Abstract not available.
. 2010 Nov;83(1 Suppl):3. doi: 10.1095/biolreprod.110.083824
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Reproduction is compromised by low body weight, due to reduction in GnRH drive to the pituitary gland. It has been shown in various species that ‘rescue’ of reproductive function in states of negative energy balance or low body weight can be effected by administration of leptin. Our studies in sheep show that this may be due to stimulation of pro-opiomelanocortin (POMC) cells by leptin, for the following reasons. POMC expression in the arcuate nucleus of the hypothalamus is upregulated in lean animals given leptin. A melanocortin agonist (MTII) also activates the GnRH/gonadotropin axis when administered by intracerebroventricular (ICV) infusion to lean hypogonadotropic animals. Since kisspeptin (product of the Kiss1 gene) is a potent stimulator of GnRH secretion, we investigated links between ‘appetite regulating’ cells and kisspeptin cells. Kiss1 expression is reduced in lean, hypogonadotropic ewes, but MTII infusion of similar animals stimulated Kiss1 expression in the preoptic area and reduced Kiss1 expression in the arcuate nucleus. We also showed that the majority of kisspeptin input to GnRH cells arises from the cells of the preoptic area, so this is a potential means by which melanocortins stimulate GnRH secretion. In the arcuate nucleus, strong reciprocal connections were found between POMC and kisspeptin cells, with weaker connections between neuropeptide Y (NPY) and kisspeptin cells. ICV infusion of kisspeptin reduced POMC expression and increased NPY expression. These and other data show the commonality of circuits in the hypothalamus that regulate appetite/energy expenditure and reproduction.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
The neurobiological mechanisms underlying steroid feedback regulation of gonadotropin-releasing hormone (GnRH) neurons are an active area of investigation. Estradiol feedback action varies depending on concentration and physiological state. Homeostatic negative feedback persists through much of the female reproductive cycle largely reducing GnRH pulse amplitude, but high levels of estradiol in the late follicular phase induce a switch in the mode of feedback initiating the GnRH surge in one of the few positive feedback events in physiology. Work over the past decade from many labs has demonstrated that estradiol acts not only through genomic mechanisms to change gene expression, but also initiates signaling cascades and thus rapidly alters cell physiology. We studied the contribution of these non-genomic effects to estradiol feedback in GnRH neurons using electrophysiological approaches. Preovulatory concentrations of estradiol increased GnRH neuron action potential firing by altering several intrinsic properties leading to increased excitability, acting via estrogen receptor beta, which is expressed in these neurons. In contrast, low physiological levels of estradiol had no effect on intrinsic properties, but tended to suppress GnRH neuron activity by altering estrogen-sensitive synaptic drive to GnRH neurons acting through the alpha subtype of the receptor. These findings add to our understanding of estradiol feedback, suggesting both rapid and genomic actions of estradiol are dependent upon level of steroid, that multiple estrogen receptor types are involved in mediating the effects, and that both direct and network mechanisms are employed to change GnRH neuron activity and ultimately hormone release.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
Primordial follicle activation (PFA) is the irreversible, metered process by which primordial follicles are selected from the reserve pool. PFA is a delicately balanced and tightly regulated process ensuring that some number of growing follicles is available during each estrus/menstrual cycle. At the same time, PFA limits the number of growing follicles, thereby avoiding premature depletion of primordial follicles and early reproductive senescence. Because the mechanisms regulating PFA in effect balance fertility with reproductive senescence, their elucidation is of fundamental importance for understanding the biological basis of female infertility and early menopause syndromes such as primary ovarian insufficiency. We initially identified the forkhead transcription factor Foxo3 as a critical regulator and suppressor of this process. Foxo3 knockout mice undergo global activation as soon as primordial follicle assembly is completed. More recently, we have shown that Foxo3 serves as an active molecular switch within the oocyte. During oogenesis, the Foxo3 protein is initially cytoplasmic, but is imported into the oocyte nucleus coincident with the completion of primordial follicle assembly. The Foxo3 protein remains within the nucleus throughout life until activation, when the protein is exported to the cytoplasm. This process is regulated by the phosphoinositide 3-kinase (PI3K) signalling pathway. PI3K-induced Akt activation promotes Foxo3 hyperphosphorylation and nuclear export, thereby triggering primordial follicle activation. Inducible genetic ablation of pathway components including Foxo3 in adult oocytes showed that this pathway functions throughout life. Thus, a principal physiologic role of the PI3K pathway is to control primordial follicle activation via Foxo3.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
In developing mouse ovaries, oocytes develop as clusters of interconnected cells called germ cell cysts. During the perinatal period, oocyte cysts break apart and granulosa cells surround individual oocytes forming primordial follicles. Mechanisms regulating cyst breakdown to establish the pool of primordial follicles are not well understood but estrogens may be involved. Treatment of neonatal mice with natural or synthetic estrogens results in abnormal multiple oocyte follicles in adult ovaries. Neonatal estrogen treatment inhibits cyst breakdown suggesting multiple oocyte follicles are cysts that did not break apart. Our model is that before birth, maternal estrogen levels are high in the fetus, generating a signal that inhibits cyst breakdown. At birth, estrogen levels drop removing the inhibitory signal and cysts begin to break apart. Estrogen elicits its action is through nuclear hormone receptors, estrogen receptor (ER) alpha and ERbeta. We examined the expression of ERs in neonatal mouse ovaries and detected ERalpha in granulosa cells and ERbeta in oocyte nuclei. ERalpha or ERbeta selective agonists inhibited cyst breakdown, suggesting that estrogen can signal through either receptor to regulate cyst breakdown. However, normal cyst breakdown occurred in mice lacking either ERalpha or ERbeta suggesting that one receptor could compensate for the other. Cyst breakdown was only slightly altered ER mutants lacking both receptors implying that another receptor may be involved. Supporting this, estradiol modified so that it can only exert effects at the membrane, was able to inhibit cyst breakdown, implying that estrogen can also function through a membrane bound estrogen receptor.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
The processes of germ cell proliferation and primordial follicle formation are critical for establishing the reproductive potential of mammalian females. Once the proliferating germ cells arrest in meiosis, the resulting germ cell nests break down in a process that involves migration and association of somatic pre-granulosa cells. While the role of steroid hormones in regulating this process has been known for some time, critical roles for other hormones like activin have recently been elucidated. Activin action is regulated in gonads by the soluble antagonist follistatin (FST). We recently created a genetically altered mouse line in which the Fst gene was altered to prevent production of 2 isoforms of FST and allowing only the FST288 isoform to be produced. Unlike the global Fst knockout which dies immediately after birth, the FST288-Only mouse survives to adulthood and displays reproductive and metabolic phenotypes reflecting important roles for FST in adult physiology. Reproductive defects include a surfeit of primordial follicles at birth, more rapid demise of these follicles before puberty, and a reduced primordial follicle pool in adults that leads to a premature ovarian failure (POF) like phenotype. Recent findings indicate that altered activin signaling is at least partially responsible for these defects that involve delayed germ cell nest breakdown and associated apoptosis. These observations expand the list of critical roles for activin and FST in regulating reproductive physiology in mammals. Moreover, the POF phenotype of the FST288-Only mouse may allow identification of mechanisms involved in idiopathic human POF.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
KRAB zinc finger proteins (KRAB-ZFPs) are the largest class of transcription factors, with over 300 encoded in the human genome. The KRAB repression motif arose recently and has rapidly proliferated within the vertebrate lineage, attesting to substantial evolutionary significance. However, while the mechanism of KRAB-ZFP repression has been elucidated, remarkably few individual gene functions are known. Identifying Regulator of Sex-limitation (Rsl) as a pair of KRAB-ZFPs mutated in rsl mice assigned a first biological role in control of sexually dimorphic liver gene expression. Dimorphic gene expression occurs in many tissues and may underlie sex differences in incidence of many diseases, including diabetes and autoimmune syndromes. The Rsl1 and Rsl2 paralogs modulate 8% of the liver transcriptome, with numerous Rsl-responsive genes residing in pathways of steroid, cholesterol and lipid metabolism. rsl mice are lean and the sexes are differentially affected by acute (fasting) and chronic (high-fat) dietary stress. Through effects on key liver enzymes (e.g., Pcd1, Scd1), Rsl accentuates sexual dimorphism in intermediary metabolism. Rsl also affects reproductive behavior and female pubertal timing, in part by regulating synthesis of Major Urinary Proteins (MUPs) and cytochrome p450s (CYPs), thereby impacting both hormonal and pheromonal cues. Overall, KRAB-ZFPs modulate expression of a broad array of genes comprising complex traits (e.g., reproduction) and underlying differential susceptibility to complex diseases (e.g., obesity). This phenotypic variation dependent on sex and environment affects individual health and ultimately determines species evolution. This research was supported by NIH DK053998 (DR).
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
Imprinting, an epigenetic phenomenon, is defined as the marking of the parental genomes of a diploid organism with regards to their parental origin. It is controlled by DNA methylation in such a way that the difference in methylation is correlated with the differential expression of the two parental alleles. The appropriate transmission of genomic imprints is essential for the control of a number of biological mechanisms in mammals, such as embryonic development and foetal growth. The embryonic period of gonadal sex determination seems to be the most sensitive for environmental effects on the methylation marks of the germ line, through an altered imprinting reprogramming. Among these environmental effects, some of the steps involved in assisted reproductive techniques, may lead to methylation defects in imprinted genes. Our work on superovulation in mice model confirms this hypothesis. Exposure of the gametes/early embryos to specific toxic environmental agents may also be detrimental. In humans, the observation of a global fall in sperm counts have suggested, among other, the hypothesis of a causative link between exposure to various toxic agents and male infertility. We have demonstrated in mice that these effects were, at least in part, mediated by imprinting defects in the sperm. Furthermore, for some of the compounds, the observed effects were transgenerationally transmitted and disappeared gradually through the generations. The reported decrease in male fertility might also be mediated by imprinting alterations.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
In mammals, including humans, the brains of males and females differ anatomically and physiologically, including sex differences in neuron size or number, synapse morphology, neural projections, and specific patterns of gene expression. Sex differences in the brain may underlie important sex differences in physiology or behavior, including several aspects of reproduction, such as the timing of sexual maturation (earlier in females than males) and the ability to generate a preovulatory gonadotropin surge (in females only). The reproductive axis is controlled by hormonal and neural pathways that converge upon forebrain gonadotropin-releasing hormone (GnRH) neurons, and many reproductive sex differences likely reflect differences in the upstream circuits and factors that regulate GnRH cells. The neuropeptide kisspeptin, encoded by the Kiss1 gene, is an important regulator of GnRH secretion. I recently determined that Kiss1 neurons in the anteroventral periventricular nucleus (AVPV) of the rodent hypothalamus are sexually dimorphic, being more abundant in females than males. The sexual differentiation and development of this sexually-dimorphic Kiss1 system has been shown to be regulated by sex steroids, in particular estradiol, during critical periods of postnatal and pubertal development. In contrast, Kiss1 neurons in the more caudal arcuate (ARC) nucleus of the hypothalamus are not sexually-dimorphic in adult rodents. However, I recently found that the regulation of ARC Kiss1 cells is sexually dimorphic during key periods of prepubertal development, with non-gonadal suppression of Kiss1 gene expression present in juvenile male but not female mice. I postulate that these various sex differences in Kiss1 neurons may relate to known sex differences in puberty onset and/or the ability to display a preovulatory gonadotropin surge. [Research support provided by the Eunice Kennedy Shriver NICHD/NIH through cooperative agreements U54 HD12303 (as part of the Specialized Cooperative Centers Program in Reproduction and Infertility Research) and NICHD grant R00 HD056157.]
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
Stem cells are necessary to maintain tissue homeostasis and the microenvironment surrounding these cells controls their ability to self renew or differentiate. For many stem cell populations it remains unclear what cells and signals comprise a niche. In this study, we identify the signaling interactions that coordinate development of a possible germ cell niche within the nascent urogenital ridges (UGRs). Using an organ culture system and live cell confocal microscopy, we identified a novel role for BMPs in controlling PGC numbers and motility as they colonize the UGRs. BMPs do not act as guidance factors for PGCs; instead BMP signaling within the urogenital ridges controls localized expression of the germ cell survival factor Kit ligand (KITL). This was confirmed in vivo by Cre mediated loss of one of the BMP receptors. Induced loss of Bmpr1a resulted in fewer PGCs, reduced expression of genital ridge marker genes including Kitl and localized cell death within the UGRs. These data demonstrate that BMP signaling in the genital ridges controls the identity and survival of the somatic cells comprising the nascent germ cell niche. Loss of BMP signaling may result in a breakdown of this permissive environment ultimately leading to fewer PGCs. This research was supported by funding from the NIH (R01 HD053900) and start up funds provided by Case Western Reserve University.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
Mammalian testes produce a huge number of sperms for a long reproduction period. This is supported by the constant differentiation of a small subset of undifferentiated spermatogonia that makes up the stem cell compartment. In the testis, differentiation and self-renew of undifferentiated spermatogonia are beautifully coordinated both temporally and spatially. Our recent investigations have suggested the essential role of somatic cell component in these regulations. Temporally, undifferentiated spermatogonia produce differentiating progeny or A1 spermatogonia periodically once every 8.6 days. Based on the analyses on Vitamin A-deficient animals, it appears that more advanced spermatocytes and spermatids modulate the local retinoic acid metabolism and this in turn regulates the timing of spermatogonial differentiation. Spatially, undifferentiated spermatogonia are shown to localize within seminiferous tubules to the vasculature-associated region. We have been investigating the cellular and molecular nature underlying this biased spermatogonial localization and found it likely that seemingly homogeneous somatic cells may be heterogeneous and provide specific microenvironment to undifferentiated spermatogonia in seminiferous tubules.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
The application of tissue-engineering principles to the study of structure-function relationships in ovarian follicle growth and maturation has led to the development of versatile in vitro culture systems in which follicles can be studied in conditions that mimic the complex 3D environment of the ovary. The use of 3D follicle culture systems has facilitated the characterization of the cell-cell interactions and intracellular signaling events that are required for follicle growth, maturation and function as well as oocyte quality. Our alginate-based 3D culture system provides an important tool for studying how mechanical forces affect cellular responses. The culture of early-stage follicles in alginate has led to the successful development of two-layer secondary follicles and mature antral follicles that contain healthy and fertilizable oocytes. In the course of our research, we observed that follicle growth was dependent upon the concentration the alginate matrix. Two alginate preparations with specific physical properties were characterized as "permissive" or "non-permissive" to follicle development and gene products identified in each condition. These studies provide evidence that the physical environment of the ovary contributes to the function of the follicle and health of the oocyte. This work was supported by the National Institutes of Health and the Eunice Kennedy Shriver National Institutes of Child Health and Human Development, grant numbers: 5U54HD041857-07 (Northwestern University Center for Reproductive Research), UL1DE019587 (The Oncofertility Consortium), and RL1HD058295 (The Oncofertility Consortium)
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
The placenta can perform functions analogous to those of the liver, lung, kidney, intestine, ovary, hypothalamus and pituitary. The mammalian placenta is the first organ to be differentiated during development, and its anatomy varies extensively across the clade of eutherian mammals. The interface between the fetally derived tissues of the definitive placenta and the maternally derived uterus ranges in invasiveness from the minimally invasive epitheliochorial placentas common in artiodactyls to the maximally invasive hemochorial placentas seen in haplorhine primates and rodents. Phylogenetic studies have demonstrated that the human hemochorial placenta is the ancestral type among placental mammals. Moreover, there is evidence for ancient adaptive evolution (i.e. dN/dS>1) during the emergence of the eutherian placenta in genes that are highly expressed in placentas of extant mammals. Further evidence that these genes are crucial in placentation comes from knockout mice models that produce abnormal placental phenotypes and/or result in embryonic or perinatal lethality (e.g., Acvr1, Adam12, Adm, Bm1, Col4a2, Fn1, Igf1r, Ncoa6, Notch2, Tfpi). Despite the great degree of conserved adaptations across many eutherian placenta expressed genes, there is also evidence for lineage specific adaptations. For example, humans and other primates have expanded gene families with placenta specific expression including gonadotropins, galectins, and growth hormones. Indeed, comparative placenta transcriptome studies highlight both evolutionarily conserved and derived patterns of gene expression in placentas from diverse mammalian species with a wide range of anatomical variation. Taken together, these studies contribute to an integrated view of the evolution of the placenta.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
Prolactin is a hormone and cytokine involved in a wide range of vertebrate biological functions. The ancestral prolactin gene also served as an evolutionary template for the generation of new genes, encoding proteins with a breadth of actions. The birth of these new genes is linked to viviparity. Prolactin loci possess considerable variation among mammalian species, including species-specific gene expansion. This expansion is most prominent in murine rodents and in ruminants. Rodent and ruminant prolactin loci expansions are largely non-orthologous, suggesting that the events were evolutionarily independent. Other species such as the human and dog only possess a single prolactin gene at the locus. The human and some other primates exhibit an expansion of the related growth hormone locus. Some species do not exhibit expansions of either prolactin or growth hormone loci. Prolactin family expansions have occurred in some species with hemochorial and synepitheliochorial placental structures. The anterior pituitary and placentation site contribute to the production of members of the prolactin family. More specifically, lactotrophs, uterine decidual cells, and various lineages of trophoblast cells are known cellular sources. In murine rodents, trophoblast giant and spongiotrophoblast cells are prominent sites of prolactin family production, whereas in ruminants, binucleate cells possess this function. Individual gene expression patterns exhibit cell type- and temporal specific characteristics. Biological activities for the prolactin family can be distinguished based on the involvement of the prolactin receptor-signaling pathway. Classical members of the family utilize the prolactin receptor, whereas nonclassical members use distinct signal transduction mechanisms. Major classical targets include the corpus luteum and mammary glands, whereas nonclassical targets include the vasculature, hematopoietic cells, and immune cells. Insights into the biology of the prolactin family have been derived from mouse mutagenesis studies. Collectively, there is compelling evidence that the prolactin family participates in the regulation of pregnancy, including acting as effectors of pregnancy-dependent adaptations to physiological stressors.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
The establishment of a stable maternofetal interface is critical in pregnancy success and hence, by inference, speciation. Amongst eutherian mammals there are three types of interface: hemochorial (as in human, rat and mouse), endotheliochorial (as in cat, mink and elephant) and epitheliochorial (as in pigs and sheep). Recently it has emerged that the last of these is evolutionarily the most recent. In epitheliochorial species a prominent glycocalyx is present at the long-lived adhesive interface between trophoblast and uterine epithelium. This leads to the question of whether fetomaternal compatibility could be specified by a type of glycocode. We have used lectin histochemistry to gather evidence that, in keeping with this hypothesis, the glycan composition of the glycocalyx in epitheliochorial species may be conserved over long evolutionary periods: for example, 35m years between pig and peccary, which have in common a strikingly low level of fucosylation at the interface. Furthermore, we suggest the glycocode may act as a barrier to interspecies hybridisation. In species exhibiting hemochorial placentation, there is some evidence for convergent evolution of the placental surface glycome as for example in human, tenrec, guinea pig, hyena and armadillo, all of which share low levels of oligosaccharides that bind the lectins from Arachis hypogaea (Galβ1,3Gal-NAcβ1-) and Glycine max (terminal GalNAcα1-), and abundant N-acetyl lactosamine. Insulin-like growth factor (IGF) action is important for placental and fetal growth, and the receptor IGF1R is N-glycosylated. We have treated explants of human first trimester villous tissue with inhibitors of N-glycosylation and shown that glycosylation regulates receptor display at the syncytial surface and, in turn, the response to a growth stimulus delivered from the mother. We thus show that there are glycan-dependent functions of trophoblast that contribute to successful reproductive outcome and may have a previously unrecognised role in speciation.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
In most organisms, primordial germ cells (PGCs) are set-aside early during embryogenesis. Subsequently, PGCs migrate through the embryo, associate with somatic gonadal cells and form the gonad. While PGCs seem highly specialized, their products are the ultimate stem cells that generate a complete organism generation after generation. We are interested in how germ line stem cell fate is established in the embryo and maintained during adult stages. Transcriptional regulation as well as translational control play an important role in controlling germ cell proliferation and germ line stem cell maintenance. We have used RNA expression screens to identify genes whose RNAs are specifically present in germ cells. We found that the 3′UTRs of these RNAs instruct the time and place when the RNAs are translated during germ cell development. This has let to the identification new targets for the translational regulators Nanos and Pumilio. These targets affect germ cell transcriptional silencing required for primordial germ cell specification and germ line stem cell differentiation in the adult.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
The transforming growth factor-beta (TGF-beta) superfamily comprises a number of ligands including the bone morphogenetic proteins (BMPs) that are collectively essential for ovarian follicle development. In developing tissues, the availability and signaling potential of BMP ligands are crucially regulated by extracellular antagonists. There are currently over 20 known mammalian BMP antagonists and in a recent PCR screen of mouse tissues we identified two factors HtrA serine peptidase 1 (Htra1) and twisted gastrulation homolog 1 (Twsg1) that are abundantly expressed in juvenile and adult ovaries. Localization of the transcripts for these genes by in situ hybridization confirmed that both were strongly expressed in granulosa cells of multilayered preantral follicles. Since preantral follicles are sensitive to FSH, as well as intraovarian growth factors, including signals from the oocyte, we proposed that these mechanisms are key regulators of BMP antagonists. To determine whether the oocyte regulates gene expression in preantral follicles, we first had to set up an appropriate model system. Preantral follicles were isolated from juvenile 12-day old mice and individually oocytectomised (OOX) by microsurgery and cultured for 24hrs. Immunofluorescent labeling of specific markers (laminin, beta-catenin) showed that the overall follicle phenotype, including the spherical shape, basal lamina integrity and granulosa cell interactions were well preserved in OOX follicles. Furthermore, the continuing health of OOX follicles following culture was demonstrated by the expression of key granulosa genes (kit ligand, inhibin alpha) and the absence of apotosis markers (TUNEL and active caspase-3) validating the use of the model system. Using quantitative RT-PCR, levels of Twsg1 mRNA were significantly elevated in OOX follicles relative to intact follicles, while Htra1 levels remained unchanged (n=5 cultures). These results indicate that under normal circumstances, the oocyte represses Twsg1 expression in adjacent granulosa cells and has little influence on Htra1 regulation in preantral follicles. To determine whether FSH contributed to the regulation of Twsg1 and Htra1, isolated intact preantral follicles were cultured in the presence of 0 (control), 1 (low dose) or 10 (high dose) ng/ml FSH for 72hrs. At this time point both doses of FSH had induced a significant increase in follicle growth relative to the controls. When follicles receiving the low or the high dose of FSH were compared against the controls (qRTPCR), no differences in the relative amounts of Twsg1 or Htra1 transcript were detectable. In summary, these results indicate the BMP antagonists Twsg1 and Htra1 are abundantly expressed in developing follicles and that the oocyte, but not FSH, is involved in regulating Twsg1 expression in adjacent granulosa cells, while the factors that regulate Htra1 remain to be established. Intriguingly, both factors are known for their affinity and specificity for BMP-2 and 4. Therefore the presence of these antagonists in ovary implies the actions of certain growth factors may be neutralized and further highlights another important level of intraovarian regulation of follicle development. Supported by the BBSRC (BB/H00002X/1).
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
Pathways regulating the formation of primordial follicles have not been well-defined. During the first four days of life in mice, membrane-enclosed cords, containing oocytes and pre-granulosa cells, break down and are remodeled to form primordial follicles. We have examined a potential role of the hedgehog (HH) signaling pathway in follicle formation. There are three secreted HH ligands, Indian, desert and sonic HH (IHH, DHH, SHH). The signal transducer smoothened (SMO) is rendered inactive by the membrane receptor patched (PTCH1). Binding of a HH ligand to PTCH1 relieves inhibition of SMO and downstream signaling occurs through GLI transcription factors. Real time RT-PCR of enriched fractions of oocytes and somatic cells showed that at birth (day 0) oocytes express all three HH ligands and Ptch1, but do not express significant levels of transcripts expected in HH target cells. In contrast, somatic cells express Ihh and Dhh as well as genes involved in signal transduction (Smo, Ptch1, Gli1, Gli2, Gli3 and Hhip). The role of HH signaling in early follicle development was studied in transgenic mice in which CRE-mediated recombination, imparted through the Amhr2cre/+ allele, induces expression of a dominant active allele of Smo known as SmoM2. In Amhr2cre/+SmoM2 mutant mice, expression of SmoM2 begins in the embryonic ovary and Mullerian duct. Amhr2+/+SmoM2 littermates lacking Cre served as controls. Mutant mice had increased degeneration of oocytes in the newborn ovary, resulting in cords deficient in oocytes and 25% fewer primordial follicles. In addition, the first wave of growing follicles in mutant mice had elevated percentages of atretic follicles and follicles containing multiple oocytes. RT-PCR was used to determine how expression of components of the HH pathway differed in ovaries of mutants and controls and to gain insight into the timing and pattern of signaling. Genes within the HH pathway for which transcription is known to be increased by HH signaling were dramatically increased in ovaries of mutant mice compared to controls from 17.5 days post coitum (dpc) through day 2 (Gli1) or day 4 (Ptch1, Hhip1). In addition, expression of Shh was dramatically increased in ovaries of mutant mice from 17.5 dpc through day 12; the mechanism whereby over-activation of HH signaling increased Shh mRNA and the significance of this have yet to be determined. Immunohistochemistry using a pan-HH antibody that recognizes all three HH ligands revealed staining of oocytes alone on 17.5 dpc and day 0 in ovaries of control mice and fainter staining in ovaries of mutant mice. Thus, it is possible that increased Shh mRNA in ovaries of mutant mice may not be translated into protein. Pan-HH staining in somatic cells increased progressively with age in both mutant and control mice and levels of Dhh and Ihh mRNA in whole ovaries also progressively increased, in a manner consistent with previous reports that expression of Dhh and Ihh in granulosa cells increases during follicle growth. In summary, HH pathway genes are expressed in a dynamic fashion in oocytes, somatic cells and whole ovary during the neonatal period. Gene expression analysis in ovaries of Amhr2cre/+SmoM2 mice, in which follicle formation and early development are impaired, are consistent with over-activation of HH signaling during the neonatal period. These results suggest that HH signaling plays a role in follicle formation and early development. Supported by NICHD R03HD057648.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
EGF supports follicle development in many species, including the hamster. However, its role on primordial follicle formation remains largely unknown. We have shown that FSH or estrogen upregulates EGF-receptor (EGFR) expression in the granulosa cells of adult hamster follicles. Further, EGF and FSH interact with each other to regulate follicle cell proliferation. We have also shown that both FSH and estradiol-17beta stimulate primordial follicle formation in the hamster. We have observed that PI3K and ERK signaling modalities were involved in hamster primordial follicle formation. The objective of this study was to determine the extent to which the EGF/EGFR signaling would utilize PI3K and ERK signaling modalities in primordial follicle formation. A low level of EGFR was expressed in the somatic cells and oocytes of the hamster ovary as early as E14. EGFR expression increased significantly, especially in the oocytes from P6 and onwards. EGFR expression in the granulosa as well as in interstitial cells increased significantly after the formation of primordial follicles, but the expression in the oocytes decreased significantly. EGFR was localized primarily in the plasma membrane of ovarian cells, but the cytoplasm also showed a modest localization. Interestingly, a dense accumulation of EGFR was noted in the oocytes undergoing apoptosis. EGF at 10 ng/ml dose significantly increased phosphorylation of Akt and ERK1/2 in cultured P8 ovaries within 10 minutes. Whereas the Akt phosphorylation decreased by 1 hour, ERK1/2 phosphorylation decreased by 30 min and then increased again by 2h to remain high for 48 hours. EGF also rapidly stimulated phosphorylation of EGFR, Raf and MEK-1 within 10 minutes, suggesting that it signaled through the EGF/EGFR/Raf/MEK/ERK pathway. EGF stimulated the migration of somatic cells from the ovary proper in the organ culture. EGF at 10 ng/ml dose significantly increased the percentage of primordial follicles in cultured ovaries, but higher doses (50 ng and100 ng/ml) had a deleterious effect, primarily on the oocyte viability. In fact, many primordial follicles in 100 ng/ml group became atretic because of the oocyte apoptosis. In conclusion, the results suggest that EGF via EGFR regulates somatic cell migration and primordial follicle formation by using the Akt and ERK-1/2 signaling. Whereas a physiological dose of EGF supports the oocyte survival, higher doses induce apoptosis possibly by premature activation of the oocytes.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
Cyp26b1 (cytochrome P450, family 26, subfamily b, polypeptide 1) encodes an enzyme that degrades the potent morphogen retinoic acid (RA). It has well studied roles in neural development, and has recently been shown to function as a meiosis inhibitor and germ cell survival factor in the male gonad. However, its expression is largely lost in the female mouse embryonic gonad around E12.5. In an effort to identify activin regulated genes in the mouse ovary and in granulosa cells, we discovered that Cyp26b1 is expressed in the postnatal ovary (although in much less abundance than in the testis) and its expression is significantly suppressed by activin treatment. The purpose of this study was to investigate the expression, regulation and functions of Cyp26b1 in the mouse ovary. Cyp26b1 mRNA was found to be expressed in granulosa cells of follicles at all developmental stages. A striking inverse correlation between Cyp26b1 and activin beta A mRNA expression within ovarian follicles and during postnatal development was observed. Activin regulation of Cyp26b1 levels in vivo was also confirmed in a transgenic mouse model that has decreased activin expression, and as a consequence exhibits highly elevated Cyp26b1 expression. Functional studies revealed that the Cyp26 inhibitor R115866 had a stimulatory effect on the proliferation of primary cultured mouse granulosa cells. As expected, activin A increases the proliferation of these cells, and a similar effect was observed with RA. Thus, some of the proliferative effects of activin may be mediated by decreased Cyp26b1, leading to increased RA levels. A pan-retinoic acid receptor (RAR) inhibitor, AGN194310, abolished the stimulatory effect of either RA or activin on granulosa cell proliferation, indicating an involvement of RAR-mediated signaling in both RA and activin-induced granulosa cell proliferation. We conclude that Cyp26b1 is expressed in the postnatal mouse ovary and is regulated by activin, and that the activin and RA pathways may interact to regulate mouse granulosa cell proliferation. This study suggests novel roles for Cyp26b1 in regulating the development and function of the mammalian ovary.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
Most studies of follicular maturation and ovulation in vitro utilize isolated follicles or whole ovaries through perfusion systems. Frequently, ovulation is established in vivo by the presence of an empty follicle. This study presents a method for real-time visualization of ovulation using an in vitro slice protocol that preserves the 3 dimensional structure of potentially key ovarian components. Ovulation is viewed from intact follicles within ovarian slices by either capturing daily images to examine progression at chosen time points or by using video microscopy to capture the continuity of events. Ovaries were collected from prepubertal (<5 wks) mice (C57 background), embedded in 8% agarose, cut at 200 µm and plated 3 slices per dish. Several different treatment combinations were investigated qualitatively, including follicle-stimulating hormone (FSH), luteinizing hormone (LH), pregnant mare serum gonadotropin (PMSG), and PG600 (PMSG and hCG). For quantitative analysis, ovaries were treated with either no hormone, 5IU PG600, 5IU hCG, or 5IU PG600 followed 48 hours later by 5IU hCG. Results showed that dishes treated with both PG600 and hCG released approximately 22% of an average of 4.2 readily releasable oocytes per slice, while no oocytes were released without hCG. Ovulation was observed within 12 hours after the addition of hCG following PG600 pretreatment based on video microscopy or end point image comparisons. For other treatment groups, there was no evidence of stimulated oocyte release. Health and viability of the released oocytes was assessed by examining DNA labeled with 1mM Hoechst stain. Video microscopy indicated that one of the first steps in stimulated release is mobilization of the oocytes within the follicles within minutes. In summary, this study characterizes a novel method for studying the mechanisms of ovulation, the ability to examine and alter the progression of ovulation in a defined serum free media in vitro, and for the analysis of oocyte behavior within follicles.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
An oocyte's competency for generating viable offspring begins during folliculogenesis. Several endocrine and paracrine factors are suspected to play important roles in controlling oocyte competency. One candidate molecule is fibroblast growth factor 10 (FGF10). In cow, it is expressed by thecal cells and the oocyte, and its cognate receptor, FGFR2b, is expressed by granulosa cells. Moreover, FGF10 expression in theca cells is associated with follicle health in cattle. The aim of the present work was to describe how FGF10 impacts oocyte maturation and subsequent embryo development after in vitro fertilization (IVF). Cumulus-oocyte complexes (COCs) were collected and cultured with serum-free or serum-containing oocyte maturation medium supplemented with various concentrations of FGF10 and/or FGF10 neutralizing antibody. In vitro fertilization was performed by co-culture of matured COCs with a pool of frozen-thawed semen from three bulls. After 8-10 h, putative zygotes were cultured with modified synthetic oviductal fluid for 8 days. To evaluate whether FGF10 improves oocyte competence, FGF10 was added to maturation medium containing or lacking bovine steer serum. Subsequent fertilization rates were not affected by FGF10 treatment but increases (P<0.05) in the proportion of 8-16 cell embryos on d 3 and the blastocysts on d 7 and 8 post-IVF were evident when supplementing 50 ng/ml FGF10 in serum-containing medium and 0.5 ng/ml FGF10 in serum-free medium. In addition, FGF10 supplementation (50 ng/ml) in serum-containing medium increased the total number of cells in blastocysts (P=0.05) and number of inner cell mass cells (P=0.004). Addition of an antibody against FGF10 during oocyte maturation did not affect fertilization rates but prevented FGF10 from increasing the proportion of zygotes that formed blastocysts at d 7 (P<0.05). Moreover, the effect of anti-FGF10 could be reversed by providing a 3.9-fold molar excess of FGF10 (50 ng/ml). FGF10 supplementation acted in several capacities to impact oocyte maturation. Addition of FGF10 in serum-free maturation medium increased (P<0.05) first polar body extrusion rates, percentage of oocytes reaching telophase I and metaphase II (TI/MII), and cumulus expansion. Blocking endogenous FGF10 with anti-FGF10 decreased (P<0.05) the percentage of oocytes reaching MII and impeded cumulus expansion. Lastly, expression profiles of putative cumulus and oocyte competency markers were examined for their involvement in FGF10-mediated responses. FGF10 affected (P<0.05) the expression of several genes, including CTSB in cumulus cells and BMP15 and GDF9 in oocytes. In summary, FGF10 improved oocyte developmental competence and subsequent embryo development. This activity may be explained, at least in part, by benefiting meiotic and molecular maturation in COCs. This work provides new insights into how FGFs, and FGF10 in particular, function to control oocyte competency in cattle and potentially other animals. This project was supported by NRICGP number 2008-35203-19106 from the USDA CSREES.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
The Sry high mobility group box containing protein, Sox3, is a transcription factor that is involved in spermatogenesis in mice. Male mice that lack Sox3 have fewer germ cells within the seminiferous tubules by two weeks after birth. This spermatogenic block occurs during spermatogonial differentiation in prepubertal animals. The targets of Sox3 during spermatogenesis have not been discovered. The receptor tyrosine kinase, Kit, is expressed in differentiated spermatogonia and has been used as a marker for this germ cell population. Through fluorescent-activated cell sorting, we determined Sox3 knockout male testes contain fewer Kit-expressing cells than their wildtype littermates. Kit signaling is important for proliferation and differentiation in many cell types, including differentiated spermatogonia. Bioinformatic analysis of the Kit promoter revealed five putative SOX binding sites. Addition of Sox3 to a differentiated spermatogonia cell line induces Kit expression and activation. These results led to the hypothesis that Sox3 may directly regulate Kit expression. Here we determine if Sox3 directly affects Kit expression by binding to its promoter. We also investigate how Sox3 and Kit affect germ cell proliferation and differentiation in vitro. This research is supported by NIH grant U01HD043425.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
Male and female germ cells differentiate to enter meiosis in response to an extrinsic cue provided by retinoic acid (RA). However, the specific factors and pathways downstream of RA signaling involved in the regulation of early gametogenesis are largely unknown. We identified Rhox13, a novel reproductive homeobox gene that is transcribed in the prenatal ovary and testis by embryonic day (E) 13.5. While translation begins in differentiating oogonia/premeiotic oocytes at E13.5, RHOX13 protein is not detected in male germ cells until postnatal day (P) 3. RHOX13 protein is present in differentiating spermatogonia/premeiotic spermatocytes from then on through adulthood. Since the temporal appearance of RHOX13 protein coincides with the onset of RA signaling in both the testis and ovary, we tested the hypothesis that RA would induce expression of RHOX13 protein in the testis. P1 testis explants were incubated in the presence or absence of all-trans RA for 24 hours. These experiments showed that Rhox13 transcripts were translated only in gonocytes from explants grown in the presence of RA. The results suggest that RA signaling is involved in alleviating the block to translational of preexisting Rhox13 transcripts in the neonatal testis. We therefore sought to identify a factor(s) involved in mediating the translational repression of Rhox13 transcripts downstream of RA signaling. Four lines of evidence suggested the involvement of the male germ cell-specific NANOS2 protein in repression of endogenous Rhox13 transcripts in the testis: 1) NANOS2 is present in gonocytes only at times when Rhox13 is NOT translated, 2) RA signaling both represses Nanos2 expression in the embryonic testis in vivo and induces translation of RHOX13 production in neonatal testis explants in culture, 3) a sequence resembling a Nanos response element consensus sequence resides in the Rhox13 3’ UTR, and 4) immunoprecipitation experiments using an antiserum to NANOS2 revealed an interaction of NANOS2 with Rhox13 mRNA. To test whether the repression of endogenous Rhox13 transcripts was dependent upon NANOS2, we performed immunohistochemistry using a RHOX13 antiserum on embryonic testis sections from Nanos2-heterozygous and -null mice. As in wild-type mice, RHOX13 was undetectable in sections from Nanos2-heterozygous mice. However, in E15.5 Nanos2-null gonocytes, RHOX13 protein was precociously expressed, revealing that NANOS2 represses the translation of Rhox13 transcripts in the embryonic testis. Translation of endogenous Rhox13 transcripts is repressed in the prenatal and neonatal testis by NANOS2, and the signal provided by RA alleviates this block, allowing the expression of RHOX13 protein. These results indicate that RHOX13 is the earliest known germ cell line-specific transcription factor expressed during differentiation of both oogonia and spermatogonia, making this homeobox protein a strong candidate for being a key regulator of genes involved in meiotic initiation. This research was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
