incorporation. The above findings suggest a scenario in which sperm-egg contact triggers activation of PYK2 which functions to trigger assembly of the actin-rich fertilization cone. Subsequently, PYK2 and FAK activation progresses through the entire egg cortex where they could be involved in remodeling the cortical actin layer. The factors that stimulate FAK / PYK2 activation as well as the contribution of this event toward developmental competence are currently under investigation.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Kiss1 cDNA Cloning and Induction of Ovulation by Peripheral Administration of Various Kisspeptins in the Musk Shrew (Suncus murinus), a Reflex Ovulator.

Naoko Inoue 1, Junko Tomikawa 1, Yuki Sasaki 1, Akira Uchida 1, Shinya Oishi 2, Nobutaka Fujii 2, Yoshihisa Uenoyama 1, Naoyuki Yamamoto 1, Kei-ichiro Maeda 1, Hiroko Tsukamura 1

Kisspeptin (firstly named metastin) is considered to play a crucial role in reproductive functions. The present study aims to investigate whether kisspeptin plays a role in reproductive functions in the musk shrew (Suncus murinus), a reflex ovulator, through stimulation of follicular development and ovulation. For this purpose, we first cloned suncus Kiss1 gene and determined the amino acid sequence of suncus kisspeptin. Second, the effect of peripheral injection of various kisspeptins and agonists on induction of follicular maturation and ovulation was investigated. Cloning of Kiss1 cDNA from the suncus hypothalamus revealed that kisspeptin precursor consists of 133 amino acid residues, which is predicted to be cleaved into two forms of suncus kisspeptin, sKp-40 and sKp-29. The C-terminal decapeptide, designated kisspeptin-10 (Kp-10), shows high homology with those of other mammals. A single subcutaneous injection of sKp-29 (0.5 nmol), but not sKp-40 (0.5 nmol), promoted follicular developments and induced ovulation in musk shrews. The sKp-29 injection mimicked the mating stimulus to induce follicular maturation and ovulation. Human Kp-54 (hKp-54, 0.05 nmol) was more potent than sKp-29 (0.5 nmol) and rat Kp-52 (rKp-52, 0.5 nmol) in terms of potency to induce ovulation. hKp-54-induced ovulation (0.5 nmol) was completely blocked by pretreatment with gonadotropin-releasing hormone (GnRH) antagonist, suggesting that kisspeptin activates GnRH neurons, and then induce ovulation in the musk shrew. To the contrary, suncus, rat and human Kp-10 had little or no effect on induction of ovulation. Synthetic GPR54 agonists (FTM080 and FTM145), which are designed to improve stability in the peripheral circulation, induced ovulation at 5 nmol. The present results indicate that kisspeptin might be involved in the mechanism mediating the reflex ovulation via activation of GnRH neurons in the musk shrew. This work was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences of Japan (PROBRAIN).

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Gonadotropins and Their Receptors in the Hermaphroditic Ricefield Eel Monopterus albus: cDNA Cloning and mRNA Expression.

Lihong Zhang, Yumei Yang, Jianfei Shou, Yang Zhang, Weimin Zhang 1

The ricefield eel is a protogynous hermaphroditic teleost fish that changes sex from functional female, through an intersexual stage, to the functional male phase during its life cycle, and a good model organism to study sex determination and differentiation of vertebrates. Previous studies suggested that HPG axis may play important roles in the initiation of sex change in ricefield eel; however, the gonadotropins and their cognate receptors remains to be elucidated in this fish. By using reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE), cDNAs encoding Cgalpha, Lhbeta, Fshbeta, Fsh receptor (Fshr), and Lh receptor (Lhr) of ricefield eel were cloned in the present study. Homology analysis showed that ricefield eel Cgalpha, Lhbeta, Fshbeta, Fshr, and Lhr shared highest identities with their counterparts in perciform fish,which conforms to the lineage relationship. A lhr pseudogene was identified from the genome of ricefield eel, and what is interesting is that phylogenetic analysis categorized teleost lhrs into two clusters, and ricefield eel lhr gene and pseudogene are grouped into one of the two clusters, respectively, which suggests that either one of the duplicated lhr genes became a pseudogene during evolution of different teleost lineages. During ovarian development, realtime PCR analysis showed an increasing trend of fshbeta mRNA levels in the pituitary from previtellogenesis to vitellogenesis, but then a decreasing trend toward maturation. As for lhbeta mRNA levels in the pituitary, a continuous increasing trend was observed from previtellogenesis to maturation. The ovarian fshr mRNA levels were significantly increased from previtellogenesis to early vitellogenesis, but then decreased significantly thereafter. However, the ovarian lhr mRNA levels were increased significantly from vitellogenesis to the mature stage. The above results may suggest that Fsh signal pathway is more important for vitellogenesis, whereas Lh signal pathway more important for oocyte maturation in ricefield eel. During sex change from female through an intersexual stage to male, fshr and lhr showed lower expression levels in transitional gonads than either ovary or testis. In the pituitary, fshbeta and lhbeta mRNA levels were significantly lower during the intersexual and male stages than the female stage. However, lhbeta /fshbeta mRNA ratios were increased significantly during sex change, suggesting that the changes in the ratio of LHbeta to FSHbeta may be important for the gonadal differentiation of ricefield eel. The present study provided a foundation for further elucidation of the roles of HPG axis in the gonadal development and sex change of ricefield eel. This research was supported by Natural Science Foundation of China (30970359, 30771651) and National High Technology Research and Development Program of China (863 Program) (No.2008AA09Z406).

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

In Vivo Biological Activity of a Recombinant Salmon Follicle-Stimulating Hormone Analog in Prepubertal Male Coho Salmon.

Koichiro Gen 1, Jon T Dickey 2, J Adam Luckenbach 3, Yoji Yamamoto 2, Olga Mirochnitchenko 4, Michael P Bernard 4, William R Moyle 4, Penny Swanson 3

The duality of pituitary gonadotropins in fishes has been established largely through molecular characterization of the genes that encode subunits of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). In contrast, little is known about the functional duality of piscine LH and FSH due to the lack of sufficient quantities of highly purified native proteins or well characterized recombinant proteins that maintain activity in vivo for physiological studies. In this study, we examined in vivo biological activity of recombinant coho salmon FSH analog (csFSHa) comprised of human glycoprotein hormone alpha subunit and coho salmon FSH beta subunit expressed as a single chain in stably transfected COS-7 cells. Media containing csFSHa was dialyzed and concentrated by ultrafiltration. The concentrations of immunoreactive and bioactive csFSHa (13.5 mg/L) were estimated by comparison to native csFSH standards in a salmon FSH radioimmunoassay and an in vitro steroidogenesis bioassay. Previous studies from our laboratory have suggested that onset of puberty in male salmon is controlled by FSH because plasma levels of FSH increase and LH is non-detectable in plasma at this stage. Thus, to test whether csFSHa could stimulate spermatogenesis in vivo, the analog (1.35 micrograms/fish) was injected intraperitoneally twice a week for 2 weeks into prepubertal male fish (n=8 per group). For comparison, other groups of fish received either saline (control) or a preparation of partially purified native salmon gonadotropins (ppGTH, mixture of FSH and LH, 1.5 micrograms/fish) due to lack of sufficient amounts of highly purified native proteins for in vivo studies. Fish were sampled for blood and tissues 6-8 h after the last injection. Pieces of testis were frozen for analysis of mRNA levels or fixed for histological staging. The mean gonadosomatic index (GSI; gonad weight/body weight x 100) of analog-injected males was 1.5-fold higher than either controls (p < 0.001) or ppGTH-injected fish (p < 0.01). The expression of putative Sertoli cell transcripts, anti-Mullerian hormone (amh) and gonadal-soma derived growth factor (gsdf), which decline with onset of spermatogenesis in salmon, were suppressed by the csFSHa compared to controls (p < 0.05). In contrast, csFSHa had no effect on plasma levels of 11-ketotestosterone (11-KT) or mRNA levels for steroidogenesis-related genes, such as star and hsd3b. This suggests that the change in testis mass induced by the csFSHa may not be mediated by 11-KT. Interestingly, ppGTH increased 11-KT levels 2-fold, had no effect on GSI, and caused a slight, but not significant elevation in testicular hsd3b mRNA levels. The difference in in vivo steroidogenic activity between ppGTH and the csFSHa may be due to the presence of high amounts of LH in the ppGTH preparation. In conclusion, the present study has shown that a recombinant csFSHa elicits biological activity in vivo that is consistent with the proposed role for FSH in onset of puberty in male salmon. The results also indicate that this strategy for production of piscine FSH analogs will be useful for basic physiological studies as well as practical applications for controlling timing of puberty in commercially important species for aquaculture. Research supported by National Research Initiative Competitive Grant no. 2007-35203-18088 from the USDA CSREES.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Long-Chain Fatty Acids Regulate GnRH Receptor mRNA Expression Level in the Gonadotrope of the Mouse Anterior Pituitary Gland.

Ryutaro Moriyama, Chikaya Deura, Nobuyuki Fukushima 1

Free Fatty acids are not only energy fuels but also signaling molecules. In fact, long-chain fatty acids (LCFAs) have been identified as the ligands for G protein-coupled receptor 120 (GPR120). We have previously observed that GPR120 is richly expressed in the gonadotropes of the mouse anterior pituitary gland and 48hr fasting induces increase in GPR120 mRNA level in the mouse pituitary. Further, in circadian variations, GPR120 mRNA expression levels have shown transient increase at 17:00, GnRH-R mRNA expression levels have decreased between 15:00 and 21:00 and plasma non-esterified fatty acids and triglyceride concentrations have increased between 11:00 and 15:00 in the ad libitum-fed matured male mice. These diurnal variations of GPR120 and GnRH-R in the ad libitum-fed were blunted by time-restricted feeding (feeding time between 06:30-18:30 at light period for 10 days). Based on these results, we hypothesized that the nutrients such as LCFAs act as signaling molecules that regulate gonadal functions at the pituitary level. The aim of present study is to clarify whether LCFAs act as the regulator of gonadotropine releasing hormone receptor (GnRH-R) mRNA in the mouse pituitary. For this purpose, we tried two experiments in this study. First, the effects of high-fat diet for 2 or 30 days on GPR120 and GnRH-R mRNA expression levels were examined in the matured mail mouse pituitaries. Second, the direct effects of palmitate on GPR120 and GnRH-R mRNA expression levels in the mouse pituitary were observed in vivo and in vitro . In the first study, high-fat diet significantly induced increase in GPR120 mRNA expression levels compare to low-fat diet groups in both second and 30 days of high-fat diet and increase in GnRH-R mRNA expression levels in 30 days of high-fat diet. Next, 24 hr treatment of 0.1 mM palmitate directly induced increase in GPR120 mRNA expression levels and decrease in GnRH-R mRNA expression levels in the primary culture cells of the matured male mouse pituitary and gonadotroph cell line, LβT2 in vitro, and GPR120 mRNA expression levels in the mouse pituitary were significantly increased compared with the control in the experiment that administered the palmitate into blood for the short term in vivo. Taken together, these results suggest that LCFAs especially palmitic acid might regulate GnRH receptor mRNA expression levels in the gonadotropes of the mouse anterior pituitary via a specific receptor such as GPR120. Research supported by Grant-in-Aid for Young Scientists (B) 20780202 from the Ministry of Education, Culture, Sports, Science and Technology, Japan and research grant (RK20-028) from Kinki University to R.M.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Dynamic Change in Circulating Pituitary and Gonadal Hormones from Birth to Puberty in the Thoroughbred Colts and Fillies.

Pramod Dhakal 1, Akiko Otsuka 2, Yasuo Nambo 3, Takehiro Harada 2, Tsunoda Nobuo 4, Masahiro Itoh 1, Watanabe Gen 2, Kazuyoshi Taya 2

The equine fetal gonads of both sexes have attracted much interest because of remarkable enlargement of gonadal size resulting from increased number of interstitial cells, but the size of gonad reduces at birth and re-starts development for puberty. The present study was designed to elucidate the changes in circulating gonadotropins and gonadal hormones from birth to puberty and thus explicate relationship between gonadal seasonality and hormonal secretion in both sexes of thoroughbred horses. In the first experiment (1st Exp), daily point sampling of jugular blood was done using spring born thoroughbred colts (n=636) and fillies (n=694) from birth to 30 weeks of age. To further expand, in the second experiment (2nd Exp), spring-born colts (n=6) and fillies (n=9) were used for continuous weekly jugular blood samples from birth to 15 months of age. To confirm the gonadal seasonality, in the third experiment (3rd Exp), fully adult stallions (n=10) were used for monthly jugular blood samples for two years. To clarify the relationship between the photoperiod and reproductive endocrinology, in the fourth experiment (4th Exp), geldings males (n=10) were used for continuous monthly jugular blood samples for one year. In the final experiment (5th Exp), for determination of effect of extended photoperiod on reproductive endocrinology, yearlings (colts; 31, fillies; 31) were used. Horses (n=30) were subjected to an artificial photoperiod (14.5 h daylight and 9.5 h of night from December 20th to April 10th). Rest received natural light as the control group (n=32). In both sexes, the 1st Exp showed that levels of steroid hormones (estradiol, testosterone, progesterone) were remarkably high at birth, followed by rapid drop within 48 hr, and remained at the lower level until six months of age. The level of ir-inhibin began to decrease after 3 days, dropping by more than half in the first 20 days after birth, and then declined slowly until six months of age. The 2nd Exp showed a clear increase in serum levels of testosterone, estradiol, and ir-inhibin in colts from around 44 weeks later (April) and remained high until 60 weeks later (August). In fillies, serum levels of progesterone and estradiol increased at around 48 weeks later (April and May), and remained high until 60 weeks. The increase in ir-inhibin was less clear in fillies. In colts, levels of LH, FSH, and PRL showed the first increase by around 16 weeks of age (June to September) and the second large increase at around 50 weeks of age (April to July in the next year). Prolactin exhibited most clear seasonal changes in both sexes. In both sexes, the seasonal increase in circulating PRL was remarkable even in the first year. In the 3rd Exp, clear seasonal changes were observed in gonadotropins and testicular hormones of the adult stallions. The highest concentration of LH, FSH, PRL, ir-inhibin, testosterone and estradiol were noticed in the breeding season (March to July), and the lowest being noted in the non-breeding season (October to February). In the 4th Exp, a similar seasonal change was observed in circulating PRL of gelding males. In the 5th Exp, the light treatment to yearlings stimulated secretion of LH, FSH, and prolactin in both the sexes. The light treatment also induced early ovulation in fillies and early secretion of testosterone and estradiol in colts. These results clearly demonstrated that the hypothalamo-pituitary axis is already responsive to changes in photoperiod and secrete PRL similar to adult horses, but pituitary gonadotrophs for LH and FSH secretion is less sensitive. In addition, the responsiveness of gonads, especially testes of weanling are less sensitive to LH and FSH for testosterone and estradiol secretion.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Molecular and Physiological Characterization of the Porcine Kisspeptin.

Junko Tomikawa 1, Tamami Homma 2, Satoshi Ohkura 1, Yoshihisa Uenoyama 1, Hiroko Tsukamura 1, Kei-ichiro Maeda 1

Kisspeptin-GPR54 signaling plays an essential role in normal reproduction in mammals via stimulation of gonadotropin secretion. Here we identified the porcine Kiss1 gene expressed in the hypothalamus. The full length of the cDNA was 857 bp encoding the kisspeptin of 54 amino acids with the C-terminal active motif designated kisspeptin-10 (Kp-10), which is identical to that of mouse, rat, cattle and sheep. To determine the action of kisspeptin on gonadotropin secretion in the pig, we intravenously injected Kp-10 at doses of 30 and 300 nmol/head (in saline) into the mature Landrace sows in the luteal phase of the estrous cycle. Blood samples were collected every 10 min for 8 hours before and 4 hours after injection, and plasma concentrations of luteinizing hormone (LH) were determined by RIA. Plasma LH concentrations increased immediately after the Kp-10injection at both doses. Furthermore, the area under the curves for 60-min postchallenge period was significantly higher than the prechallenge value in Kp-10-treated groups (P < 0.05), indicating that exogenous Kp-10 at all dose tested effectively stimulated LH secretion in sows. In situ hybridization analysis revealed that endogenous Kiss1-expressing cell populations were mainly distributed in the hypothalamic periventricular nucleus (PeN) and arcuate nucleus (ARC). Kiss1 mRNA was barely detected in the PeN of ovariectomized (OVX) females, while a large number of Kiss1-expressing cells were found in the EB-treated animals. The number in the PeN was significantly higher in EB-treated OVX pigs than in OVX females. On the other hand, Kiss1-expressing cells were widely distributed throughout the ARC regardless of estrogen status, but a small number of the neurons restricted in the most caudal part of the ARC disappeared by EB treatment. These results suggest that the PeN kisspeptin neurons could be responsible for the estrogen positive feedback regulation to induce GnRH/LH surge in the pig, and that the caudal ARC kisspeptin neurons could be involved in the estrogen negative feedback regulation of GnRH/LH release. This work was supported in part by PROBRAIN of Japan.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Effects and Molecular Mechanism of Isoliquiritigenin on Suppression of Metastasis of Human Breast Cancer Cells.

Paulus Wang 1, Chia-Jung Chan 1, Shih-Min Hsia 2, Kai-Lee Wang 1, Ching-Han Yu 1, Ru-Lian Hsu 1

Up-regulations of both vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP) are important factors involved in tumor progression and metastasis by stimulating cell migration. In the present study, isoliquiritigenin (ISL) isolated from licorice was employed to investigate the anticancer potency via modulation of the expression of VEGF and MMP in the highly metastatic human breast cancer cell line MDA-MB-231. We have demonstrated that ISL treatment for 24 h and 48 h dramatically reduced secretions and protein levels of VEGF in a time- and dosage-dependent manner from 0.1, 0.5, 1, 5 and 10 µM as determined by ELISA and Western blot analysis. The decrease of protein levels of VEGF was from down-regulation of hypoxia-inducible factor (HIF-1α) protein expression assessed by immunoblot analysis. Since several studies have demonstrated that up-regulations of metalloproteinase-9/-2 (MMP-9/MMP-2) is associated with breast cancer metastasis, we further studied the effects of ISL on migration and the protein levels of related MMP-9 and MMP-2. The migratory response to the administration of ISL was measured in MDA-MB-231 cells using the modified chamber assay and wound-healing assay. Results showed that ISL at 10 µM significantly suppressed the migration of MDA-MB-231 cells around 24 h and 48 h time intervals, respectively. Furthermore, ISL was able to decrease the protein levels of MMP-9 and MMP-2 at 48 h. These results revealed that degradtive expressions of MMP-9 and MMP-2 may correlate with a decrease in ISL-treated migration in MDA-MB-231 cells. To further clarify the molecular signal pathway for ISL-downregulated cell migration in MDA-MB-231 cells, SB203580 (p38 MAPK inhibitor), LY294002 (Akt activation inhibitor) and PD98059 (extracellular signal-regulated kinase inhibitor) were used to examine the effect of ISL on secretion of VEGF. ELISA results revealed either SB203580 or LY294002, but not PD98059, can abolish the decrease of VEGF release from ISL-treated MDA-MB-231 cells. Western blot assay indicated that ISL suppressed the phosphorylation of p38 mitogen-activated protein kinase and Akt kinase. Taken together, these findings suggested the ISL could be a potential candidate as a novel therapeutic agent in the treatment of breast carcinoma by inhibiting p38 and Akt signaling with their downstream targets VEGF, HIF-1α, MMP-9 and MMP-2.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Effect of a High Energy Diet on Steroidogenic Capacity of Dominant Follicles, Secretion of LH and Estradiol, and Precocious Puberty in Beef Heifers.

Martin G Maquivar, Lucas Souto, Leandro H Cruppe, David E Grum, Martin L Mussard, Michael L Day 1

The objectives of the present study were to evaluate the impact of a high energy diet in early weaned heifers on estrogenic capacity of the dominant follicle and related changes in systemic hormone concentrations. Twenty heifers were weaned at 86 ± 2 d of age and 137 ± 3.3 kg body weight. Heifers were randomized to receive isonitrogenous diets (14.1% crude protein) that contained either a high energy content (HIGH; 2.2 Mcal/kg; n = 10) or a lesser energy content (control diet; CONT; 1.70 Mcal/kg; n = 10) from weaning until approximately 300 d of age. Target rates of gain were 1.50 kg/d for the HIGH and 0.75 kg/d for the CONT treatment. Within treatments, heifers were designated to be ovariectomized (OVX) to assess follicular function, or to remain intact (INT) for assessment of secretion of LH and precocious (≤ 300 days of age) puberty. In the CONT-OVX and HIGH-OVX treatments, daily ultrasonographic evaluation of ovarian follicles was initiated at 130 days of age and continued through two complete follicular waves. In the subsequent (3rd wave), the ovary bearing the dominant follicle was removed the day after it achieved a diameter of 9 mm to assess intrafollicluar estradiol concentrations. Daily blood samples were collected to evaluate systemic estradiol concentrations. In the CONT-INT and HIGH-INT treatments, serial blood samples were collected at 15 min intervals for 12 hr at 145, 170, 208 and 236 days of age to assess secretion of LH. Weekly blood samples were collected and analyzed for progesterone concentrations to determine age at puberty. Average daily gain was greater (P < 0.05) in the HIGH than CONT treatment (1.57 ± 0.16 vs. 1.21 ± 0.13 kg/d respectively) and body weight was greater (P < 0.05) in the HIGH than CONT treatment from 186 d of age (treatment x age, P < 0.05). Precocious puberty occurred in 3/6 heifers in the HIGH-INT and 4/5 heifers in the CONT-INT treatment. For heifers that reached puberty, age at puberty was greater (P < 0.05) in the CONT-INT (253 ± 1 d of age) than HIGH-INT (194 ± 9 d of age) treatment. Body weight at puberty was also greater (P < 0.05) in the CONT-INT than HIGH-INT treatment. Mean LH concentration was greater (P < 0.05) in the HIGH-INT than CONT-INT treatment at 170 and 208 days of age but did not differ on other days of serial blood sample collection. Neither frequency or amplitude of LH pulses differed between treatments. Systemic estradiol concentrations in the CONT-OVX were greater (P < 0.05) than in the HIGH-OVX treatment on the day of ovariectomy (1.68 ± 0.21 and 0.94 ± 0.25 pg/ml, respectively) and the day preceding ovariectomy (2.16 ± 0.37 and 0.86 ± 0.32 pg/ml, respectively). Follicular fluid concentrations of estradiol did not differ between the HIGH-OVX (91.4 ± 36.4 ng/ml) and CONT-OVX (110.0 ± 88.49 ng/ml) treatments. In conclusion, the HIGH dietary treatment increased body weight gain and LH secretion and resulted in an earlier age at puberty, however, the incidence of precocious puberty did not differ between treatments. Intrafollicular concentrations of estradiol did not differ between the HIGH-OVX and CONT-OVX treatments. It is unknown whether the lack of difference due to diet is due to the unexpectedly high incidence of precocious puberty in the CONT treatment, or if the mechanism of action of the HIGH treatment is exerted at alternative sites within the reproductive axis.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

FGF21 Is Epigenetically Regulated by a Methyl Donor Rich Diet and a Transgenerational Model of IUGR.

Min Hu, Melissa Suter, Philip Bocock, Cynthia Shope, Lori Showalter, Kjersti Aagaard-Tillery 1

Fibroblast growth factor 21 (FGF21) is an atypical member of the FGF family that functions as an endocrine hormone. It has been reported that FGF21 transcription levels increase in murine liver after either fasting or exposure to a ketogenic diet. The developmental origins of adult disease hypothesis postulates that metabolic and growth perturbations in utero can cause susceptibility to adult dieases, In a transgenerational rat model of intrauterine growth restriction (IUGR) we have demonstrated that second-generation rats born small for gestational age will develop obesity and metabolic disorders in adulthood; this morbidity can be prevented with supplementation of nutrients along the 1-carbon pathway (ENS diet). In order to better understand the molecular physiologic basis for these observations, we studied liver tissue from the F2 generation at both day 21 (d21) and day 160 (d160). Employing standard assays for IGF1 expression and differential methylation of its promoter, we have previously demonstrated that IGF1 is epigenetically downregulated in formerly IUGR animals destined to become obese in adult life. In these studies, we have extended our characterization to FGF21, an upstream regulator of the IGF1 signaling cascade implicated as a mitigater in the development of diabetes. We observed that among females, prevention of adult metabolic disease in IUGR lineage F2 exposed and later fed an ENS diet (Teklad8640+folic acid/betaine, choline/B12/L-methionine/L-arginine/zinc) had an associative significant increase in FGF21 expression by d160 (3.419 fold, p<0.001). This dramatic induction differed by gender, as male rats under the same conditions demonstrated no significant change in FGF21 expression at d160 (0.469-fold, p=.132). Of interest, when we examined expression of FGF21 at d21, prior to the onset of adult morbidity, we observed the inverse association to be true: relative to their non-IUGR lineage counterparts on a regular diet (Teklad8640), FGF21 expression among males at d21 was modestly affected (3.419 fold, p .087), with no significant affect among females . Taken together, these findings by virtue of gender bias suggested to us that sex hormones may be influencing FGF21 expression in our rat model. To formally explore this possibility, we examined expression of FGF23, FRAG1, FGF receptor, and βklotho with QPCR. Significant increases of βklotho and FRAG1 expression accompanied our FGF21 expression observations d160 rats (p<0.001). In summary, while recent reports would suggeset that FGF21 mitigates adult metabolic disease, our observations in a transgener model indicate that FGF21 expression is altered in a gender specific, methyl donor diet amenable fashion. Research supported by NIH Director New Innovator Pioneer Award DP2120OD001500-01(K.A.T) and NICHD-March of Dimes reproductive Scientist Development Program (K.A.T).

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Regulation of Inducible Nitric Oxide Synthase Expression in Bovine Granulosa Cells and Its Involvement in Follicular Dominance.

Gustavo Zamberlam 1, Valerio Portelaj 1, Joao FC Oliveira 2, Paulo BD Goncalves 3, Christopher Price 1

Understanding the paracrine events that regulate fertility in the cow is necessary not only because of the agricultural importance of this species, but also its potential use as a model for humans. Nitric oxide (NO), a free-radical gas, has been implicated in follicular growth in rodents, but the cow is an intriguing enigma: NO is produced by bovine granulosa cells and is regulated by FSH, but the enzymes responsible are reported to be absent. Three genes encode NO synthase proteins, one of which - iNOS - is widely considered to be regulated by extracellular ligands. We hypothesize that granulosa cells express the gene encoding iNOS, and that the expression of this gene is under hormonal regulation. The objectives of the present study were to determine the abundance of iNOS mRNA in growing and non-growing follicles in cattle, to determine the regulation of iNOS expression by FSH and growth factors, and to determine the action of NOS in bovine granulosa cells. The two largest follicles on days 1 to 5 of the first follicular wave of the cycle were collected from each pair of ovaries from six cows. Messenger RNA encoding iNOS was detected in granulosa and theca cells of bovine follicles by real-time PCR. The granulosa cells of dominant follicles contained higher levels of mRNA for iNOS compared with subordinate follicles (P<0.01). The regulation of iNOS expression was evaluated in a serum-free granulosa cell culture system. FSH (P<0.05) and IGF1 (P<0.01) stimulated E2 secretion iNOS mRNA abundance in a dose-dependent manner. Moreover, both FGF2 and EGF (P<0.01) decreased iNOS expression and E2 secretion in FSH and in IGF1-treated cells. Owing to the parallel changes in estradiol secretion and iNOS mRNA, we assessed the role of estradiol in the regulation of iNOS mRNA; replacement of estrogen precursor with a non-aromatizable androgen (DHT) or the addition of an estrogen receptor antagonist inhibited FSH-stimulated iNOS mRNA abundance, and addition of E2 alone stimulated NO production in vitro. To assess the role of endogenous iNOS, cells were treated with an iNOS-selective inhibitor, which resulted in increased FasL mRNA levels (P<0.01), induction of caspase-3 activation (P<0.01) and an increase in the incidence of cell death (P<0.05). In conclusion, bovine graulosa cells express iNOS, and increased iNOS mRNA levels are associated with growth of the dominant follicle. Gonadotropic factors stimulate iNOS mRNA levels through increased E2 secretion, and physiological levels of NOS activity may contribute to growth and survival of granulosa cells. Supported by NSERC Canada and CNPq Brazil.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Effect of Feed Restriction on Early Avian Follicular Development.