Maternal RNAs, deposited by the mother into the egg, drive early embryogenesis when the newly formed embryo is transcriptionally inactive. Multiple mechanisms regulate synthesis of the zygotic RNAs and degradation of maternal RNAs during the maternal-zygotic transition. Recent studies in zebrafish have identified the role of microRNAs during the maternal-zygotic transition. MicroRNAs are short RNAs that bind to the 3'UTR of target mRNAs to repress their translation and accelerate their decay. Newborn ovary homeobox gene (NOBOX) is a transcription factor that is preferentially expressed in oocytes and essential for folliculogenesis in mice. NOBOX knockout mice are infertile and lack of NOBOX disrupts expression of many germ-cell specific genes and microRNAs. We recently reported the cloning and expression of bovine NOBOX during early embryonic development and our gene knockdown studies indicate that NOBOX is a maternal effect gene essential for early embryonic development. As NOBOX is a maternal transcript critical for development and NOBOX is depleted during early embryogenesis, we hypothesized that NOBOX is targeted by microRNAs for silencing and/or degradation. Using MicroInspector, an algorithm for detection of possible interactions between microRNAs and target mRNA sequences, a microRNA binding site (miR-196a) in the 3'-UTR of the bovine NOBOX mRNA was identified. Expression analysis of miR-196a during bovine oocytes and early embryonic development indicated that it is expressed both in oocytes and embryos and tends to increase at the 4-cell and 8-cell stages. To test if miR-196a regulates bovine NOBOX protein expression, HeLa cells were co-transfected with pcDNA3.1-bta-NOBOX with either pcDNA3.1-bta-miR-196a or pcDNA3.1 vector. Forty-eight hours after transfection, protein extracts were prepared and western blot analysis was performed using antibodies against NOBOX. Six independent transfection experiments were performed. Expression of bovine NOBOX protein was reduced in cells expressing bta-miR-196a compared to the control cells without bta-miR-196a, indicating that translation of NOBOX is repressed by miR-196a. To investigate the role of miR-196a in the regulation of endogenous NOBOX, we microinjected mature miR-196a mimic into bovine embryos at the presumptive zygote stage. Effect of miR-196a on bovine NOBOX protein was determined by immunostaining of 4-cell and 8-cell stage embryos. Expression of bovine NOBOX protein was reduced both at 4- and 8-cell embryos compared to uninjected embryos. Collectively these results suggest a functional role of miR-196a in regulating the expression of maternal (oocyte-derived) NOBOX during the maternal to zygotic transition in bovine embryos.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
Proper maintenance of germ cells and their differentiation are essential determinants of reproductive life. We discovered two novel germ cell specific transcriptional regulators, Sohlh1 and Sohlh2. Sohlh1 and Sohlh2 encode basic helix-loop-helix transcriptional regulators and are essential in early folliculogenesis and spermatogonial differentiation, as shown by individual knockouts. Sohlh1 and Sohlh2 individual knockouts show remarkably similar phenotypes. Here we examined the phenotype and gene expression in mice that lack both Sohlh1 and Sohlh2 transcriptional regulators. Antibodies against Sohlh1 and Sohlh2 show similar pattern of expression in both male and female germ cells. In males, Sohlh1 and Sohlh2 are expressed in spermatogonia, and co-expressed with Plzf and c-Kit. In females, antibodies against Sohlh1 and Sohlh2 label the same population of oocytes. In order to determine whether Sohlh1 and Sohlh2 physically interact, we utilized testes extracts to show that Sohlh1 and Sohlh2 co-immunoprecipitate, sugessting that Sohlh1 and Sohlh2 heterodimerize. In addition, Sohlh2 expression is not completely abrogated in Sohlh1-/- mice and vice versa. These data imply that residual Sohlh1 and Sohlh2 proteins were produced in each others mutant, possibly alleviating a more severe phenotype. We therefore analyzed Sohlh1-/-Sohlh2-/- double knockout mice. Sohlh1-/-Sohlh2-/- newborn ovary retains as many oocytes as wild-type. By 3-week of age, the double mutant ovary contains few if any oocytes as single mutants. We also analyzed testes development in double mutants. In postnatal day 7 double mutant testes, there is no obvious difference as compared to the wild type but spermatogonial differentiation is disrupted at 2-week of age and beyond, a finding similar to single gene mutants. The spermatogonias of single or double mutant retain Plzf expression, and continue to proliferate as shown by BrdU incorporation. Thus histological analysis revealed that male and female double mutants are a phenocopy of Sohlh1-/- and Sohlh2-/-. However, microarray analysis of double mutant and single mutants show a synergistic effect of the combined Sohlh1 and Sohlh2 deficiency on gene expression. In testis, although there is statistically no significant difference between Sohlh1-/- and Sohlh2-/-, Sohlh1-/-Sohlh2-/- versus wild-type shows nearly 4-fold probe change as compared to the case of single mutants versus wild-type. These studies indicate that Sohlh1 and Sohlh2 are purely essential for oocyte and spermatogonia differentiation, and function either cooperatively or independently in those pathways.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
The balance between mRNA synthesis and degradation determines the steady-state amount of mRNA abundance. In eukaryotic cells, removal of the 5' cap (decapping) exposes mRNAs to XRN1 endonuclease and triggers mRNA degradation. This critical step in cytoplasmic mRNA metabolism is catalyzed by DCP2 and regulated by DCP1A. In contrast to somatic cells, messenger RNAs are extremely stable during oocyte growth but oocyte maturation triggers a transition from mRNA stability to instability in which maternal mRNAs are extensively degraded. Little is known, however, about how the mRNA degradation machinery is regulated during oocyte growth and maturation. We found Dcp1a and Dcp2 transcripts are maternal mRNAs that are recruited during oocyte maturation leading to a dramatic increase in the amount of DCP1A and DCP2 protein in MII eggs. Dcp1a and Dcp2 mRNAs each contain putative cytoplasmic polyadenylation sites(CPEs) embedded in the 3' UTRs. Luciferase reporter assays, coupled with mutagenesis of the potential CPEs, confirmed that these sequences are responsible for the maturation-associated recruitment of Dcp1a and Dcp2 transcripts. In addition, a polyadenylation assay also revealed that the polyA tails of both Dcp1a and Dcp2 are elongated during maturation. No significant changes in the protein levels of other key components of mRNA degradation machinery involved in deadenylation (e.g., CNOT6) or 5'-3' degradation following decapping (e.g., XRN1) were observed, suggesting that an increase in decapping activity may be a key step controlling maternal mRNA degradation. Consistent with this proposal is that siRNA-mediated knockdown of Dcp2, but not Dcp1a, stabilizes maternal mRNAs in MII egg. We also found DCP1A is phosphorylated in MII eggs and de-phosphorylated after fertilization. CDC2A (CDK1) can likely phosphorylate DCP1A, because inhibiting CDC2A activity by roscovitine partially inhibits DCP1A phosphorylation. Because DCP1A can associate with other decapping enhancers and repressors, such as EDC3 and EDC4, post-translational modification of DCP1A by phosphorylation may change the affinity of DCP1A for DCP2 (or other components of decapping complex) and thereby modulate decapping activity in mouse eggs. This research was supported by NIH HD022732 and HD022681 to RMS.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
During maternal-to-zygotic transition in mammals, proteins in the unfertilized oocyte must be degraded for eliminating of the oogenic program and remodeling of an oocyte to a totipotent zygote. After fertilization, maternal proteins are suddenly degraded and new proteins are coordinately synthesized from the zygotic genome. The ubiquitin-proteasome system (UPS) is required for the degradation of such maternally stored proteins. However, the mechanisms underlying the structure and functions of the UPS at the maternal-to-zygotic transition are poorly understood. Here, we found that a mouse germ cell-specific protein acts as a chaperon protecting a short-lived proteasome maturation protein (POMP) from degradation and leads to the 20S proteasome assembly with POMP in the mouse zygote. DD2-2 and POMP were highly expressed in matured oocytes and fertilized zygotes where maternally stored proteins are actively degraded. These expression profiles coincided with profiles of polyubiquitinated proteins. Antisense knockdown of either DD2-2 or POMP expression caused embryonic arrest at the one-cell stage, and the development of the injected zygotes were even more severely inhibited by antisense knockdown of POMP. Zygotes following antisense knockdown of either DD2-2 or POMP failed to degrade cyclin B1, Mos, and two maternal-effect gene products (OOG1 and ZAR1) and significantly increased the accumulation of polyubiquitinated forms of those proteins. These findings indicate that DD2-2 is involved in the degradation machinery of polyubiquitinated proteins for normal embryonic development.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
Bisphenol-A (BPA) is a synthetic xenoestrogen used in polycarbonate plastics, food and beverage can liners, epoxy resins, toys, and dental sealants. BPA is present in 95% of tested human urine samples. As a potent estrogenic endocrine disruptor, BPA may adversely affect female reproductive system development, especially in low, environmentally relevant doses. However, few studies have examined the effects of low-dose BPA exposure on female reproductive system development, particularly ovaries. Previous studies have shown that oocytes from low-dose in utero-BPA exposed female fetuses display gross aberrations in meiotic prophase, including synaptic defects and increased levels of recombination. Oocytes are integral parts of primordial follicles. Mammalian females are born with a finite number of primordial follicles, of which a small fraction survives, while most die via apoptosis. Further depletion of primordial follicles could have deleterious effects on fertility and lead to a wide range of hormone-related diseases such as osteoporosis, early menopause, and cardiovascular disease. This study tested the hypothesis that in utero low dose BPA exposure (0.5 μg/kg and 50 μg/kg) changes the ovarian morphology and reduces the number of primordial follicles at birth. To test this hypothesis, CD-1 dams were dosed orally with BPA (0.5 μg/kg and 50 μg/kg) or tocopherol-stripped corn oil as a vehicle during the critical period of gonad development (embryonic day 10.5 to day 17.5). Ovaries were isolated from pups delivered by Caesarian section by embryonic day 19.0. Ovaries were then processed for histological evaluation using hematoxylin and eosin stains. Primordial follicles were counted in every 10th section. The results indicate that the ovaries from BPA exposed mice were less cellular with more dense and congested vasculization than ovaries from vehicle exposed mice. The percentage of healthy primordial follicles in the BPA exposed ovaries was significantly reduced compared to the vehicle control (vehicle control = 83.81 ± 1.99%; BPA 0.5 μg/kg/day = 64 ± 3.36%; BPA 50 μg/kg/day = 72.45 ± 1.65%, n=3, p<0.05). Further, the percentage of abnormal primordial follicles in the BPA exposed ovaries was significantly increased compared to the vehicle control (vehicle control = 16.19 ± 1.99%; BPA 0.5 μg/kg/day = 36 ± 3.36%; BPA 50 μg/kg/day = 27.55 ± 1.65%, n=3, p<0.05). Collectively, these data indicate that exposure to low levels of BPA in utero could affect the development of the ovary. This ovarian developmental defect could further affect the reproductive life of females born to BPA exposed mothers. Support: NIH T32ES07326 and NIH 1P20ES018163.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
Bisphenol A (BPA) is an industrial plasticizer that is utilized in the production of many products including baby bottles, food and water containers, medical supplies and dental fillings. BPA leaches from such containers during normal usage and these sources contribute to widespread and ubiquitous human exposure. Multiple surveys have found that 95% of adults and children have detectable concentrations of total urinary BPA. Increasing exposure to environmental synthetic estrogens, such a BPA, has been speculated to be involved in the increasing incidence of breast cancer in recent years. Early and chronic exposure to such environmental contaminates raises the possibility of the potential for long term health consequences. To test the effect of BPA exposure on mammary gland development and susceptibility to mammary carcinogenesis, we employed several approaches utilizing mouse models, including FVB/N, which has been described as susceptible to mammary gland cancer. In our first study, 8 week old FVB/N female mice were mated and checked daily for vaginal plugs. When evidence of mating and potential pregnancy was determined, pregnant female mice were treated daily with 25 μg/kg BPA or vehicle control by oral gavage beginning at gestational day 8 until parturition. Once born, the litters were culled to similar sizes and the mammary glands off the female offspring were harvested at various time points including: pre-puberty, puberty, post-puberty, pregnancy, lactation, and involution. At no time point that we examined were any significant differences in mammary gland development observed between the BPA treated and the vehicle cohort of offspring. To determine if fetal BPA exposure resulted in an increased susceptibility to mammary carcinogenesis, a similarly treated group of female offspring was subsequently exposed to the known mammary carcinogen, 7,12-dimethylbenz[a]anthracene (DMBA). Tumor latency was then assessed by weekly palpation. Among the three groups: vehicle control, low dose BPA (25 μg/Kg/day), and high dose BPA (250 μg/kg/day), both the low and high dose groups revealed a statistically significant increase in susceptibility to tumor formation. In a second study, we assessed the direct impact of BPA on overt mammary tumor growth using xenografts of MCF-7 human breast cancer cells in ovariectomized NCR Nu/nu female mice. MCF-7 cells require estrogenic input to form tumors in xenografts. Recipient females were ovariectomized at 8 weeks of age, and implanted with a placebo, estrogen (1.7 mg/60 day release), or low dose BPA pellet (37.5 mg pellet/60 day release). After recovery from surgery, 1 × 106 MCF-7 cells in matrigel were subcutaneously injected into the flanks of the mice. Both the estrogen-treated and the BPA-treated cohorts formed tumors by 7 weeks of age while no tumors were detected in the placebo cohort. Following tumor formation, a small cohort of mice was also treated with the estrogen antagonist, tamoxifen (1 mg/mouse/day). Tumor regression was observed in all mice, indicating that the estrogenic actions of BPA were driving tumor formation. These results indicate that BPA may increase mammary tumorigenesis through two mechanisms: one involves molecular alterations of the fetal mammary gland in the absence of morphological changes while the other entails direct promotion of tumor cell growth. Both results indicate that exposure to BPA at various time points increases the risk of developing mammary cancer in mice.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
BPA is a monomer that is used in the manufacture of polycarbonate plastics, epoxy resins and a multitude of consumer products. It easily leaches from the inner lining of tin cans and microwave containers into food during heating, from dental sealant into saliva and into beverages from repeated usage or contact with any acidic/alkaline content in polycarbonate bottles. The detection of BPA in biological fluids like maternal plasma, fetal plasma, placental tissue, amniotic fluid and umbilical cord blood have indicated that it can easily transverse the placental barrier. Perinatal period (gestation day (GD) 12-post natal day (PND) 21) encompasses the critical period of gestation and lactation wherein development of the hypothalamus-pituitary-testicular (HPT) axis occurs, especially development of testes that occurs on GD12. Studies on perinatal exposure of BPA have documented its effect on expression of steroid receptors (SRs) in target organs like the mammary gland and brain respectively, resulting in changes in morphogenesis and disturbances in sexual and mating behavior. However, no information is currently available on the effects of BPA in the male offspring of rats exposed to environmentally relevant doses during the critical perinatal period of development and growth of the reproductive tract. The present study aimed at investigating the effects of BPA exposure during this critical window of development (GD12-PND21) on fertility of F1 offspring and understanding its mechanism. Pregnant Holtzman rats (F0) were gavaged with BPA (1.2 and 2.4 μg/Kg /day ) from GD 12-PND 22. Pregnant female treated similarly with DES served as positive control. ON PND 75, fertility assessment of F1 males was performed by cohabitating them with normal cycling adult females. Copulated females were distributed in two sets, one was allowed to deliver the pups and the other was sacrificed on GD 20. The copulated females were examined for various fertility parameters. After fertility assessment male rats (F1) were sacrificed and serum hormonal profile, epididymal sperm count and motility was determined. Histological examination of testis and staging of seminiferous tubules Immuno histochemistry for steroid receptors and their co regulators was performed in testis and quantitative image analysis was performed to assess their levels of expression. A significant increase in the number of copulated females exhibiting resorption was observed at both dose groups of BPA. The adult F1 male offspring showed significant decrease in LH, FSH T and E2. A significant reduction in epididymal sperm count and motility was observed. At the testicular level majority of the seminiferous tubules exhibited sloughing of germ cells. Immunohistchemical localization studies demonstrated significant increase in ER with no effect on AR where as a significant reduction in the expression of SRC-1 and NCoR with a parallel increase in the expression of p/CIP and GRIP -1 was observed. Perinatal exposure to environmentally relevant dose of BPA affects the male gem line leading to impairment in the fertility of F1 male offspring.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
Polybrominated diphenyl ethers (PBDEs) are the members of brominated flame retardants which have been defined as major environmental pollutants. Previous studies have found that hydroxyl-PBDEs possess similar structures to thyroid hormones, which can enhance release of sex steroid hormones. However, its effects on testosterone release from rat Leydig cells are unclear. The aim of this study was to investigate the effects of PBDE-710 on steroidogenesis of steroid hormones in rat Leydig cells both in vitro and in vivo. Primary Leydig cells were prepared from adult male rats by percoll gradient method. Leydig cells were challenged with different concentrations of PBDE-710 to evaluate its influences on testosterone steroidogenesis. The concentrations of testosterone and pregnenolone in medium and/or plasma samples were measured by radioimmunoassay and enzyme-linked immunosorbent assay, respectively. Protein expression was examined by Western blot techniques, and nuclear translocation was determined by immunofluorence assay and Western blot assay, and steroidogenic acute regulatory protein (StAR) mRNA expression was analyzed by quantitative real time polymerase chain reaction (Q-PCR). In the in vitro study, PBDE-710 (5 and 15 ng/ml) increased testosterone release via stimulation of [1] intracellular cAMP level, [2] activation of epidermal growth factor receptor and mitogen-activated protein kinase, [3] P450scc enzyme activity (by increasing pregnenolone production), and [4] PKAα nuclear translocation and stimulate StAR gene expression. Moreover, a stimulatory effect on plasma testosterone level was also found in rats injected intravenously with PBDE-710 (750 ng/kg). These results suggest that PBDEs could affect testosterone production by acting directly on Leydig cells.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
Findings from numerous recent reports have supported the Barker hypothesis by demonstrating that the exposure to endocrine-disrupting chemicals (EDCs) during early development may alter the epigenetic programming of the genome and result in adult-onset disease. MXC is a well-studied EDC, that along with its metabolites, possesses estrogenic, anti-estrogenic, and anti-androgenic activities. Previous studies from our laboratory showed that fetal and neonatal exposure to MXC [between embryonic days (E) 19 and postnatal day (PND) 7] caused adult ovarian dysfunction at PND60. Multiple reproductive parameters were altered in the MXC-treated female rats similar to symptoms observed in women exposed to pesticides. In addition, disrupted folliculogenesis with reduced number of corpora lutea and mis-regulated expression of key ovarian genes were noted. We also demonstrated that significant changes in global and gene-specific methylation patterns (for example, of ERβ) in these ovaries were associated with the observed adult ovarian dysfunction. Previous data also revealed that critical events of early folliculogenesis such as the primordial-to-primary follicle transition was delayed in the 20 μg/kg/day MXC-treated ovaries while the 100 mg/kg/day MXC dose caused an acceleration in the progression of folliculogenesis, soon after exposure at PND7. The objectives of the current study were to evaluate the potential for age- and hormone-dependent regulation of the DNA methylation and expression patterns of ERβ. Rats were exposed to 20 μg/kg/day MXC or 100 mg/kg/day MXC between E18 and PND7 and the ovaries were examined immediately after exposure. Imunohistochemical staining and quantitation of ERβ expression revealed that there was a significant increase in its level (P < 0.01) at all follicular stages contrasting with the expression pattern in adulthood. At PND60, control ovaries had similar levels of ERβ in all stages of follicles except late antral follicles wherein the levels were reduced, in agreement with previously published reports. Interestingly, we observed a dimorphism in the expression of ERβ in the PND60, 100 mg/kg/day MXC-treated ovaries: very early stage follicles which are typically independent of gonadotropin regulation had normal levels of ERβ expression while gonadotropin-responsive preantral and early antral follicles had significantly reduced levels of expression (P < 0.001). These data suggest that a gonadotropin-dependent mechanism might be in play. Furthermore, we performed methylation profiling of the known promoter of ERβ gene in PND7 ovaries using bisulfite-sequencing PCR. Developmental exposure to MXC had no significant effects on the ERβ promoter regions contrasting with the significant hypermethylation observed at PND60. These studies demonstrate a definite age-dependent effect on the ERβ promoter methylation pattern after transient developmental MXC exposure. Research funded by ES013854 and ES005022.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
Prenatal testosterone (T) excess in sheep leads to ovarian dysfunction including increased ovarian follicular recruitment and persistence culminating in an adult Polycystic Ovary Syndrome (PCOS)-like reproductive phenotype. Growth factors expressed in granulosa cells such as antimullerian hormone (AMH) and c-kit ligand (KL) and the oocyte growth differentiation factor-9 (GDF-9) have been implicated in follicular differentiation. We hypothesized that the prenatal T excess would alter the relative expression of these growth factors thus contributing to aberrant follicular differentiation. Pregnant Suffolk sheep were given T propionate (100 mg, i.m. twice weekly) in cottonseed oil from days 30-90 of gestation (term is ~147 d). Controls (C) received vehicle. Changes in developmental expression of AMH and KL were determined in two ovarian sections (5μm thickness) <5mm apart from each of 6 animals/age/treatment group at fetal (days 90 and 140), postpubertal (10 months), and adult (21 months) ages by immunohistochemistry (IHC). Changes in oocyte GDF-9 expression were evaluated only during postpubertal and adult ages. Consecutive sections were used to relate changes in AMH and KL expression within the same follicle. Follicles were classified as primordial, transitional, primary, small preantral (SPF), large preantral (LPF), small antral (SAF) and large antral (LAF). Degree of expression was scored in a 0 to 5 scale and analyzed using T-test and linear regression. Fetal day 90 ovaries had only primordial to primary follicles while day 140 ovaries had few SPF (range/animal: 2-13), LPF (1-15), SAF (0-9), and LAF (0-1) follicles. All follicles at both ages expressed KL with AMH expressed only in the preantral and antral follicles. Number of preantral follicles studied ranged from 10-45/animal in 10 mo-old and 15-79 in 20 mo-old animals. Consistent with increased follicular recruitment, number of follicles beyond the SPF stage was higher in T females compared to C (P<0.05). Prenatal T treatment did not affect KL expression at any follicular stage. AMH expression increased with follicular stage from SPF to SAF in both groups of females. There was an age effect in AMH expression with it being higher at 20 mo compared to 10 mo in LPF and LAF in C and SPF, LPF and LAF in T females (P<0.05). AMH expression decreased between SAF and LAF (P<0.05) in C but not T females at 20 mo consistent with the altered follicular growth seen in T females. As a result, AMH expression was relatively low in LPF and high in LAF in T females compared to C (P<0.01). The percentage of LPF that had lower AMH expression (scale 0-1) was 1.8 times higher in the T group (C: 15.8 vs. T: 28.4%; P<0.05) while percentage of LAF that had lower AMH expression (0-1) was 1.4 times higher in the C group (C: 52.5 vs. 37.1%; P=0.057). Because AMH is known to reduce follicular sensitivity to FSH, the increased AMH expression in LAF in T females may contribute to the follicular persistence seen in T females. Changes in GDF-9 were evident only at 10 mo with GDF-9 expression being lower in LPF of T compared to the C group (P<0.01) suggestive of altered oocyte quality. The small sample size precluded statistical comparison of GDF-9 expression in LAF. Overall, the imbalance in paracrine environment stemming from altered AMH and GDF-9 expression in T females may underlie the follicular disruptions namely, recruitment and persistence, seen in the T females. Supported by P01 HD44232.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
Secreted phosphoprotein 1 (SPP1, osteopontin) is an extracellular matrix (ECM) protein that binds integrins to affect cell-cell and cell-matrix interactions including adhesion, migration, proliferation and survival. Secreted-protein-acidic-and-rich-in-cysteine (SPARC, osteonectin) is a calcium-binding matricellular glycoprotein known to regulate ECM interaction during tissue remodeling and repair. We developed a Spp1-/-/Sparc-/- double null mouse in which 50% fewer pups are present at birth than are found in utero on days 18-20 of gestation due to postpartum maternal cannibalization of the pups, suggesting that these pups are stillborn or exhibit severe birth defects. Indeed, hemorrhages were observed prominently in lower right limbs and distal region of the tail in many embryos; in a few embryos, hemorrhages were present the intestine and right foreheads. In order to gain insight into the developmental abnormalities and decreased litter sizes seen in double null litters, we evaluated the expression of both Spp1 and Sparc in the mouse uterus throughout pregnancy. Ten-week old outbred CD-1 female mice were mated to an intact male of the same strain (copulatory plug = Day 1 of gestation). Pregnant females were sacrificed and uteri collected on Day 4, 4.5, 5, 9, 10, 11, 12, and 16 of pregnancy. Cell-specific expression of Spp1 and Sparc mRNA in serial sections of mouse uteri was determined by radioactive in situ hybridization analysis. On Day 4 of pregnancy, Spp1 was expressed in central cells of the inner cell mass (ICM) of the embryo, in scattered immune cells in uterine stroma, and in luminal epithelium (LE) directly contacting the embryo within the implantation chamber. LE expression within the implantation chamber was absent by Days 4.5-5 of pregnancy, but remained in LE adjacent to the implantation chamber and in interimplantation sites, suggesting that Spp1 may be involved in adhesion during the initial phase of embryo attachment to the LE, but subsequently must be down-regulated at sites of attachment to allow embryo invasion. Sparc was expressed diffusely in the uterine stroma, in peripheral cells of the ICM, and was not expressed in LE through Days 4.5-5. When we utilized a model of delayed implantation to determine the effect of nidatory estrogen, localization of Spp1 and Sparc was identical to that of pregnancy. On Day 9 of gestation, Spp1 was expressed by uterine natural killer (NK) cells and LE, whereas Sparc was expressed by decidual cells and LE. On Day 10, NK cells and LE continued to express Spp1, while Sparc was localized to the stroma directly beneath the LE. On Day 12, Spp1 was also expressed by trophoblast giant cells, and Sparc was expressed in the Reichert's Membrane adjacent to the trophoblast giant cells. By Day 16 of pregnancy, both Spp1 and Sparc were expressed in adjacent but non-overlapping regions of developing fetal bone and cartilage. Because Spp1 and Sparc often have antagonistic roles during tissue remodeling and are localized to adjacent but different cell types during mouse pregnancy, we hypothesize that simultaneous deletion of both Spp1 and Sparc leads to disruption of cell-cell, cell-matrix and inter-tissue communication essential to normal conceptus development that is not observed when the genes are deleted individually. This study was supported by NIH grants R01 CA90920 and RO1 CA137091.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
Leukemia Inhibitory factor (LIF) is critical for embryo implantation in number of species including humans. In the mouse, LIF is expressed prior to implantation on day 4 of pregnancy and is essential to creating a uterine milieu conducive for embryo attachment. LIF deficient females are infertile due to defects in embryo attachment and decidualization. This failure is corrected by providing exogenous LIF on day 4 of gestation, with a single injection of LIF being able to replace nidatory estrogen, in progesterone supplemented ovariectomized pregnant females. LIF is produced by the endometrial glands, and it acts on the luminal epithelium via activation of the JAK-STAT pathway. The downstream signaling pathways and changes in gene expression induced by LIF in the luminal epithelium are still an enigma. We have undertaken a global gene profiling study to identify which genes and signaling pathways are regulated by LIF in the luminal epithelium of progesterone supplemented ovariectomized pregnant mice. At least 15 pathways, having functional relevance during embryo implantation, were found to change in the luminal epithelium in response to LIF. One of the pathways identified was Notch signaling. Candidate genes involved in this pathway were validated in ovariectomized B6C3HF1 females treated with sequentially with estradiol and progesterone to induce a state equivalent to day 4 of pregnancy. LIF was administered intraperitoneally on day 11 and animals sacrificed at 0, 1, 3 and 6hrs to isolate the uterine luminal epithelium. QPCR analysis indicated that NOTCH2 rather than NOTCH1 was down regulated by LIF. Jagged 1 (JAG1), hairy and enhancer of Split 1 (HES1) and the deltex homolog 4 (DTX4) in this pathway were altered by LIF. Our study indicates a putative role of Notch 2 pathway in embryo attachment. Analysis of Notch 2 pathway in the murine uterus will provide valuable clues to further deciphering embryo implantation; an enigma that is still waiting to be fully understood. This research was funded by the Institute of Medical Biology, A*STAR, Singapore.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
The uterine decidua, which differentiates from endometrial stromal cells following implantation, plays essential roles in supporting embryonic growth before the establishment of placenta. Here we show that decidual cells express Death Effector Domain-containing protein (DEDD). We initially found that DEDD suppresses the kinase activity of Cdk1 during mitotic phase of cell cycle, thereby adjusting the cell size. Our previous work has shown that DEDD interacts with Akt, a major element within the signaling cascade involving mitogen-related phosphatidylinositol 3-kinase (PI3K). This association maintains the protein stability of Akt, contributing to maintenance of glucose homeostasis in the body. In addition, we found that female DEDD-deficient (DEDD-/-) mice are completely infertile due to intrauterine embryonic loss. To elucidate the mechanism for the sterile phenotype, we first observed by histology that development of both the decidual zone and the edematous region surrounding decidua were defective in DEDD-/- uteri. Flow cytometry and in vitro decidualization of uterine stromal cells showed attenuated polyploidy of DEDD-/- decidual cells. We also detected, by histology and flow cytometry, excess apoptotic death of such immature decidual cells, which might cause embryonic demise prior to placentation. The reduction of Akt protein appears responsible for the inefficient decidualization, as forced expression of Akt recovered the polyploidy in DEDD-/- cells. Furthermore, our data implicated that DEDD binds to and maintains the stability of cyclin D3, an important element for decidual polyploidization. These results suggest that DEDD is indispensable for establishment of adequate uterine environment to support early pregnancy. This work was supported by the Uehara Memorial Foundation (to T.M.) and the Kanzawa Research Medical Foundation (to S.A.). The authors declare that they have no competing financial interests.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
Successful blastocyst implantation requires extensive alteration of uterine epithelial cells (UECs) followed by their removal in order for the implanting blastocyst to penetrate into the underlying endometrium. The dynamics of focal adhesions (FAs) may play a role during this process because FAs are specialised structural complexes that provide adhesion between the cell and its underlying basal lamina. The present study investigated the expression and distribution of FA proteins in rat UECs at the time of implantation and their hormonal control as well as their expression in blastocyst stage embryos. Immunofluorescence microscopy showed that the principal focal adhesion proteins, talin and paxillin, were localised along the basal cell surface of UECs on day 1 of pregnancy and significantly reduced from the site of FAs at the time of implantation allowing the UECs to become less adhesive to the underling basal lamina. This is thought to be a critical process in the removal of UECs, which enables the invasion of the implanting blastocyst into the underlying decidual cells. Ovariectomised rats treated under various hormone regimes showed that talin and paxillin were tightly regulated by ovarian hormones in UECs. In particular, FA disassembly seen at the time implantation was predominantly under the control of progesterone. FA dynamics are regulated by the clustering of specific integrin subunits and immunofluorescence microscopy and immunoprecipitation have shown that integrinβ1 and β3 subunits colocalise and interact with talin at the site of FAs on day 1 of pregnancy. Integrin β1 and β3 were markedly reduced from the site of FAs at the time of implantation. However unlike the other FA proteins, localisation of integrin β3 on the apical membrane of UECs increased significantly at this time with a corresponding increase of integrin β3 expression in the membrane shown by subcellular fractionation. Like talin and paxillin, the distributional change of integrin β1 and β3 in the UECs at the time of implantation were also under the control of progesterone. Immunofluorescence microscopy have shown that in the rat blastocyst, integrin β3 was concentrated around the nuclei and was not present in the inner cell mass and integrin β1 weakly stained in the cytoplasm of the trophoblast cells. Interestingly, another FA protein, FAK was not present at the site of FAs in the UECs during early pregnancy. Instead, FAK colocalised with a tight junctional marker ZO-1 on day 1 of pregnancy and increased in the apical portion of the UECs at the time of implantation, suggesting a possible role of FAK in regulating tight junctional structures. FAK in the rat blastocyst was present in the apical membrane and did not colocalise with a tight junctional marker occludin, suggesting that FAK has a different role in the rat uterus and the blastocyst. Taken together, our results show that disassembly of FA proteins play an integral part in the removal of UECs in order to establish successful implantation and is tightly regulated by ovarian hormones.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
Classical morphological studies on preserved uterine tissue sections have provided an anatomical understanding of the uterine vascular changes that occur during early pregnancy, but little is known regarding the physiological changes (i.e. uterine blood flow) that occur in vivo during pregnancy initiation in primates. Doppler ultrasound (DUS), the prevailing technique used to detect the fetal heartbeat, lacks the sensitivity needed to measure the low velocity vascular perfusion associated with implantation and subsequent early placentation. Contrast-enhanced ultrasound (CEU) is a non-invasive method with high spatial resolution used to assess quantitative differences in vascular blood flow and volume. Thus, the goals of this study were to use CEU to characterize and quantify the temporal changes in the vascular adaptations associated with early pregnancy, and to compare the information provided by CEU and DUS. Adult female rhesus macaques (n = 5) in the time-mated breeding program at ONPRC were pair-caged with males of proven fertility for 96 hr during the window of ovulation, and mating was confirmed by video recording. DUS and CEU began 14 days after the first day of pairing (DAP) and continued every 3 days until a fetal heartbeat was detected by DUS. The uterus of sedated animals was first visualized on a Voluson Expert 730 system (GE HealthCare) with a 2D trans-abdominal probe with Doppler capability. CEU was performed with a contrast-specific imaging technique, via an Acuson Sequoia system (Siemens), during a continuous intravenous infusion of lipid microbubble contrast reagent (Definity). Vascular blood flow and blood volume were quantified for the myometrium, endometrium and placental lobes from replenishment kinetics on the intensity data obtained after a high power pulse sequence destroyed all microbubbles in the acoustical beam. A functional corpus luteum formed (P4 > 4 ng/mL) in all females and 80% (4/5) became pregnant, as confirmed by a fetal heartbeat observed on 28.0±1.2 DAP. CEU clearly delineated the primary and secondary lobes of the placenta ~2 days apart (paired t-test; P<0.05), on 19.8±1.0 and 22.0±1.2 DAP, respectively. DUS was not able to differentiate between the placental lobes and detected endometrial thickening on 22.8±1.0 DAP, 3 days after (P<0.05) CEU identified the primary placental lobe. Visualization of the primary lobe correlated with the vaginal bleeding associated with implantation. The CEU calculated vascular perfusion was greater (P<0.05) in the primary placental lobe, which exhibited a 29% increase in relative blood volume and a 40% increase in relative blood flow, compared with the secondary lobe on the day of heartbeat detection. CEU identified greater flow through the hypertrophied endometrial spiral arteries supplying the placental lobes compared with other regions of the endometrium, whereas changes in the myometrium were not seen. In sum, CEU has defined the temporospatial changes in vascular perfusion associated with the anatomical vascular remodeling during early pregnancy in primates. Thus, CEU has great potential as a tool for basic researchers examining the peri-implantation period in the primate endometrium. Furthermore, CEU has promise as a future application for obstetricians, to resolve vascular complications that arise during pregnancy, including ectopic implantation and preeclampsia. Supported by NIH grants HD055744, HD18185, HD07133 and RR000163.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
Pregnancy-induced elevations in uterine blood flow are closely correlated with concurrent increases in plasma estradiol-17β levels and the expression/activity of eNOS. Indeed during late ovine gestation a physiologic cause and effect relationship has been established in vivo in that unilateral blockade of estrogen receptors (w/ICI 182,780) or nitric oxide synthase (w/L-NAME) locally and by a similar magnitude reduces uteroplacental blood flow. Estradiol-17β binds to estrogen receptors alpha and beta (ERα/β) and binding to these receptors results in either rapid effects (non-classical, non-genomic) and/or chronic effects (classical-genomic). Interestingly, numerous proteins that are involved in the estradiol-17β-induced rapid eNOS activation and nitric oxide production are located in specialized membrane domains called the caveolae of uterine artery endothelial cells (UAECs). Furthermore, treatment with the calcium mobilizing agonist ATP induced rapid eNOS activation which is accompanied by eNOS repartitioning with alterations in its multi-site phosphorylation state. We hypothesized that estradiol-17β induces eNOS activation via the non-classical ER receptor and initiates the sequential and coordinated re-partitioning and activation of eNOS with altered multi-site phosphorylation. UAECs were isolated from uterine arteries of pregnant ewes (gestational day 120-130; term 147d). Confluent passage 4 UAECs were treated with vehicle (Control) or with estradiol-17β (10 nM) for 10 min. The caveolae were fractionated from UAECs using sucrose density gradient centrifugation, and immunoblotting was utilized to study the multi-site phosphorylation state of eNOS relative to the domain specific levels of ERs. In control UAECs, total eNOS was predominantly located in the caveolar domain, whereas it was detectable both in the caveolar and non-caveolar domains in estradiol-17β-treated cells. In control UAECs, stimulatory P635eNOS and P1177eNOS were both not detected in any of the fractions whereas inhibitory P114eNOS was detected strictly in the non-caveolar domain. In response to estradiol-17β, stimulatory P635eNOS was detected in all cellular domains whereas stimulatory P1177eNOS was detected mainly in the caveolar domain and barely visible in the non-caveolar domain. The inhibitory P114eNOS was decreased by estrogen to low levels in the non-caveolar domain and was not detected in the caveolar domain. In control UAECs, ERs were detected in a higher abundance in the non-caveolar domain compared to the caveolar domain. estradiol-17β did not alter the distribution of the ERs. Estradiol-17β produces temporal and spatial re-partitioning of eNOS from the caveolar to non-caveolar domains and alters its multi-site phosphorylation state and their distributions in UAECs. Furthermore, since estradiol-17β actions are not calcium mediated, the striking similarities of these results to the ATP-responses illustrate alternative activation pathways of calcium mobilizing agonists. These findings may be critical to understand estradiol-17β-induced gestational vascular adaptations. NIH HL49210, HD38843, HL87144, HL70562, R25GM083252.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
The Na,K-ATPase is an ion transport enzyme essential in generating the gradients of Na+ and K+ that exists across the plasma membrane of most animal cells. The Na,K-ATPase consists of a series of isozymes, characterized by the association of different isoforms of two polypeptides, the α and β subunits. Four α (α1, 2, 3, and 4) and three β isoforms (β1, 2, and 3) have been identified in mammalian cells. The α4 polypeptide is a sperm-specific isoform that has structural and functional characteristics that are highly unique and essential to sperm physiology. To establish the role of α4 in vivo, we have engineered transgenic mice that express the rat α4 isoform in male germ cells under the protamine-1 promoter. A fusion construct between rat α4 and green fluorescent protein (GFP), placed at the C-terminus of the isoform was prepared (α4:GFP). The construct, placed into the pPrCExV-1 vector, was microinjected into the pronucleus of one-cell embryos, and transgenic animals were generated. Expression of the rat α4:GFP transgene was identified by immunoblot and immunocytochemistry using anti-α4 and anti-GFP antibodies in sperm from the over-expressing mice. The α4:GFP protein distribution localized to the middle and principal piece of the flagellum, corresponding to that of the native α4 isoform, and it also extended to the distal portions of the sperm tail. In agreement with augmented α4 levels in the transgenic animals, ATP hydrolytic activity and binding of fluorescently labeled ouabain to α4 were both increased. In contrast, activity of α1, the other Na,K-ATPase α isoform present in sperm remained unchanged. In addition, α4:GFP mouse sperm showed more membrane potential values that were more negative, suggesting higher Na,K-ATPase ion transport in the cells. Importantly, male gametes from α4:GFP mice displayed higher motility than wild type mice, as determined by computer assisted sperm analysis. Not only total sperm motility, but also progressive motility, straight line, curvilinear and average path velocities and amplitude of lateral head displacement were also increased in α4:GFP mice. Animals expressing the α4:GFP transgene exhibited a normal testis morphological appearance and had similar fertility than wild type animals. Altogether, these results support the notion that the Na,K-ATPase α4 isoform plays an important role in maintaining the Na+ and K+ gradients that are necessary for motility of spermatozoa. [Supported by NIH grants HD043044 and U54 HD055763].