Francisco J Diaz, Kate Anthony, Ashley N Halfhill 1

Selection for growth and feed efficiency in meat-type (broiler) chickens has caused a substantial decrease in reproductive efficiency. In particular, rapid growth in broiler-breeder hens is associated with severe ovarian dysfunction, including internal and/or double ovulations and reduced fertility of ovulated oocytes. Consequently, breeder animals are fed a restricted diet to limit growth and control ovarian dysfunction. However, the effect of feed restriction on development of small avian follicles <0.1 to 1 mm is not known. The objective of this study was to examine effects of feed (full versus restricted) on gene expression of markers of small follicle development and on histological changes of the ovarian cortex of young hens prior to photo stimulation (13 and 16 weeks). These ages were chosen to allow the longest treatment periods and to avoid the presence of larger gonadotropin responsive follicles within the ovarian cortex. We also wanted to determine diet-induced changes in intra-ovarian processes that were independent of gonadotropins. Broiler-breeder hens (Cobb) were purchased commercially as 1 day-old chicks and fed grower diet for 2 weeks. From 2-16 weeks, animals were randomly assigned to receive full-fed (FF) or restricted fed (RF). FF birds had free access to feed at all times, while RF birds were fed once a day a limited amount of feed according to commercial management guidelines for weight gain. At 15 weeks, FF birds were more than twice as heavy as RF birds, emphasizing the extreme effect of feeding treatments and the tremendous growth potential of these birds. Ovarian cortex was collected from 3-4 animals in each group at 13 and 16 weeks of age and analyzed using qPCR to measure levels of mRNAs and for morphological examination. Ovarian histological analysis indicated that there were more activated follicles (>0.1 mm) in sections of ovarian cortex from FF hens at both 13 and 16 weeks. Throughout the cortex there were areas containing clusters of small follicles (<0.1 mm) in both FF and RF birds. These are the smallest identifiable follicles and probably represent the primordial pool. Interestingly, FF birds contained over twice as many small follicles in these clusters than RF birds. This suggests greater follicular recruitment or decreased atresia of small follicles in FF birds. Analysis of previously identified markers of follicular development in birds revealed significant increases in BMP6 and BMP15 mRNA at 13 weeks and BMP6 and AMH at 16 weeks in samples of cortex from FF birds. The convertase proteins, PCSK3 (Furin), PCSK5 and PCSK6 process BMP and other secreted products to their mature form. Both PCSK3 and PCSK5 increased, while PCSK6 decreased in FF compared to RF birds, suggesting increased biological secretion or processing of intra-ovarian growth factors in FF birds. Finally, there were significant increases in two transcription factors, FOXL2 and GATA4 in FF versus RF hens. These transcription factors are critical for ovarian function in mammals and our present data indicate they are altered by diet in small follicles. These results demonstrate a clear diet-induced change in early follicular development analyzed by histological and gene expression measures. We conclude that overfeeding induces changes in ovarian function beginning at the earliest stages of follicular development.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Role of Prohibitin in Regulating FSH-Induced Steroidogenesis During Follicular Development.

Qi Wang 1, Benjamin K Tsang 2

With the advances in gonadotropin research, treatment of human infertility with gonadotropin preparations has become a common practice. It plays critical roles in stimulating ovarian cell proliferation, differentiation and suppressing apoptosis, which ultimately induces ovarian follicle growth and ovulation. The action of gonadotropin during follicular development is however complex and is stage-dependent, thus making optimization of gonadotropin treatment for various ovarian disorders often difficult. Prohibitin (PHB) is an intracellular protein important for cell cycle regulation, apoptosis and cellular differentiation. Ovarian PHB is up-regulated by equine chorionic gonadotropin (eCG) in vivo. Although PHB prevents cell apoptosis induced by the alkaloid staurosporine in undifferentiated granulosa cells (from primarily preantral/early antral follicles), whether the expression and action of PHB are similarly regulated in differentiated granulosa cells (large antral and preovulatory follicles) are unknown. We hypothesize that PHB is an important regulator of granulosa cell fate and plays a role in regulating steroidogenesis during follicular development. Our objective is to better understand the function and regulation of PHB during follicular development, especially during FSH-induced antral follicle growth. Using granulosa cells from eCG-primed immature rats, we observed that (1) FSH up-regulates granulosa cell PHB expression in a follicular stage-dependent manner in vitro. (2) Akt content and its phosphorylation are required for the regulation of PHB by FSH. (3) Exogenous PHB significantly reduces FSH-stimulated estradiol and progesterone production in granulosa cells. (4) cyp11a1 (p450 side-chain cleavage enzyme) mRNA and protein expression in granulosa cells are down-regulated by exogenous PHB. (5) The mRNA levels of other steroidogenic enzymes, such as cyp19 (aromatase) and Steroidogenic acute regulatory protein (StAR), are also down-regulated by exogenous PHB. Conclusion: This study demonstrates that granulosa cell PHB is regulated by FSH in a follicular stage-dependent manner and that PHB regulates FSH-induced steroidogenesis in granulosa cells. Our findings raise the possibility that the dysregulation of PHB expression and function may be linked to infertility associated with compromised follicular growth and differentiation (e.g. Polycystic ovarian syndrome) and that PHB may be a therapeutic target for the treatment of infertility. Further experiments are required to test these possibilities.Supported by Canadian Institutes of Health Research (CIHR).

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

NOT PRESENTED.

NOT PRESENTED.

Biol Reprod. 2010 Nov;83(1 Suppl):2.

WITHDRAWN

WITHDRAWN.

Biol Reprod. 2010 Nov;83(1 Suppl):2.

The BMP Antagonist Gremlin Is Not Essential for Folliculogenesis in the Mouse.

Michelle Myers, Brooke S Middlebrooke, Robert W Cook, Stephanie A Pangas 1

Members of the transforming growth factor beta superfamily are renowned for their diverse roles in developmental processes, including folliculogenesis. Bone morphogenetic proteins (BMPs) are regulated by specific BMP antagonists and gremlin, a member of the DAN family of BMP antagonists, has been implicated in reproduction. We hypothesized that ablation of gremlin in the ovary would disrupt BMP antagonism. Gremlin is present in neonatal ovaries and continues to be expressed throughout folliculogenesis. To investigate the potential role of gremlin throughout follicle development, we utilized two mouse models, each unique to a different stage of folliculogenesis: i) mice globally deficient in gremlin (denoted Grem1-/-); and ii) mice with granulosa cell deletion of gremlin (denoted Grem1 cKO). Grem1-/- mice die within 24-48 hours of birth with no obvious differences observed between wildtype and Grem1-/- neonatal ovaries. Additionally, we transplanted Grem1-/- neonatal ovaries into ovariectomized adult wildtype mice to study later follicle development in ovaries devoid of gremlin. Transplanted Grem1-/- ovaries revealed that folliculogenesis could proceed to large antral follicle development, suggestive that the loss of gremlin did not alter folliculogenesis. These findings were further confirmed utilizing Grem1 cKO mice, which demonstrated normal ovarian folliculogenesis and had comparable fertility to control mice. Analysis of other BMP antagonists in the ovary demonstrates that multiple related proteins are co-expressed with gremlin. These data suggest that gremlin is not required for folliculogenesis and loss of Grem1 in the ovary may be compensated by other BMP antagonists. Funded by a Burroughs Wellcome Career Award in Biomedical Sciences to S.A.P.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

NOT PRESENTED.

NOT PRESENTED.

Biol Reprod. 2010 Nov;83(1 Suppl):2.

In Utero and Lactational Exposure of Sprague-Dawley Rats to Diadzein Disrupts Ovarian Follicle Maturation.

Monika G Baldridge, Stanislaw K Miaskowski 1

The consumption of dietary phytoestrogens, such as isoflavones, has grown increasingly popular due to their potential health benefits. Of particular interest is the isoflavone diadzein that is found in a variety of plants, primarily soybeans. Isoflavones are naturally occurring plant estrogens that may provide health-related benefits in some tissues; however, they may have deleterious side effects on estrogen-sensitive reproductive tissue such as the developing ovary. Several studies have documented that an in utero and lactational exposure to estrogenic chemicals alters normal reproductive tissue development. This study was designed to determine (1) if an in utero and lactational exposure to a low or high dose of pure diadzein (LDiad, HDiad) exerts detrimental effects on ovarian follicle maturation in the Sprague-Dawley rat and (2) whether estrogen and progesterone concentrations are altered following exposure. Animals were treated via subcutaneous injection on gestational days (GD) 7-21 with LDiad (0.5 mg/kg/day) or HDiad (5 mg/kg/day). Female pups were euthanized on postnatal day 28 (chosen in order to follow the pups' exposure through lactation). Following euthanasia, the ovaries were excised, fixed for histology, and analyzed. The analysis included a count, measurement, and classification of preantral and antral follicles in the greatest cross-sectional area of the ovary. Blood samples collected at the time of death were analyzed for estrogen and progesterone concentrations via radioimmunoassay. No statistical change in estrogen (p=0.729) and progesterone (p=0.511) concentrations was observed among the treatment groups. Increased atresia and a decrease in the number of antral and preantral follicles were observed in the LDiad and HDiad treatment groups (p<0.001) as compared to control. These data support the hypothesis that diadzein reduces the number of follicles of certain size classes in rats exposed during the critical period of development. Atresia of preantral and antral follicles may illustrate one of the underlying mechanisms by which folliculogenesis is modulated by diadzein. The results of this study elucidate the effects of diadzein on reproductive parameters in the female, perinatal Sprague-Dawley rat. The results of this research may have profound implications for female human reproductive health.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Follicle-Stimulating Hormone Restores Growth in Follicles Lacking the Aryl Hydrocarbon Receptor.

Stacey L Dehlinger, Isabel Hernandez-Ochoa, Mallikarjuna S Basavarajappa, Jackye Peretz, Jodi A Flaws 1

The aryl hydrocarbon receptor (Ahr) is a ligand-activated transcription factor that mediates the toxicity of dioxins. The Ahr has also been shown to regulate physiological functions, including ovarian function. Previous studies have shown that Ahr-deletion (AhrKO) results in slow growth of antral follicles, which are the functional units of the ovary. Further, we previously showed that growth of follicles is slowed in AhrKO antral follicles compared to wild-type (WT) antral follicles between post-natal day (PD) 30 and 53, times when mice undergo puberty and start cyclicity. We also showed that growth of follicles in AhrKO ovaries is restored to WT levels after PD 53, but the reasons are unknown. Since follicle-stimulating hormone (FSH) promotes follicle growth, we hypothesized that slow growth in AhrKO antral follicles from PD 30 to 53, when mice undergo cyclicity, is mediated by FSH. To test this hypothesis, antral follicles from WT and AhrKO ovaries on PD 30, a time when follicle growth is impaired in AhrKO follicles, were mechanically isolated and cultured in a-minimal essential media with or without recombinant FSH (rFSH; 0 – 15 IU) for 7 days. Follicle growth was measured every 24 h and presented as percent change compared to that of day 1. AhrKO follicles treated with 0 and 5 IU rFSH had decreased growth compared to WT follicles by the end of culture (0 IU rFSH: WT=142±5.5% change, AhrKO=117±2.4% change and 5 IU rFSH: WT=169±8.9% change, AhrKO=137±5.9% change; n=3 cultures; p<0.05). Interestingly, AhrKO follicles treated with 10 and 15 IU rFSH grew in a similar manner to WT follicles by the end of culture (10 IU rFSH: WT=145±9.4% change, AhrKO=154±12.3% change, and 15 IU rFSH: WT=142±12.7% change, AhrKO=166±17.3% change; n=3 cultures; p<0.05). Since estradiol mediates follicle growth, we then compared estradiol production in WT and AhrKO follicles treated with rFSH. We further evaluated the levels of Cyp19a1 (aromatase) mRNA, a steroidogenic enzyme that converts androgens to estradiol. Cultured antral follicles were subjected to RNA and real-time PCR for Cyp19a1. Media samples were collected from the follicle culture and subjected to enzyme-linked immunosorbent assays to measure levels of estradiol. AhrKO follicles treated with 5 IU rFSH had significantly decreased estradiol levels compared to WT follicles (5 IU rFSH: WT=13564.74±1917.99 pg/ml, AhrKO=7414.95±1052.18 pg/ml, p=0.048). AhrKO follicles treated with 10 IU rFSH had increased estradiol levels compared to the WT (10 IU rFSH: WT=7989.09±1123.86 pg/ml, AhrKO=32382.69±10185.14 pg/ml, p=0.050). The levels of Cyp19a1 mRNA were significantly higher in the AhrKO follicles treated with 10 IU rFSH, as well as the follicles treated with 15 IU rFSH compared to WT follicles (10 IU rFSH: WT=5.54±0.62 SQ, AhrKO=10.03±0.94 SQ, p=0.029; rFSH 15 IU: WT=3.58±0.75 SQ, AhrKO=9.59±0.99 SQ, p=0.001). Collectively, these data suggest that FSH may restore growth in AhrKO follicles by promoting an increase in estradiol levels. These data further suggest that slow growth in AhrKO antral follicles between PD 30 to 53 may be mediated by FSH. Support: NIH HD047275.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Gene Bionetwork Analysis of Ovarian Primordial Follicle Development.

Eric E Nilsson 1, Marina I Savenkova 1, Ryan Schindler 1, Bin Zhang 2, Eric E Schadt 2, Michael K Skinner 1

Ovarian primordial follicles are critical for female reproduction and comprise a finite pool of gametes arrested in development. A systems biology approach was used to identify regulatory gene networks essential for primordial follicle development. Transcriptional responses to eight different growth factors known to influence primordial follicles were used to construct a bionetwork of regulatory genes involved in primordial follicle development. Over 1500 genes were found to be regulated by the various growth factors and a network analysis identified critical gene modules involved in a number of signaling pathways and cellular processes. A set of 55 genes was identified as potential critical regulators of these gene modules, and a subnetwork associated with development was determined. Within the network two previously identified regulatory genes were confirmed (i.e. Pdgfa and Fgfr2) and a new factor was identified, connective tissue growth factor (CTGF). CTGF was tested in ovarian organ cultures and found to stimulate primordial follicle development. Therefore, the relevant gene network associated with primordial follicle development was validated and the critical genes and pathways involved in this process were identified. This is one of the first applications of network analysis to a normal developmental process. These observations provide insights into potential therapeutic targets for preventing ovarian disease and promoting female reproduction.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Mechanical Isolation of Ovarian Preantral Follicles from Cows Cryopreserved with Dimethylsulfoxide and Sucrose Plus Alpha-Tocopherol.

Carolina RJ Jimenez, Ciro AA Torres 1

In most mammals, 90% of ovarian follicles are preantral and approximately 99.9% of these suffer atresia before ovulation, and 0.1% will ovulate and could be fertilized or not. The biotech, manipulation of oocytes enclosed in preantral follicles (MOEFOPA) is characterized as the study of pre-antral follicle (oocyte and its surrounding cells), isolated from the ovarian environment, involving its in vitro culture and cryopreservation. The preantral follicles can be collected at any age, stage of the cycle or even from animals with sudden death, making possible to collect a large number of preantral follicles. These oocytes enclosed in preantral follicles have several characteristics which make them less susceptible to cryoinjury than mature oocytes. They may have more potential to be cryopreserved in situ or isolated from the ovarian environment. The success of cryopreservation has been shown in mice, rabbits, sheep, goats, cattle and even in women, thereby increasing this developing the area in order to achieve a better cryopreservation protocols for their subsequent isolation and culture. The ovaries from 51 cows were chopped at 5mm3 section (fragments). The sections were randomly divided into: control (isolated from fresh ovary) and 8 treatments with in situ vitrification containing the cryoprotectors dimethyl sulfoxide, sucrose, and/or α-tocopherol. Each treatment suffered two different thawing process: 1) sucrose and 2) minimum essential medium, in order to determine if there is interference of the thawing method on each treatment. After thawing, the fragments were placed in 50-ml tubes with maintenance medium and 5% of bovine serum albumin before their mechanical dissociation, filtration, in order to obtain the suspended isolated preantral follicles. The viability of preantral follicles was examined with Trypan blue, dead and alive, stained or unstained, respectively, and observed in inverted microscope at 400×. Fifteen hundred live or dead preantral isolated follicles were analyzed per treatment for a total of 13,500 observed preantral follicles. The results showed that among the nine treatments the viability of isolated preantral follicles, after cryopreservation, the control ones, without vitrification, differed from all cryopreserved treatments, showing that cryopreservation affects the viability of isolated preantral follicles, but among the vitrification treatments, the dimethylsulfoxide with sucrose and 10 mM alpha-tocopherol, regardless of the thawing type, showed the best viability after cryopreservation. This work contributes to the formation of future female gamete banks, making possible the multiplication of animals with high livestock value or endangered species, showing that a large number of viable preantral follicles from the same ovary could be developed and matured in vitro.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Differential Expression Patterns of GnRH and GnIH in Ovary of Mouse During Estrous Cycle.

Padmasana Singh 1, Amitabh Krishna 1, Rajagopala Sridaran 2, Kazuyoshi Tsutsui 3

Gonadotropin releasing hormone (GnRH) has now been suggested as an important intraovarian regulatory factor. Gonadotropin inhibitory hormone (GnIH) a hypothalamic dodecapeptide, acts opposite to GnRH. GnRH, GnIH and their receptors have been demonstrated in the gonads. In order to find out the physiological significance of these neuropeptides in the ovary, we aim to investigate changes in the abundance of GnRH I and GnIH in the ovary of mice during estrous cycle. GnRH I and its receptor and GnIH were localized immunohistochemically during different stages of estrous cycle in the ovary. Relative intensities of these peptides were estimated from the contralateral ovary using immunoblot followed by densitometry. The immunostaining of both GnRH I and GnRH I-receptor proteins were mainly localized in the granulosa cells of the healthy and atretic antral follicles during proestrus and estrus phases. Granulosa cells located around oocytes or lining antral cavity and theca cells showed positive immunostaining. Oocytes of the large antral follicles showed no immunostaining. During diestrus 1 and diestrus 2 phases, moderate immunostaining was observed in the luteal cells. The immunoblot analysis showed, intensity of immunostaining of GnRH I decreased gradually from 10.00 h to 14.00 h during proestrus phase. Its expression increased sharply in the evening at 19.00 h of proestrus and remains high during the estrus phase. Subsequently, it gradually declined during diestrus 1. A sharp increase in the concentration of GnRH I was found in the afternoon of diestrus 2. The intensity of expression of GnRH I-receptor increased sharply during gonadotropin surge at 14.00 h on proestrus and remained high until late evening in proestrus. Subsequently, intensity of GnRH I-receptor declined during estrus and remained unchanged until early diestrus 1. GnRH I-receptor expression increased again during late diestrus 1 and remained high during diestrus 2. GnIH immunoreactivity was mainly observed in the granulosa cells of large antral follicles during proestrus. Strong GnIH immunoreactivity was observed in GCs and TCs of the antral follicles during 14.00 h while low immunoreactivity was observed at 19.00 h of proestrus. Mild GnIH immunoreactivity was observed in the follicles during estrus phases. During diestrus 1 and diestrus 2 phases, GnIH immunoreactivity was also mainly observed in the corpus luteum. The immunoblot analysis of GnIH showed gradual increase from morning to attain a peak at 14.00 h on proestrus and subsequently it declined sharply to a low level at 19.00 h. The GnIH concentration showed no significant change from late proestrus until early diestrus 2 phase. Thus, the relative abundance of GnRH I, GnRH I-receptor and GnIH undergo significant variation during proestrus and thus may be responsible for selection of follicle and atresia. A significant increase in the concentration of GnIH during late diestrus 2 coincides with the decline in corpus luteum activity and initiation of follicular growth and selection. In general, GnRH I and GnIH were found in close vicinity suggesting functional interaction between these peptides. It is thus, hypothesized that interaction between GnRH I-GnIH neuropeptides may be involved in the regulation of follicular development and atresia. This work was supported from UGC, New Delhi to AK; Ministry of Education, Japan to KT and NIH, USA to RS.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Rhox Homeobox Gene Cross-Regulation in Granulosa Cells.

James A MacLean, Raquel M Brown, Matthew G Davis, Kanako Hayashi 1

Mammalian ovulation is a complex, hormone-dependent developmental program in which several events must take place in an ordered progression to ensure that the oocyte is competent for fertilization. This process requires the coordinated expression of many genes which must be turned on and off in the right place at the right time for proper development of the follicle. While the hormone signals from the brain that initiate ovulation are known, the master control genes which regulate this process are not well known. Homeobox genes encode DNA-binding proteins that are master regulators of a myriad of developmental programs. The reproductive homeobox (Rhox) homeobox genes are expressed in the ovary, placenta, testis, and epididymis, and thus are good candidates to regulate both male and female reproductive tissue development and physiology. In this report, we investigate altered expression patterns of the Rhox cluster during ovulation using an eCG primed, hCG induced superovulation model in Rhox5-null animals. Rhox5-null female mice do not exhibit compromised fertility suggesting that another RHOX factor may compensate functionally. None of the Rhox genes sharing peak expression at 4h post hCG with Rhox5 were found to be upregulated in the knockout. Interestingly, loss of RHOX5 resulted in reduced expression of Rhox8 during this window although Rhox8's and a lower progesterone-induced peak expression at 8h post hCG. Supported by NIH HD055268.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

GATA6 Depletion Reduces Cyclic AMP-Stimulated IGF1 mRNA and Free Protein Levels in Luteinizing Porcine Granulosa Cells.

Holly A LaVoie, Jonathan B Nguyen, Richard J Kordus, Yvonne Y Hui 1

Ovarian transcription factors GATA4 and GATA6 have been shown to regulate genes mediating steroidogenesis but few other gene targets have been identified. To discover other novel targets of ovarian GATA4/6 we coupled RNAi-mediated knockdown in luteinizing porcine granulosa cells with gene microarray analysis. RNAi-mediated depletion of GATA4, GATA6, or their combination in granulosa cells was followed by treatment with cAMP analog, 8-Bromo-cAMP (1 mM; 8Br-cAMP) for 24 h. Total RNA was subsequently subjected to microarray analyses using the Affymetrix Porcine Genome Array. Array analyses revealed the mRNA for the insulin-like growth factor 1 gene (IGF1) was the most highly stimulated cAMP-responsive target under Control RNAi conditions and was decreased by GATA6 and GATA4+GATA6 RNAi-mediated depletion. Real-time PCR confirmed IGF1 mRNA was highly stimulated by cAMP and GATA6 or GATA4+GATA6 knockdown conditions significantly reduced the amplitude of this cAMP effect by 35-40% (n= 8; P<0.01). Basal (vehicle treatment) IGF1 mRNA expression was also reduced by combined GATA4+GATA6 reduction (P<0.05). GATA4 reduction alone did not affect IGF1 mRNA levels. We also examined mRNAs for IGF binding proteins (BPs) -2, -3, -4, and -5 and assessed their binding activity by ligand blotting. Treatment with 8Br-cAMP increased IGFBP2 and IGFBP4 mRNA (P<0.05), decreased IGFBP5 mRNA (P<0.05), and did not affect IGFBP3 mRNA under Control RNAi conditions (n=6). GATA4+GATA6 coreduction increased basal and cAMP-stimulated IGFBP2 and IGFBP4 mRNA levels and decreased mRNA for IGFBP5 (P<0.05). GATA6 reduction also led to increased basal expression of IGFBP4 and decreased expression of IGFBP5 (P<0.05). GATA4 reduction alone decreased basal IGFBP5 mRNA (P<0.05). In contrast to effects on IGFBP mRNAs, IGF-1 binding capacity of IGFBPs 2-5 in concentrated media did not show differences with GATA reduction. Free IGF-1 measured in concentrated culture medium by ELISA showed cAMP-stimulated IGF-1 levels were decreased under GATA6 and GATA4+GATA6 knockdown conditions (n=3, P<0.05). In addition, EMSAs utilizing two oligonucleotides containing GATA consensus sequences corresponding to regions of the 5'-UTR of exon 1 of the IGF1 gene and granulosa nuclear extracts revealed that both GATA4 and GATA6 could bind the more proximal GATA sequence. The intensity of protein-DNA complexes with the proximal GATA site oligonucleotide was reduced by GATA6 knockdown to a greater extent than by GATA4 knockdown. In summary, GATA6 depletion alone or in combination with GATA4 depletion reduces IGF1 mRNA and its free protein, which may be due to reduced binding of GATA6 to an enhancer in the 5'-UTR of the IGF1 gene. This study provides the first evidence to support GATA6 as an ovarian specific transcriptional regulator of the IGF1 gene.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Androgens Stimulate Aromatase and P450scc in Primary Rat Granulosa Cells Independently of Follicle Stimulating Hormone.

Jill Bennett, Cassandra Richards, Kimberley Mullen, Carlos O Stocco 1

It is recognized that follicle stimulating hormone (FSH) induction of aromatase expression in granulosa cells is potentiated by androgens. Evidence suggests that this synergism may be mediated by a direct androgenic effect or by estrogenic effects through androgen conversion to estrogen. Whether androgens have any effect on aromatase expression independently of FSH is a matter still under debate. In addition, whether androgens and/or estrogens participate in the regulation of other FSH target genes such as P450scc and StAR has not been examined. Primary rat granulosa cells were used to investigate the effect of various steroids on the expression of aromatase, P450scc and StAR in the absence or presence of FSH. Cells were cultured in defined medium for 96 hours with increasing concentrations (1, 10 and 100 nM) of estradiol or testosterone. Results showed a dosage response to testosterone as well as to estrogen for aromatase. However, a larger induction of aromatase expression was observed with testosterone treatment when compared to estradiol. Additional groups were also treated with FSH (50 ng/ml) during the last 48 hours of incubation. As expected, testosterone synergized with FSH for the induction of aromatase; but, no synergism was observed between estradiol and FSH. Similar findings were observed when the expression of P450scc was studied. In contrast, co-incubation of FSH with testosterone did not increase StAR expression above the levels stimulated by FSH alone. To gain insights on the mechanism that may mediate testosterone stimulation of aromatase, cells were treated with androstenedione, dihydrotestosterone, (DHT) a pure androgen or two metabolites of DHT: 3αdiol and 3βdiol. It was found that androstenedione was equivalent to testosterone in its ability to induce aromatase. However, DHT, 3αdiol and 3βdiol did not stimulate aromatase to the same degree that testosterone and androstenedione did. Next, we examined whether testosterone affects the expression of transcription factors known to be involved in the regulation of aromatase expression such as SF-1 and LRH-1. It was observed that testosterone stimulates LRH-1 expression in a dose dependent manner but it has no effect on SF-1 levels. To investigate whether these factors mediate the effect of testosterone, cells were transduced with lentivirus expressing small interference RNA (siRNA) against LRH-1 or SF-1. Testosterone stimulation of aromatase expression was observed in cells treated with control siRNA or with anti-SF1 siRNA. However, testosterone could not stimulate aromatase in the presence of an anti-LRH-1 siRNA. Taken together these results demonstrated that androgens not only synergize with FSH on the stimulation of aromatase and P450scc but they may also have a direct effect on the expression of genes directly involved in the differentiation of granulosa cells. Research supported by NIH R01HD057110.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Involvement of EGFR and Smad2/3 Activation in FSH-Induced Porcine Oocytes-Cumulus Cell Complex Maturation.

Eva Nagyova 1, Radek Prochazka 1, Lucie Nemcova 1, Antonella Camaioni 2, Alzbeta Mlynarcikova 3, Sona Scsukova 3, Antonietta Salustri 2

It has been shown that the activation of both EGF receptor and SMAD2 and SMAD3 in mouse cumulus cells is essential for their preovulatory maturation. In the present study we investigated whether these changes are also required for the final differentiation of pig cumulus cells. Porcine oocyte-cumulus complexes (OCCs) were stimulated in vitro with FSH in the absence or in the presence of the following inhibitors: AG1478 (an EGFR tyrosine kinase inhibitor), SB431542 (a specific inhibitor of SMAD2 and SMAD3 activation) and SIS3 (a specific inhibitor of SMAD3 activation). We found that EGFR inhibitor AG1478 completely blocked FSH-induced cumulus expansion by preventing hyaluronic acid production and its incorporation within the extracellular matrix. In addition, in the presence of AG1478, most of FSH-stimulated OCCs were in germinal vesicle stage (GV) and did not mature. Conversely, AG1478 did not affect the stimulation of progesterone synthesis by FSH. In agreement, EGF was unable to stimulate progesterone synthesis by OCCs cultured in vitro. The SB431542, that inhibits the activation of SMAD2 and SMAD3, only partially inhibited cumulus expansion by slightly decreasing both total production and matrix accumulation of hyaluronic acid. Surprisingly, SIS, that inhibits only SMAD3, produced an inhibitory effect higher than SB431542. A divergent effect of the two inhibitors was observed on steroid synthesis: FSH-induced progesterone synthesis increased in the presence of SB431542 and decreased in the presence of SIS. Both inhibitors did not affect the induction of oocyte meiotic resumption promoted by FSH. These results suggest that in pig, like in mice, FSH stimulates cumulus cells to produce EGF that acting in an autocrine way induces the formation of the expanded matrix and the signaling for oocyte meiotic resumption. The results also suggest that SMAD2 and SMAD3 activation is required for optimal stimulation of hyaluronic acid and proteins involved in the organization of this polymer in the pig OCC matrix. Finally, FSH stimulation of progesterone synthesis seems to be independent from EGF, while it appears to be counteracted by SMAD2, but not SMAD3 activation, thereby suggesting that specific SMAD2 signaling pathway is probably indispensable to prevent luteinization of cumulus cells. Supported by grant GACR 523/08/0111 and VEGA 2/0153/08.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Human Ovarian Expression and Localization of Tissue Inhibitor of Metalloproteinase 3 (TIMP3) During the Periovulatory Period.