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
Luminal acidification in the epididymis is critical for sperm maturation and storage. Clear cells express the vacuolar H+-ATPase (V-ATPase) in their apical membrane and are major contributors to proton secretion. We showed that this process is regulated via recycling of V-ATPase-containing vesicles. We now report that RhoA and its effector ROCKII are enriched in rat epididymal clear cells, where they are located in the cortical region and on intracellular structures. In addition, cortical F-actin was detected beneath the apical membrane and along the lateral membrane of "resting" clear cells using a pan-actin antibody or phalloidin-TRITC. In vivo luminal perfusion of the cauda epididymal tubule with the ROCK inhibitors, Y27632 (10-30 µM) and HA1077 (30 µM), or with the cell permeable Rho inhibitor, Clostridium C3 transferase (3.75 µg/ml) induced the apical membrane accumulation of V-ATPase and extension of V-ATPase-labeled microvilli in clear cells. However, these newly formed microvilli were devoid of ROCKII. ROCKII was also absent from microvilli that were induced by luminal perfusion with the cAMP permeant analogue, cpt-cAMP. In addition, Y27632 (30 µM) or HA1077 (30 µM) induced a decrease in the amount of F-actin versus G-actin detected by Western blotting. These results provide evidence that depolymerization of the cortical actin cytoskeleton via inhibition of RhoA or its effector ROCKII favors the recruitment of V-ATPase from the cytosolic compartment into the apical membrane in clear cells. In addition, our data suggest that the RhoA/ROCKII pathway is not directly involved in the formation of apical microvilli. We propose that inhibition of RhoA/ROCKII might be part of the intracellular signaling cascade that is triggered upon agonist-induced apical membrane V-ATPase accumulation and subsequent activation of V-ATPase-dependent proton secretion in clear cells. This research is supported by NIH grant DK38452.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
During male germ cell development, the vast majority of mRNAs is translationally repressed at the post-meiotic stage. While individual components implicated in the transcriptional and posttranscriptional control of specific germ cell gene expression have been identified, the mechanism that coordinates widespread translational suppression during germ cell differentiation remains unclear. In this study, we established that the cross-talk between miRNAs and actin-associated protein sets the tone for the mRNA-specific as well as general uncoupling of transcription and translation during post-meiotic germ cell maturation. Our study reveals that loss of the miRNA-processing enzyme Dicer function in haploid spermatids causes altered translational activation of several post-meiotic germ cell-specific transcripts, resulting in abnormal chromatin compaction and impaired fertility. We identified a specific miRNA-targeted actin-associated protein 2/3 complex (Arpc) subunit that plays a crucial role in this process as it sequesters germ cell-specific transcripts through their 3' UTR into translationally inert RNP complexes, and making them inaccessible to translational machinery. Interestingly, this actin-associated protein mediates translational suppression by interacting with GW182, a component of processing and chromatoid bodies, discrete cytoplasmic foci in the somatic and germ cells, respectively, believed to play an instrumental role in the storage and degradation of translationally suppressed mRNA. Collectively, our findings suggest that miRNAs are important checkpoint regulators that control timely expression of genes critical for post-meiotic germ cell differentiation and add a new dimension to the increasing role of actin and actin-associated proteins in post-transcriptional gene regulation. In light of recent studies showing an association between altered expression of actin-binding proteins and poor semen quality in human males, we propose that miRNA(s) targeting this Arpc subunit may serve as novel biomarkers for defective sperm and impaired male fertility. This research is funded by the NIH (HD053774-03).
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
Activin has a major impact on male and female reproduction by stimulating the synthesis and secretion of follicle-stimulating hormone. Non-pituitary effects of activin have also been reported, although the paracrine effects of this growth factor in several reproductive tissues are not well understood. The glycoprotein, follistatin (FST) binds to activin and prevents its interaction with the activin Type II receptor. Thus, to identify the paracrine functions of activin, we produced transgenic mice that overexpress FST, an intrinsic inhibitor of activin, under control of the mouse mammary tumor virus (MMTV) promoter. Although the MMTV-FST mice were constructed to assess the role of activin in mammary development and tumorigenesis, expression of the transgene was also observed in the testis and epididymis using real-time PCR and immunohistochemistry. FST protein expression occurred in the Leydig cells as well as the principal cells of the epididymis. This expression resulted in infertility. Of the 26 transgenic founder males that were obtained, only 3 males produced offspring, despite the appearance of vaginal plugs. Five transgenic males were then housed with proven FVB/N female breeders and subsequently 2 separate sets of super-ovulated females. None of these approaches resulted in pregnancies, while 100% of the wild type littermate males produced pups. Serum hormone analysis revealed a moderate elevation of follicle-stimulating hormone in the transgenic males (68.90 ± 6.82 ng/ml) as compared to wild type (WT) (58.26 ± 6.53 ng/ml) in one FST line examined. Similarly, luteinizing hormone levels were also significantly elevated in the same line (WT: 0.30 ± 0.21 ng/ml vs. FST 3.37 ± 2.25 ng/ml). Serum testosterone levels were also increased in a large cohort of FST transgenic founders compared to their WT littermates (9.19 ± 7.5 vs. 3.36 ± 4.8ng/ml, respectively). Although MMTV-FST males were infertile, females remained fertile, permitting expansion of transgenic mouse lines. Five different lines were obtained from the female transgenic founders and F1 offspring were similarly analyzed for fertility defects. Four of these lines maintained the male-specific fertility defect. To assess the basis for infertility, testes and epididymides were collected and examined histologically. Of the five lines examined, only one had a significant reduction in testicular weight while no difference in seminal vesicle weight occurred in any lines examined. Sperm collected from the caudae epididymides of 5 FST males had detached heads and/or were immotile. Light and electron microscopic examination of the testes and epididymides revealed impairment of fluid resorption and sperm transit in the efferent ducts and initial segment of the epididymis, as indicated by accumulation of fluid and sperm stasis. Consequently, a variety of degenerative lesions were observed in the seminiferous epithelium, such as vacuolation and early stages of mineralization and fibrosis. Together, these data suggest that activin may not only function in spermatogenesis, but also in fluid homeostasis in the male gonads and excurrent ducts, possibly via regulation of androgen-binding protein and/or contractility of these organs.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
Doublesex and Male abnormal-3 (Mab-3)-related transcription factor 1 (DMRT1) is an evolutionary conserved transcriptional factor that is expressed only in the testis, where it is produced in both Sertoli cells and germ cells. While deletion of Dmrt1 demonstrated its required role in postnatal testis development and fertility, less is known of its cell-specific functions within the Sertoli cells and germ cells. We hypothesized that DMRT1 participates in distinct signaling pathways in these cells. To elucidate these pathways, Dmrt1 transgenic mice (Dmrt1+/-;Tg [male]) carrying a yeast artificial chromosome transgene, in which the rat Dmrt1 cDNA is directed to Sertoli cells by the Wilm Tumor (Wt1) locus, were bred to Dmrt1-null mice (Dmrt1-/- [female]) to generate mice with specific rescue (Dmrt1-/-;Tg) of DMRT1 in Sertoli cells. Animals were screened by PCR and classified into 3 groups based on genotype: rescue (Dmrt1-/-; Tg), wild type (Dmrt1+/+) and knockout (Dmrt1-/-). Animals were evaluated at postnatal day 7 (P7) and, for each, one testis was processed for RNA isolation and the other for immunohistochemistry. Immunohistochemistry and quantitative polymerase chain reaction were used to confirm expression of DMRT1 and microarray expression profiles, respectively. RNA expression profiling was performed on murine Affymetrix gene chip (430V.2; 2/genotype) and data analyzed with Partek genomic suite (6.4). Probe intensity measurements were corrected for background, normalized and summarized using Robust Multi-array Average (RMA). Gene expression data sets were mapped into prospective pathways using functional annotation and identified molecular interactions datasets within Ingenuity Pathway Analysis (IPA) systems (version 8.5). Analyses of biological pathways specific to Sertoli cells or germ cells were assessed by comparing data between each of the genotypes. This revealed 22 networks shared by rescue and knockout, 11 networks unique to knockout and 6 networks unique to rescue (Score ≥ 3). Data unique to the knockout, which represents DMRT1 in germ cells, showed significant representation of pathways involving histidine, tyrosine and phenylalanine metabolisms, 4-1BB signaling (T-lymphocytes), germ-cell & Sertoli cell junction signaling and cAMP-mediated signaling (p-value ≤0.05). Likewise, the presence of Dmrt1 in Sertoli cells, represented by data unique to the rescue, showed significant representation of pathways involved in human embryonic stem cell pluripotency, PI3K/AKT signaling, PXR/RXR signaling and PTEN signaling (p-value ≤0.05). Top biological functions common to rescue and knockout included cell morphology, cellular development and lipid metabolism; while cell death and genetic disorders were top functions unique to knockout and molecular transport, cell growth and proliferation were top functions specific to rescue. The approach has provided insight on cell-specific pathways influenced by Dmrt1 in postnatal testis differentiation.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
Persistent Mullerian Duct Syndrome (PMDS) is a rare form of human male pseudohermaphroditism that presents with retained female reproductive tract tissue. Shortly after commitment of the bipotential embryonic mammalian gonadal ridge to develop along the male pathway, Sertoli cells differentiate in the testes and produce anti-Mullerian hormone (AMH; also known as Mullerian-inhibiting substance (MIS)), a TGFbeta family member that causes Mullerian duct (MD) regression in males. Because the embryonic female gonads do not produce AMH, the MDs differentiate into the oviduct, uterus, cervix, and cranial portion of the vagina. Homozygous deletion or knockdown of either AMH or its receptors, BMPR1A, ACVR1, and AMHR2 in mice leads to retention of the MDs and mutations in either AMH or AMH receptors are thought to account for 85% of patients with PMDS. The Wnt/beta-catenin plays an important role in the differentiation of the Mullerian duct. However, essential roles for Wnts or beta-catenin in normal Wolffian duct (WD) differentiation into the epididymus, vas deferens, and seminal vesicles have not been described. In this study, we have expressed a constitutively activated (CA) form of beta-catenin from a floxed allele that lacks the 3rd exon (Ctnnb1tm1Mmt) in the Mullerian mesenchyme of male mice under the control of Amhr2tm3(cre)Bhr/+ and observed focal retention of MD duct remnants in postnatal mice in both the epididymides and vasa deferens. Histological and immunohistochemical (alphaSMA and vimentin) analyses confirmed the presence of uterus in Amhr2tm3(cre)Bhr/+;Ctnnb1tm1Mmt/+ males. Further, histological analyses showed that the remaining MD-derived tissue in Amhr2tm3(cre)Bhr/+;Ctnnb1tm1Mmt/+ inhibited coiling of the efferent ducts in the caput and caudal regions of the epididymides. Analysis of cytokeratin expression also showed that the integrity of the pseudostratified columnar ductal epithelium in the cauda epididymides was compromised near the retained MD tissue in mutant mice. To confirm that testes do not influence the phenotype observed in mutant mice, we observed normal expression pattern of Sertoli (AMH and Sox9), Leydig (3beta-hsd), and germ (vasa) cell markers in testes of both control and mutant mice. To ensure that the action of CA nuclear beta-catenin in the MD mesenchyme is directly related to the inhibition of AMH signaling during MD regression, we performed organ culture assays with embryonic day E13.5 female urogenital ridges from control and mutant mice. We showed that addition of AMH to the female urogenital ridges causes nearly complete MD regression by the lack of expression of Wnt7a, an MD epithelial marker as evidenced by whole mount in situ hybridization. In contrast, we observed significant retention of Wnt7a expression in the mutant ridges with added AMH as predicted. These studies suggest that dysregulated Wnt/beta-catenin signaling in the MD mesenchyme might also be a contributing factor in this rare form of male pseudohermaphroditism.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
The corpus luteum (CL) undergoes dynamic changes during the estrous cycle. After the onset of luteolysis, the drastic decrease of progesterone secretion and disruption of endothelial and luteal cells are induced by prostaglandin F2alpha (PGF). Various types of immune cells such as neutrophils, eosinophils, macrophages, and CD4-positive and CD8-positive T-cells exist in the bovine CL and have essential roles in luteal function. In addition, many studies have examined to configure the molecular and cellular response of the CL to PGF from 0.5 h or later period after administration. However, PGF can reach the CL much earlier because oxytocin is released from the CL within 5 min. Therefore, we investigated the possible very rapid change in the number of neutrophils and eosinophils as well as mRNA expressions of chemoattractant for neutrophils (interleukin-8 [IL8] and CXCL1) and eosinophils (CCL5), CD68 as a macrophage marker, tumor necrosis factor alpha (TNF) as a major cytokine, and GM-CSF as a stimulator of growth and differentiation of granulocytes after PGF administration. Cows (n=5 for each time point) at the mid luteal phase (Days 10-12; Day 0 = estrus) were injected with PGF (0 min) and CLs were collected by ovariectomy at 0, 5, 15, 30 min, and 2 h. In chemoattractants, mRNA expressions of CXCL1 and IL8 (neutrophils) and CCL5 (eosinophils) increased at 15, 30, and 5 min after PGF administration, respectively. The mRNA expression of TNF increased at 15 min but CD68 mRNA decreased at 15 and 30 min after PGF administration. Also, mRNA expression of GM-CSF drastically decreased at 5 min and maintained at low levels. Interestingly, number of both neutrophils and eosinophils significantly increased already at 5 min after PGF administration when the level of their chemoattractants has not yet increased or just started to increase. This very rapid response of immune system after PGF injection led us to hypothesize that PGF may directly affect immune cells to migrate into the CL. To investigate the effect of PGF on the migration activity, blood samples were collected at 0, 5, 30 min and 2 h after PGF administration, and we tested the migration of neutrophils using transmigration assay with IL8 as a representative chemoattractant. Although IL8 (10 ng/ml) stimulated neutrophil migration in all time points after PGF injection, the response levels of migration was constant in all groups. Additionally, these neutrophils as well as peripheral blood mononuclear cells did not express PGF receptor mRNA, indicating that PGF can not directly attract immune cells into the CL. On the other hand, cell adhesion molecules (CAM) such as E-selectin, P-selectin, intercellular CAM (ICAM) and vascular CAM (VCAM) are essential when leukocytes attach to endothelial cells and migrate within the tissue. Contrary to expectation, mRNA expressions of E-selectin and ICAM were decreased at 5-30 min, although P-selectin and VCAM mRNA did not change at 5-30 min after PGF injection. These findings indicate that PGF rapidly activates migration of neutrophils and eosinophils without stimulation of their chemoattractants within 5 min after PGF administration. Taken together, immune cells such as neutrophils and eosinophils and their chemoattractants IL8, CXCL1 and CCL5 may be involved in the rapid response of luteolytic cascade in the cow, but some unknown mechanisms appear to exist to attract these immune cells so rapidly by PGF into the CL. Supported by Global COE and JSPS programs.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
The testis is a site of immune privilege, thereby providing protection for the developing germ cells from immune responses such as inflammation which can reduce fertility. Testicular macrophages are proposed to play a role in the maintenance of testicular immune privilege, but the mechanisms by which they might do this are incompletely understood. Macrophages are classified into two general phenotypes based on their responses to stimulation; "classically activated" (M1) macrophages, which undergo a strong inflammatory response, and alternatively activated (M2) macrophages, which have anti-inflammatory properties and can promote tissue repair. Our aim was to characterize the phenotype of rat testicular macrophages in order to define their potential role in immune privilege. We isolated purified populations of rat testicular macrophages by non-enzymatic dissociation and characterized their phenotype during short-term culture (2 to 16 hours), comparing to bone marrow-derived macrophages (BMMs). Real-time PCR and ELISAs were used to measure production of inflammatory regulators. M1 responses were stimulated with bacterial product lipopolysaccharide (LPS) and cytokine interferon-γ (IFNγ). M2 responses were stimulated using the anti-inflammatory cytokine interleukin-4 (IL-4). Testicular macrophages constitutively expressed mRNA for the anti-inflammatory cytokine Il-10 (6-fold higher than BMMs) and produced Il-10 protein at 8pg/ml whereas BMMs produced no detectable IL-10 protein. Il-10 mRNA was highest in testicular macrophages stimulated with LPS and IFNγ, at 6 hours and continuing to 16 hours, with protein peaking after 16 hours (500pg/ml). Testicular macrophages therefore are producing an anti-inflammatory factor in response to what is usually a pro-inflammatory stimulation. The anti-inflammatory cytokine Tgfβ was produced by testicular macrophages in relatively low amounts compared to BMMs, and this did not increase after stimulation. Testicular macrophages had a greatly diminished response to LPS/IFNγ at 2 hours, in terms of the pro-inflammatory products tumor necrosis factor α (Tnfα) and Il-1β with 172- and 109-fold increases in mRNA, respectively, compared to 1900- and 1330-fold increases measured in BMMs. Testicular macrophages had little Il-10, Tnfα, or Il-1β response to IL-4 at 2 hours, but produced the suppressor of cytokine signaling, Socs1, suggesting a novel role for this factor in the testis. Interestingly, at the longest culture time-point, IL-4 treatment of testicular macrophages lead to a downregulation of Il-1β mRNA, but no significant change in IL-10 levels. These results provide evidence that testicular macrophages have an M2 or alternatively activated phenotype, involving IL-10, but not TGFβ, consistent with a role in supporting testicular immune privilege. M2 macrophages contribute to fibrosis in other tissues, implicating a possible involvement of these macrophages in testicular fibrosis, which is a major cause of testicular damage in conditions such as vasectomy, cryptorchidism and infertility. This research was supported by ARC Discovery Grant (DP0664729).
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
SPP1 is secreted by activated lymphocytes, is chemotactic for monocytes, and is expressed at implantation sites that undergo extensive angiogenesis, specifically by uterine stromal immune cells beginning on Day 10 of pregnancy in pigs. Angiogenesis is classically thought to be derived from endothelial cells that line the pre-existing vasculature. However, recent studies have revealed the existence of endothelial progenitor cells (EPCs) that are mobilized to populate newly-vascularized tissue, differentiate into mature endothelial cells, and incorporate into newly-forming blood vessels. We previously detected fragments of SPP1 known to be angiogenic in endometria from Days 9, 12, and 15 of pregnancy using Western blot analyses. We therefore investigated whether SPP1 deposited into the uterine stroma facilitates adhesion, migration and recruitment of EPCs for new blood vessel growth during pregnancy in pigs. EPCs were obtained from blood in piglets within 6 h of birth. Blood samples were diluted in PBS and layered over a Ficoll-Paque density gradient from which the mononuclear cell layer was collected, washed, and cultured on flasks precoated with human fibronectin. Cell populations were characterized using RT-PCR analyses using primer sets specific for CD14, CD31, CD34, CD45, CD133, vascular endothelial growth factor receptor 2 (VEGFR2), vascular endothelial cadherin (VE-cadherin; CDH5), and integrin a4 (ITGA4), integrin a5 (ITGA5), integrin av (ITGAV), integrin B1 (ITGB1) and integrin B3 (ITGB3) subunits. Expression of CD31, VEGFR2, CDH5, and ITGA5 increased from passage 1 to passage 16, while expression of CD14, CD45 and ITGB3 decreased with passage. CD133 and ITGA4 were undetectable, while CD34, ITGAV and ITGB1 remained constant. These changes appear to show a phenotypic change from circulating monocytes to mature endothelial cells. Expression of von Willebrand factor (vWF), CDH5, and vascular endothelial growth factor receptor 1 (VEGFR1) were confirmed using indirect immunofluorescence. In vitro adhesion and migration assays demonstrated that SPP1 stimulated dose-dependent migration and adhesion of EPCs, and the adhesion was integrin-dependent. Affinity chromatography experiments revealed direct binding of integrin avB5 on EPCs to SPP1. Invasion assays using three-dimensional collagen matrices demonstrated that EPCs did not penetrate when seeded onto the matrices alone. However, when combined with human umbilical vein endothelial cells (HUVECs), EPCs successfully incorporated into HUVEC networks and were periodically located at the leading edge of invading sprouts. Collectively, these results indicate that EPC adhesion and migration is directed by SPP1, suggesting a potential role for cross-talk of EPCs and extracellular matrix in angiogenic events during pregnancy. Supported by National Research Initiative Competitive Grant 2006-35203-17199, USDA Cooperative State Research, Education, and Extension Service.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
Ovulation is likened to an inflammatory response, consisting of prostaglandin production, cytokine secretion, increased vascular permeability and the infiltration of immune cells; with many of these changes having been shown to contribute to the process of follicle rupture. Mice that are null for the progesterone receptor (PRKO) exhibit a complete and specific failure to ovulate. To determine whether inflammatory processes linked to follicle rupture are altered in ovaries of PRKO mice at the time of ovulation, we examined the expression of inflammatory genes and localized immune cells within ovaries of female PRKO mice that had received eCG followed by an ovulatory dose of hCG. Immediately prior to ovulation, 10h post-hCG, the expression of macrophage cell surface markers CD68, CD80 and CD86 was not different in ovaries from PR+/- compared to PRKO mice. In addition detection of macrophages by immunohistochemistry showed their localization around large pre-ovulatory follicles was similar in PR+/- and PRKO ovaries. T cell abundance and activation status was investigated by measuring ovarian expression of specific T cell surface markers 10h post-hCG. Expression of CD4 and CD8 were not different; however CD28, a T cell co-stimulatory receptor, was 2-fold higher in ovaries of PRKOs indicating that T cell activation may be heightened. In support of this, CCR4- a T cell receptor that regulates Th2 polarity, was decreased 3-fold in PRKOs. Localization of neutrophils by immunohistochemistry showed that there are low numbers of ovarian neutrophils in mice treated with eCG only, or 12h post-hCG; however neutrophil numbers are dramatically increased in the ovary at 16h post-hCG. The increase in ovarian neutrophil numbers at the time of ovulation did not occur in PRKO mice. For comparison, neutrophil localization in COX-2 (Ptgs2) null mice which also fail to ovulate was normal. To investigate ovarian production of neutrophil chemotactic factors we performed Boyden chamber chemotaxis assays and found that extracts of ovulating ovaries (16h post-hCG) produced neutrophil chemotactic factors while extracts of ovaries prior to hCG treatment did not. However, extracts of ovaries from PR+/- and PRKO mice at this timepoint exhibited similar neutrophil chemotactic activity indicating that lack of chemotactic factors is not responsible for reduced neutrophil numbers in PRKO ovaries. Further, expression of neutrophil growth factors G-CSF (Csf3) and GM-CSF (Csf2) were not different in PRKO compared to PR+/- ovaries 10h post-hCG. In contrast, expression of both E-selectin and VCAM-1, leukocyte adhesion receptors expressed on endothelial cells in response to inflammatory stimuli, was significantly decreased in PRKO ovaries. PRKO ovaries at 10h post-hCG also exhibited dramatically reduced mRNA expression of cytokines IL-1b, IL-6 and IL-10, as well as Ptgs2, which is likely to contribute to the lack of E-selectin and VCAM-1 and other inflammatory processes within the ovaries of these mice. These results clearly demonstrate that specific aspects of the inflammatory profile induced at ovulation are disrupted in PRKO mice, namely cytokine expression, leukocyte adhesion receptor expression, neutrophil infiltration and possibly T cell activation; and provide evidence for cellular mechanisms by which immune cells contribute to the process of ovulation.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
Thymectomy on day 3 after birth depletes regulatory T-cells leading to multiple independent organ-specific autoimmune diseases in specific strains of mice. Autoimmune diseases of the ovary and the stomach are components of the autoimmune polyendocrinopathy syndrome that occurs in mice following experimental perturbation of the normal immune system. Neonatal thymectomy is a well established mouse model shown to have similarities with human autoimmune primary ovarian insufficiency or POI (earlier called premature ovarian failure or POF). In the present study we identified several ovarian antigens involved in this model. Sera from neonatally thymectomized (NTx) mice (A/J and B6 x A/J strains) on day 3 after birth (n = 12, labeled as NTx1, NTx2...NTx12) was used to screen for the presence of anti-ovarian antibodies (AOA) in ELISA against total ovarian extracts. Sera from 3 sham operated mice served as control. Five of the 12 mice (NTx1, NTx2, NTx6, NTx11 and NTx12) showed the presence of circulatory AOA. Using mouse oocyte extracts as targets in Western blot analysis, multiple antigenic targets were observed. Proteins with approximate molecular weights viz., for animal NTx1 was 70 and 100 kDa; for NTx2 was 30 and 120 kDa; for NTx6 was 60 kDa; NTx11 was 25, 80 and 120 kDa and for NTx12 was 25, 68 and 80 kDa targets were identified. The 25 and 80 kDa protein targets were common to animals NTx11 and NTx12 and protein of 120 kDa was common to animals NTx2 and NTx11. None of the 3 sham operated mice showed any immunoreactivity to any of the proteins in the oocyte extract. Using total ovarian extracts from mouse, rat and human ovaries in Western blot analysis, proteins 120 kDa and 25 kDa were conserved in all the 3 species; protein 30 kDa in mouse and human; and protein 60 kDa in mouse and rat. By indirect immunofluorescence (IIF), 4/5 sera (NTx1, 2, 6 and 11) demonstrated that the presence of anti-oocyte antibodies (AOcA) in primordial, primary, secondary as well as tertiary follicles. Sera from 1 mouse (NTx12) also showed the presence of anti-zona pellucida antibodies in the secondary follicle stage onwards. None of the 5 sera immunoreacted to cells of the corpora lutea, thecal layers and granulosa cells. Using IIF on early mouse embryos 4/5 sera immunolocalized to the 1-cell, 2-cell, 4-cell, 8-cell embryos and morula. None of the 3 sham operated mice showed immunolocalization to any development stage of embryogenesis. Animal models of autoimmune POI/POF may yield important insight into both potential mechanisms of autoimmune disease development and ovarian antigens that may affect disease progression. Specific immunodominant antigens may provide screening reagents for early stages of POI/POF in humans.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
The initiation of pregnancy is marked by a number of immune modifications occurring in both the uterus and the embryo, which can affect embryo viability and therefore the maintenance of pregnancy. During the peri-implantation period, immune cells are exposed to endocrine and paracrine signals, such as conceptus-derived interferon-tau (IFNtau), which support feto-placental development. The aim of the current study was to characterize the immune expression signature in bovine endometrium during early pregnancy. To this end, beef cross heifers were synchronized and assigned to either a cyclic group or were artificially inseminated with fertile bull semen to generate a pregnant group. Half of the heifers in each group received a PRID on Day 3 after estrus, which elevated progesterone (P4) concentrations from Day 3.5 to 8 (p< 0.05). Endometrial tissue samples (n=5 animals per group) were collected at day 5, 7, 13, 16 from the resulting four treatment groups: (i) cyclic, normal P4 (ii) cyclic, high P4 (iii) pregnant, normal P4 (iv) pregnant, high P4, and the mRNA expression profile of 28 cytokines and non classical MHC class I genes were analysed by quantitative real-time PCR. Results showed that 21 of the 28 genes studied were significantly (P < 0.05) modified by pregnancy especially at day 13 and day 16: 10 were up-regulated (e.g. Interleukin IL-12alpha, JSP-1, radical S-adenosyl methionine domain containing-2 RSAD2) and 8 were down-regulated (e.g. Interferon IFNgamma, IL-11, and Tumor necrosis factor TNFalpha). Strikingly, we observed a shift from a down-regulation of expression during the morula/blastocyst stage (day 5/day 7) to an up-regulation during conceptus elongation (day 13/day 16) in 3 genes; Colony stimulating factor CSF-1, IL-12beta and pentraxin PTX3. Furthermore, our data revealed that these genes were also regulated by P4; 14 genes were up-regulated (e.g. IL-15, IL-18, Leukemia inhibitory factor LIF) and 5 genes (IL-11, IFNalpha, IFNgamma) were down-regulated by high P4. Some of these genes (IL-15, LIF) are known to be T helper 2 (Th2) promoting cytokine production. For 9 of the genes, this up-regulation by P4 occurred around the time of maternal recognition of pregnancy, which may be correlated to copious IFNtau secretion at that time. For example, TGFbeta and TNFalpha expression was decreased on day 13 of pregnancy, which could be associated with a repressive effect of the conceptus on the endometrium, exerted by IFNtau. It is well known that these two factors act on cellular growth and differentiation, so this quelling could limit endometrial adhesive properties and therefore prevent a precocious attachment of the embryo to the endometrium before the window of implantation. Furthermore, TNFalpha, is associated with a Th1 response and is considered as a pro-inflammatory cytokine. We also observed an up-regulation of the non classical MHC class I genes JSP-1, NC-2 and NC-4, on day 16 of pregnancy. Again, this may be due to the action of IFNtau and may help to create a favourable immunological environment, which would protect the implanting embryo from lysis by uterine NK cells. In conclusion, the present study details immunomodulatory events of early pregnancy and confirms the Th1/Th2 paradigm by highlighting some new immune factors including JSP-1, NC-2, NC-4, PTX3 and RSAD2, that may play a crucial role during early pregnancy and in particular around the time of maternal recognition of pregnancy. Research supported by SFI-PICA Program B813 and SFI-PI Program 06/IN1/B62.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
It is well-established that mammalian embryos produced in vitro have a reduced developmental potential as compared to embryos produced in vivo. It is known that embryos cultured in the laboratory can display aberrant gene expression, altered epigenetic states, reduced developmental potential and perturbed patterns of fetal growth. Mammalian embryos undergo a tremendous amount of chromatin remodeling during cleavage development, from protamine-histone exchange at fertilization to global changes in DNA methylation profiles. We hypothesize that the window of cleavage development may mark a critical time when the embryo is particularly sensitive to environmental impacts that affect its epigenetic state. Understanding the link between epigenetic states and embryo developmental competence is therefore critically important to improving in vitro embryo production. To this end, our laboratory focuses on investigating the roles that chromatin modifications serve during cleavage development in the porcine embryo. We find that porcine embryos possess a unique epigenetic signature with regard to two histone modifications, methylation at the lysine 9 and lysine 27 residues of histone protein H3 (H3K9 and H3K27, respectively). Questions that we are currently addressing include: Which histone modifying enzymes can access the nucleus during cleavage development? Do all histone modifying enzymes function during cleavage development? Understanding how chromatin is modified during cleavage development, how these modifications differ between mammalian species and how these modifications can be perturbed during in vitro embryo manipulation are central to developing methods that improve embryo production.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
It has long been assumed that the human placenta has significantly reduced CYP17 expression that limits its capacity to produce estrogens without the continuous supply of androgenic precursors from the fetus. Immunohistochemical studies from our laboratory have demonstrated immunoreactivity for CYP17 in the cytoplasm of human syncytiotrophoblasts, which we have confirmed by western blot analysis. OBJECTIVES: To assess the biosynthetic capacity of the human placenta to express CYP17 and generate androgens de novo, and to elucidate under which signaling pathways these may be regulated. METHODS: Two cell types were utilized in all experiments, human primary placental cells extracted from mid-trimester placentas and Jeg3 from a human placental line. Cells in baseline experiments were grown in media without the addition of steroid precursors. The media was exchanged and collected daily for steroid measurement via radioimmunoassay. To optimize the maximal enzymatic activity, experiments were duplicated in the presence of 22-hydroxycholesterol (5 M). CYP17 expression was quantified by RT-PCR. Protein kinase A pathway activator and inhibitor, forskolin (10 M) and H89 (10 M), respectively, were used to assess this pathway's role in placental androgen modulation. RESULTS: 17-hydroxyprogesterone, androstenedione and estradiol were all easily detected and quantified in both cell types. The daily production of 17-hydroxyprogesterone and androstenedione ranged from 0.05-0.5 ng/mg of cellular protein, with a higher production in primary placental cells. Estradiol generation was similar in both cell types, 100-200 pg/mg of protein per day. Dehydroepiandrosterone was undetectable throughout. No discrete trends in the rate of steroid production were noted over five days of cell culture. In the presence of 22-hydroxycholesterol, 17-hydroxyprogesterone and androstenedione production increased 5-8 fold. CYP17 mRNA was easily detected by standard RT-PCR. When treated with forskolin, CYP17 mRNA levels increased 10-fold from baseline, meanwhile H89 suppressed its expression. Co-treatment with forskolin and H89 completely inhibited the inductive effect of forskolin. CONCLUSION: Human placental cells are capable of producing androgens and estradiol de novo in the absence of a fetal compartment. CYP17 mRNA is easily detected and its expression is regulated at least in part by the PKA pathway.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
Modification of oocyte-glycoproteins by oocyte-specific ablation of complex N- and O-glycans has revealed new roles for the oocyte in the regulation of fertility and ovarian function. Floxed alleles of T-syn and/or Mgat1 were deleted specifically in the oocyte using a ZP3Cre recombinase transgene thus preventing the generation of complex O- and N-glycans respectively. In mice that generated oocytes lacking complex O-glycans, ovulation rate was sustainably increased revealing an unexpected role for oocyte O-glycans in regulating fertility. Analysis of T-syn mutant ovaries revealed increased numbers of follicles although follicle atresia was not decreased. Interestingly, prepubertal females treated with exogenous gonadotrophins had a reduced ovulation rate. Therefore, we propose a model of slowed follicle development which results in follicles accumulating. Deletion of T-syn also resulted in the generation of numerous multiple-oocyte follicles (MOF). However, MOFs do not ovulate multiple eggs and thus do not contribute to the increase in fertility. These MOFs are generated by follicles joining revealing a role for oocyte-glycoproteins in the regulation of follicle integrity. Mice generating oocytes lacking both T-syn and Mgat1 (double mutants; DM) undergo premature ovarian failure by 3 months of age. DM female fertility was markedly reduced with only 30% producing a single small litter. By 12-week, gonadotrophin levels were elevated, ovarian steroids were decreased and ovaries were almost completely devoid of growing follicles. These mice have revealed new roles for oocyte-generated glycoproteins in the regulation of ovulation rate, follicle integrity and ovarian function. (This research was supported by both the NIH and the MRC).
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
Although the health of all Americans increased significantly during the twentieth century, significant gaps in health status and in access to health care continue to exist between white Americans and African Americans, Native Americans and Hispanic Americans. There are multiple causes for these disparities, including low income, lack of health insurance, low health literacy, inadequate utilization of the health care system, and insufficient racial and ethnic diversity of the nation's health professionals. The Sullivan Alliance was formed in 2005 to increase the racial and ethnic diversity of health professionals in the United States. The methods developed to accomplish this goal and the progress to date will be presented, along with plans for future activities.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
The corpus luteum (CL) secretes the hormone progesterone which is essential for the establishment and maintenance of pregnancy in mammals. Prostaglandin (PG) F2alpha is secreted by bovine endometrial and luteal tissue late in the estrous cycle to cause regression of the CL. Often the presence of an embryo fails to control secretion of PGF2alpha after breeding leading to regression of the CL and termination of the pregnancy. The omega-3 fatty acids eicosapentaenoate (EPA) and docosahexaenoate (DHA) have been shown to reduce PG synthesis in several tissues. The objective of the current study was to determine if the addition of EPA and DHA when added to the diet would be incorporated into luteal tissue. Seventeen 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 EPA and DHA; n = 9 cows) or corn gluten meal at 6% dry matter intake (n = 8 cows). Jugular blood samples were collected immediately before the start of supplementation and every 7 days thereafter for 49 days to monitor changes in plasma omega-3 fatty acids. Body weights were also taken immediately before start of supplementation and weekly thereafter throughout the study. 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 approximately 0.5 g of luteal tissue was stored at -80°C until analyzed for omega-3 fatty acid content. Initial and ending body weights did not differ (P > 0.10) between cows supplemented with fish meal and those with corn gluten meal. Plasma EPA was greater (P < 0.05) beginning at day 7 of supplementation and DHA was greater (P < 0.05) beginning at day 28 of supplementation for those cows receiving fish meal in the diet. Fish meal supplementation resulted in greater than 260% increase in luteal omega-3 fatty acid content (P < 0.05). Further, there was a 12% reduction in luteal arachidonic acid (AA) in tissue obtained from cows supplemented with fish meal (P < 0.05). Fish meal supplementation resulted in a reduction in luteal -omega6:-omega3 ratio (P < 0.05). Our data show that fish meal supplementation increases luteal omega-3 fatty acid content and reduces AA, the precursor for PGF2alpha. The increase in luteal omega-3 fatty acids may reduce PGF2alpha secretion after breeding resulting in increased reproductive performance in dairy and beef cows. This project was supported by National Research Initiative Competitive Grant no. 2008-35203-19099 from the USDA Cooperative State Research, Education, and Extension Service and Omega Protein Corporation.
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Biol Reprod. 2010 Nov;83(1 Suppl):3.
Prostaglandin F2alpha (PGF) and reactive oxygen species (ROS) have been implicated in the functional and structural regression of the corpus luteum (CL). However, the role of ROS in the regulation of bovine CL function remains unclear. Treatment of luteal tissue with PGF increases the generation of ROS and induces apoptosis. Superoxide dismutase (SOD) is one of the most important antioxidative enzymes that control the local generation of ROS. Interestingly, a decrease in intracellular SOD activity has been associated with an increase in luteal ROS generation and decrease of progesterone production by the CL. Thus, SOD seems to play important roles in regulating the local mechanisms that control the luteal function and the luteolytic action of PGF in bovine CL. The present study was performed to determine whether SOD is regulated in bovine CL throughout the estrous cycle and during PGF-induced luteolysis. In addition, to determine the physiological relevance of PGF and ROS in the regulation of luteal endothelial cell (LEC) function, we examined the effects of PGF and hydrogen peroxide (H2O2) on SOD expression and SOD activity in cultured bovine LECs. In the first experiment, we examined the changes in the expressions of SOD mRNA and protein, and total SOD activity in bovine CL. Stage-dependent changes: CL tissues were collected from Holstein cows by colpotomy at five different luteal stages; early (Days 2-3 after ovulation), developing (Days 5-6), mid (Days 8-12), late (Days 15-17) and regressed (Days 19-21), n=4 CL/stage. Time-dependent change after PGF treatment: The levels of SOD mRNA and protein expression, and SOD activity in CL tissues were analyzed at 0, 0.5, 2, 12 and 24 h after administration of a luteolytic dose of PGF analogue (0 h) in cows on Day 10 of the estrous cycle (n=5, cows/time-point). SOD protein was detected in CL tissue throughout the estrous cycle. The level of SOD protein was greater in the developing- and late-luteal stages than in the other stages (P<0.05), and the lowest in the regressed-luteal stage (P<0.05). The lowest SOD activity was detected at the early luteal stage, and increased from the early to the mid luteal stage, the highest activity was observed at the late luteal stage. An injection of PGF induced a significant increase in protein expression of SOD protein between 0.5 and 12 h, but inhibited SOD expression at 24 h (P<0.05). SOD activity increased after PGF injection and remained higher between between 0.5 h and 24 h than at 0 h (P<0.05). In the second experiment, cultured LECs were exposed to PGF or H2O2 for a short-term (2 h, mimicking functional luteolysis) and a long-term (24 h, mimicking structural luteolysis). SOD mRNA expression was stimulated by PGF (1-10 µM) and H2O2 (10-100 µM) at 2 h (P<0.05). PGF and H2O2 stimulated SOD protein expression and total SOD activity at 2 h (P<0.05), whereas PGF and H2O2 inhibited SOD mRNA and protein expressions at 24 h (P<0.05). In addition, H2O2 stimulated PGF biosynthesis at 2 h and 24 h, suggesting the presence of a local amplificatory system for PGF and ROS in bovine LECs. The overall findings suggest that SOD plays important roles in controlling the intraluteal PGF and ROS concentrations in the bovine CL during the estrous cycle as well as during luteolysis.This research was supported by JSPS Grants (No. 19580326)
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
The corpus luteum (CL) is a transient endocrine gland in which angiogenesis and vessel regression occur during cyclic ovarian function in adult females. The CL differentiates from the follicle wall after ovulation by tissue remodeling and extensive neo-vascularization. Moreover, the integrity and function of the luteal vasculature decline during regression of the CL near the end of the cycle. The Notch family pathway, particularly Delta-like ligand 4 (Dll4) and its receptors Notch1 and 4 were recently identified as novel factors involved in angiogenesis. This system regulates cell fate decisions, including proliferation and death. The transmembrane receptors are cleaved upon binding of their ligands, leading to the release of the intracellular domain, which translocates to the nucleus where it functions as a transcriptional coactivator of regulatory genes of cellular fate. This processing requires the activity of two proteases, namely tumour necrosis factor alpha-converting enzyme (TACE) and presenilin/gamma-secretase. Given the implication for angiogenesis in the CL lifespan, we investigated the role of the Notch pathway in luteal function and regression of the CL during pregnancy in rats. To determine if the Notch signaling pathway is implicated in CL regression during pregnancy, pregnant rats were injected with a luteolytic dose of PGF2alpha (400µg, ip) or vehicle on day 19 of pregnancy. Ovaries were removed 4 and 24 hours after the treatment and CL's collected by microdissection. Total RNA was isolated and protein extraction was performed for real-time PCR and western blot assay respectively, of Dll4, Notch1 and 4. Dll4 mRNA levels significantly decreased at 4 hours after PGF2alpha administration, whereas no changes were detected at the protein level. In contrast, both mRNA and protein expression of Notch1 and 4 receptors significantly decreased 4 hours after PGF2alpha administration. However, by 24 hours after treatment there were no further changes in the expression of the ligand and receptors. To elucidate if the Notch system regulates luteal function during pregnancy, either vehicle (control) or the gamma-secretase inhibitor DAPT (10 µg/ovary) was injected into the bursa of both ovaries on day 19 of pregnancy. Twenty-four hours after treatment, blood samples and CLs were collected. Luteal function was evaluated by measuring serum progesterone (P4) by RIA and apoptosis was examined by DNA fragmentation in agarose gels. The levels of P4 significantly decreased after DAPT administration. However, apoptotic DNA fragmentation in the CL was not affected by DAPT treatment. These results suggest that the Notch signaling pathway promotes CL function and is acutely suppressed during PGF2alpha-induced CL regression in pregnant rats. Supported by: ANPCYT (BID 1201 OC-AR PICT 99:05-06384), NIH-FIRCA RO3-TW007041 and NIH-NCCR RR00163.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Luteolysis is initiated by uterine secretion of PGF2alpha, which is delivered locally from the uterine vein to the adjacent ovarian artery to the luteal-containing ovary. Some conclude that uterine secretion of PGF2alpha induces luteal PGF2alpha secretion to complete luteolysis. Most studies have elucidated mechanisms involved in luteolysis within the corpus luteum (CL) by first inducing luteolysis with an exogenous luteolytic dose of PGF2alpha and then quantifying changes in the CL in vivo or in vitro to describe mechanisms involved in luteolysis. Circulating progesterone declines slowly to less than 1 ng/ml during spontaneous luteolysis at the end of the estrous cycle; however, circulating progesterone decreased precipitously to less than 1 ng/ml within 4 hours after manual removal of the corpus luteum or when luteolysis was induced with a luteolytic dose of PGF2alpha in cows, indicating that mechanisms of luteolysis during a spontaneous luteolysis and PGF2alpha-induced luteolysis may not be similar. These experiments were conducted to examine luteal secretion of progesterone and PGF2alpha in vitro during spontaneous or PGF2alpha-induced luteolysis in ewes. In Experiment 1, jugular venous blood was collected for progesterone analysis and day-12 or day-16 ovine corpora lutea were removed and weighed. Luteal slices were incubated in vitro in M199 for 4 and 8 hours and concentrations of progesterone and PGF2alpha were quantified by RIA. In Experiment 2, ewes received Vehicle or a luteolytic dose of PGF2alpha on day 9, jugular venous plasma was collected daily, and corpora lutea were collected 48 hours (day-11) after treatments were given, weighed, and luteal slices were incubated in vitro in M199 for 4 and 8 hours and progesterone and PGF2alpha in media and progesterone in jugular venous plasma were quantified by RIA. In Experiment 1, jugular venous progesterone on days 12 and 16 and luteal weights were analyzed by a One Way ANOVA and data for progesterone and PGF2alpha in culture media were analyzed by a 2X2 Factorial Design for ANOVA. In Experiment 2, jugular venous progesterone was analyzed by a 2X3 Factorial Design for ANOVA and progesterone and PGF2alpha in culture media were analyzed by a 2X2 Factorial Design for ANOVA. In Experiment 1, jugular venous progesterone and luteal weights were greater (P<0.05) in day-12 than day-16 ewes and progesterone secretion in media from day-12 ewes was greater (P<0.05) than day-16 ewes, while luteal PGF2alpha secretion in vitro by day-12 and day-16 did not differ (P>0.05). In Experiment 2, jugular venous progesterone and luteal weights in PGF2alpha-treated ewes over the daily sampling period were lower (P<0.05) than in Vehicle controls, and progesterone secretion in media from Vehicle-treated ewes was greater (P<0.05) than PGF2alpha-treated ewes, while luteal PGF2alpha secretion in vitro by PGF2alpha-treated ewes was greater (P<0.05) than Vehicle-treated ewes. In conclusion, at least one mechanism of spontaneous luteolysis differs from PGF2alpha-indused luteolysis in ewes.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Loss of progesterone (P4) secretion by the corpus luteum (CL) at the end of an estrous cycle is via uterine PGF2a secretion; however, PGF2a is not decreased during early pregnancy in ewes. Instead, the embryo imparts resistance to PGF2a during early pregnancy via increased uterine PGE1 and PGE2. PGE1 or PGE2 given every 4 hr intra-uterine prevents spontaneous or induced luteolysis in ewes. PGE1 prevents luteolysis in ewes by increasing CL mRNA for LH receptors and occupied and unoccupied LH receptors, while PGE2 prevents their loss. Estradiol or PGE2 infused intra-uterine every 8 hr does not inhibit luteolysis, but estradiol+PGE2 inhibited luteolysis in heifers. However, intra-luteal implants of PGE1 or PGE2 prevented decreased CL weights and circulating P4. The objective was to determine how intra-luteal implants of PGE1 or PGE2 prevent luteolysis in Brahman or Angus cows. Cows received intra-luteal implants containing Vehicle, PGE1, or PGE2 from D-13-D-19 post-estrus via a flank laparotomy. CL were recovered on D-19, weighed, quartered, and frozen in liquid nitrogen until analysis for mRNA for LH and PG (FP, EP1, EP2, EP3, EP3A, EP3B, EP3C, EP3D, and EP4) receptors by RT-PCR. D-13 Angus CL weights served as pre-luteolytic controls. LH receptors on CL were quantified by RIA and RRA. LH and PG receptor mRNA were analyzed by REST-2008 software and expressed as relative expression ratios compared to control groups. Changes in LH mRNA copy number and occupied and unoccupied LH receptors were analyzed by a Factorial design for ANOVA (breed & PGE subtype). Data for breed, PGE1 or PGE2 affecting LH and PG mRNA receptors did not differ (P>0.05) and were pooled. PGE1 or PGE2 prevented (P<0.05) loss of mRNA and receptors for LH compared to controls. mRNA for FP receptors decreased (P<0.05) in D-19 controls compared to D-13 controls regardless of breed. PGE1 and PGE2 up-regulated (P<0.05) FP gene expression compared to Vehicle controls regardless of breed, but gene expression was greater (P<0.05) in Angus treated with PGE2. EP1 was not regulated by any condition. PGE1 and PGE2 down regulated (P<0.05) EP2 and EP4 compared to Vehicle regardless of breed. PGE1 up-regulated (P<0.05) EP3, EP3A, and EP3B and EP3C and EP3D (P<0.10) compared to Vehicle regardless of breed. PGE2 only up-regulated (EP3A), but EP3A was up-regulated in Brahman by PGE1 or PGE2. We conclude that both PGE1 and PGE2 may prevent luteolysis by up-regulating expression of LH receptors and may involve PG receptor regulation. The similarities in relative gene expression profiles induced by PGE1 and PGE2 support their agonistic effects, although the actions of PGE1 seem to impact a larger number of genes.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Corpora luteal (CL) defects contribute to reproductive failure issues and are believed to account for approximately 65% of miscarriages. Common luteal defects include abnormal luteal development and decreased progesterone production, which are attributed in part by abnormal luteal angiogenesis. Leptin is an important metabolic hormone known to have both reproductive and angiogenic properties. Previously, we reported the expression of both leptin and its receptor in the developing caprine CL, a period of time during which angiogenic activity is highest. Furthermore, it was determined that leptin influenced angiogenesis in the developing CL by upregulating the expression of angiogenic stimulators, i.e., VEGF and FGF. Collectively, the evidence suggests that leptin may be involved in CL development. Therefore, it is hypothesized that blocking the action of ovarian leptin will disrupt luteal development in the caprine ovary. Nine cycling crossbred does of similar age were randomly allocated to one of three treatment groups: Control [C; saline + 2.5U heparin; n=2], rabbit Anti-Leptin antibody [AL; AL antibody (1:10 dilution) + 2.5U heparin; n=5], and rabbit IgG antibody [IgG; rabbit antibody (1:10) + 2.5U heparin; n=2]. All does were unilaterally ovariectomized to control for site of ovulation. Females were checked twice daily for classical, behavioral estrus, using a cryptorchid male, for 2 consecutive cycles. On day 10 of the 3rd estrous cycle does were synchronized (PGF2α administration) for the surgical insertion of an osmotic, infusion pump at estrus (day 0). Pumps containing treatments, C, AL, or IgG, were placed in apposition to the ovarian-uterine vascular plexus for a 14-day infusion of treatment at a flow rate of 0.5 µl/hr. After 14 days, the osmotic pump and the remaining ovary were removed for analysis. Gross morphology of CL tissue was recorded and either snap frozen in liquid nitrogen or paraffin embedded for microscopic evaluation. Blood samples were collected via, jugular venipuncture, from day 0 until day 14 for analysis of serum progesterone and leptin during the infusion period. Effect of treatment on CL development was analyzed using the Chi Square procedure of SAS. Serum hormones were analyzed using the MIXED procedure of SAS for repeated measures. Gross morphological evaluation revealed that the infusion of AL increased the frequency (P<0.05) of abnormal luteal formations (56.25% abnormal vs. 43.75% normal) in the remaining ovary compared to C and IgG ovaries (16.67% abnormal vs. 83.33% normal and 0.0% abnormal vs. 100% normal, respectively). However, lutea cellular structure and uniformity exhibited abnormal morphology in both the abnormal and normal CLs in the AL infusion group, which was not evident in either the C or IgG infusion groups. Neither serum concentrations of leptin nor progesterone differed relative to infusion treatment. Although progesterone did not differ between infusion groups during the treatment period, the abnormal cellular morphology in AL group may be indicative of unhealthy tissue, which may impact the longevity of the CL. Collectively, the evidence supports the supposition that leptin appears to be involved in normal luteal development in the caprine ovary.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
The objective of the present study was to determine whether PGF2a and PGE2 are selectively transported from the uterus to the ovary at the time of luteolysis and during the establishment of pregnancy in sheep. Suffolk cross-bred ewes that exhibited estrous cycle lengths of 17 days were used in this study. A laparotomy was performed on days 12 (n=4), 14 (n=4) and 16 (n=4) of the estrous cycle or pregnancy. To determine the selective transport of PGF2a and PGE2 between the uterus and the ovary, blood samples were collected simultaneously from the utero-ovarian vein (UOV) and the ovarian artery (OA) adjacent to the corpus luteum (CL), as well as from a jugular vein (JV). After blood sampling, ovariohysterectomy was performed and uterine flushing (UF) were obtained. Tissue samples were also collected from the uterus, CL, and vascular utero-ovarian plexus (UOP) adjacent to the CL. Blood and tissue samples were analyzed for PGF2a and PGE2 using ELISA while tissue protein expression was measured using western blot and immunohistochemistry. A. Studies on PGF2a transport indicated that: (i) The concentration of PGF2a in UF was higher on days 14-16 of pregnancy (2000-2500 pg/ml) compared to that of estrous cycle (1000-1500 pg/ml); (ii) the concentration of PGF2a in the UOV on days 14-16 of estrous cycle was 900-1350 pg/ml while on the same days of pregnancy it was higher at 1800 to 2250 pg/ml; (iii) the concentration of PGF2a in the OA on days 14-16 of estrous cycle was 100-150 pg/ml while on days 14 to 16 of pregnancy it was higher at 200-250 pg/ml; and (iv) the concentration of PGF2a in JV plasma on days 14-16 of estrous cycle and pregnancy ranged from 40 to 80 pg/ml. B. Studies on PGE2 transport indicated that: (i) the concentration of PGE2 in UF was higher on days 14-16 of pregnancy (10000-12000 pg) compared to that of estrous cycle (1000-3000 pg/ml); (ii) the concentration of PGE2 in UOV plasma on days 14-16 of estrous cycle was 600-1800 pg/ml while in pregnancy it was higher at 6000- 7200 pg/ml; (iii) the concentration of PGE2 in the OA on days 14-16 of the estrous cycle and pregnancy was 50-250pg/ml and 4800-5300pg/ml, respectively; and (iv) the concentration of PGE2 in JV plasma on days 14-16 of estrous cycle was 25-100 pg/ml and pregnancy was 2000-4000 pg/ml, suggesting that PGE2 is much more resistant to catabolism in the pulmonary circulation than PGF2a. C. Studies on PGF2a versus PGE2 transport indicated that both PGE2 and PGF2a levels were higher in UF, UOV, and OA on days 14-16 of pregnancy compared that of estrous cycle. D. Results on PGF2a and PGE2 biosynthetic machinery in the uterus indicated that COX-2, PGES-1, PGFS-AKR1C1, PGFS-AKR1C3, PGT, EP1, EP2, EP4, and FP were distinctly expressed in endometrium on 12-16 of the estrous cycle and pregnancy and selectively directed towards PGE2 production in pregnancy. PGT was expressed in the UOP on days 14-16 of the estrous cycle and pregnancy. DISCUSSION: Together, these results suggest that PGF2a and PGE2 are selectively transported from the uterus to the ovary through the UOP during the estrous cycle and early pregnancy. Such a transport mechanism is most likely regulated in the endometrium as well as UOP by the prostaglandin transporter protein, and its function may be modified by cell signaling cross-talk. Supported by USDA award 2008-35203-19101 to JAA and in part by USDA award 2004-35203-14176 to JAMcC.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
NOT PRESENTED.
Biol Reprod. 2010 Nov;83(1 Suppl):3.
MicroRNAs (miRNAs) are small noncoding RNAs that regulate many biological processes via gene silencing and/or transcript degradation. Their precise role(s) in preimplantation embryo development remains largely unclear. Here, we describe the miRNA expression profile of mouse preimplantation embryos by multiplex real-time polymerase chain reaction and identify 16 miRNAs that are specifically up-regulated in the zygotes. One of these miRNAs is miR-34c, which is weakly expressed in the oocytes but is increased by 4-fold in the zygotes. Microinjection of miR-34c inhibitor, but not non-specific miRNA inhibitor in zygotes leads to an inhibition in DNA synthesis and significant suppression of the first cleavage division. 3'UTR luciferase assay and Western blotting with or without inhibition of miR-34c showed that miR-34c regulates the expression of Bcl-2 in the zygotes and the trophoblast cell line JAR. Microinjection of anti-Bcl-2 antibody to zygotes partially reverses, while microinjection of Bcl-2 protein mimics the effect of miR-34c inhibition. In addition, microinjection of either miR-34c inhibitor or Bcl-2 reduced c-myc expression, which is known to be important for early embryonic development. Our findings provides direct evidence that miR-34c is important for zygotic development and that Bcl-2 and c-myc are downstream molecules of miR-34c modulating the first cleavage division.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
The zona pellucida (ZP) is the extracellular matrix that surrounds the oocyte. The biosynthesis of ZP requires an integrate participation of the oocyte and the granulosa cells, being that canine ovaries have oocytes that synthesize ZP proteins in response to the expression of specific genes responsible for the folliculogenesis. A structural model of the zona pellucida, based on the biochemical and ultrastructural analysis of the mouse ZP, involves long filaments of a repeating ZP2 and ZP3 heterodimer. ZP1 provides structural integrity for the zona pellucida by cross-linking with disulphide bonds the ZP2/ZP3 filaments, however, it is not known if the model proposed for the mouse can be applied for others mammals, since differences between species were often observed and few specific studies was to carried with canine embryos. The objective in this study was to identify and verify the composition of the zona pellucida in canine embryos in different stages of development using Schiff periodic acid (PAS). The embryos collection was performed by flushing the oviducts and uterine horns after ovariohysterectomy using PBS with heparin and polyvinyl alcohol. All embryos were fixed in 2.5% glutaraldehyde and included in historesin. Histological sections with 3 μm were stained with hematoxilin-eosin (HE) and Schiff periodic acid (PAS). Light microscopy observation showed that canine ZP has two defined acellular stratus. The inner part was PAS positive indicating that this stratus was formed by glycoproteins and, the outer stratus was PAS negative and barely stained with HE indicating neutral proteins. It is known that many sugars are associated with ZP which can be added during its formation in the ovarian follicle or during the oocyte transport through the oviduct. In rabbits, a mucoprotein cover is added to the oocyte by the oviduct cells, which have an important role in the relationship between ZP and the endometrium during implantation. The bilaminar structure observed in this experiment of the canine ZP can be related to the addition of components during oviductal transport, since this feature was not observed on oocytes that were harvested from sliced ovaries in previous studies performed at the same laboratory. Financial support: FAPESP.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
NOT PRESENTED.
Biol Reprod. 2010 Nov;83(1 Suppl):3.
During the pre-implantation period of embryonic development, the mammalian embryo exhibits dramatic changes and many key events take place. The developmental stage at which porcine embryos are most commonly manipulated is the blastocyst, and the quality of the embryos used is critical to the success of reproductive technologies such as embryo transfer. Despite the importance of this critical period, our ability to determine early embryonic quality based on morphological criteria is limited. As an alternative, characterization of the gene expression profile of the early porcine embryo could identify gene markers of embryonic quality. The objective of this study was to perform comparative gene expression profiling analysis of in vivo-derived expanded and hatched porcine blastocysts using two-colored microarray (Pig-Oligo Array, USDA Pig Genome Consortium). Microarray data were interpreted using WebArray and DAVID functional annotation tools. The microarray results identified 1783 genes that were commonly expressed in both expanded and hatched porcine blastocysts. A further 148 genes were differentially expressed (p<0.05) and in comparison to expanded blastocysts, 35 genes were down-regulated and 113 genes were up-regulated after hatching. Many of the differentially expressed genes are related to important molecular mechanisms, such as macromolecule metabolism and energy related pathways. A total of 14 genes of interest (LDHA, LDHB, SLC16A7 (previously known as MCT2), BSG, SLC2A1, SLC2A3, SLC2A5, POU5F1 (previously known as OCT4), ACTB, GAPDH, GRB2, SETX, SPG7, and YWHAZ) were selected from the gene list generated by the microarray analysis, and their expressions were verified using SYBR Green-based real-time PCR. For the genes tested, the real-time PCR results were consistent with the microarray results. The differences detected in the gene expression profiles between in vivo-derived expanded and hatched porcine blastocysts increases our understanding of the molecular mechanisms involved in the hatching process and identifies potential candidate gene markers of porcine embryo quality. This research was supported by the Natural Sciences and Engineering Research Council (NSERC) and is a part of the activities of the EmbryoGENE NSERC Strategic Research Network.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
In mice, blastocysts become dormant as a result of ovariectomy on day 4 1000 h of pregnancy, before ovarian estrogen secretion induces implantation. This condition, termed delayed implantation, can be maintained by continued progesterone treatment but is terminated upon injection of estrogen which leads to implantation-competent (activated) blastocysts and subsequent implantation. Using these delayed and activated blastocysts, we previously demonstrated that both breast cancer 1 (Brca1) mRNA and protein levels were upregulated in activated blastocysts as compared to delayed ones. BRCA1 was identified based on its linkage to hereditary early-onset breast-ovarian cancer families. This protein is multifunctional in a variety of molecular pathways such as DNA repair and recombination, cell cycle, apoptosis, and gene transcription. Therefore, we hypothesize that BRCA1 may play an important role in the activation of blastocysts. To test this hypothesis, we first examined the spatiotemporal BRCA1 expression in mouse blastocysts collected at different times on day 4 of pregnancy: 1000 h (before estrogen secretion), 1800 h (after estrogen secretion), and 2300 h (just prior to implantation). BRCA1 expression was primarily localized to the trophectoderm (TE) from day 4 1000 h to 2300 h. Interestingly, the BRCA1 expression level is strongly upregulated in the blastocysts at day 4 2300 h. These results again indicated that BRCA1 expression level correlates to the activation of blastocysts. While in vitro fertilization is currently a widely utilized technique, successful implantation rates are typically low. One of the possible reasons for this low success rate may be that the blastocysts from in vitro fertilization have insufficient implantation ability. To further investigate this possibility, we examined the expression and localization of BRCA1 in blastocysts derived from in vitro fertilization compared to blastocysts from day 4 of pregnancy. While BRCA1 localization was similar between the two samples, BRCA1 levels were slightly low in blastocysts derived from in vitro fertilization. It has been shown that 4-hydroxyestradiol mediates activation of blastocysts. It has been also shown that prolactin faciliates blastocyst formation from the 2-cell stage in vitro and that BRCA1 is upregulated by prolactin in human breast cancer cell lines. In light of this, we examined whether 4-hydroxyestradiol and prolactin influenced BRCA1 expression level in blastocysts derived from in vitro fertilization. As we expected, both 4-hydroxyestradiol and prolactin upregulated BRCA1 levels in these blastocysts. Our results suggest that BRCA1 most likely plays a physiological role in directing implantation-competent blastocysts in the mouse.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Previously, we found growth arrest specific gene 6 (Gas6) is highly expressed in germinal vesicle (GV) oocytes than metaphase II (MII) oocytes using Annealing Control Primer (ACP)-PCR technology. The present study was undertaken to investigate the role of Gas6 and its receptor Mertk during oocyte maturation and early embryogenesis using RNA interference (RNAi). Expression pattern of Gas6 and its 3 types of receptors, Axl, Tryo3, and Mertk was evaluated in oocytes and embryos at different developmental stages. Gas6 mRNA was highly expressed in oocytes and PN stage embryos. But it dramatically decreased between 2C and 4C, and increased again at 8C thereafter. Expression of Mertk was relatively constitutive in oocytes and embryos, while the other receptors, Tyro3 and Axl, were not detected in oocytes. After Gas6 or Mertk RNAi by microinjection of dsRNA for Gas6 (561 bp) or Mertk (543 bp), respectively, changes in oocyte maturation, knockdown of mRNA and protein, spindle and chromosome status, parthenogenetic activation, embryo developmental, and activities of two important kinases, M-phase promoting factor (MPF) and mitogen-activated protein kinase (MAPK), were measured. Interestingly, despite of the specific and marked decrease in Gas6 or Mertk mRNA and protein, maturation rates, spindle and chromosomal organizations, and MAPK activity were not affected. However, only discernible difference was found in MPF activity by Gas6 RNAi in oocytes. In addition, all MII oocyte after Gas6 RNAi were not developed further by parthenogenetic activation but arrested at MII oocyte. Gas6 RNAi at PN stage embryos resulted in arrested preimplantational embryo development at 2C (82.0%). Results of the present study suggest that Gas6 signaling is not related to the oocyte nuclear maturation, but to cytoplasmic maturation that may impinge on early embryogenesis.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Developmental abnormalities of the male GU tract are the most common birth defects. These birth defects include cryptorchidism and hypospadias. Cryptorchidism is the failure of testis descent into the scrotum. It occurs in 6% of full term newborn males and this reduces to about 3% by 6 months of age, and results in impaired spermatogenesis. Hypospadias is a midline fusion defect of the male ventral urethra and occurs in nearly 1 in 125 live male births. The etiology of these congenital GU birth defects is poorly understood and likely arises from multifactorial origins including genetic and environmental factors. Karyotype analysis can detect chromosomal defects in patients with congenital genitourinary defects, although results are not always informative. A karyotype has limited resolution and cannot detect submicroscopic, clinically significant chromosomal rearrangements. We hypothesize that subtle chromosome aberrations are present in patients presenting with both hypospadias and cryptorchidism and are causative in the etiology of these birth defects. Genome wide high-resolution comparative genomic hybridization arrays (aCGH) provide a novel approach to detect these previously unrecognized variants. Genes encoded in regions of copy number variation may be dosage sensitive and critical for GU tract development. Genomic DNA was analyzed by aCGH using 720K NimbleGen arrays (Roche) and analyzed using Nexus Copy Number software (BioDiscovery). Fluorescent in situ hybridization (FISH) and/or qPCR were employed to validate putative regions of duplication or deletion that were distinct from common copy number variants (CNV) found throughout the genome. Sex-matched genomic DNA from men of proven fertility served as a control. Patients with combined hypospadias and cryptorchidism exhibited distinct submicroscopic chromosome duplications or deletions not detected in normal pregnancy proven fertile controls or in public copy number variation databases. These regions include chromosome microdeletions at 5p13, 7p14, 9p11, 12q21, 10p13, 15q14, 22q11 (copy number losses) and chromosome duplications at 7q32-35, 10q24, 11q12, 15q11, 16p13, 16q21, 22q13 and Xq28 (copy number gains). We hypothesize these regions encode genes required for normal GU tract and male external genitalia development (and perhaps function). TaqMan Copy Number Assays targeting 7q35, 22q13 and Xq28 confirmed the presence of 3 duplications in patients with midshaft and penoscrotal hypospadias. Further analysis of 22q13 revealed a functional overlap in genes associated with signal transduction and biosynthesis of glycosphingolipids at loci 1p35 and 9q34 respectively. Moreover, we identified additional submicroscopic chromosomal aberrations within the previously reported candidate locus, Xq28. Gene expression analyses and functional studies of candidate genes in these and other regions are needed to define the significance of these findings for patients with combined hypospadias and cryptorchidism. Array CGH represents an accurate high-resolution method to diagnose clinically significant chromosomal aberrations associated with congenital GU tract defects. Supported in part by K12 DK0083014, the Multidisciplinary K12 Urologic Research (KURe) Career Development Program to DJL (SL and CJ), 1R01DK078121 from the National Institute of Kidney and Digestive Diseases to DJL, and by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (P01HD36289) to DJL
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Studies conducted under temperate latitudes with Bos taurus cows concluded that pregnancy losses are the most important components of reproductive failure in cattle. Such losses occur mainly during the early embryonic period of gestation due to non-infectious causes. It was hypothesized that similar losses occur in Zebu cattle (Bos taurus indicus) raised in the tropics. The objectives were (1) to evaluate a non-surgical transcervical collection (EC) technique of day-18 concepti to further study pregnancy losses in Zebu cows and (2) use EC to accurately establish embryonic mortality up to the critical period for maternal recognition of pregnancy without having to slaughter the inseminated dams. Seventy-two Nelore beef cows were artificially inseminated (day zero = D0) after natural estrus observation and submitted to EC on D18. To recover the concepti, a 24G Foley catheter was trespassed through the cervix to flush the uterus with PBS. The catheter was coupled to a custom-made embryo collection device and the fluid was collected, filtered (75 micrometers mesh) and then swirled and poured into a searching Petri dish containing PBS medium with 0.4% BSA. Examination was carried out with a stereomicroscope at 40× magnification. Prior to the evaluation, debris were removed, conceptus was isolated and, in certain cases, rolled and stretched with a pipette. When present, D18 concepti were elongated structures and were classified according to integrity (whole = WH, fragmented = FR or severely fragmented--no piece >2mm= SF) and length. In addition to EC, ultrasound examination of reproductive organs and blood samples were taken on D18. Ultrasonography was performed using a MindRay 3300VET equipment (transrectal B-mode with a 7.5 MHz transducer). Antral follicles and corpora lutea (CL) were measured. Plasma concentrations of progesterone ([P4]) were determined using a solid phase radioimmunoassay kit (Coat-a-Count, DPC, Los Angeles). Data were submitted to ANOVA and differences were considered at the P < 0.05 level. Overall recovery rate was 68.1%. The 49 recovered concepti were classified as follows: 21 WH (42.8%), 9 FR (18.4%) and 19 SF (38.8%). On D18, respectively for cows with WH, FR, SF and none conceptus the [P4] (ng/mL) was: 8.20 ± 0.47 a, 7.99 ± 0.72 a , 6.02 ± 0.85 b and 4.76 ± 1.10 c and the diameter (mm) of the largest follicle was: 4.10 ± 1.00 b, 5.60 ± 1.50 b, 8.50 ± 0.07 a and 6.20 ± 1.30 b. In cows from which a WH or a FR conceptus was retrieved, [P4] was above 5.42ng/mL. [P4] below 4ng/mL on D18 were only observed in cows with SF embryos or contained no conceptus. In conclusion, not all cows bearing conceptus on D18 presented ovarian function compatible with pregnancy maintenance. Indeed, the presence of larger follicles coupled with low [P4] on D18 associated with the retrieval of fragmented embryos indicated that poorer quality concepti were less effective to signal their presence in the uterus towards the maternal unit.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
In the past few years, microarray platforms have been developed and used to study RNA abundance in early embryos. Although reports are numerous, the integration of this information is still awaited. The peculiar nature of early mammalian development constitutes a hurdle in the interpretation of results produced by different teams. The nature of oocytes and early embryos differ drastically from somatic cells as their number, size, transcriptional potential, RNA and protein contents fluctuates across development and therefore need to be considered for any interpretation of data. The RNA content and its adenylation status are factors that profoundly impact downstream microarray results. As such, we benchmarked the mains sample processing steps and we identified several sources of methodological imprecision, some of which strongly influence the output of microarray studies, making datasets incompatible. These results are the foundation of the establishment of a Canadian Research Network called EmbryoGENE dedicated to the study of embryonic gene expression. One of the mandates of the Network is to provide technological platforms to be shared amongst members, therefore ensuring full compatibility between datasets. EmbryoGENE has been engaged in the development of a novel microarray platform that will take into consideration the peculiar nature of mammalian oocytes and early embryos. The first steps was to survey the pre-hatching transcriptome (aside from small non-coding RNAs) using a deep sequencing approach. Bovine oocytes and pre-hatching embryos were pooled and handled to generate a normalized cDNA library that was submitted to 454 Titanium sequencing. A little over 1.2 million reads with an average read length of 323 bases were obtained. Sequence analysis allowed the generation of an augmented catalog of transcripts. Overall, pre-hatching development seems to include mRNAs from 14,433 genes. The survey also highlighted the presence of an additional 1,600 novel uncharacterized transcripts. Emphasis was then directed towards the identification of RNA isoforms. Sequences allowed the identification of 1,100 variants of the exon skipping type. Further data mining suggests the presence of protein coding transcripts bearing 3' UTRs of variable length. The transcript catalog was utilized to design microarray probes that account for varying 3' UTR as well as for sample amplification bias. The platform utilizes the Agilent SurePrint 44K format. A data analysis pipeline was also developed integrating a laboratory information management system (LIMS), a microarray quality control module, data pre-processing tools and statistical packages. EmbryoGENE microarray platform provides standard operation procedures (SOPs) for sample handling and processing. Users input their data in the database that will be used to study the impacts of assisted reproductive technologies and maternal nutrition on embryonic gene expression.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
In vitro produced embryos have lower cryotolerance than the in vivo counterparts, resulting in lower pregnancy rates when they are cryopreserved and transferred; thus, transporting fresh embryos (not frozen) is a viable alternative to obtain good pregnancy outcomes. The aim of this work was to evaluate different systems for transportation of fresh in vivo- and in vitro- produced embryos up to 8h. In vivo produced embryos were collected nonsurgically from superovulated cows. Also, oocytes were collected from slaughterhouse ovaries, matured, fertilized, and the resulting embryos cultured in vitro by standard procedures in a chemically defined medium. Day 0 of culture was ~18 h after the onset of in vitro fertilization. Seven days after fertilization or insemination, respectively, in vitro- and in vivo-produced embryos were blocked by quality and stage and allocated to different treatments in a 2 x 2 x 2 factorial design, with factors: embryo origin (in vivo (n=32) vs. in vitro (n=69)), media (mPBS with 0.05% PVA vs. HCDM2 with 0.4% BSA), and temperature (25 vs. 37°C). The embryos were blindly and subjectively evaluated for stage (5=early blastocyst, 6=blastocyst, 7=expanded blastocyst and 8=hatched blastocyst) and quality (1=excellent, 2=good, 3=fair and 4=dead) by a single person every 2 h during an 8 h period (Time). An ANCOVA model to adjust storage time as a covariate was used; main effects and first, second, and third order interactions of fixed factors were included in the model. Second order interactions were not significant and dropped from the model. Two way significant interactions for embryo quality were: temperature X embryo, media X time and temperature X time (P < 0.05). In vitro-produced embryos were better kept at 25°C (quality 1.56 vs. 2.00) whereas in vivo produced embryos quality was best at 38°C (quality 2.02 vs. 1.75). HCDM2 medium resulted in better quality embryos with time. Quality of embryos with time was better under 25°C. There were no embryo X media or time interactions (P > 0.1). For stage, significant interactions were temperature X embryo and media X time (P < 0.05). In vivo produced embryos progressed better in stage at 25°C whereas in vitro embryos performed better at 38°C. Embryos placed in mPBS progressed faster than in HCDM2. In summary, HCDM2 was better for long time embryo transportation, 25°C was best for in vitro produced embryos, whereas for in vivo produced embryos 38°C was best. For short time of transportation (2 h) both temperatures are appropriate. However, 25°C is better suited for long time transportation (8 h). Research supported by Fondos Mixtos del Estado de Chihuahua-CONACYT, Grant No. CHIH-2008-C01-91923.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
NOT PRESENTED.