Katherine L Rosewell 1, Feixue Li 1, Matts Brannstrom 2, Thomas E Curry 1

Alterations in the interaction between the follicle and the matrix must occur before follicular rupture and ovulation can take place. Such interactions are controlled in part by the delicate balance between the matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). We hypothesize that the TIMPs would increase prior to ovulation in order to keep MMP activity in check to provide localized degradation at the apex and prevent wholesale destruction of the follicle wall. In the present study we focused on expression and localization of TIMP3, which is the only TIMP bound to the extracellular matrix, during the periovulatory period in the human. Follicles were collected from patients undergoing elective surgery at different stages of the ovulatory process. Patients were divided into four groups (preovulatory, early ovulatory, late ovulatory, and post-ovulatory phase). Surgery was performed at the preovulatory stage without administering hCG. The remaining women received an injection of 250 µg hCG (Ovitrelle, s.c.) when the diameter of their dominant follicle was ≥14mm and ≤ 20mm on trans-vaginal ultrasound. These patients had surgery during the following time intervals after hCG-injection; early ovulatory phase 12h to ≤18h, late ovulatory phase >18h to ≤ 34h and the postovulatory phase 44h to 70h. For the localization studies, the intact follicle with the apical orientation was embedded and processed for immunohistochemistry (IHC) using a TIMP3 antibody. For the quantitative studies, the follicle was bisected and the granulosa and theca cells separated and collected for real time PCR. Real time PCR analysis for Timp3 mRNA in granulosa and theca cells revealed a significant increase in Timp3 gene expression in granulosa cells from the early to the late ovulatory stage (n=5). Thecal Timp3 expression was constitutive across the periovulatory period. TIMP3 protein was localized by IHC to the granulosa cells and theca cell layers. In the preovulatory follicle there was positive staining in the granulosa cells, no staining in the theca. In the early ovulatory stage there was positive staining in both the granulosa and theca interna. The staining was most intense in the granulosa and theca cells; interna and externa of 2 follicles which were in the late ovulatory group. There was staining of TIMP3 in the lumen of some of the blood vessels and it was particularly intense in one early ovulatory follicle that had a large vascular bed surrounding the follicle. Several primordial follicles were seen in the sections and these were positively stained by the TIMP3 antibody. By using human follicles collected throughout the periovulatory period, we have been able to validate our hypothesis that Timp3 gene expression increases as the follicle progresses toward ovulation. We have shown that localization of TIMP3 protein appears first in the granulosa cell layer and then steadily accumulates in the granulosa-thecal layers as ovulation approaches. This localization places TIMP3 in the appropriate cellular layer to control the degradation of the follicular wall by the MMPs.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Methoxychlor and Methoxychlor Metabolite HPTE Regulate Estrogen Receptors Expression in Rat Ovarian Granulosa Cells In Vitro.

Craig N Harvey, Mehmet Uzumcu 1

The sensitivity of the ovary to endocrine disrupting compounds (EDC) has become an area of increased interest in reproductive biology. Methoxychlor [1, 1, 1,-trichloro-2, 2,-bis-(p-methoxyphenyl) ethane; MXC] is a weak estrogenic organochlorine pesticide that induces reproductive dysfunction. The major metabolite of MXC, 2,2-bis-(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), is not only a stronger estrogen receptor α (ERα) agonist, but also acts as an estrogen receptor β (ERβ) antagonist. We have shown that transient fetal and neonatal exposure to MXC reduces the levels of ERβ and cytochrome P450 (CYP) 11A1 protein in the adult ovary. HPTE inhibits steroidogenesis by down-regulating CYP enzymes (CYP11A1 and CYP19A1) in follicle-stimulating hormone (FSH)-induced granulosa cells (GC) in vitro. Studies have shown that there is a close regulation between ER signaling pathways and expression of members of estrogen-metabolizing CYPs family, including CYP 1A1 and 1B1 in various tissues types. The objective of this study was to determine if MXC or HPTE regulates ERα, ERβ, CYP1A1 and CYP1B1 expression in GC. To this end, GC were cultured in a serum-free media with FSH and treated with MXC or HPTE (10 µM) for 48 h. The expression levels of ERα, ERβ, CYP1A1, and CYP1B1 were measured using quantitative reverse transcription-PCR. Both MXC and HPTE significantly inhibited Eβ expression in the GC (n≥5, P<0.05) while FSH did not stimulate ERβ expression. In contrast, FSH stimulated ERα expression, which was inhibited by both MXC and HPTE (n=4, P<0.001). In addition, HPTE induced CYP1B1 (n=4, P<0.05) while no effects were observed on CYP1A1 by MXC or HPTE. Both MXC and HPTE inhibited ERα and ERβ, whereas only HPTE stimulated CYP1B1, implying that the modulation of estrogen-metabolizing CYPs requires a stronger estrogenic effect. These results suggest that modulation of CYPs is associated with inhibition of ERs in the ovary, which may have a role in MXC-induced ovarian dysfunction and female infertility. Supported by ES013854, ES005022, and Bristol Myers Squibb.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

A Role of the Catalytic Activity of Protein Kinase CK2 During FSH Stimulated Estradiol Biosynthesis in Rat Granulosa Cells.

Kerri S Kluetzman, Barbara A Weaver, James A Dias 1

Activation of the human follicle stimulating hormone receptor (hFSHR) by its cognate ligand, follicle stimulating hormone (FSH), induces maturation of ovarian follicles, resulting in generation of mature eggs and production of estrogen and progesterone. Canonical cAMP mediated protein kinase A (PKA) defined signaling pathways are known to regulate these processes. However, divergent pathways are also at play in FSH mediated FSHR function. We hypothesized that coordination of these divergent pathways may involve adapter proteins which are associated with the occupied FSH:FSHR forming a signaling complex or signalosome. We have previously demonstrated that protein kinase casein kinase 2 (CK2) co-immunoprecipitates with hFSHR. However, a role for CK2 in FSH action has not previously been evaluated. To determine the effect of CK2 catalytic activity on FSH induced granulosa cell function, the specific CK2 inhibitor tetrabromocinnamic acid (TBCA) was used at 10µM. Primary rat granulosa cells derived from DES treated rats were stimulated with hFSH in the presence and absence of TBCA. cAMP, progesterone and estradiol were measured at 1 hr, 48 and 96 hr respectively. TBCA did not affect FSH induced total cAMP production (P=0.4098) when cells were treated with FSH in the presence of 1nM 3-isobutyl-1-methylxanthine (IBMX). Moreover, progesterone production was unaffected by TBCA (P=0.7143). Unexpectedly, TBCA almost completely blocked estradiol production (P=0.0002) in these cells. The effect of TBCA was also evaluated in HEK-293T cells, which were transiently transfected with hFSHR. TBCA had no effect on total cAMP production (P=0.4935) when IBMX was present. Internalization of 125I-hFSH in HEK-293T cells transiently transfected with FSHR was also unaffected by pre-incubation with TBCA (P=0.2355). Several important observations have been made. First, it is clear that cAMP is necessary but not sufficient for full estradiol production. Second, there appears to be a key regulatory point of estradiol production, which requires CK2 catalytic activity. Future studies will aim at understanding how the catalytic activity of protein kinase CK2 coordinates with the FSHR signalosome and is necessary for granulosa cell estradiol production. Understanding this process could provide for a better understanding of ovarian responsiveness to gonadotropin stimulation as well as develop targets for estrogen dependent disease and cancer. Supported by HD18407.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Temporal Expression Pattern of Genes Involved in Steroidogenesis, Cell Cycle, and Mtor-Signaling in the Ovarian Somatic Cells During Follicular and Luteal Growth.

Anitha Kalaiselvanraja, Raj Duggavathi 1

Mammalian target of rapamycin (Mtor) is a master regulator of cellular processes including cell-cycle, protein synthesis, transcription and apoptosis in response to nutrient and metabolic signals. Since follicular and corpus luteum (CL) growth involves integration of numerous nutrient and growth-factor signals within follicular and luteal microenvironment, Mtor is a potential candidate for the molecular hub of such integration. Recent studies indicate that Mtor may play a role in the regulation of granulosa cell proliferation. However, the potential roles of Mtor in granulosa-luteal differentiation and steroidogenesis have not been studied. Thus, the objective of our study was to assess temporal relationship among the expression pattern of genes involved in Mtor pathway, granulosa cell proliferation and steroidogenesis. We used superovulation of immature mice and laser microdissection to collect granulosa and luteal cells at specific stages of follicular and CL growth. The expression pattern of cell cycle (Ccnd2 and Cdkn1b), steroidogenic (Cyp19a1, Star and Cyp11a1) and Mtor-pathway (Mtor and Rptor) gene was analyzed by real-time PCR. The expression of Cyp19a1, Ccnd2 were high during follicular growth and low during CL growth; that of Star, Cyp11a1 and Cdkn1b was low during follicular growth and high after ovulation induction through CL growth. This pattern of expression reflected granulosa cell proliferation and estrogen synthesis during follicular growth, and luteal cell differentiation and progesterone synthesis during CL growth. As expected, the expression of Mtor and Rptor was high during follicular growth. Surprisingly, the expression of Mtor increased after ovulation during CL growth, in correlation with that of Cyp11a1 and Cdk1nb). These results indicate that in addition to granulosa cells proliferation, Mtor may also play a significant role in luteal cell steroidogenesis and maintaining their cell-cycle inhibition. Supported by NSERC Discovery Grant.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Thyroid Autoantibodies and Thyroid Stimulating Hormone (TSH) in Recurrent Miscarriages; Implication of the Number of Abortion.

Myunghee Kim, Hyunjeong Jeoung, Heesun Lee, Dachung Chung, Sungil Roh 1

The association of thyroid autoimmunity to miscarriages is an interesting issue. Many studies show the significant positive association between the presence of thyroid autoantibodies and abortion. Most of them are focused on the group of women with history of 3 or more consecutive pregnancy losses. Since 2008, ASRM (American Society of Reproductive Medicine) extended the definition of recurrent pregnancy loss from 3 or more to 2 or more failed pregnancies. As this new broad meaning is applied, it calls for the quest for the different implications in the number of abortion related to thyroid autoantibodies and TSH. In this aspect it is needed to compare the incidence of thyroid autoantibodies and TSH level in recurrent miscarriage patients according to the number of miscarriages. In this study, we retrospectively analyzed the data of a total of eighty-four women with recurrent miscarriage history in community-based fertility clinic. Data were divided into two groups according to the number of abortion. One is the group of 2 consecutive failed pregnancies. The other is the group of 3 or more consecutive failed pregnancies. Thyroid autoantibodies including antithyroglobulin antibody and antimicrosomal antibody and the level of TSH are compared between two groups. Thyroid autoantibodies are present in 8 of 51 patients (15.7%) with two miscarriages history and 8 of 33 patients (24.2%) with three or more miscarriages history (P=0.398). TSH level was 2.24mU/L in two miscarriage group and 2.12mU/L in 3 or more miscarriage group (P=0.077). The incidence of high TSH over 2mU/L is 24 of 51 patients(47.1%) vs 11 of 33(33.3%) patients in each group(P=0.260). In conclusion, these observations show that thyroid autoantibodies and serum TSH are not significantly different in the miscarriage patients depending on the number of abortions In sight of this result, it can be suggested that thyroid autoantibody and TSH screening has the similar implication in the group of two miscarriages compared to the group of 3 or more miscarriages. This hypothesis should be verified in further randomized prospective studies and the research extended to clinical outcome is needed for better management of recurrent miscarriages in the future.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

High Affinity Binding of PSP94 to Human Immunoglobulin Suggests Its Possible Role in Sperm Function.

Smita D Mahale 1, Dhanashree D Jagtap 1, Deepak N Modi 1, Mukesh Kumar 2, Bhakti R Pathak 1

Prostate secretory protein of 94 amino acids (PSP94) is one of the most abundant proteins from prostatic secretions and a major constituent of human seminal plasma. It has also been referred to as beta-microseminoprotein and protein homologues to this have been reported in other species. The exact role of this protein in prostate pathphysiology or sperm function has not been unequivocally established. We had earlier reported that PSP94 has the ability to bind to human IgG. The aim of the present study is to delineate the molecular interaction of PSP94-IgG and to understand its possible role in sperm function. Ability of human IgG and its sub-domains to bind to PSP94 was monitored by ELISA. Kinetics of this interaction was measured by surface plasmon resonance (SPR) technique. Recently solved X-ray crystal structure of PSP94 (PDB 3IX0) by our group was used to model the structure of PSP94-IgG complex. Binding kinetics of PSP94-IgG interaction revealed high affinity binding of IgG to PSP94. This interaction was found to be through the Fab domains of IgG. In-silico molecular modeling of PSP94-IgG complex showed that amino and carboxyl terminal beta strands of PSP94 to be most plausible region involved in IgG interaction. To understand the significance of PSP94-IgG interaction in sperm function, in-vitro experiments were carried out. The results reveal that PSP94 is able to prevent the binding of IgG to human spermatozoa suggesting that PSP94 present in seminal plasma might have an important role in-vivo, in protecting spermatozoa from immune attach in the female genital tract. Research supported by grant from Department of Biotechnology, Government of India and Indian Council of Medical Research (SDM).

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Lymphocyte Subsets in the Primate Testis: Implications for Studies of Local Cellular Immunity.

Mark P Hedger 1, Richard D Hayes 2, Stefan Schlatt 3, Robert I McLachlan 4, Moira K O'Bryan 1

The frequency and types of lymphocytes that appear within a tissue are an indication of local immunological conditions. Although immune cells and immune responses in the testes of experimental rodents have been well-studied, the human and non-human primate testis has received much less attention, and baseline quantitative data are lacking. The question as to whether immunological studies in the rodent testis can be considered representative of the human needs to be considered, because such studies are frequently extrapolated to the human testis under assumptions that may or may not be completely justified. Lymphocyte subsets were identified and quantified in Bouin’s-fixed paraffin-embedded adult human and rhesus or cynomolgus monkey testes, using a panel of anti-human antibodies and routine stereological methods. Significant numbers of cells expressing the specific T cell marker CD3 were observed within the interstitial tissue (excluding vascular profiles), but not the seminiferous epithelium, of normal human and monkey testes (0.7 - 2.6 x 103/mg tissue). No B cells (CD19+) were observed. Two antibodies against the natural killer (NK) cell marker CD56 (Leu-19), or neural cell adhesion molecule (N-CAM), gave strikingly different results. An antibody directed specifically against the mature 140 kDa isoform of N-CAM that is expressed by NK cells labelled a number of interstitial cells in the human testis (1.0 - 2.8 x 103/mg tissue), which were clearly distinguishable from either Leydig cells or macrophages based on their characteristic morphology and nuclear diameter. Using an antibody that recognises several alternate N-CAM splice variants, however, this molecule also was found to be expressed on the surface of human Leydig cells, Sertoli cells and early germ cells. Frequency of cells expressing the circulating Fc receptor γIII (CD16) in the human testis was more than one order of magnitude lower than the CD56 staining, indicating that most testicular NK cells or macrophages do not express this marker. Relatively few interstitial tissue cells in the monkey testes (< 10/mg tissue) were labelled by the NK cell antibodies examined, although it remains to be established whether this indicates that NK cells are absent, or that the N-CAM marker is not optimal for this species. These data indicate that the interstitial tissue of the mature primate testis normally contains significant numbers of lymphocytes (T cells and NK cells). The numerical density of the major lymphocyte subsets in the human testes was similar to that observed in the adult rat in previous studies, suggesting that the rat is a suitable model for studying testicular cellular immune responses.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Simple Protocol to Cryopreserve Bovine Endometrial Epithelial Cells.

Yasser Lenis Sanin, Juliana Bedoya, Zulma Tatiana Ruiz-Cortes 1

The bovine endometrium is a high specialized tissue composed by epithelial endometrial cells (EPEC) the main prostaglandin F2alpha (PGF2alpha) producers and by stromal endometrial cells. The isolation and culture of EPEC cell biotype is an important model for the study of cellular and molecular events that occur during luteinization and/or pregnancy recognition. Our laboratory has already standardized the EPEC cultured during 32 days. The objective of this study is to characterize an EPEC primary culture protocol after cryopreservation. Uterus from abattoir were 4°C transported and processed. Isolated EPEC were centrifuged and seemed in 24 well dishes at 600.000 cells/mL of supplemented RPMI-1640 medium. Cells were divided in 2 treatments: control and rapid cryopreservation method of 10 minutes at 0°C followed by immediate -80°C in 15% DMSO at 11 and 23 days in culture. After 4 days cryopreservation, cells were thawed and re-seemed as described before. Cryopreserved cells presented conglomerates and pseudopodia cytoplasm prolongations after 9 days in culture characteristic presented in control at days 3-4. At day 11 in culture preserved EPEC presented a very low confluence compared to control value. At day 23, an important increase in confluence of cells post-cryopreservation was observed, indicating a time depending cells recuperation. We propose a simple EPEC cells cryopreservation protocol followed by a minimum of 20 days incubation period as a model of viable bovine endometrial cells. Further researches on functional (PGF2alpha production) features are necessary.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

A Comparison Between HTF and SOF as Extenders for Freeze-Drying Tomcat Sperm.

Luis Carlos Oña Magalhães, Cely Marini Melo, Mateus José Sudano, Daniela Martins Paschoal, Ana Izabel Silva Balbin Villaverde, Fernanda da Cruz Landim-Alvarenga, Maria Denise Lopes 1

As an alternative method to preserve spermatozoa, many groups have been using the lyophilization technique. Attempts to freeze-dry spermatozoa are not new. The objective of the present study was to test whether Human Tubal Fluid (HTF) and/or Synthetic Oviductal Fluid (SOF) are able to protect tomcat sperm cell nucleus and thus be used as extenders for the freeze-drying process. Sperm-rich fraction from ejaculates collected using an artificial vagina was divided into two aliquots. Aliquots were then added with either HTF or SOF having a final concentration of 2x103 sperm/mL. Aliquots were then put into 2.0mL Cryogenic Vials, being 0.5 mL the maximum volume per vial in order to aid the freeze-drying process. Vials were kept at 4°C for 60 minutes, then kept in N2 vapor for 8 minutes, and finally plunged into N2. Samples were then taken to the freeze-drying machine in order to obtain a stable product and remove the liquid fraction. With a view to analyzing DNA integrity it had been used acridine orange, and to analyze membrane integrity it had been used the association of 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl carbocyanine iodide (JC-1), Pisum sativum agglutinin conjugated with fluorescein isothicyonate (FITC-PSA), and propidium iodide. Both media (SOF and HTF) showed that they may be used as freeze-drying extenders since they both somehow protected the sperm cells’ DNA (84.5% and 86.2% of DNA integrity for HTF and SOF media, respectively) despite the fact that they both showed extensive cell membrane and acrosome damages, when analyzed via epifluorecense microscopy. However, SOF was considered of better performance due to the fact that sperm cells were easier to isolate for the intracytoplasmic sperm injection (ICSI) procedure when samples were reconstituted. Samples that used HTF as media for both freeze-drying procedure and reconstitution required the use of Vortex followed by the use of Pasteur pipettes because sperm cells were strongly bonded. Bearing in mind the fact that both media would provide a defined extender for freeze-drying, the SOF medium needed less effort to be handled if compared to the HTF because sperm cells were easier to separate from one another. Cells that were immersed in HTF needed extra care since they required vortexing and pipetting to separate spermatozoa from one another. This reserach was supported by CAPES.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

A Follicle-Stimulating Hormone Agonist Promotes Follicular Development During Controlled Ovarian Stimulation in Rats and Macaques.

Pieter Verbost 1, Ruud Vande Lagemaat 1, Mary Zelinski 2

Recent developments in gonadotropin therapy for follicular stimulation/ovulation induction in infertile women include creating recombinant gonadotropins with longer half-lives in vivo, or orally active gonadotropin mimetics to replace daily injections currently required. The compound Org 210071-0 is a low molecular weight, FSH-receptor agonist based on in vitro characterization in CHO cells carrying the FSH-receptor and growth of mouse follicles in culture. This study determined the efficacy of Org 210071-0 (FSH agonist) to stimulate growth and maturation of multiple preovulatory follicles and their enclosed oocytes in rats and rhesus monkeys in vivo. Female rats (n=6/group) received recombinant human (rec-h)FSH (0.5 µg/kg sc, bid on cycle day A1 and A2, once on pro-estrous morning) or various doses of Org 210071-0 (sc or orally) along with a GnRH antagonist (ganirelix, 40 μg/kg sc, same dosing scheme as FSH) and rec-h luteinizing hormone (LH) (0.2 µg/kg sc, same dosing scheme as FSH). Human chorionic gonadotropin (hCG, 75 U/kg sc, proestrus afternoon) was given to induce ovulation. Beginning at menses, rhesus monkeys (n=5/group) received either rec-hFSH (30 IU, IM bid) or Org 210071-0 (6 mg/kg, SC sid every other day for 4 days) until follicles ≥4 mm in diameter were observed with ultrasound. A bolus of hCG (1000 IU, IM) was given 27 h prior to follicle aspiration to determine oocyte maturation as well as fertilization and embryonic development in vitro. In rats, 0.3 mg/kg Org 210071-0 given sc stimulated follicle growth to the same extent as rec-hFSH (0.5 µ/kg sc) based on ovulation rate (stimulation to 350% and 300% of placebo, respectively). When Org 210071-0 was given orally, the hCG-stimulated ovulation rate following 3 mg/kg (3 times/day) or 1.5 mg/kg (6 times/day, was similar to rec-hFSH (340% stimulation) in 2 and 3 out of 6 animals, respectively. In macaques, the interval required to achieve multiple follicular growth was longer (P<0.05) with Org 210071-0 (17±1 days) relative to rec-hFSH (9±1). The percentage of follicles >4 mm was similar between Org 210071-0 (22±7) compared to rec-hFSH (30±6). In both groups, serum estradiol increased steadily during treatment; levels were greater (P<0.05) in rec-hFSH (925±210 pg/ml) than FSH agonist (528±267 pg/ml) on the day of hCG administration. Of the total cohort, the percentage of germinal vesicle-containing oocytes retrieved from rec-hFSH (8±3) was less (P<0.05) than FSH agonist (35±10). In rec-hFSH, the percentage of oocytes resuming meiosis to metaphase I (61±6) was greater (P<0.05) than FSH agonist (38±9). However, similar percentages of metaphase II oocytes were retrieved in rec-hFSH (16±8) and FSH agonist (9±2). A greater percentage of oocytes fertilized in rec-hFSH (68±10) compared to FSH agonist (21±14). Blastocysts developed in 4/5 animals in rec-hFSH, and 2/5 in FSH agonist. In summary, a FSH-mimetic induced multiple follicular development in rats when given sc and orally, and in macaques when given sc. Although less effective than rec-hFSH, mature oocytes capable of fertilization and embryonic development in vitro were obtained in macaques following Org 210071-0. A higher dose of Org 210071-0 may be required in primates to achieve results equivalent to those of rec-hFSH. Whether this FSH agonist is orally active in primates is under investigation. Thus, development of FSH-mimetics for the treatment of infertility in women is feasible.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

NOT PRESENTED.

NOT PRESENTED.

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Optimization of Nonviral Methods to Produce Transgenic Common Marmoset (Callithrix jacchucs) Embryos.

Ikuo Tomioka, Akiko Shimada, Erika Sasaki 1

The common marmoset (Callithrix jacchus) is a non-endangered New World primate that is native to Brazil. Marmosets offer many advantages compared with other laboratory primates for studying reproductive biology: they are the only anthropoid primates that routinely ovulate multiple oocytes per ovarian cycle, have a short gestation period and reach sexual maturity at around 1 year of age. Moreover, it is possible to synchronize the ovarian cycle, and efficient protocols for superovulation have been developed over the last few decades. Last year, we successfully generated the first transgenic primates able to pass on a foreign gene to their offspring. We used self-inactivating lentiviral vectors to introduce the foreign gene, but lentiviral vectors have a number of drawbacks as gene transfer vectors, including limited insert size (8 kb) of the transgene, and the risks involved in using viruses. Hence, we compared two nonviral methods to introduce foreign genes into marmoset embryos, namely, transgene injection into the pronucleus of embryos (pronucleus gene injection method) or transgene injection into oocytes at the same time as intracytoplasmic sperm injection (ICSI-mediated gene transfer method). We constructed the gene of monomeric red fluorescence protein (mRFP) carrying CMV promoter named CMV-mRFP and used it at a concentration of 5 ng/µl in both methods. Oocytes at the germinal vesicle stage were collected by follicle aspiration and cultured in Waymouth medium containing 5% fetal bovine serum, 1% insulin-transferrin- selenium, 100IU/ml FSH, 0.5mM sodium pyruvate, 10mM sodium lactate, 4mM hypotaurine, and 1mM glutamine. After 25 to 30 hours of culture, in vitro matured oocytes were subjected to ICSI-mediated gene transfer or in vitro fertilization. The day after in vitro fertilization, the CMV-mRFP gene was introduced into the pronucleus of fertilized embryos. Embryos with the CMV-mRFP gene introduced by both methods were cultured in ISM1 medium until the 8-cell stage followed by incubation in ISM2 medium after the 8-cell stage. With the pronucleus injection method, the CMV-mRFP gene was introduced in a total of 214 embryos at the pronucleus stage and 23% of embryos (50/214) developed to the 16-cell stage. Seventy-two percent (36/50) of the 16-cell stage embryos expressed mRFP fluorescence. In contrast, a total of 202 oocytes were subjected to the ICSI-mediated gene transfer method and 91% (183/202) of embryos formed pronuclei. Fourteen percent (25/183) of embryos that formed pronuclei developed to the 16-cell stage embryos, and 28% (7/25) of the 16-cell stage embryos expressed mRFP fluorescence. In summary, the pronucleus gene injection method is more suitable for producing transgenic embryos of common marmosets than ICSI-mediated gene transfer in terms of high developmental ability of embryos in vitro and high transduction efficiency of foreign genes in the 16-cell stage. Non-human primates are valuable experimental animals as a model for human disease and regenerative medicine. With the recent development of reproductive technologies including the present study based on the common marmoset, this species has many advantages for studying reproductive biology and should lead to a surge of interest among biological researchers.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

In Vitro Versus In Vivo Effects of NB-DGJ and NB-DNJ as Lead Compounds for Development of Non-Hormonal Reversible Oral Male Contraceptives.

Vijayalaxmi G Gupta 1, Sheri Hild 2, Sudhakar Jakkaraj 3, Gunda I Georg 3, Joseph S Tash 1

The development of safe, effective and reversible male contraceptive methods is an important goal in expanding the current choices that, in practice, fall short in their reliability and effectiveness. Hormonal approaches to male contraception are currently in clinical trials, but non-hormonal approaches also need to be developed as viable alternatives. Although, N-butyldeoxynojirimycin (NB-DNJ) is a potent reversible anti-spermiogenic oral male contraceptive agent in C57Bl/6J mice, it shows poor or no efficacy in rabbits and humans. As such, it remains a good lead compound to develop new analogues if species specificity can be overcome and potency improved. Whether these compounds as part of their contraceptive action, in fact, inhibit their putative enzyme targets namely, testis ceramide-specific glucosyltransferase (CGT) and/or glucosidase activity, in vitro is not known. To address this question, we report relative in vivo antispermatogenic efficacy of NB-DNJ and NB-DGJ (N-butyldeoxygalactonojirimycin) in mice and rats following daily single gavage administration, as opposed to mixing the compound in the food as performed in previous studies by others. We compared their anti-spermiogenic efficacy against their biochemical potency as inhibitors of the likely target enzymes in vitro. Our studies show that daily single oral administration of 15 mg/kg NB-DNJ and/or 50 mg/kg NB-DGJ over 35 days resulted in an anti-spermiogenic effect in C57Bl/6J mice. Our in-vitro enzyme assays standardized for testicular CGT and glucosidase activity indicated that C57Bl/6J enzyme activity is more sensitive to NB-DNJ than the enzyme activity in rats. Rat mating trials showed that NB-DGJ, but not NB-DNJ induced reversible infertility. Both NB-DGJ and NB-DNJ inhibited testicular CGT and glucosidase activity in vitro. Thus we observed that the in vitro effects of NB-DNJ in rat model did not coincide with its effect in vivo. This raises questions whether the target enzyme is not accessible to NB-DNJ in vivo, or there are target enzymes hitherto unknown which are affected by iminosugars that explain the contraceptive activity. Our in vivo studies, rat mating trials, and the in vitro enzyme assay results collectively indicate a need to identify contraceptive targets for iminosugars other than testicular CGT and glucosidase and the need to determine the relative bioavailability of new analogues to the targets in vivo. This research was supported by grant U54 HD055763.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Immunological Genetic Identity among Cloned Dogs with the Same Genetic Background in MHC Class II DLA-DQA1 and DLA-DQB1.