Biol Reprod. 2010 Nov;83(1 Suppl):3.
NOT PRESENTED.
Biol Reprod. 2010 Nov;83(1 Suppl):3.
The potential adverse effects of parabens were reported in both in vivo and in vitro systems, but the molecular mechanism(s) and long-term consequences of parabens exposure are largely unknown. In this study, we further examined the induction of an estrogenic biomarker gene-calbindin-D9k (CaBP-9k) to investigate the estrogenic activity of parabens (methyl-, ethyl-, propyl-, isopropyl-, butyl-, and isobutylparabens) in the rat pituitary GH3 cell line. Following 24 h exposure, significant increases in CaBP-9k transcript and protein were observed depending on the concentration treated and the linear length of the alkyl chain from methyl- to isobutyl parabens, whereas co- treatment with fulvestrant, a pure antiestrogen, largely reversed the paraben-induced expressions of CaBP-9k mRNA and protein in GH3 cell line. To better understand the mechanism(s) of CaBP-9k induction by these endocrine disrupting compounds, we measured the levels of estrogen receptor (ERalpha) and progesterone receptor (PR) expression following parabens exposure. In the GH3 cells, a great increase in PR mRNA and protein was observed in a concentration-dependent manner after parabens treatment. Paraben-induced expression of CaBP-9k was effectively blocked in the presence of antagonist of 17beta-estradiol (fluvestrant). To confirm whether progesterone receptor signaling is involved in parabens derived induction of CaBP-9k mRNA and protein, we treated GH3 cells with antiprogesterone (mifepristone). In the study, parabens-induced up-regulation of CaBP-9k was completely reversed by mifepristone. Taken together, these results indicate that CaBP-9k may be induced by parabens via the PR-involved pathway in addition to ERα pathway in GH3 cell line.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
The endocrine disruptor (ED) is an exogenous substance that acts on the endocrine system and modulates normal physiological functions of the body. Although EDs such as 2,3,7,8-Tetrachlorodibenzodioxin (TCDD) affect normal reproductive function in humans and affects the growth and reproductive functions in rodents, the underlying mechanism that modulates these changes remains unclear. Accumulating evidence suggested preimplantation embryo development is controlled by concerted expression of microRNAs (miRNA) that regulate mRNA stability and protein translation. We hypothesized that EDs affect pre-implantation embryo development by modulating miRNA expression, as well as attachment (an initial step on implantation process) of embryo onto endometrial epithelium. Treatment of mouse zygote with 10nM TCDD did not affect the blastulation rate of mouse embryo developed in vitro. Yet, analysis of the expression of 238-plex miRNA by RT-PCR at the blastocyst stage revealed 9 and 11, up- and down-regulated miRNA (>2-fold changes; p<0.05) between the TCDD-treated and control embryos. RT-PCR confirmed the differential expression on some of the selected miRNA including miR-133a and 199a expression in single blastocyst. Computation analysis (picTar) identified putative implantation-related targets for these miRNAs including cadherin, integrin and GSK3beta. An in vitro choriocarcinoma (Jeg-3 and BeWo) and endometrial epithelial (Ishikawa and RL95-2) cells co-culture system was established and used to study the effect of TCDD on spheroids attachment. The receptor of TCDD, aryl hydrocarbon receptor (AhR), as well as estrogen receptor (ERalpha) alpha and (ERbeta) beta were detected in all the cell lines. TCDD dose-dependently (1pM - 10nM, for 24 hours) induced CYP1A1 (a biomarker gene for TCDD exposure) transcript and protein expression in these cell lines. In addition, TCDD dose-dependently suppressed attachment of BeWo spheroid onto the RL95-2 monolayer (from 82% to 53%, n=6). Yet, TCDD has no effect on cell proliferation. Our results are the first demonstrating that ED modulates the miRNA expression of preimplantation embryos, and may in turn affect the normal expression of genes that are important for embryo attachment and implantation in vitro. This research was supported in part by a RGC Collaborative Research fund (CRF) Group Research Project (HKBU 1/CRF/08).
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
The endocrine disruptor methoxychlor (MXC) is a chlorinated organic pesticide which was first registered for use in 1948 and now is widely used in many countries against various species of insects that attack field crops, trees, vegetables, fruits, gardens, stored grain, livestock and domestic pets. MXC exposure is highest in people working in manufacturing industries, people living near farms, livestock, and in pet owners treating pests with MXC. MXC exposure causes adverse effects on reproductive functions in adult females, including altered estrous cyclicity, reduced fertility, and ovarian atrophy. MXC has been shown to target antral follicles. Specifically, it decreases the number of healthy antral follicles and increases the number of atretic antral follicles in adult female mice. Previous studies have shown that MXC induces atresia and decreases estradiol levels after 96 hours of follicle culture. The goal of the present study was to determine whether MXC induces atresia and decreases estradiol levels earlier than 96 hours of culture. Further, the present study was designed to determine whether MXC-induced changes in atresia precede MXC-induced changes in estradiol levels or vice versa. To complete these goals, antral follicles were mechanically isolated from ovaries of CD1 female mice aged 35-40 days. The isolated antral follicles (10-15 per treatment) were cultured in supplemented alpha-minimum essential media in the presence of dimethylsulfoxide (control; DMSO) or MXC (1, 10, 100 µg/ml) for 24 and 48 hours at 37°C and 5%CO2. After culture, the media was collected and follicles were fixed in Dietrick solution. Fixed follicles were embedded in plastic blocks, cut into 2 µm sections, stained with Lee's methylene blue-basic fuchsin, and graded for atresia on a scale of 1 to 4 based on the percentage of apoptotic bodies (1=0%, 2=<10%, 3=10-30%, and 4=>30% apoptotic bodies). The media was subjected to measurements of estradiol levels by enzyme-linked immunosorbent assays. The results show that MXC does not induce atresia at 24 hours, but it does induce atresia by 48 hours (24 hours: DMSO = 1.73 ± 0.11, MXC1 µg/ml = 1.59 ± 0.16, MXC10 µg/ml = 1.78 ± 0.20, MXC100 µg/ml = 1.90 ± 0.16; 48 hours: DMSO = 1.25 ± 0.14, MXC1 µg/ml = 1.79 ± 0.09, MXC10 µg/ml = 2.22 ± 0.15, MXC100 µg/ml = 4 ± 0; n=3 separate experiments; p ≤ 0.05 for DMSO versus MXC at 48 hours). The results also indicate that MXC does not alter estradiol levels at 24 hours, but it does decrease estradiol levels by 48 hours (24 hours: DMSO = 17.10 ± 4.98, MXC1 µg/ml = 18.73 ± 2.50, MXC10 µg/ml = 26.46 ± 7.95, MXC100 µg/ml = 18.63 ± 4.27 pg/ml; 48 hours: DMSO = 599.38 ± 68.97, MXC1 µg/ml = 225.65 ± 18.79, MXC10 µg/ml = 120.48 ± 35.31, MXC100 µg/ml = 32.85 ± 2.97 pg/ml; n=3 separate experiments; p ≤ 0.05 for DMSO versus MXC at 48 hours). Collectively, these data suggest that MXC decreases estradiol levels and induces atresia as early as 48 hours and that these effects of MXC on antral follicles occur simultaneously. Support: NIH R01 ES012893 and the Environmental Toxicology Program at UIUC.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Methoxychlor (MXC) is an organochlorine pesticide that has been shown to target the mouse ovary. Exposure of female mice to MXC results in decreased antral follicle number and increased follicular atresia. Upon entering the body, MXC is readily metabolized into various metabolites which have been hypothesized to be more toxic than the parent compound. One such metabolite is 1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(4-methoxyphenyl)ethane also referred to as mono-OH MXC. Mono-OH MXC has been shown to inhibit follicular growth and cause follicular atresia at doses lower than those causing the same effects as MXC. Previous studies have also shown that exposure to mono-OH MXC decreases production of the sex steroid hormones androstenedione, testosterone and 17beta-estradiol by cultured mouse antral follicles. We therefore hypothesized that mono-OH MXC decreases the levels of sex steroid hormones by reducing the expression of the enzymes involved in ovarian steroidogenesis. To test this hypothesis, antral follicles isolated from female CD-1 mice (ages 32-35 days old) were either untreated or exposed to dimethylsulfoxide (DMSO, vehicle control) or mono-OH MXC (0.1-10 µg/ml) in culture for 96 hours. At 96 h, follicles were collected and processed for gene expression analysis by quantitative real-time PCR (n=11-14 follicles/group). Expression of mRNAs encoding steroidogenic acute regulatory protein (Star), cholesterol side-chain cleavage enzyme (Cyp11a1), 3beta-hydroxysteroid dehydrogenase 1 (Hsd3b1), 17alpha-hydroxylase/17,20 lyase (Cyp17a1), 17beta-hydroxysteroid dehydrogenase 1 (Hsd17b1) and aromatase (Cyp19a1) was determined. Expression was reported as the average ratio of the starting quantity value (SQ) of each gene normalized to that of beta-actin (Actn; n=3 separate experiments). There were no differences in expression of Star (DMSO: 18.91 ± 2.19 SQ; 0.1 µg/ml: 23.19 ± 2.26 SQ; 1 µg/ml: 18.49 ± 3.13 SQ; 10 µg/ml: 28.49 ± 2.28 SQ; n=3, P>0.05), Cyp11a1 (DMSO: 5.57 ± 1.01 SQ; 0.1 µg/ml: 6.66 ± 1.07 SQ; 1 µg/ml: 6.11 ± 1.40 SQ; 10 µg/ml: 3.84 ± 0.32 SQ; n=3, P>0.05), Hsd3b1 (DMSO: 1.10 ± 0.16 SQ; 0.1 µg/ml: 1.06 ± 0.12 SQ; 1 µg/ml: 1.39 ± 0.18 SQ; 10 µg/ml: 1.05 ± 0.89 SQ; n=3, P>0.05) and Hsd17b1 (DMSO: 0.15 ± 0.04 SQ; 0.1 µg/ml: 0.08 ± 0.01 SQ; 1 µg/ml: 0.15 ± 0.01 SQ; 10 µg/ml: 0.06 ± 0.001 SQ; n=3, P>0.05) in mono-OH MXC-treated follicles when compared to DMSO-treated follicles. A significant trend for decreased Cyp17a1 mRNA was observed in follicles exposed 0.1-10 µg/ml mono-OH MXC (DMSO: 3.61 ± 1.57 SQ; 0.1 µg/ml: 2.55 ± 1.19 SQ; 1 µg/ml: 1.94 ± 0.50 SQ; 10 µg/ml: 0.13 ± 0.18 SQ; n=3, P<0.05). Levels of Cyp19a1 were also reduced with exposure to 10 µg/ml mono-OH MXC (DMSO: 0.59 ± 0.09 SQ; 0.1 µg/ml: 0.48 ± 0.07 SQ; 1 µg/ml: 0.59 ± 0.11 SQ; 10 µg/ml: 0.004 ± 0.001; n=3, P<0.05). Collectively, these results suggest that mono-OH MXC reduces levels of androstenedione, testosterone and 17beta-estradiol by reducing the expression of two of the key enzymes required for their synthesis (Cyp17a1 and Cyp19a1). Reduced levels of 17beta-estradiol production by antral follicles may be responsible for the reduced growth and increased levels of atresia observed in mono-OH MXC-treated follicles. NIH R01 ES012893.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Alachlor [2-chloro-N-(2,6-diethyl phenyl)-N-(methoxymethyl) acetamide] is a commercialized effective herbicide in control of the growth of most grasses, most annuals and some broad leaved plants. It is one of the legal pesticides in Taiwan. Previous studies had demonstrated that alachlor had estrogenic effects and could affect reproductive system. Previous studies indicated that alachlor bound to not only estrogen receptors but also progesterone receptors. It had also been found that serum steroid hormones including testosterone and estrogen were influenced by long-term alachlor exposure in Crucian Carp (Carassius auratus). However, the mechanisms of alachlor action on male reproductive endocrinology have not been investigated yet. Primary Leydig cells prepared from adult male rats were incubated with or without human chorionic gonadotropin (hCG, 0.05 IU/ml), 8-bromo-adenosine 3',5'-cyclic monophosphate (8-Br-cAMP, a permeable analogue of cAMP, 10-4 M), 25-OH-cholesterol (10-5 M) and androstenedione (10-6 M) at 34 °C for 60 min. The concentrations of medium testosterone and pregnenolone were measured by radioimmunoassay and enzyme-linked immunosorbant assay, respectively. Alachlor did not affect basal testosterone release, but markedly inhibited hCG- as well as 8-Br-cAMP-induced testosterone release in vitro. Neither 25-OH-cholesterol- nor androstenedione-induced testosterone release was altered by administration of alachlor. These results suggest that alachlor could affect testosterone production at upstream of steroidogeneic enzyme activities. Our results implied that alachlor may be an endocrine disrupting compound.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Environmental toxicants with the potential to alter fetal development of the reproductive system are of major concern to scientists and the general public alike. In particular, the estrogenic plastic monomer bisphenol A (BPA) is a compound of tremendous concern due to its inclusion in many everyday consumer items including baby bottles and canned foods. While some animal studies have indicated exposure to extremely low doses of BPA (less than 50 µg/kg/day) can result in significant alteration of reproductive endpoints, other studies have found no effects of BPA exposure on reproductive system development. To investigate whether low doses of BPA could affect testis morphogenesis in mice, we conducted a gestational exposure study. Pregnant CD-1 dams (n=3 per treatment group) were orally dosed with corn oil vehicle, 50 µg/kg/day BPA, or 0.5 µg/kg/day BPA from embryonic day 10.5 (E10.5) to E17.5. This time period coincides with sex determination and covers subsequent testis cord organization and expansion. A fourth treatment group received 0.05 µg/kg/day of the known ERalpha agonist diethylstilbestrol (DES) to serve as a positive control. Pups were collected via caesarean section at E19.0, shortly before the time of birth. In utero exposure to BPA or DES did not significantly alter litter size, pup body weight, or anogenital distance compared to vehicle-treated controls. Histological analysis revealed abnormal testis morphology in 50% of testes exposed to 50 µg/kg/day of BPA and 80% of testes in the 0.05 µg/kg/day DES and 0.5 µg/kg/day BPA treatment groups. Dysgenic BPA- and DES-exposed testes contained fewer testis cord cross-sections and increased interstitial area compared to vehicle controls. Underdevelopment of the testis cords was particularly evident near the rete testes, where uncoiled lengths of testis cord appeared as finger-like projections. Thus, our study indicates in utero exposure to BPA doses equal to or below the FDA acceptable daily intake value of 50 µg/kg/day can alter fetal testis development in the mouse. Furthermore, the histological similarities between DES- and BPA-exposed testes suggested BPA-induced changes in testis cord development are likely mediated by ERalpha. Surprisingly, altered testis histology following low-dose BPA or DES treatment was remarkably similar in appearance to the fetal testis cord dysgenesis we observed in conditional knockout (cKO) mice lacking expression of inhibin betaA (Inhba), a subunit of activin A, in the fetal Leydig cells. Loss of fetal Leydig cell-derived activin A leads to decreased Sertoli cell proliferation and subsequent impairment of testis cord expansion. Murine fetal Leydig cells express ERalpha, suggesting exposure to estrogenic compounds could potentially influence fetal Leydig cell expression of Inhba/activin A. We therefore hypothesized that BPA- and DES-induced testis cord dysgenesis results from alteration of fetal Leydig cell expression of Inhba and/or disruption of activin A signaling. We are currently testing our hypothesis by performing quantitative real-time PCR analysis for genes involved in the testicular activin A signaling pathway. This research was supported by NIH 1P20ES018163 and NIH T32ES07326.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Hundreds of chemicals in the environment have been identified that interfere with the body's own estrogen and thousands of chemicals remain to be tested. In 1996 congress passed the Food Quality and Protection Act that mandated the United States Environmental Protection Agency develop screening and testing methods to detect these types of environmental chemicals that can interfere with the endocrine system, termed endocrine disrupting chemicals (EDCs). Thus far, chemicals that interfere with estrogen signaling are the largest group of EDCs. Thirteen years later screening and testing methods are still not in place. This endeavor has been complicated by the fact that estrogen receptor ligands (ER) have tissue specific effects. Estrogens function within target tissues by binding to specific estrogen receptors (ERs), alpha and beta, which are ligand activated transcription factors. Different ER ligands can elicit tissue specific responses. It is known that different ligands induce unique conformations in ER, and it is thought that ER conformation determines the specificity of the biological response. Currently, crystallography is used to map ER conformation on single ligand-ER complexes. These studies are arduous, time consuming and expensive and are not amenable to more than one ligand at a time. This study aims to test the hypothesis that ER conformation, measured by peptide profiling, can be used to predict in vivo biological activity. Using a cell culture based reporter gene assay, we have measured the unique peptide profiles for 8 different ligand-ER complexes (estradiol, ICI, bisphenol A, methoxychlor, tamoxifen, resveratrol and and genistein) with 15 different ER interacting peptides. Results support the hypothesis that ligands with similar biological activity can be grouped based on their peptide profiles. The next steps are to measure the in vivo bioactivity and tissue specificity of ER ligands and use to identify peptides most predictive of ER biology and to develop a prediction model to use the peptide profile to predict tissue specific ER activity. To date, methods exist to only predict a chemical's estrogenic activity on a tissue-by-tissue basis. We have developed a novel method to predict tissue specific biological responses of ER ligands for multiple tissues in one assay based on ER conformation. Development and validation of this peptide-profiling assay will likely provide a rapid high-throughput assay to effectively identify chemicals that may have a negative impact on human health by disrupting ER activity.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Exposing postpartum, anovular, suckled, beef cows to bulls reduces interval from calving to resumption of ovulatory cycles. Likewise, exposing, postpartum, anovular cows to excretory products of bulls for 12 h daily produces the same reduction in interval to resumption of ovulatory activity as the presence of bulls. Thus, the "biostimulatory effect" of bulls appears to be mediated by a pheromone(s) excreted by bulls into the environment. Bull urine applied oro-nasally to prepubertal heifers has been reported to hasten the onset of puberty; furthermore, urine is the medium for delivering many male pheromones in species other than bovids. The objective of this experiment was to determine if daily exposure to bull urine alters resumption of luteal activity and artificial insemination pregnancy rates of primiparous, postpartum, anovular, suckled, beef cows. The null hypotheses were that: 1) interval from exposure to urine to resumption of luteal activity; 2) proportions of cows cycling at the end of the exposure period; and 3) artificial insemination pregnancy rates do not differ between cows exposed for 12 h daily to mature bull urine or saline. Anovular cows were stratified by calving date, body weight, calf body weight, calf sex, dystocia score, and body condition score 44 ± 10 d after calving and assigned randomly to be exposed to mature bull urine (n = 20) or physiologic saline (n = 21). Each cow was evaluated for the presence of a corpus luteum (CL) once weekly by transrectal ultrasonography, and jugular blood samples were collected every 3 d beginning on the day of exposure for assay of progesterone. A rise in progesterone concentrations of > 1.0 ng/mL in 2 consecutive samples confirmed by the presence of a CL was used to determine resumption of luteal activity. Cows were exposed to freshly-collected urine or saline dripped at a rate of 2 L/h over a 12-h period daily (2200 to 0800 h) for 52 d before initiation of an estrous synchronized protocol that included a 7-d controlled internal drug release device (CIDR), PGF2a, and fixed-time artificial insemination. Cows were exposed to urine or saline during the 7 d that CIDRs were in place and were discontinued at CIDR removal; 72 h before fixed-time artificial insemination Interval from the start of urine or saline exposure to resumption of luteal activity by 52 d did not differ between bull urine- and saline-exposed cows. Likewise, there was no difference in the proportions of cows that resumed luteal activity by the end of the 52-d exposure period. Although artificial insemination pregnancy rates 35 d after insemination of bull urine- and saline-exposed cows did not differ significantly, there appeared to be a benefit to exposing cows to bull urine (65 and 48% for bull urine- and saline-exposed cows, respectively). In conclusion, exposing primiparous, postpartum, anovular, suckled, beef cows to mature bull urine for 12 h daily does accelerate resumption of ovulatory activity but it does appear to improve artificial insemination pregnancy rates. Furthermore, bull urine per se may not be the medium that carries the biostimulatory pheromone(s) that accelerates resumption of ovulatory activity in the bovine. However, bull urine may carry an additional pheromone(s) that is associated with fertility in the bovine.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
The biostimulatory effect in sheep is known to cause a relatively rapid increase in LH pulse frequency in ewes that accelerates resumption of ovulatory activity during the transition into the breeding season. Recently, it was reported that cortisol pulse frequency decreased and pulse amplitude increased in postpartum, anovular, suckled cows exposed to bulls. It was suggested that alterations in hypothalamic-pituitary-adrenal axis activity may play a role in the physiologic mechanism of the biostimulatory effect of bulls to accelerate resumption of ovulatory activity in the bovine. It is not known if activation of the hypothalamic-pituitary-adrenal axis is involved with the ram effect in sheep. The objective of this experiment was to determine if temporal patterns of cortisol concentrations are altered in 18-mo-old virgin Targhee ewes exposed to rams during the transition into the breeding season. The hypotheses were that exposing seasonally anovular ewes to rams would alter patterns of cortisol concentrations and that these changes are related to changes in temporal characteristics of LH concentrations that accelerate the occurrence of ovulation. Seasonally anestrous ewes were randomly assigned to be exposed to rams (RE; n = 12) or exposed to wethers (NE; n = 12). Blood samples were collected at 15 min intervals beginning 120 min before introduction of males (time = 0 min), and continued for 360 min of male exposure. Sera were assayed for cortisol and LH by RIA. Characteristics of temporal cortisol and LH concentrations included: mean and baseline concentrations, pulse amplitude, duration, and frequency. The difference between mean and baseline concentrations, pulse amplitude or frequency of cortisol peaks before and during exposure to males did not differ between RE and NE ewes; however, cortisol pulse duration was longer in RE than in NE ewes. The difference between mean and baseline concentrations of LH, LH pulse amplitude and duration of LH pulses before and during exposure to males did not differ between RE and NE ewes; however, number of LH pulses during exposure to males was greater in RE ewes than in NE ewes. Similarly, the change in LH pulse frequency increased in RE ewes after exposure but decreased in NE ewes after exposure. There were no linear relationships for change in pulse amplitude, duration, and frequency between cortisol and LH concentrations before and during exposure to males. In NE ewes, as the change in mean cortisol concentrations increased there was a linear decrease in the change of mean LH concentrations; likewise, as the change in baseline cortisol concentrations increased the change in baseline LH decreased before and during exposure to wethers. However, there were no linear relationships for the change in mean or baseline cortisol and LH concentrations before and during exposure to rams in RE ewes. These results are consistent with current knowledge that cortisol has a suppressive effect on LH concentration patterns in ewes. It appears that when ewes are exposed to rams this relationship is quite different; instead, ram exposure increased cortisol pulse duration and LH pulse frequency. We postulate that ram exposure during the transition into the breeding season alters hypothalamic-pituitary-adrenal axis activity by permitting an increase in LH pulse frequency which in turn stimulates the ovulatory cascade and accelerates ovulatory activity.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
The purpose of this study was to re-examine the pattern of cytosine methylation (5MeC) in mouse preimplantation stage embryos. It is currently considered that progressive demethylation of the genome occurs throughout the preimplantation stage and new methylation occurs at or soon after implantation. Given the essential requirement for DNA methylation for genomic stability this prolonged relative demethylation creates a biological paradox. We have therefore undertaken a reassessment of this phenomenon. The immunofluorescence staining method was used to test the 5MeC in different stages of preimplantation stage embryos. We found that global demethylation of both the paternal and maternal genome progressively occurs throughout the zygote stage and is complete by syngamy. This demethylation can be perturbed after IVF or prolonged culture. By the 2-cell stage, embryos collected direct from the oviduct showed some remethylation high levels of cytosine methylation, and interphase nuclei 4-cell and 8-cell embryos stained extensively for 5meC. The remethylation during the early 2-cell stage was inhibited by the selective DNMT inhibitor, RG108 (5 µM) - late stage zygotes where incubated for 16h with or without RG108 and the resulting 2-cell embryos stained for 5meC. The embryos that developed in the presence of the DNA methyltransferase inhibitor showed significantly less methylcytosine staining than the embryos in the untreated culture conditions (p<0.001). Treatment of embryos during this period with RG108 significantly reduced their capacity to develop to normal blastocysts, indicating that this early DNA re-methylation reaction was important for the normal development of the embryo. These results indicate that the current paradigms of epigenetic reprogramming in the early embryo require reassessment.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Folate is a nutrient that is essential for cell proliferation and involved in the biosynthesis of nucleotides and S-adenosylmethionine (SAM). SAM is the methyl donor for numerous cellular biochemical reactions including DNA methylation, an important epigenetic modification implicated in the control of gene expression, genomic imprinting and normal fertility. Although folate supplementation is used as a treatment for human male infertility, little is known about the effects of low or high dietary folate concentrations on sperm DNA methylation patterns. Our goal was to use a mouse model to examine the effects of folic acid deficiency and supplementation on epigenetic patterns in male germ cells. Male Balb/c strain mice were fed, from weaning age (day 21) to one year of age, either a control diet containing an adequate amount of folic acid (2 mg/kg diet), a 7X low folic acid diet (0.3 mg/kg diet) or a 20X high folic acid diet (40 mg/kg diet). The mice were sacrificed at 12 months and reproductive organs (testes, epididymides and seminal vesicles) collected and weighed. Sperm were collected from the cauda epididymides and genomic DNA extracted. Restriction landmark genomic scanning (RLGS) was used to assess genome-wide levels and patterns of DNA methylation (n=4 samples/group). Although weight gain and reproductive organ weights were not affected by the folate deficiency or supplementation, for both treatment groups, RLGS demonstrated evidence of epigenetic abnormalities in sperm. Mice in the folic acid supplemented group exhibited six consistent changes in their methylation profiles, all of which were hypermethylation. Interestingly, among the 14 loci showing methylation alterations in all of the folic acid deficient mice, 10 were hypermethylated and four were hypomethylated. The loci with altered methylation are in the process of being identified using a virtual RLGS profile. Together, the results indicate that DNA methylation patterns in mouse sperm can be altered by long term exposure to both low and high dietary folate. (Supported by CIHR)
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Unexplained human pregnancy loss accounts for 25% of spontaneous miscarriages, with a considerable number occurring during the early stages following implantation. The first organ to form during pregnancy is the placenta, whose function is vital to the growth, well-being and survival of the future developing fetus. Genomic imprinting is one of the most important regulatory mechanisms involved in the normal function of the mammalian placenta. Imprints are established differentially, in a parent specific manner during gametogenesis, by DNA methylation of CpG sites in differentially methylated regions (DMRs) of imprinted genes that confer either transcriptional repression or gene expression. Following fertilization and activation of the embryonic genome, imprinted genes are expressed monoallelically from either the paternal or maternal allele. Hypo- or hyper-methylation which could result in biallelic expression or gene silencing respectively, can adversely affect fetal and placental growth, often causing lethal phenotypes during early development. The aim of this study was to investigate DNA methylation patterns of three known imprinted loci in resorption sites following early pregnancy loss, compared to viable healthy placenta. Pregnant 8-10 week old female BDF1 mice were sacrificed on d16 of fetal development. Biopsies of viable healthy placenta (n=6) and resorption sites (n=5) were collected for DNA methylation studies. Quantitative analysis using methylation sensitive (Hha1) and dependant (McrBc) restriction enzymes followed by real-time PCR was performed for the investigation of DNA methylation in the DMRs of three known imprinted genes: Igf2r, Kcnq and Peg1. Expected methylation values close to 50% were observed for the methylation dependant restriction enzyme, McrBc in both viable placenta and resorption biopsies for all three imprinted genes. In contrast, the imprinted gene Peg1 had a lower degree of methylation observed in viable placenta (34.89%) and a higher degree of methylation observed in resorption biopsies (62.05%) for the methylation sensitive restriction enzyme, Hha1 (P<0.05). Additionally, a higher degree of methylation was observed for the imprinted genes Igf2r and Kcnq in resorption biopsies (63.85% and 63.82% respectively) compared to viable placenta (44.04% and 50.97% respectively) for the methylation sensitive restriction enzyme, Hha1 (P<0.05). This data would indicate that viable placenta possess hypo-methylation at certain CpG sites within the DMR of the imprinted gene, Peg1. This is in concordance with previous studies that have shown lower levels of methylation at repeat sequences in placental tissues, as well as for some restriction enzymes in the DMRs of imprinted genes. Our results have also revealed potential hyper-methylation at certain CpG sites in the DMRs of all three imprinted genes relative to early pregnancy loss. Interestingly, imprinted loci disruptions have been observed in several human developmental disorders including partial hyper-methylation at the DMR of Peg1 in Silver-Russell syndrome. Further investigation into the role of DNA methylation patterns in both normal placental function and pregnancy loss is warranted and may assist with future patient management.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
H19 is an imprinted non-coding RNA known to be transcribed from the maternally inherited allele. Genomic imprinting is defined as parent-specific gene expression. Previous research has shown maternal-specific expression of H19 in mouse blastocysts developed in vivo and biallelic expression in embryos cultured in vitro. In those studies embryos were collected at a particular stage of embryonic development (i.e. late blastocyst stage) despite the time disparity for embryos in different treatment groups to reach the desired developmental stage. For this study, we determined H19's allelic expression in embryos produced after ovarian hyperstimulation at specific times (84 hours, 96 hours, or 108 hours) following presumed ovulation, as opposed to specific stages of development. In addition, we determined the effects of two culture conditions (KSOM augmented with amino acids or Whitten's medium) on imprinted H19 expression in embryos cultured from the 2-cell stage, collected at 32 h post presumed ovulation, until 84, 96, or 108 h post-ovulation. RNA was collected and cDNA was synthesized from single embryos. Results showed that approximately 25% of embryos in all groups expressed biallelic H19 at 84 and 96 hours post-ovulation. Surprisingly, 50-77% of blastocysts expressed H19 from the normally silent paternal allele in all three treatment groups (i.e. in vivo produced, KSOM and Whitten's) at 108 hours post-ovulation. From these results we concluded that biallelic H19 expression is a normal event around the time of implantation in mouse embryos. However, recent data from our laboratory shows that superovulation can affect the level of global DNA methylation in growing oocytes. Therefore, we set out to determine if the high level of biallelic H19 expression was really the result of a developmental cue or simply an adverse outcome of the superovulation scheme. For the second study, we followed the same design as that described for embryos from superovulated females but this time the embryo donors did not receive hormonal stimulation. Briefly, females were observed for the presence of a copulatory plug to establish approximate time of ovulation. Embryos were either collected at the 2-cell stage and cultured as above or flushed from the uterus at 84 hours, 96 hours, or 108 hours. As in the previous experiment, cultured embryos were collected at equivalent times post ovulation. Again, RNA was collected and cDNA was synthesized from single embryos. Following PCR, products were restricted with an enzyme unable to cut the paternal allele allowing us to differentiate expression of each parental allele. Preliminary results show a similar pattern of biallelic expression in embryos produced after natural ovulation as those produced after superovulation. We suggest that during the peri-implantation stage, biallelic expression of H19 may be attributed to a molecular clock and not to be the result of embryo culture as has been previously concluded.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Oogenesis in mammals was historically believed to cease in the perinatal period, thus endowing females with a finite supply of oocytes. Ovarian failure, whether by natural or pathological means, was consequently attributed to irreversible depletion of this finite oocyte reserve. These traditional concepts have been challenged recently, with accumulating evidence supporting the existence of female germline stem cells (fGSCs) in the postnatal mammalian ovary. While unexpected and highly controversial, these findings raise the distinct possibility that ovarian failure could be a target of therapeutic modification, with significant clinical implications for women's health. Due to the prevailing dogma regarding mammalian oogenesis, progress in exploring the function and regulation of vertebrate fGSCs and their role in postnatal oogenesis has been slow. Among the many limitations has been a lack of genetically relevant, experimentally tractable models of vertebrate oogenesis. Herein, we describe development of a novel vertebrate model of fGSC biology, designed specifically to provide insights into postnatal oogenesis and ovarian regeneration. We have combined chemical and genetic tools to generate a unique transgenic zebrafish model of targeted oocyte ablation that permits direct observation of fGSC-mediated regeneration. The approach employs a reversible, gene-directed enzyme prodrug therapy system, using the zona pellucida C (zpc) gene promoter to drive oocyte-specific expression of a bacterial nitroreductase enzyme. Oocyte-specific expression of nitroreductase catalyzes the reduction of a prodrug (metronidazole) to a cytotoxic alkylating agent, designed to induce oocyte-specific cell death with no deleterious effects on fGSCs. Prodrug removal is predicted to result in fGSC-mediated oogenesis to replace ablated oocytes. Preliminary studies have confirmed that a seven day exposure to metronidazole results in rapid oocyte ablation. We now propose to use this approach to investigate postnatal oogenesis, with the goal of identifying regulatory factors underlying fGSC function. This unique genetic resource will provide valuable insights into the molecular and cellular control of postnatal oogenesis, while providing a novel model of ovarian regeneration. We are hopeful that this work will assist in resolving the debate surrounding the existence of fGSCs and postnatal oogenesis in mammals, and contribute to the development of novel therapeutic strategies urgently required to treat or postpone premature ovarian failure.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
The mouse and rat have been important model organisms for elucidating meiotic regulatory mechanisms in mammals. However, rarely are parallel studies carried out with these two species that allow direct comparison of experimental results. The present study was conducted with just this purpose--to compare meiotic behavior in vitro in isolated oocytes from primed mice and rats under a variety of meiosis-arresting and -inducing conditions. Cumulus cell-enclosed ocytes (CEO) were isolated from 20-23-day-old mice and 24-26-day-old rats primed with pregnant mare serum gonadotropin. Denuded oocytes (DO) were obtained by removing cumulus cells with a small-bore mouth-operated pipet. The culture medium used was Eagle minimum essential medium with 3 mg/ml bovine serum albumin. When CEO were cultured overnight in medium containing increasing concentrations of hypoxanthine, dbcAMP, or IBMX, meiotic arrest was maintained much less effectively in rat oocytes than in mouse oocytes. Since the best inhibition in rat CEO was obtained with IBMX (37% germinal vesicle breakdown, or GVB, at 100 µM in rats versus 8% GVB at 50 µM in mice), this inhibitor was used at these concentrations for meiotic induction experiments. Follicle-stimulating hormone (FSH), amphiregulin (AR) and 3-h dbcAMP pulsing induced GVB in mouse CEO (mCEO) but had no effect in rat CEO (rCEO). Human chorionic gonadotropin (hCG) was ineffective in both groups. Interestingly, cumulus cells of both mCEO and rCEO responded to FSH, AR and dbcAMP by undergoing expansion in serum-supplemented medium. When IBMX-arrested mCEO were treated with 1 mM pyruvate, meiosis was induced, but 5.5 mM glucose was inhibitory; surprisingly, these energy sources had the opposite effects in rCEO. In inhibitor-free medium, polar body formation in pyruvate-supplemented medium was augmented in mCEO by glucose but suppressed in rCEO. Further, GVB was stimulated in mCEO by the IMP dehydrogenase inhibitor, mycophenolic acid, with no effect in rCEO. Finally, the AMP-activated protein kinase (PRKA) activator, AICAR, was a potent stimulator of maturation in mCEO and mDO, but only marginally stimulatory in rCEO and ineffective in rDO. When oocytes were stressed by PRKA-activating stresses (heat pulsing and treatment with 2-deoxyglucose), GVB was induced in mouse oocytes but not rat oocytes. Moreover, 2.5 h after hCG injection, a high percentage of freshly isolated mouse oocytes exhibited GV-positive staining for active PRKA but no such staining was observed in rat oocytes, supporting a causal role in GVB in mouse, but not rat, oocytes. Yet immunofluorescent staining revealed that later in maturation, active PRKA was localized to the MI and MII spindle poles and anaphase midzone in both mouse and rat oocytes. Rat oocytes were not physiologically compromised, because they readily underwent spontaneous maturation in vitro to MII and exhibited levels of GVB comparable to mouse oocytes in vivo 2.5 h after hCG injection (~65%) and in vitro following hCG stimulation of preovulatory follicles for 6 h. These data indicate significant metabolic differences between mouse and rat CEO and suggest that the cumulus cell is a more important meiotic regulatory site in mice than in rats.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Following germinal vesicle breakdown (GVBD), the genome of an immature oocyte is transcriptionally inactive and any changes in mRNA and protein repertoire until the maternal to zygote transition (MZT) are controlled through interactions with the surrounding cumulus oophorus and post transcriptional events occurring within the oocyte and early embryo. MicroRNAs have a demonstrated ability to regulate both mRNA and protein repertoires through their ability to confer post transcriptional gene regulation through interactions with the 3' untranslated region (UTR) of mRNA molecules. The outcome following an miRNA:mRNA interaction can depend largely on the expression of several critical RNA binding proteins, such as dead end homolog 1 (DND1). DND1 is critical for germ cell development and has more recently been demonstrated to prevent miRNA mediated transcript degradation through interactions with the 3' UTR of miRNA targeted transcripts. Our working hypothesis is that numerous miRNAs are expressed during oocyte maturation and that the presence of RNA binding proteins such as DND1 influences miRNA impact on mRNA stability and translation during oocyte maturation and prior to MZT following in vitro fertilization. The objective of the current study was to determine the expression of miRNAs in the oocyte during in vitro maturation and the presence of DND1 during oocyte maturation and early embryo development following in vitro fertilization. We utilized one microgram of total RNA from germinal vesicle (GV) stage oocytes and metaphase II (MII) arrested oocytes for miRNA microarray analysis and identified several hundred mature miRNAs are expressed in the oocyte, including miR-21, miR-23, miR-24, miR-34, miR-483-5p, and numerous others consistent with those expressed in mice oocytes during meiotic progression. We then utilized pools of 25 GV, MII and 4- to 8-cell stage embryos (n=4 per stage) for quantitative real-time PCR and pools of 100 GV and MII oocytes (n=3 per stage) for western blot analysis of DND1. Absolute quantification of DND1 mRNA indicated that abundance was greatest in GV stage oocytes and was approximately 2-fold (P > 0.05) less in MII arrested oocytes following in vitro maturation. Embryos in vitro fertilized and cultured to the 4- to 8-cell stage demonstrated an approximate 7.9 fold (P < 0.05) reduction in DND1 mRNA compared to MII arrested oocytes. Based on western blot analysis DND1 (~34 kDa) was present in both GV stage and MII arrested oocytes and was not significantly different (P > 0.05) between the two stages. Collectively, these data demonstrate the presence of numerous mature miRNAs in the pig oocyte during maturation temporal to the relative abundance of DND1 mRNA and protein expression. The presence of numerous miRNAs in the maturing oocyte suggests post transcriptional gene regulation may be partially controlled by DND1 interactions with mRNAs possessing the appropriate RNA recognition motif in the 3'UTR. This work was supported in part by the USDA National Institute of Food and Agriculture (2008-35205-05309) and the Iowa State University Center for Integrated Animal Genomics.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
We have shown that like starvation, ingestion of the bacterial product Rapamycin (RAP) interferes with egg production in female Drosophila. RAP ingestion results in posterior follicle cells (PFC) in Stage 8/9 egg chambers losing epithelial polarity, after which PFC invade and phagocytose the oocyte. Germ cell apoptosis was only detected after PFC lost polarity, after which total egg chamber destruction occurred. Strikingly, knockdown of the RAP receptor FKBP12 specifically in PFC rescues oogenesis in flies fed RAP and also the laying of embryos that can develop into normal offspring. This result showed that somatic cells can be induced to destroy intact oocytes of normal ‘quality,’ without a requirement for prior oocyte compromise. A screen of candidate genes has so far revealed that the induction of oocyte destruction by RAP involves genes that control apicobasal epithelial polarity. In flies bearing heterozygous mutations for discs large, merlin, or warts, PFC epithelia fail to lose polarity upon RAP treatment. Embryo laying and offspring development to adulthood are rescued in these mutants when housed on RAP concentrations that entirely block oogenesis in wild-type flies. The response to RAP was found to be conserved in mammals, as mouse ovarian follicles cultured in vitro with RAP show the rapid destruction of the oocyte by adjacent granulosa cells. The demonstration that ‘healthy’ oocytes can be destroyed by adjacent somatic cells activated by extrinsic stimuli is a novel finding that has implications as we consider mechanisms that result in oocyte loss over time.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
The oocyte has limited capacity to utilize glucose on its own and relies on the surrounding cumulus cells to metabolize glucose and provide metabolic intermediates. Type I diabetes results in multiple defects in both oocyte and cumulus cell metabolism including decreased ATP and citric acid cycle metabolites and mitochondrial abnormalities. NAD is as a coenzyme involved in redox reactions during cell metabolism. Recent studies have investigated NAD synthesis as well as NAD-dependent changes in gene expression. Nicotinamide phosphoribosyltransferase (NAMPT) is the rate limiting enzyme in the synthesis of NAD that catalyzes the synthesis of nicotinamide mononucleotide (NMN) from nicotinamide and 5-phosphoribosyl-pyrophosphate (PRPP). NAMPT-mediated NAD synthesis plays a role in many cellular processes such as metabolism, stress, and aging. The Sir2 (silent information regulator 2) family of NAD-dependent deacetylases, known as sirtuins, couple NAD availability to deacetylation of target proteins, and changes in gene expression. In these studies we investigated the effects of Type I diabetes on expression of NAMPT and the Sir2 family member, SIRT1 in cumulus cells and oocytes of mice. To generate hyperglycemia, female B6SJL mice were injected with streptozotocin (STZ) at a dose of 85 mg/kg for 3-4 days until glucose levels greater than 250 mg/dl were detected. Akita mice have an autosomal dominant mutation resulting in hyperglycemia and β-cell dysfunction and were used as a genetic model of Type I diabetes. Cumulus enclosed oocytes were collected from control or diabetic mice 48 h after injection of 10 IU equine chorionic gonadotropin. Oocytes and cumulus cells were separated by manual pipetting and prior to freezing. For Western blot analysis, 200 denuded oocytes or 5 µg of protein isolated from cumulus cells were used per lane. For protein analysis NAMPT (1:3000), SIRT1 (1:2000), or GLUT1 (1:3000) antibodies were incubated overnight at 4°C. All proteins were normalized to β-ACTIN (1:5000) and analyses were performed using ImageJ software. NAMPT was elevated in the denuded oocytes of both STZ-induced and Akita mice compared to non-diabetic controls. We also observed increased SIRT1 in denuded oocytes of STZ-treated mice. In cumulus cells the reverse was true. There was a decrease in both NAMPT and SIRT1 in cumulus cells from STZ-treated mice. GLUT1 content in cumulus cells from STZ-treated mice was also decreased, which could result in decreased glucose in these cells. These are the first studies to investigate the roles of NAMPT and SIRT1 in oocytes and cumulus cells, and indicate that these proteins respond differently in cumulus cells and oocytes in response to hyperglycemia. The cumulus cells, which metabolize most of the glucose in the cumulus-oocyte-complex have decreased NAMPT, and a subsequent decrease in SIRT1 as a result of hyperglycemia. However, in the oocyte both NAMPT and SIRT1 are increased. Others have reported changes in gene expression as a result of diabetes in cumulus cells from both mice and women. This study identifies changes in the NAD-dependent deacetylase SIRT1 in cumulus cells and oocytes, a possible link between metabolism and altered gene expression. Future studies will continue to investigate the role of NAD biology in cumulus cells and oocytes.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Calcium (Ca2+) is known to be an important regulator in the apoptotic signaling. Calbindin-D9k (CaBP-9k) and -D28k (CaBP-28k) have a high affinity to Ca2+ ions. Uterine calbindins have been shown to be involved in the regulation of myometrial activities by intracellular Ca2+. In addition, uterine calbindins are expressed in the mouse endometrium and highly regulated during implantation and development. The aim of the present study is to evaluate the regulation of apoptosis in the uterus of immature CaBP-9k, CaBP-28k, and CaBP-9k/28k knockout (KO) mouse models. Our finding indicate that the Bax protein levels were enhanced in the uterus of CaBP-28k and CaBP-9k/28k KO mice compared to wild-type mice, and no difference was observed in Bcl-2 protein expression. Also, the levels of caspase 3, 6 and 7 protein were induced in both CaBP-28k and CaBP-9k/28k KO mice compared to wild-type mice. These results suggest that absence of calbindins may induce an increase in the apoptotic signaling. In addition, we investigated the expressions of the endoplsmic reticulum stress genes by western blot analysis in calbindin KO mice. These results showed that CHOP and Bip proteins were increased in CaBP-28k and CaBP-9k/28k KO mice compared to wild-type mice, and no difference was observed in the expression of PDI and IRE1alpha proteins. It is of interest that the expression of CaBP-28k blocked up-regulation of apoptosis-related gene expression. In summary, this study demonstrates that CaBP-28k decreases the apoptosis-related gene expression in uterine mice.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Ovarian cancer is the fifth leading cause of cancer death among women in the United States, and is the most lethal of the gynecologic cancers. Although it has long been believed that ovarian cancer originates from the ovarian surface epithelium, advanced serous ovarian carcinoma shares several biomarkers with secretory epithelial cells of the Fallopian tube, leading to new research indicating that some forms of ovarian carcinoma may originate in the Fallopian tube. It is been proposed that neoplastic tubal epithelial cells metastasize to the ovary, which is rich in hormones and growth factors that further encourage cancer growth. Regardless of the cell type responsible for ovarian cancer, it is likely that the ovary plays a role in development and progression of disease. Current hypotheses concerning the etiology of ovarian cancer propose that over a lifetime of ovulation, inflammation at the site of ovulation generates DNA-oxidizing free radicals. Combined with increased rates of cell proliferation that occur as the ovarian surface epithelial cells repair the wound induced by ovulation, neoplastic changes in the ovarian surface epithelium or tubal epithelium may occur. As both the ovarian surface epithelium and the tubal epithelium express receptors for the gonadotropins, high levels of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) that occur during menopause may then act on neoplastic cells to encourage cancer growth. To better understand the roles that the ovary and Fallopian tube play in ovarian cancer, we have analyzed the impact of ovulation on tubal epithelial cells by superovulating CD1 mice and examining cell proliferation and DNA damage. Tubal epithelial cells from superovulated animals do not exhibit increased proliferation as compared to non-superovulated animals. However, superovulation induces increased levels of phospho-histone H2A.X in tubal epithelial cells, indicating that these cells are susceptible to double-stranded DNA breakage in response to ovulation. To begin to separate the components of ovulation that may contribute to DNA damage in the Fallopian tube, we have developed both an immortalized baboon tubal epithelial cell line and a three-dimensional organ culture system for mouse oviduct and baboon Fallopian tubes. Both of these culture systems maintain secretory and ciliated epithelial cell types as analyzed by immunohistochemical staining and Western blotting for cell markers. To determine the effect of the gonadotropins on tubal epithelial cells, FSH or LH was added to either the baboon tubal epithelial cell line or to the baboon or mouse 3D organ cultures. Mouse and baboon tubal epithelial cells did not proliferate in response to FSH or LH as analyzed by MTS cell proliferation assay or incorporation of BrdU into proliferating cells. It is therefore likely that inflammation induced by ovulation may play a role in generating oxidative stress leading to DNA damage in tubal epithelial cells. Tubal epithelium is susceptible to ovulation-induced DNA damage, indicating that ovulation may play a role in the initiation and progression of serous carcinoma in the Fallopian tube.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Phospholipase A2 (PLA2) constitutes a family of enzymes that hydrolyze fatty acids from the sn-2 position of phospholipids with the concomitant formation of lysophospholipids. The fatty acid, arachidonic acid serves as the major precursor of eicosanoids, such as prostaglandins, prostacyclins and thromboxanes. Lysophospholipids serve as precursors of other lipid mediators such as platelet activating factor and lysophosphatidic acid. Based largely on structural and enzymatic properties, PLA2s have been grouped into four main subfamilies: low molecular weight secreted PLA2s (Groups 1-3, 5, 10 and 12); cytosolic PLA2G4; calcium-independent PLA2G6 and platelet activating factor acid hydrolases (PLA2G7 and PLA2G8). Secreted (s) PLA2 enzymes are of particular interest in feto-maternal interactions because they may act extracellularly at the level of the outer leaflet membranes, plasma lipoproteins present in the uterine lumen and membrane receptors. The objective of this study was to identify the sPLA2 enzymes produced by ovine uterine endometrium and conceptuses during the peri-implantation period of pregnancy. Uteri were collected surgically from sheep on days 13 through 24 of pregnancy. Conceptuses were flushed from uteri with sterile saline and several pieces of endometrium from the central regions of uterine horns ipsilateral to the corpus luteum were collected. Total RNA was isolated from tissues and both semi-quantitative PCR and quantitative real-time PCR analyses were performed using primers specific for PLA2G1B, PLA2G2A, PLA2G5 and PLA2G10. Proteins from tissues and uterine washings were extracted and submitted to Western blot analysis using specific antibodies to the secreted PLA2 enzymes listed above. Both conceptus tissue and endometrium expressed mRNA for all sPLA2 enzymes analyzed. Conceptus expression of PLA2G1B mRNA increased significantly between days 15 and 17 of pregnancy and remained elevated whereas endometrial expression was relatively uniform over the same time period. Conversely, expression of PLA2G10 mRNA by endometrium increased between days 15 and 17 but conceptus expression if this sPLA2 was relatively unchanged. Secreted PLA2 proteins and activity were observed in uterine washes. Protein expression tended to reflect mRNA results. Changes in expression of sPLA2 enzymes occurred between days 15 and 17 a time coincident with rapid conceptus growth and attachment to the endometrium indicating that sPLA2s may regulate expression of lipid mediators involved with these processes.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Treatment of hamsters on the day of birth with the synthetic estrogen and prototypical endocrine disruptor, diethylstilbestrol (DES), results in a 100% incidence of uterine hyperplasia/dysplasia in adulthood and a large proportion of the disrupted organs progress to neoplasia (endometrial adenocarcinoma). Extensive histomorphological investigation of this pathological phenomenon established that, in accordance with the classic two-stage model of carcinogenesis, the neonatal DES exposure event directly and permanently disrupts the developing hamster uterus (initiation stage, IS) so that it responds abnormally when it is stimulated with the natural ovarian estrogen, estradiol (E2), in adulthood (promotion stage, PS). Molecular elements involved in both stages of this phenomenon were first profiled by microarray and immunoblot analyses of total RNAs and proteins extracted from whole uteri. Those analyses at the whole-organ level revealed a number of genes whose expression at both the mRNA and protein level were at least 2-fold and significantly different in neonatally DES-exposed vs. control uteri. Our follow-up immunohistochemical analyses based on those profiling findings generated the following observations: Progesterone Receptor: Nuclear staining in both epithelial and stromal cells up-regulated in IS but down-regulated in PS. E-Cadherin: Epithelial cell membrane staining up-regulated in both IS and PS. Insulin Receptor Substrate-1 (IRS-1): Nuclear staining in stromal cells up-regulated in IS. Specificity Protein 1 (Sp1): Epithelial and stromal cell nuclear staining down-regulated in PS. Signal Transducer and Activator of Transcription 5A (Stat5A): Cytoplasmic staining in epithelial cells up-regulated in IS. Tenascin: Stromal extracellular matrix staining up-regulated in IS (altered distribution in PS). These results provide important new tissue/cell-specific information about altered proteomics in our experimental system. They also confirm that: 1) progression of the neonatal DES-induced disruption/neoplasia process in the hamster uterus involves a spectrum of gene expression alterations, and 2) the complex of genes and their manner of altered expression is quite different during the initiation vs. promotion stages of the phenomenon. Supported by the Flossie E. West Memorial Foundation and NIH grant P20 RR016475.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Amplification and overexpression of centrosome-regulating pathways occurs in many human cancers. Small molecule inhibitors targeting different components of these pathways are now being tested in different clinical scenarios. Several investigators have explored whether overexpression of centrosome-regulating Aurora kinases occurs in cervical cancers. We hypothesized a novel, small molecule inhibitor highly specific for Aurora A kinase could be utilized to arrest the growth and proliferation of cervical cancers. To test this hypothesis, we treated established cervical cancer cell lines with varying doses of AURKA inhibitor, MK-5108. We found that two cervical cancer cell lines, HeLa and Caski, responded to increasing concentrations of MK-5108 with decreased proliferation and cell viability with an IC50 of 150 nM and 300 nM, respectively. However, MK-5108 appeared to have little impact on the growth on SIHA cells. Introduction of MK-5108 resulted in an arrest of HELA cells at the G2-M checkpoint, associated with a significant decrease in phosphorylation of the downstream AURKA target, CEN-PA. Decreased levels of CEN-PA phosphorylation could be observed starting at 10ng/ml of MK-5108 in vitro. Treatment of both Caski and Hela cells potentiated the effects of docetaxel, particularly at low doses. Surprisingly, MK-5108 decreased the cytotoxic impact of doxorubicin, gemicitabine or cisplatin on both Caski and Hela cells. To better assess the effects of MK-5108 in vivo, we established a xenograft model that relied on the IP injection of 1 x 106 HELA cells. Using this model, we found that weekly administration of 60 mg/kg of MK-5108 for 4 weeks resulted in a significant decrease in both total and individual tumor weights, an observation absent at the 30 mp/kg dose when compared to sham gavaged controls. This was accompanied revealed a 2-fold greater number of apoptotic cells in sections of tumor retrieved from MK-5108-treated mice compared to untreated animals. In conclusion, our observations indicate that MK-5108 may offer a novel therapeutic strategy for clinically targeting cervical cancers either as a single agent or in combination with docetaxel. However, MK5108 does not appear to potentiate the proven cell killing effects of established chemotherapeutic agents that require an intact G2-M cell cycle checkpoint for their therapeutic effects.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Elevated circulating P4 advances conceptus elongation via alterations in endometrial gene expression including the early loss of the nuclear progesterone receptor (PGR) from the luminal epithelium (LE) and superficial glands (SG). In heifers with low P4, conceptus development is delayed following embryo transfer on Day 7. We aimed to identify 1) if the expression pattern of previously identified progesterone-regulated genes is altered in cyclic heifers with low P4 and 2) if low P4 delays the down-regulation of the PGR from the LE and SG. Fifty-two beef heifers in standing estrus were randomly assigned to (i) high P4, (ii) normal P4 (control) or (iii) low P4 treatments. Elevated P4 was achieved by insertion of an intravaginal P4 device on Day 3 post estrus while low P4 was achieved by repeated administration of PGF2α. Heifers were slaughtered on Day 7 or 13. Uterine cross sections and endometrial tissue were removed from the uterine horn ipsilateral to the corpus luteum (n=5 per time point in the high and normal P4 groups and n=7 per time point in the low P4 group). Quantitative real-time PCR was performed to determine if Low P4 affected the expression of genes previously shown to be altered by elevated P4. Immunohistochemistry was performed to localise the PGR. Heifers in the low P4 group had lower (P<0.05) serum P4 concentrations than those with normal or high P4. Heifers in the high group had higher (P<0.05) P4 on Days 4-7 compared with controls. Low P4 did not alter the expression of SLC2A5, PCR1 or CYP26 expression. DKK1, ASGR2 and MGC137099 expression was lower (P<0.05) in Low P4 heifers on Day 7 compared with controls. Both ANPEP and LPL expression was lower on Day 7 and their down regulation on Day 13 was delayed in low P4 heifers (P<0.05). In contrast, the increase in CTGF expression on Day 13 in normal heifers was delayed in the Low P4 group (P<0.05). DGAT2 expression was lower in Low P4 on Day 7 and higher on Day 13 compared with controls. Analysis of the triglyceride pathway revealed GPAT, DGKA, MOGAT1, MOGAT2, AGPAT3 and AGPAT4 genes were not affected by Low P4. AGPAT 1, 2, 5 and 6 expression was higher (P<0.05) in heifers with Low P4 on Day 7 compared with controls. On Day 7, heifers with low P4 displayed higher (P<0.05) PGR mRNA expression and increased (P<0.05) PGR protein in the LE and SG compared with the normal and high P4 groups. In conclusion, low P4 in cyclic heifers alters temporal changes in gene expression in the endometrium, concomitant with a delay in the down-regulation of the PGR from the LE on Day 7. This altered gene and protein signature may contribute to delayed conceptus development observed in the presence of low P4. Funded by Science Foundation Ireland research grants 06/IN1/B62 and 07/SRC/B1156.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Spermatozoa can remain viable during hours or even days in the oviductal environment and some of them undergo several metabolic and functional changes to become fertilizing-competent (process known as capacitation). We have found that proteins from conditioned medium (CM) of human oviductal tissue cultures modulate some sperm functions. Thus, the aim of the present study was to isolate and identify proteins from CM with capacity to interact with human spermatozoa. One of the isolated proteins, identified as calgranulin B, was detected in the native human oviductal fluid (nOF) and immunolocalized in the oviductal tissue and spermatozoa. Human tubal tissues were obtained from pre-menopausal women (n= 20) with no clinical history of infection or cancer disease, scheduled for routine hysterectomies. The nOF were recovered by flushing the tubes with DMEM/Ham F12 medium. Explants of tubal tissues were cultured in DMEM/Ham F12 medium at 37°C and 5 % CO2 for 24 h, followed by a further incubation with [35S]Met (30 µCi/ml) during 24 hs, to obtain de novo [35S]Met-proteins. After incubation, CM was collected and centrifuged 5 min at 700 × g, to remove debris. The CM were then dialysed, lyophilised and stored at -70°C until use. Total protein concentration in CM was determined using the Bradford assay. Human spermatozoa were obtained from normozoospermic samples of healthy donors (n=4) after 3 days of sexual abstinence. Motile sperm were recovered by swim-up and were incubated under capacitating conditions in Ham F10 medium supplemented with 3.5% BSA for 2 h in the absence or the presence of [35S]Met-proteins from CM. At the end of incubations, sperm membrane proteins (SMP) were extracted, analysed by SDS-PAGE (10%) and electrophoretically transferred to nitrocellulose membranes. In addition, a chromatographic affinity column was prepared with motile sperm membrane extracts coupled to Sepharosa 4B, in which CM was seeded. The eluted protein fractions were subjected to SDS-PAGE 10%. The protein bands were analysed by LC-MS/MS. The presence of the identified protein in nOF and CM was examined by western blot analysis using specific antibodies. A protein band with an estimated molecular weight (MW) of 14 kDa was detected in the autoradiographies of SMP from spermatozoa that were incubated with [35S]Met-labelled oviductal proteins. At least five [35S]Met-protein were detected in the eluted fractions obtained by affinity chromatography, with estimated MW of 127 kDa, 94 kDa, 79 kDa, 17 kDa and 14 kDa. The later protein was identified as human calgranulin B by LC-MS/MS analysis. Calgranulin B was detected in CM and nOF by western blot and immunolocalized (with a second antibody coupled to Cy3 fluorochrome) in the oviductal epithelial cells from sections of tubal tissue and on spermatozoa. In conclusion, a protein of 14 kDa was detected in the protein extract from human sperm incubated in the presence of CM and isolated by affinity chromatography columns of sperm membrane extracts coupled to Sepharosa 4B. This protein, identified as calgranulin B and immunodetected on spermatozoa, was present in nOF and in the tubal epithelia. The data support the idea that some human oviductal proteins could modulate the sperm function through direct binding on spermatozoa. Study supported by grants from FONCyT and SecyT UNR.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Postnatal uterine development involves differentiation and development of the endometrial glandular epithelium from the luminal epithelium or adenogenesis as well as development of the mesenchyme into the endometrial stroma and myometrium. The success of developmental mechanisms regulating postnatal uterine morphogenesis dictates, in part, the embryotrophic potential and functional capacity of the adult uterus. The Wnt gene family is critical in guiding the epithelial-mesenchymal interactions that direct postnatal uterine development. Wnt7a is expressed in the epithelium of the fetal Mullerian duct, which gives rise to the vagina, cervix, uterus and oviducts. Global disruption of Wnt7a in mice causes posteriorization of the uterus and oviductal anomalies due to developmental defects in Mullerian duct patterning and differentiation in the fetus. The central hypothesis is that Wnt7a is a critical regulator of postnatal endometrial gland morphogenesis and stromal growth and uterine receptivity to the embryo during pregnancy. In order to understand the physiological roles of Wnt7a, a conditional allele, Wnt7a(fl), was generated by introducing LoxP sites around Exon 2. A targeting construct was made using a bacterial artificial chromosome (BAC) library clone containing mouse Wnt7a from BACPAC Resources (Children's Hospital Oakland Research Institute, Oakland, CA). Homologous recombination was performed in embryonic stem (ES) cells. Correctly targeted ES cells were identified by PCR and Southern blot analysis and used to generate chimeric animals by blastocyst microinjection. In order to circumvent the developmental defects when Wnt7a is deleted in the embryo, we conditionally ablated Wnt7a in the uterus after birth using the Cre/LoxP system and the progesterone receptor (PR)-cre recombinase knock-in mouse model. The PR is expressed in the uterus after birth and is not expressed during Mullerian duct differentiation. Floxed Wnt7a homozygote mice were then bred to PR(cre/+) heterozygote mice to generate mice with the PR(cre/+)Wnt7a(fl/+) genotype. These mice were then mated to generate PR(cre/cre)Wnt7a(fl/fl) mice. Female PR(cre/cre)Wnt7a(fl/fl) were necropsied on postnatal day (P) 3, 6, 12 or 15 (P0 = birth). Histological analyses found that endometrial glands were absent in the uteri of PR(cre/cre)Wnt7a(fl/fl) mice as compared to wildtype controls. Thus, Wnt7a plays critical roles in both patterning of the Mullerian duct during fetal differentiation and uterine adenogenesis in the neonate. (Supported by NIH R21 HD054679)
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Uterine fibroids are benign tumors that affect 70% of women by age 50 and can cause debilitating symptoms (pain, bleeding, and infertility). Their etiology is poorly understood, but recent research suggests that fibrosis (a form of altered wound healing) plays a role in the formation of uterine fibroids. We first developed an in vitro injury model in primary myometrial cells to study the initiation of the wound healing process after induced injury (serum starvation [SS] followed by serum back [SB]). Cells developed a myofibroblast-like phenotype and expressed thrombospondin-1 (THBS1). THBS1 is a marker of early wound healing that also activates transforming growth factor beta (TGFB), a key mediator in fibrotic disease processes. To improve upon the issues of tissue availability, variability, and cell number associated with primary culture, our current objective was to validate the cell injury model in the immortalized myometrial cell line M53 (a gift from Dr. William Catherino, USUHS). Cells were cultured in 10% fetal bovine serum until 80% confluent, then subjected to injury (72 h SS, 0% serum) or control (10% serum) followed by SB. Cells were lysed at 0, 0.5, 2, 4, 8, 16, 24, and 48 h after SB (duplicate wells). We repeated the experiment 3 times, performed real-time PCR for THBS1, and analyzed THBS1 and pSMAD3 protein (an indicator of activated TGFB) by western blot and densitometry. All data were normalized to GAPDH mRNA or protein. SS cells expressed 2-3 fold more THBS1 mRNA than controls at 2, 4 and 8 h after SB (P<0.05). THBS1 protein in control cells remained constant for 16-24 h after SB while in SS-SB cells THBS1 protein gradually increased, peaked at 4-8 h, and then declined gradually. A late increase in THBS1 protein was sometimes observed at 24-48 h in both groups. pSMAD3 was increased in controls (5 fold over 0 h) at 0.5 to 8 h after SB; in contrast, in SS-SB cells the maximum increase occurred at 0.5 h (13 fold over 0 h; 4 fold over control, P<0.02). pSMAD3 decreased over time but remained higher than in controls until 16 h after SB (1.8 fold over control, P<0.05). In conclusion, the immortalized myometrial cells responded to injury (SS-SB) with a transient increase of the early wound healing marker THBS1 and a distinct prolonged response in pSMAD3, a mediator of activated TGFB. These results concur with findings obtained from primary cultures and support the use of this cell line in the further development of our fibrosis-based in vitro model to study the early development of uterine fibroids. Supported by the Charles B. Hammond Research Fund, Duke University Dept. of Obstetrics and Gynecology.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
The oviduct is an organ whose function is characterized by cyclic changes to both the ciliated and secretory epithelial cells that line this tissues lumen. Infections of the oviduct by a variety of sexually transmitted microorganisms can lead to pelvic inflammatory disease, tubal occlusions, ectopic pregnancies and/or infertility. Such infections are known to disrupt apoptotic pathways within the oviduct, suggesting key apoptotic processes may be targeted for the prevention and/or treatment of sexually transmitted diseases. The objective of this study was to determine the normal, daily changes in apoptotic gene expression in the oviducts of regularly cycling mice, thus generating the requisite background information for the future study of disease-induced oviductal pathology. The estrous cycle was monitored in CD1 female mice by analysis of vaginal cytology and three females were sacrificed at 1 pm on each day of the estrous cycle. Peripheral concentrations of estradiol and ovarian levels of mRNA for Cyp17 and Cyp19 were determined to confirm correct staging of the cycle. Oviducts were collected for the analysis of 84 key apoptotic genes using real-time PCR based superarrays. Three independent arrays were generated for each day of the estrous cycle (oviducts collected from one mouse / array). The expression of mRNA for eleven genes (Trp73, Bag3, Bok, Card6, Akt1, Polb, Trp53bp2, Atf5, Traf2, Nfkb1 and Casp9) was down-regulated at proestrus when compared to diestrus (P < 0.05). In contrast, the expression of mRNA for one (Bcl2l10), six (Cd40, Fas, Prdx2, Cflar, Lhx4 and Cd40lg) and nine (Tnfrsf11b, Casp14, Tsc22d3, Trp73, Tnfsf10, Card6, Polb, Akt1 and Bag3) genes was upregulated at estrus, metestrus and diestrus, respectively, when compared to the previous day of the estrous cycle (P < 0.05). These results suggest regulation of apoptotic pathways in the oviduct by ovarian-produced steroids and represent the first pathway wide analysis of apoptosis in the normal mouse oviduct. Supported by P20RR015592 (COBRE) and K12DA014040 (BIRCWH) from the NIH.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Post-breeding endometritis in mares is related to inflammatory process, which is stimulated by the presence of sperm cells inside the uterus. However, when this inflammatory response takes the persistent form, it becomes one of the most frequent causes of sub-fertility and this persistent form usually occurs in mares older than 14 years of age. With the efficiency shown by nonsteroidal anti-inflammatory drugs in immunomodulation of uterine inflammation and the possibility of increasing the productivity of these affected animals, this study aimed to evaluate the impact of the dexamethasone systemically administered in the rate of embryo recovery from mares resistant and susceptible to persistent post-breeding endometritis. This study comprised 30 mares which were divided into two groups: 15 mares between 14 and 18 years of age with a history of intra-uterine fluid accumulation after mating determined by ultrasound, low fertility rate, poor perineal conformation and uterus in the abdominal cavity designed. The remaining 15 mares selected to compose the group of resistant mares ranged from 4 to 8 years old, had no history of intra-uterine fluid accumulation, decrease in fertility rate or anatomical changes cited above. These 30 mares had 2 consecutive estrous cycles accompanied by rectal palpation and ultrasonography, and when a follicle with at least 35 millimeters of diameter and a compatible uterine edema were detected, the mares underwent induction of ovulation with 1 milligram of deslorelin acetate (Intra-Muscular). In the interval between 1 and 2 hours prior to artificial insemination 40 milligrams of dexamethasone was administered intravenously. Fertility was measured by the rate of embryo recovery on the eighth day after ovulation confirmation. Estrous cycles with a unique artificial insemination were used in this study. Due to the small number of animals in the experiment, the rates of embryo recovery did not differ between resistant (Control group: 73.33%; Treated group: 80%) and susceptible mares (Control group: 33.33%; Treated group: 69.23 %) regardless of treatment (p> 0.05). However, systemic treatment doubled the number of embryos recovered per cycle when compared to the control group for the mares susceptible to PPBE. Thus, further studies should be conducted using a larger number of subjects so that the results of this work are better understood. Financial support: FAPESP.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Relaxin, acting through its G-protein receptor, RXFP1, has important functions in the female reproductive tract. Relaxin is produced and secreted by the corpus luteum, which leads to cell proliferation, growth and increased extensibility of the lower reproductive tract thereby facilitating parturition. Although RXFP1 was identified in 2002, to date the key regulatory events that govern the ontogeny and adult expression of this receptor remain poorly understood. The goal of the present study was to use a mouse model with an IRES-LacZ reporter cassette incorporated into the Rxfp1 gene to understand the ontogeny and the factors controlling the expression of Rxfp1 in the reproductive tract of female mice during the neonatal and juvenile periods. Uteri, cervix, vagina, and oviducts from LacZ reporter and wild-type controls female mice were collected, prepared and processed for beta-galactosidase (LacZ) activity using X-gal as the substrate. LacZ staining, indicative of RXFP1 expression, was not observed at day 0, 10, or 20 postpartum (PP) in the oviduct, uterus, cervix and vagina. However, LacZ staining was present by the age of 35 days at levels similar to those seen in adults (> 60 PP). RXFP1 expression was localized in the subepithelial stroma and myometrial compartments of the cervix, uterus, vagina and oviduct, while the epithelium was negative. To determine whether normal RXFP1 ontogeny is influenced by the ovary, mice with the reporter gene were ovariectomized at 21 PP, and LacZ staining was examined at 35 PP. Rxfp1 expression is not regulated by the ovary, since LacZ expression was present at 35 PP in ovariectomized females, indicating that removal of the ovary does not preclude RXFP1 ontogeny. In summary, RXFP1 appears pubertally in female reproductive organs and has similar distribution in the various organs examined. These data provide new insights into the sexually dimorphic expression, ontogeny, and potential sites of action of RXFP1.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Mammalian uterus has drastic tissue remodeling during the cycling, implantation, and pregnancy. The tissue remodeling requires a fine-tuned balance between the levels of proteases and their cognate inhibitors. The plasminogen activators involve in the tissue remodeling by converting the abundant extracellular plasminogen into plasmin, an active protease, which degrades the extracellular matrix. The activity of plasminogen activators is modulated by several protease inhibitors that belong to the serine protease inhibitor (SERPIN) superfamily, such as SERPINE1, SERPINB2, and SERPINE2. In this study, we investigated the expression patterns of SERPINE2 in the mouse uterus during the estrous cycle, pregnancy, and lactation period. SERPINE2 was purified from the mouse seminal vesicle secretion using liquid chromatography and identified by liquid chromatography/tandem mass spectrometry. The antiserum against the SERPINE2 protein was raised in rabbit. Western blot was used to verify the specificity of the antibody. To reveal the uterine SERPINE2 expression pattern, uterine tissues at various stages were collected for real-time PCR quantification and immunohistochemistry staining. Serpine2 was the major inhibitor of plasminogen activators in the mouse uterus as demonstrated by the real-time PCR quantification. The Serpine2 mRNA was detected in the cycling and early pregnant uterus. However, the mRNA expression levels were predominantly high during the lactation period. The antiserum against SERPINE2 was specifically recognized the SERPINE2 protein from thousands of protein components in the tissue extract as demonstrated by Western blotting. SERPINE2 was primarily localized in the luminal and glandular epithelial cells but it also detected at circular and longitudinal smooth muscle cells. Interestingly, although the Serpine2 mRNA expression levels had no significant fluctuations in the uterus from 0.5 to 5.5 days postcoitus, the SERPINE2 protein was less expressed in the uterus at 0.5 to 2.5 days postcoitus during the preimplantation period; however, the protein expression was highly expressed in the luminal and glandular epithelial cells at 3.5 to 5.5 days postcoitus during the implantation period. These findings indicate SERPINE2 is a major plasminogen activator inhibitor and may participate in the tissue remodeling process during the murine embryo implantation.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
There are several membrane calcium (Ca2+) channels, transient receptor potential cation channel subfamily V member 5/6 (TRPV5/6), Na+/Ca2+ exchanger (NCX1) and plasma membrane Ca2+-transporting ATPase1 (PMCA1b). They have been turned out to be as important regulators which mediate the transport of Ca2+ between cytosol and blood-stream in various tissues, such as duodenum, kidney and uterus,. TRPV5 and TRPV6 are known as taking part in absorbing Ca2+ selectively. On the contrary, NCX1 and PMCA1b are functionally involved in extruding Ca2+. The expressions of these four genes in other vertebrates including laboratory animals and humans have been confirmed thoroughly. The purpose of this study is to examine and compare their expression patterns in dogs compared with those in other animals to confirm the expressions of four major Ca2+ related genes in a canine model. To perform this study, we euthanized two adult (2.5 year old) female dogs and prepared tissue samples by incising abdomens. Then we perform reverse transcription/polymerase-chain reaction (RT-PCR), western blot analysis and immunohistochemistry (IHC) in canine tissues. By RT-PCR, the mRNAs of TRPV5, TRPV6, and NCX1 were highly expressed in the kidney, but PMCA1b mRNA is mostly detected in the uterus. As shown in RT-PCR, we confirmed the similar expression patterns of these proteins by both western Blot analysis and IHC. Taken together, these results indicate transcriptional and transitional expressions of TPRV5, 6, NCX1, and PMCA1b in the duodenum, kidney and uterus of a canine model.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Plasma membrane Ca2+-pumping ATPase (PMCAs) play a critical role in maintaining cellular Ca2+ homeostasis. PMCA mRNAs are encoded on four genes designated PMCA1-4. In the previous study, both PMCA1 (Ca2+-pumping ATPase 1) and PMCA4 (Ca2+-pumping ATPase 4) are expressed at similar levels in astrocytes and in neurons. Although PMCA1b is expressed in the uterus of rats during estrous cycle, the expression of PMCA1 and its potential roles has not been elucidated yet during menstrual cycle in human endometrium. Thus, in the current study, the expression pattern of PMCA1 was examined to predict its roles in human endometrium during the menstrual cycle. Using real-time PCR and western blot analysis, uterine expressions of PMCA1 mRNA and protein increased to 1.5 fold at early-, mid- and late-proliferative phase in the endometrium of human uterus, compared to other menstrual phases. In addition, uterine PMCA1 was abundantly localized in the cytoplasm of the luminal and glandular epithelial cells in menstrual phases, indicating that this protein may participate in uterine calcium balance of human endometrium during menstrual cycle. Taken together, these results suggest that a high level of uterine PMCA1 expression may be involved in reproductive function during the cycle of human.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
There is growing interest in the use of androgens as therapeutics in women. Steroids, however, can regulate adipose tissue development, fat depot distribution, adipose cell function, and whole-body insulin sensitivity. Gender-related differences in insulin sensitivity and adipose tissue function are attributable to the action of estrogens and androgens, and alterations in the levels of sex hormones are associated with metabolic syndrome. In females, hyperandrogenic conditions that occur during menopause and in obese women with Polycystic Ovary Syndrome (PCOS) correlate with the development of insulin resistance, whereas, in males, hypoandrogenism is associated with insulin resistance. It has been reported that androgen treatment of in vitro-differentiated adipocytes derived from preadipocytes from human females inhibited insulin-stimulated glucose uptake. The role of adipose tissue in lipid metabolism, however, is generally thought to be more significant than its role in glucose disposal. Therefore, in this study, we assessed the effect of androgens on basal and insulin-dependent fatty acid uptake in ex-vivo cultures of visceral (retroperitoneal) adipose tissue of adult female Rhesus macaques. To do this, we used a fluorescent microscopy-based screening procedure in which cellular fluorescence intensity correlates with the level of fatty acid uptake. Freshly isolated retroperitoneal fat explants were immobilized at the bottom of incubation/imaging chambers, pre-treated with 0.1-10 nM insulin for 2 hours, and then labeled with the fluorescent fatty acid analogue BODIPY-500/510 C1, C12 (BODIPY-C12). In this system, BODIPY-C12 accumulation, assessed by confocal microscopy at the single-cell level, was stimulated by prior insulin treatment in a dose-dependent fashion. To test the effect of androgens on insulin-dependent fatty acid uptake, fat explants isolated from female animals were pre-incubated with various concentrations of testosterone or dihydrotestosterone for 48 hours prior to determination of insulin-stimulated fatty acid uptake. Short-term androgen treatment (less than 24 hours) had no effect on the basal or insulin-stimulated fatty acid uptake in adipose tissue, while 48 hours exposure to androgens evoked a dose-dependent inhibition of insulin-dependent fatty acid uptake in explants from female animals. Dihydrotestosterone concentrations as low as 10 nM significantly inhibited insulin action in adipocytes. Testosterone was less potent in inhibiting insulin-stimulated fatty acid uptake. Both androgens had a slightly stimulatory effect on basal insulin-independent fatty acid uptake in adipocytes. Taken together, the present screening procedure establishes the inhibitory role of androgens on insulin-dependent fatty acid uptake in visceral fat of non-human primates. These data demonstrate that androgens can modulate the insulin sensitivity of fatty acid as well as glucose uptake in adipose tissue; thus, hyperandrogenism associated with PCOS can directly modulate glucose and lipid metabolism in peripheral insulin target tissues. Moreover, this technique could be used to test the metabolic actions of novel androgens being developed for reproductive disorders prior to initiating extensive clinical trials. Supported by NIH grants HD18185 and RR000163.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
NOT PRESENTED.