Ji Eun Kim, Hyun Ju Oh, Jung Eun Park, Min Jung Kim, So Gun Hong, Goo Jang, Byeong Chun Lee 1

The canine major histocompatibility complex (MHC) is dog leukocyte antigens (DLA), which is known to be the most polymorphic genetic system in canine species. Although many cloned dogs have been produced, there was no research about genetic identity of MHC among cloned animals. Recently in Lee's group, two cloned beagles (BG1, 2) were produced by somatic cell nuclear transfer (SCNT) using fetal fibroblast 3 (BF3). Also, four transgenic cloned beagles (Ruppy 1-3, 5) were generated using transgenic BF 3 infected Red fluorescent protein (RFP) gene. We hypothesize that non-transgenic (BG1, 2) and transgenic (Ruppy 1-3, 5) cloned beagles derived from identical donor cell have same genetic characteristic except for RFP gene insertion in immunological site of genome. Thus, the aim of this study is to confirm the immunological identity of DLA class II in cloned beagles produced using same nuclear donor cell. Genomic DNA was extracted from blood of BG1, BG2, and Ruppy 1, 2, 3 and 5. Forward and reverse primers used for DLA-DQA1 and DQB1 respectively were DQAF: 5'-TAAGGTTCTTTTCTCCCTCT-3' and DQAR: 5'-GGACAGATTCAGTGAAGAGA-3' DQBR: 5'- CTCACTGGCCCGGCTGTCTC-3' and DQBR: 5'-CACCTCGCCGCTGCAACGTG-3'. Polymerase Chain Reaction (PCR) products were purified, sequenced directly using the Big Dye Terminator kit. Sequencing analysis was performed on an automated 3730xl DNA analyzer. In experiment 1, sequence of DLA-DQ alpha 1(DQA1) and DLA-DQ beta 1(DQB1) exon 2, hypervariabel region, was compared in BG1 and BG2. Experiment 2 also compared the sequence of DQA1 and DQB1 among Ruppy 1, 2, 3 and 5. Experimental 3 compared sequence of DQA1 and DQB1 among all cloned dogs (BG1, BG2 and Ruppy 1-3, 5). As a result, BG1 and BG2 have same allele for DQA1 and DQB1 as we expected. They share DQA1*00101 and DQB1*02901 in experiment 1. In experiment 2, Ruppy 1, 2, 3 and 5 also have identical DQA1*00101 and DQB1*02901 allele. No discrimination between transgenic dogs and cloned dogs was seen in DQA1 and DQB1 Allele in experiment 3. DQA1, DQB1 allele was identified as *00101 and *02901 in all dogs. We provided the allele identity of DQA1and DQB1 in cloned beagles, which can be used as preliminary data for immunological related studies. In conclusion, transgenic cloned dogs despite of being inserted red fluorescent protein genes in their nuclear DNA were immunologically compatible with non-transgenic cloned dogs. We demonstrated that cloned beagles produced using identical nuclear donor were immunologically compatible. This study was financially supported by KOSEF, SNU foundation (Benefactor; RNL BIO), Nestle; Purina PetCare, and the Korean MEST, through the BK21 program for Veterinary Science.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Functional Mechanism of WNT7A in Ovarian Carcinomas.

Shin Yoshioka, Sophia Ran, Horoshi Okuda, James A MacLean, Mary E McAsey, Kounosuke Watabe, Kanako Hayashi 1

The WNT system is known to play an important role in the regulation of developmental events where they govern cellular proliferation and differentiation. WNT signaling plays a key role in the normal embryonic development of the ovary as well as normal follicular development and ovarian function. Abnormal activation the WNT/beta-catenin signaling pathway has been associated with ovarian tumorigenesis. It is likely that the WNT signaling pathway is involved in ovarian cancer development via multiple, diverse mechanisms such as gene mutations and changes in pathway components such as extracellular inhibitors and nuclear transcription cofactors. To date, the ligand partners associated with WNT signaling have not been examined in ovarian cancers. The present study investigated the potential role of WNT7A in the ovarian carcinomas including its functional mechanisms. Although WNT7A has not been detected in normal ovary, WNT7A became abundantly expressed in ovarian malignant tumors, and patients had worse overall survival rate in the group with high expression of WNT7A. Frizzled receptors (FZDs), co-receptors (LRPs), WNT inhibitors and antagonists (DKKs and SFRPs) were also detected in ovarian carcinomas. To determine whether WNT7A activates the canonical CTNNB1-TCF/LEF and/or the non-canonical JNK signal transduction pathways in ovarian tumors, SKOV3 cells, which do not express WNT7A, were transfected with the TOP-FLASH reporter or pFR-Luc reporter with pFA2-c-Jun, then overexpressed WNT7A. The activity of the TOP-FLASH reporter was significantly stimulated by cells transiently transfected with WNT7A (6.2-fold). WNT7A significantly activated JNK by a 2.3-fold induction of luciferase activity compared with control cells. Overexpression of WNT7A stimulated the expression of MMP7 (8.8-fold), MMP9 (2.4-fold) and CDH1 (5.2-fold) in the SKOV3 cells compared to control. MMP7 promoter activity was conferred by 237 bp 5'-flanking sequence which contains 2-TCF binding sites. Further, stable overexpression of WNT7A in SKOV3 cells increased cell adhesion and migration. The directional cell migration decreased by 60% in WNT7A knocked down SKOV3.ip1 cells, which are highly expressed WNT7A, using siRNA. Therefore, these results suggest that WNT7A is a critical regulator of ovarian tumorigenesis and progression via the canonical WNT/β-catenin and/or non-canonical JNK pathway. Supported by ACS-IL 139038.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Effects of Ovarian Steroids on Gastrointestinal Motility in Ovariectomized Rats.

Wei-Ju Huang 1, Cai-Yun Jian 2, Han-Wei Lin 2, Full-Young Chang 3, Paulus S Wang 2

Many women have the problems in gastrointestinal (GI) tract including nausea, vomit, abdominal distension, heart-burn, constipation and diarrhea. These clinical observations have led to speculation about the possible roles of several hormones on Gl function such as human chorionic gonadotropin (hCG), estrogen, progesterone (P4) and oxytocin. However, the current conclusions from the studies about sex- and pregnancy-related alterations in gastric emptying and Gl transit are based on indirect evidence, and the role of female steroid hormones is unclear. The purpose of this study is to examine the effect of ovarian steroid hormones on gastric emptying, Gl transit, and the cyclooxygenase (COX-2) expression in GI tract under the administration of estrogen and P4 in ovariectomized (Ovx) rats. The diestrus rats were used as normal control. After ovariectomy, rats were divided into several groups. Ovx rats were s.c. injected with sesame oil (group 1), estradiol benzonate (EB, 10 μg/ml/kg, group 2), progesterone (P4, 20 mg/kg, group 4), or EB plus P4 (group 4). After administration for 7 days, rats were fasted 16-20 hrs before GI experiment. Our results showed that diestrus rats have slower gastric emptying than that in Ovx rats. Ovarian steroid hormones have different effects on the GI motility, in which estradiol delayed emptying but P4 stimulated it. Moreover, when combined treatment with oxytocin, the stimulatory effect of P4 on gastric emptying was attenuated. COX-2 expression was decreased under treatment of EB, P4, or EB plus P4. We concluded that the gastric emptying delayed by estrogen was at least in part via the decreased expression of COX-2 although the stimulatory effect by P4 was irrelevant. We suggest that the results will be helpful to understand the GI problems and their action mechanisms under normal or during the pregnancy in women.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Novel Glycogen Synthase Kinase 3beta Inhibitors Induce Apoptosis in Ovarian Cancer.

Tyvette Hilliard, Amanda Muehlbauer, Irina Gaisina, Arsen Gaisin, Franck Gallier, Alan Kozikowski, Joanna Burdette 1

Ovarian cancer is the most lethal gynecological malignancy among US women. Paclitaxel/carboplatin is the current drug therapy used to treat ovarian cancer, but most women develop drug resistance and recurrence of the disease arises which leads to death within 5 years. Combination therapy or alternative strategies are of great importance. A possible target is glycogen synthase kinase 3beta (GSK3beta), which is a protein that is overexpressed in serous ovarian cancer. Recent studies have shown that GSK3beta inhibitors (GSKi) are known to induce apoptosis in pancreatic cancer cells, and can destabilize p53, which may promote cellular death in response to DNA damaging agents, much like chemotherapy. Inhibitors of GSK3beta, a downstream kinase in the Wnt signaling pathway, were evaluated as a probable drug therapy using serous ovarian cancer cell lines. A series of maleimide-based GSK3beta inhibitors were synthesized and then tested in vivo and in vitro using SKOV3 and OVCA432 ovarian cancer cell lines. From a panel of 10 GSKi, the GSKi 9ING41, was found to be the most effective in vivo and in vitro for slowing ovarian cancer cell growth. 9ING41 induced apoptosis as indicated by DAPI positive nuclear condensation, DNA laddering, and TUNEL analysis. Activation of caspase-3 and cleaved PARP, as depicted using western blot analysis on ovarian cancer cells, indicated that the mechanism for apoptosis is through caspase activation. Additionally, GSKi significantly downregulated phosphorylation of the alpha and beta subunits of GSK in the OVCA432 cell line, reduced the levels of cyclinD1, and inhibited phosphorylation of the downstream target glycogen synthase. An in vivo xenograft study using SKOV3 cells was completed to test the efficacy of two commercial inhibitors, LiCl and SB216763 compared to 9ING41. Tumor progression was hindered by 9ING41 in vivo and phosphorylation of downstream molecules including the beta subunit of GSK and phosphorylated glycogen synthase was blocked. The non-selective commercially available GSK3beta inhibitor, LiCl, was not able to induce apoptosis completely in vitro or significantly reduce tumor growth in vivo. Novel GSK3beta inhibitors were shown to be more potent than commercially available inhibitors by reducing tumor growth by the induction of apoptosis. Therefore, GSK3beta inhibitors alone or in combination with existing drugs may provide an alternative treatment strategy to serous ovarian cancers.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Caspase 3 Expression in Equine Granulosa Cells and Oocytes in Young and Old Mares.

Bernardo L Rodrigues, Juliano C Silveira, Jason E Bruemmer, Gerrit J Bouma, Elaine M Carnevale, Colin M Clay 1

Age-related infertility is a multifactorial process, involving depletion of the follicular reserve, hormonal dysregulation and anatomical and histological changes. The change in balance between pro-survival and pro-apoptotic factors within the ovary of aged females has been demonstrated. Cross-talk between the oocyte and surrounding cells is essential for follicular development and oocyte maturation, and the viability of all cell types is of utmost importance. Apoptosis is a highly conserved process of programmed cell death, essential for a variety of physiological mechanisms. In the ovary, apoptosis begins during fetal life and continues after birth, occurring during every reproductive cycle with follicular atresia. Apoptosis can be initiated either by the germ cells or the follicular cells, and the fate of the follicle relies on the balance between factors promoting cell proliferation, growth, differentiation or death. This process is tightly regulated and requires a sequential activation of multiple caspases. Caspase 3 (CASP3), an effector caspase, has been identified in the ovary of several species and is considered the main effector caspase involved in granulosa cell apoptosis, but conflicting evidence exists for its role in germ cell apoptosis. The number of apoptotic granulosa cells is negatively correlated with pregnancy rates after in vitro fertilization treatment in women. Evidence of apoptosis in the oocyte has been suggested as one of the reasons for poor oocyte quality and lower fertility in aged mice. Moreover, an active form of CASP3 has been identified in human oocytes that failed to cleave after fertilization. Therefore, we postulated that aged mares would have increased CASP3 expression, which is correlated with increased granulosa cell apoptosis, when compared to young mares. The objectives of this study were to 1) demonstrate CASP3 expression in equine granulosa cells and oocytes, and 2) compare CASP3 mRNA levels between young and old mares as an indicator of apoptosis. Pre-ovulatory follicles (≥35 mm) from young (<10 years; n=10) and old (≥20 years; n=11) mares were aspirated 30-36 hours post hCG administration, using a transvaginal ultrasound-guided technique. Oocytes and granulosa cells were separated from each and RNA was isolated using PicoPure RNA isolation kit. Qscript cDNA synthesis kit was used to generate cDNA templates for oocyte and granulosa cells. Primers were designed based on the equine sequence, and confirmed by product sequencing. Real time PCR analysis was performed using the LightCycler480 PCR system, and TUBULIN and GAPDH were used as normalizer genes for oocyte and granulosa cells, respectively. CASP3 expression was detected in oocytes and granulosa cells, but there was no difference (p>0.30) in mRNA levels between young and old mares. This suggests that apoptosis mediated by CASP3 in granulosa cells and oocytes does not play a role in the reduced fertility of oocytes from old mares. However our samples only represent cells from pre-ovulatory follicles after induced maturation by hCG. Thus, further studies are required to evaluate CASP3 expression at different follicular stages, as well as the active form of CASP3 protein, in addition to other members of the caspase family in order to elucidate the involvement of apoptosis as a mechanism of age-related infertility in the mare. This work was supported by the Cecil and Irene Hylton Family Foundation.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Generation of Putative Induced Pluripotent Stem Cells from Adult Canine Fibroblast by Three Factor Reprogramming (Oct3/4, Klf4, and Sox2).