Biol Reprod. 2010 Nov;83(1 Suppl):3.
MicroRNAs (miRNAs) alter translation in nearly every biological process examined to date, but little is known as to the identity of miRNAs in porcine gametes and embryos, or the role of miRNA in porcine reproductive processes. Assisted reproductive technologies such as somatic cell nuclear transfer and intracytoplasmic sperm injection frequently lead to deviant embryonic growth; however, the underlying mechanisms responsible for aberrant development remain largely unknown. The miRNA regulatory pathway may contribute to the failure of these embryos to develop or establish and maintain pregnancy. The objective of this study was to determine the presence and identity of miRNAs in porcine oocytes and pre-implantation embryos. Total RNA enriched for small RNA was isolated using the mirVana miRNA Isolation Kit (Ambion Inc) from three pools of matured oocytes (n= 200, 50, and 5), three pools of in vivo produced 8-cell embryos (Day 4; n= 4, 3, and 4), and three pools of in vivo produced blastocysts (Day 7.5; n= 5, 4, and 5). A total of 275 ng RNA from each pool was subjected to qRT-PCR probing for 88 mature human miRNAs and 8 controls (RT2 miRNA PCR Array System; SABiosciences) and, because many miRNAs are highly conserved among species, allowed for efficient cross-species amplification. Specific miRNA abundance was classified according to manufacturer's recommendations and miRNAs exhibiting a Ct of ≥35 were classified as non-detectable, and detectable miRNAs were categorized as displaying low expression (Ct= 30-34.9), moderate expression (Ct= 25-29.9), or high expression (Ct ≤ 24.9). Data were analyzed using the RT2 Profiler PCR Array Data Analysis online tool. A total of 71 miRNAs (80.7% of the 88 miRNA's probed) were detected in the oocytes, 36 (40.9%) in the 8-cell embryos, and 81 (92%) in the blastocysts. Chi square analysis showed differences in proportions of detectable vs. non-detectable miRNAs between oocytes and 8-cell embryos (P< 0.05), oocytes and blastocysts (P< 0.05), and 8-cell embryos and blastocysts (P< 0.01). A 3x3 Chi square analysis showed significant differences in proportions of high, moderate, and low miRNA expression between oocytes and 8-cell embryos (P< 0.01) and 8-cell embryos vs. blastocysts (P< 0.01). The let-7 family, a highly conserved group of miRNAs shown to play critical roles in developmental timing in non-mammalian species, was present in porcine oocytes and embryos, but consistently exhibited higher expression in the oocytes. A 2x2 Chi square analysis showed significant differences in proportions of moderate/ high (Ct ≤ 29.9) vs. low/ non-detectable (Ct ≥ 30) let-7 expression between oocyte and 8-cell embryos (P< 0.01). These data indicate that, like mRNA, miRNAs from maternal sources are depleted prior to the 8-cell embryonic stage and that miRNA expression post-8-cell embryonic stage is due to the activation of the fetal genome. Although there is no single cause for early embryo loss, the identification of specific miRNAs that are associated with embryo growth, differentiation, or survival could lead to the development of a PCR-based diagnostic assay to provide an assessment of oocyte or embryo quality. Future studies are being directed at validating specific miRNA expression levels from individual embryos at various stages of pre-implantation development via qRT-PCR.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Gonadal aromatase (Cyp19a1a) plays a pivotal role in gonadal sex differentiation, especially in lower vertebrates like teleosts. The transcription of cyp19a1a gene in the ovary has been shown to be up-regulated by Ftz-f1 homologues, which can be enhanced by Foxl2 synergistically. In the present study, a foxl2 gene (efoxl2) and two Ftz-f1 homologue genes, designated as eff1a and eff1b respectively, were cloned from the gonad of ricefield eel. Furthermore, a C-terminal truncated form of eff1a transcripts, designated as eff1a-s, was also identified in the gonad. Sequence analysis showed that eff1a-s lacked the ligand binding domain including motifs for I-Box, DRD, and AF-2, and is probably generated by 3'-end alternative splicing and polyadenylation of eff1a transcripts. RT-PCR analysis detected the expression of efoxl2, eff1b, eff1a, and eff1a-s in the gonads of ricefield eel, with higher expression levels of eff1a in the ovary than testis, and higher expression levels of eff1b and eff1a-s in the testis than the ovary. As potential binding sites for Ftz-f1 and Foxl2 were observed in the proximal promoter region of ricefield eel cyp19a1a, an in vitro transient transfection assay was employed to examine the effects of eFoxl2, eFf1b, eFf1a, and eFf1a-s on the promoter activity of ricefield eel cyp19a1a gene. Both eFf1b and eFf1a activated the transcription of cyp19a1a, whereas eFf1a-s repressed the basal as well as eFf1b and eFf1a-mediated transcription of cyp19a1a. eFoxl2 alone seemed to activate the transcription of cyp19a1a with higher potency than either eFf1b or eFf1a, and the activation was further enhanced very significantly in the presence of eFf1a, but not eFf1b. These results indicated that Foxl2 could interact with Ftz-f1 homologues to up-regulate the transcription of cyp19a1a gene differentially, and the molecular basis for this differential interaction is worth further study. 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):3.
The phenotypes of calbindin-D9k (CaBP-9k) and -28k (CaBP-28k) single knockout (SKO) and double KO (DKO) mice are similar to wild-type (WT) mice due to the compensatory action of other calcium transport proteins. However, in this study, we investigated the proteins expression of forebrain, midbrain, and hindbrain in CaBP-9k and CaBP-28k SKO and CaBP-9k/CaBP-28k DKO mice in order to characterize the difference of brain in the importance of CaBP-9k and CaBP-28k. Under normal dietary conditions, 4-weeks SKO and DKO mice did not exhibit any changes in phenotype or the expression of active calcium transport genes as compared to wild type (WT) mice. First, we identified CaBP-9k and CaBP-28k expressions in brains of 4-weeks SKO and DKO mice. Reverse-transcription (RT)-PCR data showed CaBP-9k expression in hindbrain of WT and CaBP-28k SKO mice, but protein did not show its expression in hind-brain. This result suggests a little expressions of CaBP-9k in hindbrain of WT and CaBP-28k SKO mice. Prion proteins (PrP) are hypothesized to infect and propagate by refolding abnormally into a structure which is able to convert normal molecules of the protein into the abnormally structured form. All known PrP induce the formation of an amyloid fold, in which the protein polymerizes into an aggregate consisting of tightly packed beta sheets. This altered structure is extremely stable and accumulates in infected tissue, causing tissue damage and cell death. Recent reports suggest that imbalance of brain metal homeostasis is a significant cause of PrP. In this study, PrP were significantly expressed approximately two fold in forebrain, midbrain, and hindbrain in CaBP-9k and CaBP-28k SKO and CaBP-9k/CaBP-28k DKO as compared with WT mice. Phosphor-Bad (S136) was highly expressed, but phosphor-Bad (S155) was reduced in forebrain, midbrain, and hindbrain, respectively. Especially, phospho-ERK expression was decreased, proposing that the increased PrP expression may associated with the decrease of phospho-ERK expression. Cell survival marker, Bcl-2 expression did not change, but cell death marker, Bax expression reduced in forebrain, midbrain, and hindbrain. This data may present the relation with the phospho-ERK expression. In addition, Cu/Zn-superoxide dismutase (Cu/Zn-SOD) and Mn-SOD expressions were declined in forebrain, midbrain, and hindbrain. Phospho-GSK alpha expression was decreased, but phospho-GSK beta expression was increased. These results suggest that CaBP-9k and CaBP-28k SKO and CaBP-9k/CaBP-28k DKO mice may be vulnerable to influence the environmental stresses.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Subfertility affects 10-15 percent of couples worldwide, and male factor is one of the dominant causes accounted for one third of these cases. Notably 40-90 percent male subfertility is due to idiopathic defective sperm production. Today, hundreds of genes have been shown to be involved in the regulation of spermatogenesis. Previously, we identified from the rat testis a novel acrosome-specific gene Vad1.3 in a retinol-treated Vitamin A-deficiency (VAD) model. VAD1.3 was expressed in the testes from postnatal day 25 and its immunoreactivity was localized to the acrosomal cap of the rat, mouse, human, monkey and porcine spermatids. To explore the functional role of VAD1.3 in spermatogenesis in vivo, we used the gene-targeting approach to generate Vad1.3 knockout mice. Vad1.3 targeted allele was germline transmitted. No gross defect in spermatogenesis was found in the heterozygous mice testes. Crossing of heterozygous mice produced wild-type and heterozygous offsprings in a ratio of 1 : 2.6 (n = 92), but no Vad1.3-/- offspring was found. There were no significant difference in corpus luteum numbers between the wild-type and heterozygous mice, suggesting these animals ovulated comparable number of oocytes. However, the number of implantation site on day 8 and 15 post coital (dpc) (n = 7) as well as the litter size (n = 9) was consistently lower in the crossing of the heterozygous (8.0 ± 0.8; 7.3 ± 0.8; 7.2 ± 0.6, respectively) than the wild-type (9.1 ± 0.4; 8.3 ± 0.3; 8.1 ± 0.6, respectively) mice. Since no resorption site was found on 8 dpc in both groups, the difference in implantation site may result from failure in fertilization, preimplantation embryonic development or implantation of homozygous knockout embryos. To study the time of loss of these homozygous embryos, genotyping of individual preimplantation embryos after whole genome amplification was performed. It was found that Vad1.3-/- embryos existed at the zygotic and 2-cell stages but was absent at the blastocyst stage, suggesting that the Vad1.3-/- embryos died before blastocyst formation. Interestingly, spermatogenesis in 9-14 months old heterozygous mice exhibited higher incidence of germ cell loss when compared with the control mice of the same ages (4/15 vs. 1/15, respectively). Yet, spermatogenesis were comparable between the wild-type and heterozygous mice on post-natal day 25, 35, 45 and 60. In sum, our results indicate that VAD1.3 is critical for early preimplantation embryo development and spermatogenesis in adult mice. The use of tissue-specific knockout approach may help to answer the functional role of VAD1.3 in fertility. This project is supported in part by an RCG grant HKU7537/05M to KFL.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Germ cells are the only cells that are possible to communicate to next generation and having pluripotency. In the development of primordial germ cells (PGCs), specific molecular mechanisms such as epigenetic regulation and signal transduction are involved. Recently, several reports have showed that PGCs specific expression genes are essential for PGCs development and the mutants lack the normal PGCs development. We have identified the novel gene named as GSE (gonad specific expression gene), which are expressed at the ovary and testis of adult mice. Subsequently, we have isolated a novel gene named as GIAP (GSE-interacting protein), which are identified by the yeast two hybrid system. GSE are confirmed to interact with GIAP in both ovary and testis of adult mice by immunoprecipitation. To investigate the involvement of GSE-GIAP interaction in the development of PGCs, we performed the expression analysis of GSE and GIAP in the fetus stage. Additionally, we obtained presumed biological characters of GIAP by the public database. As a result, we showed that GSE expresses from embryonic day 7.5 (E7.5) to E11.5 fetus and both of ovary and testis at E12.5 fetus. We also showed that GSE expresses in E11.5 PGCs by in situ hybridization. Furthermore, immunohistochemical analysis indicated that GSE and GIAP co-localized in E 9.5 and E11.5 PGCs. Therefore, it suggested that GSE also interacts with GIAP in PGCs. The informatics analysis indicated that it is possible for GIAP to have PRMT activity. PRMT family is protein arginine methyltransferase and is known to play an important rule for the development of PGCs. BLIMP1, master regulator of PGCs development, interacts with PRMT5 and regulates the development related factors. We will address the function of GSE and GIAP in the development of PGCs.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Factor VIII/von Willebrand factor (vWF), a major glycoprotein of plasma proteins, is a regulation complex of coagulation system in blood, so involved in hemophilia, von Willebrand disease and heyde's syndrome. To develope blood protein bioreactor in animals, we reported human vWF gene carrying transgenic pigs which produced vWF proteins in their milk via activity of bovine casein promotor. However, three different lines of vWF transgenic pigs did not produced same amount of vWF proteins in their milk, even thought the same gene was used in the process of DNA injection into the pronucleus of zygote. To select the line/individuals of vWF transgenic pigs which could be used as a bioreactor, quantitative PCR and FISH analysis was performed in the progeny of three vWF founders. These transgenic lines were nominated as V1, V2 and V3, and showed their loci on the chromosome number of 11, 15 and 4, respectively by a result of FISH analysis. The copy numbers of transgenes in the progeny of three lines were conjectured as 4.5 ± 1.47, 6.82 ± 4.29 and 15.42 ± 5.72 by a result of qRT-PCR analysis. The expression patterns of vWF from individual milk of lines showed significant different results of 26, 17 and 280,000 ng/ml by ELISA assay respectively. These results show that V3 line has a ability of vWF expression in the mammary glands and its progeny could be used as a bioreactor to stabilize other blood clotting factors like Factor VIII. With data on qRT-PCR, FISH and ELISA of protein expression in milks, transgene integration locus could be a key factor of transgene expression level, vice versa, our vWF gene construct which consists of bovine alpha S1 casein promotor/human vWF cDNA/bovine growth hormone poly A tail could be affected by other factors like gene expression in mammary glands or its local expressions of another gene at least in pigs. In the NIAS of RDA, bioreactor transgenic pigs of blood regulating proteins like EPO, tPA and Factor VIII has been produced since 1998. So this line of vWF could be used as a founding transgenic line of multiple gene carrying pigs and subsequent sublings of vWF and other lines should be examined to confirm ability of expression and functional differences of wanted proteins.This work received grant support from the Agenda Program (No.PJ006702), Rural Development Administration, Republic of KOREA.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Our laboratory recently generated a cystic fibrosis transmembrane conductance regulator gene (CFTR) knockout (KO) ferret model of cystic fibrosis (CF) using viral gene-targeting and somatic cell nuclear transfer (SCNT). Here we describe the phenotype of CFTR null ferrets and methods for genetic complementation of disease using tissue-specific transgenesis in CFTR-KO fibroblasts by SCNT. Neonatal CFTR-KO ferrets demonstrated many of the characteristics of human CF disease, including defective airway chloride transport, variably penetrant meconium ileus (MI), pancreatic and liver disease. 75% of all CFTR-KO kits die from meconium ileus with in 36-48 hrs after birth. Severe malabsorption by the gastrointestinal tract was the primary cause of death in the 25% of CF kits that escaped MI. Since a major reason for generating the CFTR-KO ferret model was to study the progression of lung disease in adult animals, methods to overcome the limitations imposed by the severe gut phenotype in this model were needed. To this end, we cloned a gut-corrected CFTR-KO ferret by somatic cell nuclear transfer using transgenic CFTR-KO fibroblasts expressing fCFTR under the direction of the intestine-specific fatty acid protein (FABPi) promoter. A transgenic construct containing the rat FABPi promoter driving the fCFTR cDNA and a floxed PGK promoter driven zeocin resistant gene cassette was engineered and transfected into ferret fibroblasts generated from a 28 day CFTR-KO embryo. The transfected cells were selected in zeocin and the pooled cells (i.e., non-clonal) were used for SCNT. Three clones were born, but only one survived the early post-natal period and passed meconium. The two additional clones born had to be euthanized within 36 hrs after birth due to severe MI with microcolon. The surviving clone was euthanized to determine the expression patterns of recombinant fCFTR and this clone had a grossly normal intestine lacking any signs of MI. Fibroblasts, liver, lung, and intestine were harvested from this MI-complemented founder clone for analysis of recombinant fCFTR tissue expression patterns prior to nuclear transfer recloning and expansion of the line. Results from analysis of this MI-complemented FABPi-fCFTR/CFTR-KO clone demonstrated the presence of the transgene cassette in the genomic DNA and recombinant fCFTR protein expression in the intestine but not in the lung and liver (endogenous fCFTR protein was expressed in all these organs from wild type ferrets but not CFTR-KO ferrets). In a separate set of experiments, we cloned non-transgenic CFTR-KO kits with fibroblasts derived from a CFTR-KO kit that suffered from severe MI with microcolon; all four clones born from this experiment suffered from MI with varying degrees of microcolon. Thus, the intestinal phenotype of cloned CFTR-KO kits appears to closely reflect that of the CFTR-KO founder. Cumulatively, these findings demonstrate the successful generation of a transgenic FABPi-fCFTR/CFTR-KO founder for which tissue-specific expression of recombinant fCFTR in the intestine can correct the MI phenotype. Currently, nuclear transfer is being performed to expand the FABPi-fCFTR/CFTR-KO line with and without CRE-excision of the PGK-Zeocin cassette. This first demonstration of generating a transgenic ferret proves the feasibility of promoter-directed complementation in the ferret by SCNT, and may significantly enhance the utility of the CF ferret model for research.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
GATA4 is an essential transcription factor required for the development and function of multiple tissues derived from mesoderm and endoderm. Among these tissues, GATA4 is prominently expressed in the gonads of both sexes and has been shown to be particularly important for normal testis development and gene expression. In Sertoli cells, GATA4 regulates expression of the Sry, Sox9, and Amh genes whereas in Leydig cells, GATA4 appears to control the expression of several key genes in the steroidogenic pathway such as Star, Cyp11a1, HSD3B2, and Cyp19a1. In spite of its crucial role, the molecular mechanisms that regulate Gata4 expression in vivo remain poorly understood. We recently found that the GATA4 gene in both humans and rodents is expressed as multiple transcripts with distinct 5' origins (exons 1a and 1b being the most prominent). Moreover, we previously showed that an E-box element located near the transcription initiation site, upstream of exon 1a, is important for Gata4 transcription in multiple gonadal cell lines. To confirm the importance of this element in vivo, we mutated the endogenous E-box element (CACGTG) of the Gata4 promoter by homologous recombination in ES cells and derived mice containing the resulting Gata4EboxKO-Neo allele. Heterozygous Gata4EboxKO-Neo intercrosses failed to produce viable homozygous offspring. Indeed, we found that homozygous Gata4EboxKO-Neo animals, which retained the floxed Neo selection cassette, recapitulate the Gata4-null phenotype and die around embryonic day (E) 9.5. To determine if this phenotype was related to the presence of the Neo cassette, we crossed heterozygous Gata4EboxKO-Neo mice with mice ubiquitously expressing Cre recombinase to remove the floxed Neo cassette. Interestingly, the resulting heterozygous as well as homozygous Gata4EboxKO mice did not show any obvious phenotype and were fertile. We have begun a systematic characterization of Gata4 expression in Gata4EboxKO homozygotes in comparison to wild-type littermates. To this end, Gata4-expressing tissues (testis, ovary, heart, liver) were collected at different developmental stages (E15.5, P14 and in adult) and expression of transcripts 1a and 1b was assessed by quantitative real-time PCR. In homozygous Gata4EboxKO mice, we detected a significant decrease of 1a transcript expression in all examined tissues while 1b transcript expression was unchanged. We are currently analyzing the impact of reduced 1a transcript expression on GATA4 protein levels and gonad morphology. In conclusion, our data to date prove that the E-box element of the proximal Gata4 promoter is specifically involved in the regulation of Gata4 1a transcript expression in vivo. This research was supported by CIHR grant MOP-14796 to RSV.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
A role for RNA regulation in male germ cell development is well established and evolutionarily conserved. The significance of RNA regulation in germ cell biology is enhanced by recent identification of unique small RNAs (piRNAs), cellular components (nuage) and proteins (such as DAZL, NANOS3 and PIWIL2) with specific and necessary functions in spermatogenesis. Furthermore, previous microarray results from our lab indicate that RNA regulatory proteins are over-represented by mouse spermatogonial stem cells (SSCs). The Ybx1 gene identified in that study encodes the protein YBX1 which has varied cellular functions including translational regulation. RT-PCR indicated that Ybx1 mRNA was expressed in all mouse tissues analyzed, including the testis, though its specific role in the testis is unclear. In the testis, we show that YBX1 is expressed specifically by cells on the basement membrane of mouse seminiferous tubules, consistent with the location of undifferentiated spermatogonia. Additionally, in vitro (immunocytochemistry in SSC culture) and in vivo (immunohistochemistry in histological sections of the testis) studies show that YBX1 is co-expressed with the pan germ cell marker DAZL as well as the stem and progenitor spermatogonia marker, PLZF. To begin investigating the regulation of YBX1, we looked at its phosphorylation status and found that YBX1 is phosphorylated in the adult mouse testis. Interestingly previous in vitro studies show that AKT directly phosphorylates YBX1 and that this phosphorylation regulates the ability of YBX1 to bind mRNA. In SSCs the AKT pathway is activated by GDNF (glial cell derived neurotrophic factor) signaling. GDNF is the factor necessary for long-term renewal of mouse SSCs in vitro. Thus, we hypothesize that GDNF-activated AKT signaling is mediated, at least in part, by YBX1 regulation of target mRNAs. This work was supported by NIH grant RR18500 and Magee-Womens Research Institute and Foundation.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Oogenesis is characterized by a series of developmentally regulated events, which result in the matured oocyte that can give rise to a new organism after fertilization. Oocyte-specific genes play critical roles in oogenesis; however, the molecular details of oocyte-specific genes are poorly defined. Through analysis of expressed sequence tags (ESTs) from a rainbow trout oocyte cDNA library, we identified and characterized a novel oocyte-specific gene coding for an F-box protein (Fbos, F-box oocyte-specific). F-box proteins are components of ubiquitin-ligase complexes, which bind substrates for uiquitin-mediated proteolysis. F-box proteins have also been associated with cellular functions such as signal transduction and regulation of the cell cycle. To define the molecular mechanisms of Fbos-dependent biological processes during oogenesis in rainbow trout, we used Fbos as bait in a yeast two-hybrid screen system, along with a rainbow trout ovary cDNA library. More than 300 yeast colonies were able to grow on nutritional selective plates; 18 clones displayed strong LacZ activity as shown by colony lift filter assay for β-galactosidase activity. After verifying the interactions by reintroduction of the candidate prey plasmids into yeast reporter strain containing Fbos plasmid, 8 plasmids were rescued successfully and subjected to DNA sequence analysis. The sequencing data demonstrated that Fbos protein interacts with a number of proteins including histone H3.3, adenylate kinase 2, ribosomal protein L36, oocyte protease inhibitor 1, zona pellucida protein 2 (ZP2) and tissue inhibitor of metalloproteinase 2 (TIMP-2). All 5 proteins showed significant β-galactosidase activities at different levels compared to the control in a quantitative β-galactosidase assay, indicating different binding affinities of these proteins with Fbos protein. Some of these proteins like ZP2 and TIMP-2 are known to play critical roles in oocyte development in various species. Our results suggest that through protein-protein interactions, the novel oocyte-specific F-box protein may play an important role in early oocyte development by regulating other critical proteins involved in oogenesis in rainbow trout.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
During oogenesis, regulatory sequences in the 3' untranslated region (3' UTR) direct messenger RNA (mRNA) stability, localization, and silencing to control timing of protein translation during oocyte maturation. Cleavage factor Im (CFIm), comprised of subunits NUDT21 and CPSF6, directs polydenylation site selection in somatic cells by binding to a UGUA consensus sequence, therefore determining the length of, and regulatory elements present in, the 3'UTR. CFIm protein and mRNA is highly expressed in male germ cells compared to somatic cells suggesting in important role for CFIm in germ cell development. Specific oocyte transcripts, including Mos and Tacc3, have UGUA sequences in proximity to the polyadenylation signal but the developmental expression of CFIm subunits during oogenesis has not been investigated. Using 3'RACE amplification and sequencing, adult BALB/c mouse ovaries express Nudt21 and Cpsf6 transcripts with a shortened 3'UTR compared to somatic cell expression of the CFIm subunits. The ovarian Nudt21 and Cpsf6 sequences are identical to the previously reported 3'UTR for the male germ cell isoforms. Developmental protein expression of NUDT21 and CPSF6 during the later stages of oogenesis was investigated with protein that was isolated from prepubertal (day 10) female BALB/c mice and adult (8 weeks) BALB/c female mice. Adult ovary NUDT21 protein expression increased 46% from prepubertal ovary expression levels. CPSF6 protein expression increased 34% in adult ovaries when compared to prepubertal ovaries. Adult ovarian protein levels of NUDT21 and CPSF6 were elevated compared to somatic kidney protein (an increase of 55% and 50%, respectively) demonstrating elevated expression in female gonads. This increased protein expression in adult ovaries compared to prepubertal ovaries suggests a role of CFIm during onset of cyclicity in adult mice. A further understanding of the role of CFIm in regulating the 3'UTR of oocyte transcripts could contribute to the understanding of transcript arrest and initiation of translation during oogenesis.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Steroidogenic Factor-1 (SF-1) is a nuclear receptor expressed in multiple cell types within the Hypothalamic-Pituitary-Gonadal-Adrenal axis. SF-1 is known to control development, differentiation and maintenance of cells in each of these organs, as well as steroid production in the gonads (testis, ovary) and the adrenal gland. SF-1 acts as a master transcriptional regulator to control multiple genes involved in these processes. SF-1 knockout mice show complete agenesis of the adreno-gonadal primordium, precluding the use of this in vivo model to study the molecular actions of SF-1 in early development of steroidogenic cells and tissues. Embryonic stem cells can be induced to differentiate into multiple cell lineages in culture. These in vitro systems provide useful models for identification of key regulatory genes involved in differentiation and developmental processes and their mechanisms of action. We created ES cell lines that stably express SF-1 (SF-1-ESCs) and subjected these cells to multiple conditions that elicit steroidogenic differentiation. Under these conditions, we saw marked differences in cell morphology and cell proliferation/survival between native ES cells and SF-1-ESCs. Differentiating SF-1-ESCs showed induction of genes involved in cholesterol processing, steroidogenesis and adreno-gonadal development. Upon differentiation through embryoid bodies, the SF-1-ESCs also showed increased induction of early lineage markers of the mesendodermal lineage as compared to the native ES cells. The SF-1-ESCs thus provide a valuable model to study the role of SF-1 in early differentiation events towards the steroidogenic lineage. To study the molecular targets of SF-1, we created cell lines expressing biotin tagged SF-1 (Bio-SF-1). Biotin-ChIP assays using these lines showed binding of SF-1 to the promoter regions of known target genes. We are currently using high throughput analysis of Biotin-ChIP assays and expression analysis to study transcriptional control by SF-1 in these cells. (Grant Support: NIH RO1 HD044801)
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Prostaglandin (PG) F2a is the main luteolytic factor in ruminants. It has been generally accepted that there is a positive feedback loop between endometrial PGF2a and ovarian oxytocin (OT) in ruminants. On the other hand, since administration of OT antagonist together with PGF2a does not suppress uterine PGF2a release, OT may not be essential for PGF2a stimulation in cow. Based on above evidence, we hypothesized the presence of a local autoamplification system for PGF2a and that uterine PGF2a is stimulated by itself in cow. In the present study, to test the above hypothesis, we determined 1) mRNA and protein expressions of F-series-prostanoid receptor (FPr) throughout the estrous cycle and 2) effect of PGF2a on PGF2a production in bovine endometrial tissue. Endometrial tissues were collected at estrus (Day 0), the early I (Days 2-3), early II (Days 5-6), mid (Days 8-12), late (Days 15-17) luteal stages and the follicular stage (Days 19-21). FPr mRNA and protein expressions were determined by real time RT-PCR and western blotting analysis. FPr mRNA expression was higher at the follicular stage than at the estrus and early I luteal stages whereas FPr protein expression increased after the developing luteal stage and it became the highest level at the late luteal stage (P<0.05). Since the highest level of FPr protein was expressed at late luteal stage, endometrial tissue strips of the late luteal stage were used to assess the dose-dependent effects of PGF2a on the PGF2a production by the tissue. The tissues were exposed to PGF2a (0.01, 0.1 and 1 microM), or PGF2a together with cyclooxygenase (COX) inhibitor (indomethacin: 10 microM) for 4 h, media were exchanged twice to remove the treated PGF2a then the tissues were cultured for an additional 4 h. PGF2a was determined by enzyme immunoassay (EIA). PGF2a stimulated PGF2a production in a dose-dependent manner. This effect was significant at 1 microM (P<0.05). In addition, endometrial tissues from the early I and mid luteal stages were also exposed to PGF2a (1 microM) to analyze the stimulatory effect of PGF2a at different stages of the estrous cycle (early I, mid and late luteal stages). In the both stages, PGF2a stimulated PGF2a production (P<0.05). PGF2a stimulation was higher at the late luteal stage compared to the early and mid luteal stage (P<0.05). Indomethacin suppressed PGF2a-stimulated PGF2a production in endometrial tissues of the all stages studied. This result suggests that the increase in PGF2a detected in the medium is not the PGF2a added to the medium but the endometrial product, and that PGF2a is increased by stimulation of cyclooxygenase activity. The overall results suggest the presence of a local autoamplification system for PGF2a in bovine endometrium. This autoamplification system together with the positive feedback loop between OT and PGF2a may induce the drastic increase in endometrial PGF2a secretion at the late luteal stage to ensure luteolysis in cow.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
At near-term pregnancy, the levels of serum PGE2, calcitonin (CT) and progesterone are all higher. The rapid bone growth of fetus is under this period. Therefore there is a critical role of PGE2 on enhanced CT secretion during the periods of rapid bone growth; however, a cause and effect relationship between serum PGE2 and CT has not been established. We used TT cell, which come from human medullary thyroid carcinoma cell, is a parafollicular cell (C cell) of thyroid, to study the effects of PGE2 on the TT cell viability and division. TT cells were treated with PGE2, arachidonic acid (AA, a substracts of PGE2), COX2 inhibitors (such as NS398 and DUP697), Indomethancin (Indo, a COX inhibitor), SC560 (a COX1 inhibitor), PGE2 receptor antagonist (L161982, an EP4 receptor antagonist) or PGE2 receptor agonists (butaprost--an EP2 receptor agonist, 17-phenyl trinor-PGE2 (17-PT-PGE2)--an EP1 and EP3 receptor agonists, or sulprostone--an EP3 receptor agonist). After treatment, the cell number and cell division effects were observed. The cellular phosphor-STAT5a and STAT5a were also be measured by Immunofluorescence. The data were expressed as mean ± SEM, and analyzed by the analysis of variance and multiple range tests. The results indicate that the index of TT cells no. were significant increase by PGE2 or AA treatment. Indo or DUP697 treatment, decreased the index of TT cells no. After treated with AA, the mitotic index was in a dose-dependent manner in TT cells. PGE2, sulprostone and butaprost were also increased the mitotic index of TT cells, but not in 17-PT-PGE2 treatment. L161982 was decreased the mitotic index. In addition, the enhanced effects of AA on TT cell mitotic were inhibited by Indo, NS398, DUP697, and SC560. DUP697 totally blocked the AA effects. PGE2 and AA increased the cellular level of phospho-STAT5a in mitotic TT cells. The enhanced effects of AA on the cellular level of phospho-STAT5a were not inhibited by Indo, NS398 and SC560, but DUP697 blocked these effects. Treated with Indo or DUP697 decreased the cellular level of STAT5a. AA increased the cellular level of STAT5a, but Indo, NS398 and DUP697 blocked these effects. These results suggest that the PGE2 increased TT cell mitosis, and this effect may go through the EP2, EP3 and EP4 receptors, and related with cellular STAT5a phosphorelation. Research supported by MKC 98R07 to C. C. Lu.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Polycystic ovary syndrome (PCOS) is the most common cause of infertility in female. The diagnostic criteria of PCOS include irregular ovulation, hyperandrogenism, and polycystic ovaries. Besides abnormal reproductive functions, metabolic abnormalities are usually present, such as obesity, insulin resistance, and metabolic syndrome. Previous studies had developed several rat models for PCOS. However, there were only partial ovarian features and metabolic characters consistent with human PCOS. In our present study, we used dehydroepiandrosterone (DHEA)-treated rat as animal model for PCOS. DHEA is an intermediate product of sex hormone synthesis. It has little androgen activity and can be converted to testosterone or estrogen in peripheral tissues. Three-week-old female rats received daily subcutaneous injection of DHEA (30 mg/kg body weight) or sesame oil as vehicle control for 12 weeks, respectively (n=8 per group). Their reproductive cycle, body weight, adiposity, blood pressure, insulin sensitivity, plasma metabolic profiles and hormone profiles were measured. Compare with control rats, the estrus cycles in DHEA-exposed rats were totally disturbed. DHEA-treated rats had smaller ovarian size, and the pathological examination also showed increased number of cysts but absence of corpus luteum in their ovaries. Plasma testosterone levels were higher in DHEA group than in control group, but estradial levels and progesterone levels were significantly lower in DHEA group than in control group. The body weights of DHEA-treated group were higher than control group. However, DHEA-treated rats showed smaller adipose tissue mass, their adiposity indexes were significantly lower. The result of insulin tolerance test and oral glucose tolerance test showed mild elevated blood glucose levels and plasma insulin levels in DHEA-exposed rats, indicated the presence of defect in insulin sensitivity. Plasma triglyeride levels were not affected, but there were elevation in cholesterol levels, especially in LDL-cholesterol levels. Our investigation indicated DHEA-treated rats showed similar reproductive abnormalities consistent with human PCOS, including the presence of cycle irregularity, ovarian cysts, and hyperandrogenism. Although the absence of obesity, our metabolic analysis indicated that these rats showed mild insulin resistance and dyslipidemia. In conclusion, DHEA-treated rats mimicked abnormalities of reproductive and metabolic characters in human PCOS. This research is supported by NSC98-2320-B-341-001-MY2, National Science Council, Taiwan (ROC).