Sehwon Koh, Shengdar Tsai, Steve Bischoff, Natasha J Olby, Jorge Piedrahita 1

Pluripotent stem cells such as embryonic stem (ES) and iPS cells can give rise to derivatives of all three germ layers and thus have great potential for clinical applications related to regenerative medicine. Unfortunately, isolation of ES cells requires availability of embryos. Moreover, it is not possible to genetically match ES cells and patients resulting in immune rejection. iPS cells, in contrast, can be generated from somatic cells from the patients being treated, solving the issue of genetic match and availability of embryos. In mice and humans, it has been shown that embryonic and adult fibroblasts can be reprogrammed by introducing four transcription factors, Oct3/4, Sox2, Klf4 and c-Myc. The oncogenic transcription factor, c-Myc, however, hinders clinical application of iPS cells. Here we report the derivation of putative iPS cells from adult canine fibroblast without using c-Myc. The VSV-G pseudotyped retroviruses containing three transcription factors (hOct3/4, hKlf4, hSox2) were produced and transduced into lung fibroblasts isolated from a 3-year old dog. The transduced cells were seeded onto gamma-irradiated 50/50 mixture of SNL and L-Wnt3a feeder cells and cultured in DMEM containing 10% FBS and 1mM VPA for 7 days. The media was then replaced with DMEM containing 20% knockout serum replacement(KSR), non-essential amino acid (NEAA, 100 µM) and mono-thioglycerol (MTG, 0.15 mM) with zbFGF2 (10 ng/mL) until day 14. At day 15, Wnt3a conditioned ES media supplemented with leukemia inhibitory factor (LIF) and three chemical inhibitors (3 µM CHIR99021, 0.5 µM A-83-01, 5 µM kenpaullone; 3i ES media) was introduced. A few granulated colonies appeared at day 14 and ES-like colonies were picked day 21 postinfection, expanded and analyzed as described below. Canine iPS cells isolated from adult lung fibroblast were very similar to dog ES cells in morphology and alkaline phosphatase (AP) expression. The morphology and AP expression was maintained in 3i ES media. The putative iPS cells also expressed stage-specific embryonic antigen (SSEA)-1 and SSEA-4. The expression of SSEA-4 was higher than SSEA-1, consistent with what is seen in dog ES cells. Both RT-PCR and immunocytochemistry (ICC), showed that the isolated cells express the pluripotency markers, Oct3/4, Sox2 and Nanog. However, the viral transcription factors transduced in the iPS cells were not completely silenced. Spontaneous differentiation by withdrawal of LIF led to formation of embryoid bodies (EB) composed of cells representative of all three germ layers as confirmed by detection of cells positive for desmin and vimentin (mesoderm), GFAP, nestin and TuJ1 (ectoderm) and alpha-fetoproten (AFP; endoderm). Similar results were seen by directed in vitro differentiation. At present we are determining whether the isolated cells can give rise to complex teratomas after injection into nude mice. To summarize, we were able to directly isolate pluripotent cells from cultured adult somatic cells by using three transcription factors without c-Myc. The isolated dog putative iPS cells have similar morphology and marker genes expression to dog ES cells. Availability of stable canine iPS cells would have significant implications for regenerative medicine in both veterinary and human clinical applications. This work was funded by a grant to JP and NO from the Center of Comparative Medicine and Translational Research, North Carolina State University.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Cryopreservation of Cultured Mouse Spermatogonial Stem Cells.

Yong-An Lee, Byung-Gak Kim, Bang-Jin Kim, Ki-Jung Kim, Yong-Hee Kim, Buom-Yong Ryu 1

Spermatogonial stem cells (SSCs) are a documented as source for maintaining of spermatogenesis and for multipotent stem cells. Thus, the preservation of SSCs is great interest of studying for production of transgenic animal and multipotent adult stem cells. A number of culture methods what are able to preserve undifferentiated state and effectively product a large number of SSCs were developed already, but it have to cost much money and a lot of effort. Therefore, an essential prerequisite for the future widespread application of SSCs is the development of efficient preservation methods to facilitate their storage and we have studied cryopreservation of pure SSCs. Testis cells for SSCs culture were collected from a pup transgenic mouse line C57BL/6-TgEGFP that was bred into DBA/2 background (designated Green) and SSCs from the collected cells purified via Magnetic Activated Cell Sorting (MACS) technique with anti-Thy-1 antibody after enzymatic digestion using 0.25% trypsin and 7mg/ml DNase 1. Subsequently, they were cultured under serum-free conditions consisted with mitotically inactivated STO cell feeders and growth factors including GDNF(10ng/ml), GFRa1(75ng/ml) and bFGF(1ng/ml). After 4 weeks of initiation of culture for the obtaining highly purified SSCs were collected by trypsinisation and they were suspended in a cryopreservation medium, which contains 10% dimethly sulphoxide(DMSO) and 10% fetal bovine serum(FBS) without other supplement or with other supplement such as 50mM Trehalose and Sucrose. Aliquots of 1ml cell suspension, containing 5 x 105 SSCs, were transferred to 1.5ml cryovials, placed in a freezing container, and frozen at -80°C for 1 day, after which the cryovials were plunged into liquid nitrogen for 6 days. After a week, the cryovials were thawed in a water-bath at 37°C volume of 10 µl were stained with Tryphan blue solution for check of survival rate, 5ml of MEM alpha was added drop-wise. After washing by centrifugation, the cells were cultured during 7 days for comparing of proliferation ability with non-cryopreserved SSCs. The proliferation ability was measured by cell count after 7 days and compared with non-cryopreserved SSCs that were cultured from 5 x 105 SSCs. Cryopreserved SSCs with cryopreservation medium are shown survival rate of only 73 ± 6.4%, and they proliferated to 4.7 ± 0.3 x 105 cells after culture during 7 days. When supplement was added to freezing media, there are shown survival rate of 86.4 ± 2.1% in Trehaolse, and 91.1 ± 0.36% in Sucrose. And the cells number increase to 6.05 ± 0.22 x 105 and 6.25 ± 0.2 x 105 cells respectively after culture for 7 days. In conclusion, both Trehalose and Sucrose help to increase survival rate and proliferation ability of SSCs after cryopreservation. Therefore, the result of current studies will be beneficial for long term-preservation of SSCs. This study was supported by a grant of Stem Cell Research Center (SC-5140), Healthcare technology R&D Project (A084318), Ministry for Health, Welfare & Family Affairs, and BioGreen 21 Program (No. 20070401034012), Rural Development Administration, Republic of Korea.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Derivation of Putative, Metastable Induced Trophoblast Stem Cells from Pigs.

Haruyo Matsuyama, Toshihiko Ezashi, Bhanu Prakash VL Telugu, Andrei P Alexenko, R Michael Roberts 1

Authentic trophoblast stem cells (TSC) have been produced from blastocysts and ectoplacental cone of the mouse, but not from other species. Here we report the generation of trophoblast cells with stem cell-like properties during attempts to produce induced pluripotent stem cells (iPSC) from porcine embryonic fibroblasts (PEF) by lentiviral delivery of re-programming genes, OCT4, SOX2, KLF4, and c-MYC. Approximately one-fifth of the colonies that formed after the re-programming steps displayed an unusual phenotype consisting of a mixed population of cells. These colonies also tend to appear a week earlier than the "true" iPSC colonies. They consisted of cells of different sizes, including ones with a more flattened, epithelial-like morphogy than standard iPSC, and were not uniformly positive for alkaline phosphatase. After extended culture on the standard human (h) embryonic stem cell (ESC) medium (DMEM-F12, 20% Knock-out serum replacement supplemented with 4 ng/ml hFGF2) normally employed for maintenance of iPSC, regions of the colonies often developed epithelial "dome-like" structures that lifted-off of the substratum to form floating spheres. Several features of the phenotype of these cells indicated a close similarity to trophoblast rather than iPSC, so that they were tentatively named "induced trophoblast stem cells" (iTSC). Global gene expression profiling on porcine Affymetrix microarrays and routine RT-PCR performed on RNA from the iTSC and the cogenerated iPSC, as well as the parental PEF, revealed that the iTSC were a distinct cell grouping. Specifically, there was an up-regulation of transcription factors associated with trophoblast in the mouse, e.g. GATA2, PPARG, MSX2, DLX3, ETS2, HAND1, GCM1, CDX2 & TEAD4, of genes required for steroid biosynthesis, such as CYP11A1, and of some unique porcine trophoblast markers, e.g. PAG6 &10, IFND, IFNG, & IL1B. There was also partial but not complete down-regulation of OCT4 and SOX2 compared to iPSC. Interestingly, the "early" iTSC formed non-metastasizing tumors resembling teratomas in immunocompromised mice. These "teratomas" were uniform in cross section and consisted primarily of layers of epithelial cells plus some areas of striated muscle. They expressed CDX2, plasmin/trypsin Kunitz inhibitor, and PAG, indicating they were initiated from a source of stem cells with trophoblast potential. Upon extended passage, however, the putative iTSC cells lost CDX2, as well as OCT4, expression, suggesting that the TSC network was unstable under prevailing culture conditions. We then examined whether culturing the early passage cells in a medium that supports mouse TSC, i.e. containing 20% FBS and 25 ng/ml FGF4, would preserve their stemness and stabilize their phenotype. The TSC medium provided continued expression of CDX2 over extended passage numbers and a more homogeneous type of colony comprised of compact, relatively flattened cells. The co-production of iTSC with iPSC during re-programming suggests that expression of the same stemness genes that induce a pluripotent phenotype can also induce trophoblast. Possibly, relative levels of ectopic expression of these genes influence what gene network for stemness predominates. Supported by Missouri Life Sciences Board Grant 00022147 and NIH grant HD21896.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Directed Neuronal Specification of Porcine Induced Pluripotent Stem Cells.

Toshihiko Ezashi, Bhanu Prakash Telugu, R Michael Roberts 1

A recent goal of this laboratory has been to create porcine pluripotent stem cells, i.e. ones capable of differentiating into all cell types of the body thereby expanding the potential use of swine as a biomedical model for studying human disease as well as cells that might be easily modified by gene knock-in/knock-out technologies. Importantly, pig is a potentially useful model for studying human pathologies due to similarities in organ size, immunology and whole animal physiology between the two species. If the safety and efficacy of stem cell transplantation is to be tested in an animal model before being applied to humans, the pig would likely be a species of choice. The ability to derive porcine (p) iPSC lines from a particular outbred animal and conduct tissue transplantation on the same pig later and follow the success of the transplant over the course of months or even years would be a particularly valuable advance. We have recently created piPSC from embryonic fibroblasts and primary umbilical cord mesenchyme by a similar strategy used for the mouse and human, namely ectopically expressing reprogramming genes (OCT4/SOX2/MYC/KLF4) in somatic cells. The piPSC phenotypically resemble human embryonic stem cells (ESC), express the typical gene and surface antigen markers of ESC, proliferate continuously in culture, possess high telomerase activity, form embryoid bodies (EB) and teratomas, where they are able to differentiate along the three main germ line lineages. The present aim has been to demonstrate that piPSC can be directed to differentiate towards neuronal endpoints by using protocols based on those used successfully with human ESC. The piPSC, ID6, grown on a MEF feeder were mechanically detached and formed EB were cultured in ES cell medium containing knockout serum replacement but lacking FGF2 for 4 days. The EB were cultured following two days with serum free medium supplemented 0.1 mg/ml transferrin, 5 µg/ml insulin, 20 nM progesterone, 0.1 mM puterescine, 30 nM sodium selenite and 2 µg/ml heparin. The EB were then collected in the same medium and allowed to settle on dishes plated to which they attached and formed a flattened cell mass within 48 h. The cells differentiated into typical neuronal rosettes by d 9 in the same medium, which had a radially-organized columnar morphology and expressed transcripts for the neuroectodermal transcription factors, PAX6 and OTX2. These experiments demonstrate that the porcine iPSC line, ID6, is capable of being directed along a neuronal lineage by cues that work successfully on human ESC and iPSC, raising the possibility to achieve controlled differentiation of this same kind of cell along additional lineages. They provide confidence that porcine iPSC may soon be employed to provide tissue grafts that can be matched genetically to recipients and tested for their safety in transplantation. Supported by Missouri Life Sciences Board Grant 00022147 and NIH grant HD21896.

(poster)

Biol Reprod. 2010 Nov;83(1 Suppl):2.

Efforts Towards Isolation of Feline Embryonic Stem Cells.

Tatiana S Rascado, Joao F Lima-Neto, Mateus J Sudano, Daniela M Paschoal, Fernanda da Cruz Landim-Alvarenga 1

The domestic cat can be used as a biological model for humans, due to similarities in some disease and genetically transmitted conditions. Embryonic stem cells may complete nuclear reprogramming more efficiently than somatic cells and, therefore, are potentially useful for increasing interspecific cloning success. The objective of this study was to establish an effective method of isolation of the inner cell mass of cat embryos aiming to test the ability of the ICM to attach to the culture dish and to form embryonic stem cell colonies. Moreover, the ideal density of feline embryonic fibroblast feeder layer was studied. Domestic cat oocytes were matured, fertilized and cultured in vitro until the blastocyst stage. The inner cell mass was isolated by two different methods, the immunosurgery and mechanical isolation, and transferred to a monolayer of chemically inactivated cat fibroblasts with 10 µg/mL mitomicin C in two different densities. The base culture media was DMEM/F12 supplemented with 20% fetal calf serum, non essentials amino acids, glutamine, LIF, fibroblast growth factor-2, beta-mercaptoethanol and antibiotics. To the immunosurgery, the embryos were incubated in Tyrods acid solution, after they were incubated in anti cat IgG (1:10 dilution) and then they were incubated in complement from guinea pig (1:10). The group of ICM isolated by mechanical isolation (n=15) using a scapel blade shown high rates of attachment to the culture dish (95%) while the group of ICM isolated by immunosurgery (n= 15) shown 50% rates only. Culture was performed in a 5% CO2 in air incubator at 38.5°C. However until now no good colonies were formed. The densities of inactivated fibroblasts tested were 250,000 cells/well (2 cm2) and 140,000 cells/well. The higher density was harmful because the fibroblast detached of the culture dishes after the first renovation of the culture media. These experiments were repeated ten times. We concluded that mechanical isolation is better than immunosurgery to isolate ICM of cat embryos and high densities of feline inactivated fibroblast is not ideal to culture of embryonic stem cells.

(poster)

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

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