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Colostrum (first milk) serves as an important source of bioactive molecules, including antibodies and hormones. Since all neonatal mammals drink milk, such milk-borne bioactive factors (MbFs) are likely to be important for postnatal development. The lactocrine hypothesis states that MbFs delivered into the circulation of nursing infants can influence developmental programming of neonatal tissues. The peptide hormone relaxin (RLX), a prototypical lactocrine-acting factor, is present in the colostrum of several species including pigs and humans. RLX supports events required for development of the neonatal porcine reproductive tract, including expression of estrogen receptor (ER) alpha (ESR1) in the uterus and cervix. Normal ESR1 expression in these tissues is altered in neonatal pigs deprived of colostrum from birth (postnatal day = PND 0). Both ER subtypes, ESR1 and estrogen receptor beta (ESR2), are expressed in human and rodent cardiac tissue. It is not known if the neonatal porcine heart is an estrogen or RLX target tissue, or whether nursing or RLX affect ER or RLX receptor (RXFP1) expression in the heart, as demonstrated for the reproductive tract. Objectives were to determine effects of age, nursing and exogenous RLX treatment on the expression of markers of neonatal porcine cardiac development including ESR1, ESR2 and RXFP1. At birth, gilts were assigned randomly to either nurse ad libitum or to receive hormone-free milk replacer, with or without exogenous RLX treatment (20 µg/kg BW im/6h for 48 h). Left ventricular cardiac tissues were collected from newborn (PND 0) gilts prior to ingestion of colostrum and from treated gilts on PND 2. Immunoblotting was used to assess cardiac ESR1 and ESR2 protein expression, with actin used as a reference for loading. Quantitative RT-PCR was used to quantify cardiac RXFP1 mRNA expression. Cardiac ESR1, undetectable in newborn gilts, was induced in gilts that nursed, but was undetectable in replacer-fed animals at PND 2. Exogenous RLX had no effect on cardiac ESR1 protein expression in nursing gilts, but partially restored (P<0.05) ESR1 expression in replacer-fed gilts at PND 2. Cardiac ESR2 protein was detectable at birth and at PND 2 in both nursed and milk replacer-fed gilts. Exogenous RLX treatment did not affect cardiac ESR2 protein expression on PND 2. Cardiac RXFP1 mRNA levels were higher (P<0.05) on PND 0 than on PND 2 for all treatment groups. Within PND 2 groups, cardiac RXFP1 mRNA was reduced (P<0.05) in animals that nursed or received exogenous RLX in comparison to milk replacer-fed animals. Together, data suggest that factors present in colostrum are necessary for normal expression of neonatal porcine cardiac ESR1. Although exogenous RLX was bioactive in uterine tissues, treatment of milk replacer-fed gilts with RLX alone was not sufficient to restore ESR1 protein expression to levels observed in nursing animals at PND 2. Data support and extend the lactocrine hypothesis by showing that milk-borne factors affect expression patterns for developmentally important genes and proteins in the neonatal heart. Lactocrine signaling may affect neonatal cardiac tissue programming and development in a manner similar to that proposed for reproductive tract tissues. (Support: USDA-NRI 2007-35203-18098; NSF-EPS 0447675)
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Our long term goal is to test whether leptin depletion is responsible for some of the effects of maternal nutrient restriction during early pregnancy on the placenta and embryo. To test this hypothesis, it is first necessary to establish whether plasma leptin concentrations decrease in response to food restriction in pregnant female mice. Leptin, a peptide hormone produced by adipose tissue, has been found to correlate to body mass index or other measures of adiposity in multiple species. Leptin release is also known to be regulated by circadian rhythm, food intake, insulin, and corticosteroids. Accordingly, leptin levels fall even more rapidly following food restriction than would be explained by fat loss alone. However, there is reason to doubt that this is the case in female mice, particularly during pregnancy. During later pregnancy, leptin concentrations increase independently of adiposity, a process that begins around day 11 in mice. A previous study found that leptin actually increases in response to food restriction during the latter half of pregnancy in rats. Another found that restriction to 60% of ad libitum consumption led to significantly decreased plasma leptin concentrations in male, but not female mice. The goal of this study was to determine whether 50% food restriction affects plasma leptin concentrations in pregnant and non-pregnant female mice. Thirty-six female mice were fed ad libitum (ad lib) or fed 50% of the food consumed by ad lib-fed mice (restricted) at 1600h each day for 10 days. On the tenth day, terminal blood samples were taken from three animals on each diet every four hours. Leptin concentrations differed most between diet groups in the afternoon and evening, but were not different in the morning. We then compared plasma leptin concentrations in samples taken at 1600h after 10 days in each treatment group: non-pregnant, ad lib fed (n=8), non-pregnant, restricted (n=8), pregnant (d11.5) ad lib (n=7) and pregnant restricted (n=4). Two additional plug-positive mice on the restricted diet did not maintain pregnancy, and were excluded from analysis. Initial mouse weights were the same across treatment groups. Final weights were significantly higher in pregnant vs non-pregnant animals fed ad lib, but similar in pregnant and non-pregnant animals on the restricted diet. Mouse weights on the restricted diet were significantly lower than those of ad lib fed mice. Leptin concentrations were similar in pregnant and non-pregnant mice fed ad lib. Leptin was significantly lower in both pregnant and non-pregnant mice following food restriction (p<0.0001), but fell more dramatically in the pregnant animals. Preliminary results indicated that when both groups of pregnant mice were fed in the morning, and blood samples were collected in the morning, leptin concentrations were higher in the restricted group, perhaps due to observed differences in the timing of food consumption. We conclude that plasma leptin concentration does fall in response to food restriction in pregnant and non-pregnant female mice, but the timing of sampling and feeding are critical. This work is supported by NIH HD055231
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
NGF and its receptors TrkA, and p75 are expressed and can exert their biological roles in the female reproductive system including uterine growth and proliferation. However, excluding the data reported for experimental and domestic animal species, there are no studies yet available on NGF and its two receptors and on its putative involvement in the uterine function of wild animal. Regarding uterine activity, seasonal breeders may offer a useful model to study basic mechanisms on the regulation of uterine function changes in the breeding and non-breeding season without any manipulations. The wild ground squirrel is a typical seasonal breeder and has a breeding season from April to May that is followed by a long period of sexual dormancy from June to March. The aim of the present study was to clarify involvement of NGF system in the regulation of uterine function outside the nervous system, we investigated the expression and distribution patterns of NGF and its receptors in uterus of the wild female ground squirrels during the breeding season, pregnancy and the non-breeding season, and to elucidate the relationship between the role of regulation of NGF and its receptors and uterine function in wild female ground squirrels. In this study, NGF and its receptor TrkA were immunolocalized in stromal cells, luminal epithelial cells, glandular cells and smooth muscle cells during the breeding period and pregnancy, whereas p75 was immunostained only in luminal epithelial and glandular cells. In the non-breeding period, NGF and its receptor TrkA and p75 were observed in luminal epithelial cells and glandular cells, with no staining found in stromal cells and smooth muscle cells. Strong immunostaining of NGF and its receptors TrkA was observed in luminal epithelial cells and glandular cells in the breeding period and pregnancy compared with those in the non-breeding period. The intensities of the immunohistochemical signals for p75 did not appear markedly different among the breeding period, pregnancy and non-breeding period. The mean level of NGF and TrkA mRNA and protein in the breeding period and pregnancy was significantly higher than those in the non-breeding period. However, the mean level of the protein of p75 receptors was not notably altered during the breeding period, pregnancy and non-breeding period. When compared to human, mouse, and rat sequences, the nucleotide identities were 82%, 84%, and 76%, respectively, for NGF cDNA (176 base pairs [bp]); 79%, 80% and 66% for TrkA cDNA (261 bp); and 80%, 83% and 75% for p75 cDNA (295 bp). In addition, plasma concentrations of estradiol-17beta and progesterone changed seasonally: estradiol-17beta and progesterone were higher in the breeding period and pregnancy, lower in the non-breeding period. These results suggest that the patterns of NGF and its receptors TrkA expression are correlated with changes in plasma concentrations of estrogen and progesterone, and NGF and its two receptors TrkA and p75 may be involved in the regulation of uterine function changes in seasonal cycle in the wild female ground squirrel. This study is supported by Program for Changjiang Scholars and Innovative Research Team in Universities (IRT0607) from China.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Process of trophoblastic cell lineages takes a crucial role at feto-maternal interface in synepitheliochorial bovine placenta which involves fetal side trophoblast composed with two different types cells, mononucleate and binucleate cells. We developed efficient system to establish bovine trophoblast cell lines (BTs) with BMP4. There are various cell lines with different characteristics. The aim of this study was to explore the method for inducing trophoblast cell differentiation and the process of trophoblast cell lineage in cattle. We used four different origin cells which show different cell characters by a preliminarily examination. The cell characters were evaluated with genes expression profiles by quantitative RT-PCR and morphological features. First we examined the expression of two genes bCSH1 and bIFNT which posses specific feature in different cell, bi- and mononucleate, respectively. BT-B no expressed both genes, BT-C expressed both, BT-H expressed both moderately and BT-K expressed bCSH1 but no bIFNT. All four cells expressed POU5F1 and CDX2. These cells were cultured for 8 days in/on different substrates; collagen coat with/without 10% FCS, collagen gel and matrigel (Becton Dickinson), respectively. After 8 days culture on collagen gel and matrigel, BT-B expressed binucleate cell specific molecules like bCSH1, bPRP1, bPAG1 and mononucleate specific bIFNT. Similar expression profiles of these genes were found in BT-C during cell culture and the intensity of bIFNT expression was 100 times higher in BT-C. In BT-H, only on-matrigel culture stimulated bCSH1 and bPRP1 expression. BT-K expressed all these genes in/on all culture conditions. In four different culture condition, the genes expression profiles in BT-K was similar to those of BT-1 which was established previously and have been used as a trophoblast cell feature, now the generation of this cell passage has been over 300 times. The expression of POU5F1 and CDX2 were constant and very similar in all cell lines including BT-1 with any different culture conditions during 8 days culture. On the matrigel, bovine placental lactogen protein (PL, gene name bCSH1) expression was increased on days 8 of culture in BT-B and BT-C remarkably but not on days 4. The PL expressions were coincided with the gene expression of bCSH1 in any culture conditions. In previous study, on-collagen gel culture stimulated mononucleate to binucleate cell using BT-1 effectively, however on-matrigel culture was more effective to trophoblastic cell differentiation in the present study. Overall on-matrigel condition stimulated the bCSH1 expression in all cell lines and the activity of on-matrigel condition was greater those in on-collagen gel condition which has been known as an effective method for trophoblast cell differentiation from mononucleate to binucleate in BT-1 cell. These data suggest that on-matrigel culture involves active stimulators for trophoblastic cell differentiation and new established cell lines involve different cell lineages and provide a useful tool for studying trophoblast cell differentiation. This research was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports Science and Technology, Japan (Kiban-kenkyu B 20380159).
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Cell-cell communication through gap junctional connexin (Cx) channels likely plays an important role in regulation of utero-placental growth during the earliest stages of placentation. Detection of the expression of the mRNAs and proteins for connexins has been used as an indicator of the presence of structural and functional gap junctions in tissues. Gap junctional channels may contain only one type of connexin or may consist of combinations of several different connexins--including Cx26, Cx32, Cx37, and Cx43. Our objective was to determine whether these four connexins are expressed in a differential manner during early development of utero-placental tissues. To quantify the expression of Cx26, Cx32, Cx37, and Cx43 mRNA in the ovine placenta, maternal (caruncle; CAR) placental tissues were collected from ewes (n = 5-8/group) on days 14, 16, 18, 20, 22, 24, 26, 28, or 30 after mating, from non-pregnant (NP, control) ewes on day 10 of the estrous cycle, and from the fetal placenta (fetal membranes; FM) on days 16 through 30. Tissues were snap-frozen for quantitative real time RT-PCR and Western analyses. For CAR (all compared to NP ewes): Cx26 mRNA increased (P<0.01) 3-fold on day 14, 5- to 8-fold on days 18 to 28, and 12-fold on days 16 and 30. Cx32 mRNA expression tended to increase (P<0.09) on day 18. Cx37 mRNA expression increased (P<0.01) 2-fold on days 22 to 30. Cx43 mRNA expression increased (P<0.01) by 5- to 6-fold on days 20 and 22 of pregnancy. Using regression analyses, the patterns of expression of Cx mRNA across days of pregnancy in CAR were best described as: Cx26--exponential sigmoidal (P<0.001; R2=0.42); Cx37--exponential (P<0.001; R2=0.44); and Cx43--exponential (P<0.03; R2=0.10). Expression of Cx26, Cx37, and Cx43 mRNA in CAR were positively correlated with each other (P=0.02; R2=0.27). For FM: Cx26 and Cx32 mRNA expression were unchanged across all days of pregnancy. Compared with day 16, Cx37 mRNA increased 3-fold on day 18, 15-fold on day 20, and 30- to 40-fold on days 22 through 30 of pregnancy. Compared with day 16, Cx43 mRNA expression increased (P<0.01) 3-fold on day 18 and >4-fold on days 20 and 22 of pregnancy and then decreased. Using regression analyses, the patterns of expression of Cx mRNA in FM across days of pregnancy were best described as: Cx37--exponential sigmoidal (P<0.001; R2=0.59), and Cx43--cubic (P<0.001; R2=0.51). These data suggest that Cx26, Cx37, and/or Cx43 play important roles in growth and development of the maternal placenta and Cx37 and/or Cx43 in the fetal placenta. In maternal and fetal placental tissues, changes in expression of Cx, such as those evaluated in our study, may signal a need for increased cellular proliferation that may be regulated by a gain or loss of specific gap junctional communication channels. Thus, further investigation is needed to determine the functions of specific connexins in female reproductive processes including placental growth and fetal development. Supported by Hatch Project ND01712; USDA grant 2007-01215 to LPR and ATGB, NIH grant HL64141 to LPR and DAR, and P20 RR016741 from the INBRE program of the National Center for Research Resource.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Cattle form the cotyledonary placenta in the feto-maternal interface. Two specific types of trophoblast cells, trophoblast giant binucleate cells and trophoblast mononucleate cells, play a crucial role in cattle placentation. The mechanisms underlying these cells differentiation and proliferation are still unclear. In human, syncytin 1 is an human endogenous retrovirus family W (HERV-W) envelope protein that expressed in placental syncytiotrophoblast and is involved in fusion of cytotrophoblast cells to form the multinucleated syncytial layer of the placenta. In this study, we cloned the sequences similar to human syncytin (tentatively named bovine endogenous retrovirus envelope element-like transcripts, bERVEs) and confirmed the localization of these genes in bovine placenta to explore and identify the expression of bovine syncytin. We collected all tissues used in this study from Japanese black cattle. They were artificially inseminated and the day was designated as day 0 of gestation. During estrous cycle and gestation (from day 17 to 252 of gestation), conceptus, endometrium, placemtomes, fetal membranes and other somatic tissues like skeletal muscle, testis and liver were collected. Total RNA were extracted using TRIzol (Invitrogen, Carlsbad, CA, USA) and used for cloning and quantitative RT-PCR of bERVEs and also for in situ hybridization analysis. We examined bERVEs in bovine genome database in silico. The information was used for cloning, sequencing and homological analysis. In sillico analysis identified 29 bERVEs in bovine genome. Using sequences information we cloned two bERVEs; tentatively named bERVE-A and -B in bovine tissues. The clone A shared approximately 94% homological sequences to the registered as the similar to human syncytin in bovine genome database. The bERVE-A contained open reading frame (ORF) with 107 amino acids. Other fragment, bERVE-B, contained approximately 99% homology with the registered sequences. The expression profile of them was different. bERVE-A specifically expressed in the placentomes and intercotyledonary tissues. The expression of bERVE-A was slightly expressed in the conceptuses (from day17 to 19 of gestation) and the expression was increased to day 30 of gestation in fetal membranes. In addition, bERVE-A was hardly expressed in the endometrium throughout estrus cycle. On the other hand, no significant expression of bERVE-B was found in the placentomes and its was detected in other tissues. In situ hybrizidation detected bERVE-A gene in both trophoblastic cells; predominantly expressed in trophoblast giant binucleate cells throughout gestation. This is the first evidence for the expression of endogenous retrovirus-like transcripts in bovine placenta. These results suggest that the molecules derive from endogenous retrovirus genes are produced in placenta, and take a crucial role for proliferation and differentiation of trophoblast cells in cattle.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
The human placenta is an invasive tumour-like organ that invades the uterine endometrium and its vasculature to establish sufficient fetal-maternal exchange. A subpopulation of trophoblast cells, known as extravillous trophoblasts (EVT), grows out of the anchoring villi, invades the uterine decidua and remodels the uterine arteries. During this process, EVT cells adopt an endovascular phenotype and replace the endothelium of the arteries transforming these normally small muscular arteries into large, non-contractile and flaccid vessels. This transformation allows for unimpeded placental perfusion necessary for the adequate exchange of crucial molecules between the fetal and maternal circulations. We have previously shown that EVT cell invasion is negatively regulated by a decidua-derived, small leucine rich proteoglycan, decorin (DCN). Decorin was shown to bind vascular endothelial growth factor receptor (VEGFR)-2 on the first trimester EVT cell line, HTR-8/SVneo. In this study, we examined whether decorin binding to VEGFR-2 antagonises VEGF-induced EVT cell migration and endothelial-like tube formation on matrigel (an in vitro model of the endovascular phenotype); and if so, the underlying signalling mechanisms behind this inhibition. To study migration, cells were pre-treated with decorin and seeded in the upper chamber of a transwell insert, where VEGF121 was placed in the bottom chamber. Decorin inhibited VEGF-induced tube formation and migration in a concentration-dependent manner. Western blot analysis revealed that VEGF stimulated p38 Mitogen-Activated Protein Kinase (MAPK) as well as MAPK p42/p44 (ERK1/2) activation and that these activation events could be blocked by decorin. Inhibitors for p38 MAPK and ERK1/2 (SB203580 and U0126, respectively) also inhibited VEGF-induced migration. These results suggest that decorin inhibits VEGF-induced migration by interfering with VEGF induced p38 and ERK1/2 activation. Our novel finding of decorin as an antagonistic ligand for VEGFR-2 as well as its ability to block migration and acquisition of an endovascular phenotype has implications for the pathobiology of preeclampsia, a hypo-invasive trophoblast disorder in pregnancy. (Supported by funds from the Canadian Institutes of Health research to PKL).
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor which mediates a variety of biological processes including reproduction and in particular normal placentation. The AhR has been extensively studied as a receptor for environmental toxicants such as dioxin. Upon binding to these toxicants, the AhR is activated and could induce adverse developmental effects on many tissues including fetal cardiovasculature. Recently, normal physiological roles of AhR have begun to be recognized, particularly after the discovery of endogenous AhR ligands such as 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), which has a much lower toxicity as compared with the extreme deleterious effects of dioxin. However, little is known about AhR actions in endothelial cells and their underlying signaling mechanisms. Herein, we examined if ITE activated AhR and affected proliferation of human umbilical vein (HUV) endothelial (HUVE) cells in response to fetal bovine serum. The expression of AhR in HUV and HUVE cells was evaluated by immunohistochemistry and Western blot analysis, respectively. Cell proliferation was determined in vitro using the crystal violet cell proliferation assay. AhR was immunolocalized in endothelial cells of HUV and was expressed in HUVE cells in vitro. We observed that ITE dose-dependently (0.1 nM-5 μM) and time-dependently (2-6 days) inhibits (P < 0.05) HUVE cells proliferation. ITE also reduced (P < 0.05) AhR protein levels in a time-dependent manner, with an initial decrease seen at 6 hr and completely vanished after 48 hr, indicating that ITE induced robust AhR activation. AhR plays an important role in mediating HUVE cells growth and may account for the potential deleterious effects of environmental toxicants on embryonic/fetal vasculature and thus fetal development. (HL64703 to JZ, HD38843 to RRM and JZ, HL49210 to RRM)
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Although cloned animals have been successfully produced, a very low birth rate by SCNT embryos has been observed. Such low efficiency of cloning could possibly arise from abnormal or poorly developed placenta and fetus. In the present study, we present differentially regulated proteins in the conceptuses from SCNT embryos to understand the molecular nature of the tissue. Proteins expressed specifically or prominently on day 14 were identified in cloned porcine conceptuses using two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF). The conceptuses were obtained from the recipients pregnant by natural mating (control) or SCNT embryos, respectively. The size and morphology of the samples looked similar in both groups. In the proteomic analysis, 67 proteins were identified as differentially regulated proteins in the SCNT conceptuses. Among these proteins, 61 were down-regulated, whereas the other 6 proteins were up-regulated. In the down-regulated proteins, 2 elongation factors and 5 isomerase were considered to be involved in regulation of early-implantation development and peroxiredoxin-4, Oxidation resistance 1 and thioredoxin peroxidase II which is antioxidant enzymes reduce oxidative stress by scavenging reactive oxygen species (ROS). Among the up-regulated proteins, annexin V, an apoptotic regulator that interacts with the key components of apoptotic signaling, was identified. These findings demonstrate that the conceptuses from SCNT embryos showed aberrant protein expression patterns during pre-implantation development; although the morphology of the tissues looked normal and it can be suggested that the abnormal function of conceptuses in clone might be related negatively to placental formation and fetal development. This work received grant support from the Agenda Program (No. PJ006778), Rural Development Administration, Republic of Korea.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
A critical event in equine placentation is the invasion of the endometrium by the chorionic girdle (CG) to form the endometrial cups and secrete equine chorionic gonadotropin (eCG). The CG is composed of rapidly dividing uninucleate trophoblast cells that terminally differentiate into eCG-secreting binucleate trophoblast cells. Between 36 and 38 days post ovulation, the binucleate cells of the CG trophoblast take on an invasive phenotype and invade the endometrium to form the endometrial cups (EC). eCG is detected in the maternal circulation by day 40 post ovulation, peaks at approximately day 60, and is undetectable by day 120, with the death of the EC trophoblasts thought to be due to maternal immune-mediated destruction. Failure of endometrial cup formation in experimental donkey-in-horse pregnancies established by embryo transfer leads to pregnancy loss in most cases, pointing to the importance of the ECs. Day 34 CG contains a heterogeneous population of uninucleate and binucleate cells that readily adhere to tissue culture plastic when cultured in serum-containing medium, while serum-free medium promotes the spontaneous formation of free-floating three-dimensional spherical structures termed trophoblast vesicles (TV). TV are composed primarily of differentiated binucleate cells that label with monoclonal antibodies to EC trophoblast markers. The TV also secrete eCG in culture. In vitro studies of TV can be used to model the processes leading to EC formation: attachment, invasion, and migration. This study combined an in vitro model of trophoblast cell attachment to extracellular matrices (ECM) with expression array analysis of the CG transcriptome to examine the molecular events involved in CG trophoblast invasion. Day 34 equine conceptuses were collected non-surgically. CG cells were cultured in serum-free medium for 4 days to form small (0.5-2.0mm in diameter) free-floating TV. An attachment assay was performed using defined numbers (5-45) of TV placed in 2.0 mL culture wells coated with collagen type I, laminin, or bovine serum albumin (BSA). Vesicles were assessed for adherence using phase contrast microscopy at 1, 2, 3, and 4 hours of incubation. A 14,300 probe equine expression array designed on the Agilent platform was used to compare gene expression between Day 34 invasive CG and non-invasive chorion trophoblast. Absolute transcript quantification using quantitative real time PCR was conducted on mRNA from Day 34 chorion, CG, and CG vesicles to characterize eCG beta and integrin alpha 2 (ITGA2) expression. Results of assays with CG vesicles on collagen type I and laminin suggest a role for cell-ECM adhesion molecules, such as integrins, in equine trophoblast attachment. ITGA2 is known to mediate binding of collagen type I and type IV as well as laminin in mammalian cells. Microarray findings of ITGA2 upregulation in Day 34 CG and CG vesicles was confirmed by qRT-PCR analysis. This evidence supports previous findings that differentiation of human trophoblast cells to an invasive phenotype is accompanied by temporally and spatially regulated switching of their integrin repertoire. ITGA2 expression patterns mirrored eCG beta expression in Day 34 CG and CG vesicles. Further investigation is warranted to fully understand the importance of ITGA2 and its relationship to eCG beta in the CG trophoblast as it attaches and prepares to invade the endometrium. This research was supported in part by NIH T32 RR007059-15.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Extracellular matrix metalloproteinase inducer (EMMPRIN) is a transmembrane glycoprotein located on the surface of normal and pathological cells. EMMPRIN stimulates the production of several matrix metalloproteinases (MMPs) through direct cell-cell interaction and/or as a paracarine manner. Apart from its MMPs inducing ability, it also facilitates cellular differentiation, developmental process and female fertility. Regarding the expression and role of EMMPRIN in the bovine endometrium is still obscure. Therefore, the aim of this study was to 1) analyze the expression and cellular localization of EMMPRIN along with MMPs during estrous cycle and early gestation and; 2) regulation of MMPs by direct cell-cell and cell-matrix interaction using cultured cells and collagen type-I matrix. EMMPRIN mRNA was expressed in both cyclic and pregnant endometrium and significantly higher in the endometrium at day 35 of gestation than the cyclic endometrium. The fetal membrane at day 35 of gestation also expressed EMMPRIN mRNA but the intensity of expression was slightly lower than that of the endometrium. In Western blot analysis, an approximately 65 kDa band was detected in the endometrium. On the other hand, the intense band migrated approximately 51 and 32 kDa in the cultured bovine epithelial cells and BT-1 cells, respectively; whereas, a 32 kDa band was detected weakly in the stromal cells. Both in situ hybridization and immunohistochemistry data showed that EMMPRIN was primarily expressed in luminal and glandular epithelium with strong staining on day 19 conceptus. At day 19 of gestation, expression of EMMPRIN mRNA on luminal epithelium was decreased than that observed at middle of estrous cycle, however, on day 30 of gestation, slightly increased expression was found at the site of placentation. In the endometrium, expression of EMMPRIN mRNA was limited in the stromal compartment. Expression of MMP-2 and MMP-14 mRNA were mainly detected in stroma and their expression also decreased at day 19 of gestation however it was also expressed at the site of placentation at day 30 of gestation as observed for EMMPRIN. Therefore, we have expected that EMMPRIN from epithelium may stimulate stromal MMP-14 and MMP-2 expression in the bovine endometrium; and regulate the endometrial functions during early gestation. Our in vitro study showed that collagen type-1 significantly stimulated the expression of EMMPRIN, MMP-2 and MMP-14 by both the stromal and epithelial cells. Furthermore, direct cell-cell interaction of epithelial and fibroblastic cells significantly stimulated both MMP-2 and MMP-14 mRNA expression. Taken together, expression of EMMPRIN on feto-maternal interfaces may have crucial role in adhesion and fusion of embryo to luminal epithelium by directly itself through physiological tissues remodeling and developmental process, and/or stimulating MMPs to compensate endometrial functions. Therefore, this study for the first time suggests that EMMPRIN is an important glycoprotein and its differential expression regulates endometrial remodeling during early gestation in cows. Supported by a grant from the Research Project for Utilizing Advanced Technologies (05-1770) from the Ministry of Agriculture, Forestry, and Fisheries of Japan and grants (Kiban-kenkyu C 19580335; Kiban-kenkyu B 20380159) from the Ministry of Education, Culture, Sport, Science, and Technology of Japan.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Successful pregnancy requires activation and recruitment of leukocytes within the uterus. Chemokine ligands 9 (CXCL9) and 11 (CXCL11) are estrogen induced proteins that affect recruitment of immune cells and development of maternal immunological tolerance towards the embryo. Our objective was to determine the roles of these cytokines during successful porcine implantation and during early embryonic loss. In experiment 1, uterine horns were removed from both cyclic (D0, 5, 10, 12, 15, 18) and pregnant gilts (D10, 12, 15, 18), and quantitative RT-PCR was used to evaluate endometrial gene expression. Endometrial CXCL9 and 11 mRNA were greatest at estrus (D0). CXCL11 mRNA was greater in D10 and 18 cyclic gilts, but remained minimal in pregnant gilts. However on D15, CXCL11 RNA was approximately 7,000 fold greater in pregnant compared to cyclic gilts. Similarly, CXCL9 mRNA expression was 20-fold greater in D10 cyclic gilts compared to pregnant gilts. On D15, there was a 66-fold increase in concentrations of CXCL9 in pregnant gilts when compared to cyclic gilts. In experiment 2, chemokine gene expression was evaluated in endocrine disrupted pregnancy using a premature estrogen treatment to induce pregnancy loss. Uteri were harvested on D10, 12, 13, 15 and 17 of pregnancy from successful and endocrine disrupted gilts. Endocrine disruption of pregnancy did not alter gene expression of CXCL11, but concentrations of CXCL11 were greatest on D15 of pregnancy agreeing with the first study. Estrogen exposure decreased CXCL9 gene expression by 12 fold on day 15 of pregnancy. In conclusion, leukocyte populations within the uterus are greatest at D0 and on D18 of the estrous cycle, and leukocyte population is greatest on D15 of pregnancy. Also, exposure to exogenous estrogen prior to normal endogenous conceptus release decreases CXCL9. These findings suggest that CXCL9 and 11 are involved in recruitment of leukocytes to the uterine endometirum during pregnancy and may be vital to the successful implantation process. Funding was provided by INBRE grant number P20RR016478.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Establishment of pregnancy in ruminants requires blastocyst growth to form an elongated conceptus that produces interferon tau, the pregnancy recognition signal, and initiates implantation. Blastocyst growth and development is dependent upon secretions from the uterine endometrium. An early increase in circulating concentrations of progesterone (P4) stimulates blastocyst growth and conceptus elongation in ruminants. Microarray analysis was used to identify candidate genes and regulatory networks in the endometrium that govern preimplantation blastocyst growth and development in early P4-treated ewes. Ewes (n=4 -5 per treatment and day) were treated daily with either corn oil (CO) vehicle or 25 mg P4 in CO from 36 h after mating (Day 0) to hysterectomy on either Day 9 or Day 12 of pregnancy. An additional group of ewes (n=5) received P4 to Day 8 and 75 mg of mifepristone (RU486, a P4 receptor antagonist) from Days 8 through 12. Total RNA was isolated from ipsilateral endometrium collected at hysterectomy. Based on microarray analyses, levels of mRNA for the following genes in the endometria were quantified by quantitative PCR: angiopoietin-related protein 3 precursor (ANGPTL3), chromogranin A precursor (CHGA), C-type lectin domain family 4 member E (CLEC4E), chemokine (C-X-C motif) ligand 14 (CXCL14), ephrin-A1 (EFNA1), ephrin-B1 precursor (EFNB1), fatty acid binding protein 3, muscle and heart (FABP3), interferon gamma (IFNG), interleukin-6 precursor (IL6), ISG15 ubiquitin-like modifier (ISG15), lectin, galactoside-binding, soluble, 3 (LGALS3), parathyroid hormone precursor (PTH), plasma retinol-binding protein precursor (RBP4), radical S-adenosyl methionine domain containing 2 (RSAD2), slit homolog 2 protein precursor (SLIT2), slit homolog 3 protein precursor (SLIT3), and Von Willebrand factor (VWF). Relative quantification of gene expression across day and treatments was evaluated using a comparative CT method by subtracting the expression of alpha tubulin (TUBA1) CT value of each sample from the target gene CT value. All days and treatments were compared to mean CT value of the respective gene. Endometrial expression of CLEC4E, FABP3, PTH, SLIT2, SLIT3 and VWF mRNAs was not different (P>0.10) in Day 9 CO and Day 9 P4, Day 9 CO and Day 12 CO, or Day 12 CO and Day 12 P4 ewes. Endometrial ANGPTL3, CHGA, CXCL14, EFNA1, EFNB1, ISG15, LGALS3, RBP4 and RSAD2 mRNA levels were higher (P<0.05) in Day 12 CO than Day 9 CO ewes. Further, CHGA, CXCL14, LGALS3 and RBP4 mRNA levels were higher (P<0.10) in Day 9 P4 than Day 9 CO ewes. In Day 12 P4 ewes, IFNG, IL6, ISG15 and RSAD2 mRNA levels were higher (P<0.05) than in endometria from Day 12 CO ewes. All genes evaluated, with the exception of CLEC4E and PTH, had higher (P<0.05) mRNA levels in endometria from Day 12 P4 than Day 12 P4+RU486 ewes. In situ hybridization analyses determined that CXCL14 mRNA was present in endometrial luminal (LE) and glandular epithelia (GE), whereas IL6 mRNA was in immune cells within the LE. Factors encoded by genes that were regulated by day of pregnancy and P4 are hypothesized to regulate endometrial and/or trophectoderm functions that impacts conceptus growth and elongation during early pregnancy. Research supported by Agriculture and Food Research Initiative Competitive Grant No. 2009-01722 from the USDA National Institute of Food and Agriculture.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Uterine receptivity is a transient state in which the uterus can accept an embryo to implant into the uterine wall. It is controlled by the ovarian hormones progesterone (P4) and estrogen (E2). P4 and E2 exert their uterine effects mainly through their respective receptors, PR and ER. Bisphenol-A (BPA) is an endocrine disruptor that has been widely used in polycarbonate plastics and epoxy resins. It has been demonstrated that BPA has adverse effects on uterine development and can alter uterine expression of PR and ER. In addition, it has been reported that BPA treatment at ~400 mg/kg/day in early pregnancy is detrimental to embryo implantation. However, it is unknown whether the adverse effect is on the embryo or the uterus or both during embryo implantation. To investigate the potential effect of BPA on uterine receptivity, wild type C57B6/129 mixed background young adult females (4-6 each group) were mated with untreated wild type stud males. The timed pregnant mice were treated with 0, 40, 100, and 400 mg/kg/day BPA (dissolved in sesame oil) daily via subcutaneously injection from embryonic day 0.5 to 3.5. Implantation sites were visualized with Evans blue dye injection at day 4.5 (mating night as day 0, implantation normally initiates ~ day 4.0 in mice). No implantation sites were detected in both100 and 400 mg/kg/day BPA treated groups. No embryos were flushed from these uteri either, indicating that BPA at these high doses are toxic to the early stage embryos. Reduced number of implantation sites were detected in the 40 mg/kg/day BPA treated group. However, the appearance of the blue bands (implantation sites) was not as sharp as those seen in the uteri of the control group. Immunohistochemistry of PR indicated that there was no decidual zone at the implantation sites in the 40 mg/kg/day BPA treated group, and PR was highly expressed in the luminal epithelium, suggesting that implantation was abnormal in this group. PR expression disappeared from the luminal epithelium but was strong in the primary decidal zone in the implantation sites from the control group. PR expression in day 4.5 uteri from both100 and 400 mg/kg/day BPA treated groups was also dysregulated. To further define the effect of BPA on uterine receptivity at these high doses, we will transfer embryos from untreated females into BPA-treated pseudo-pregnant uterus. Our results demonstrate that high doses of BPA are detrimental to early stage embryos and their effect on uterine receptivity awaits further clarification. (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):3.
Early pregnancy diagnosis is an essential component of dairy cattle reproductive planning because long-term non-pregnancy results in longer dry periods and low milk productivity. However, available pregnancy tests are inconvenient and require specific expertise or expensive equipment. The purpose of this study was to confirm pregnancy in cows by detection of early pregnancy-specific milk proteins through an approach that combines two-dimensional gel electrophoresis (2-DE), MALDI TOF-MS analysis, generation of antibodies to synthetic peptides, and Western analysis. Milk samples were collected from pregnant (day 30 and 50) and non-pregnant cows. After acidifying to pH 4.6 to remove casein proteins, samples were centrifuged and then frozen until use. For each thawed sample, 2 mg milk protein, loaded at the anode, was separated in the first dimension based on isoelectric point using pH 3.0 - 10.0, pH 4.0 - 7.0, and pH 6.0 - 9.0 strips. Proteins were resolved in the second dimension by SDS polyacrylamide gel electrophoresis on 8% - 16% linear gradient gels. The gels were stained with colloidal Coomassie brilliant blue and then scanned. The images of stained gels were analyzed to detect differences in protein spots between non-pregnant and pregnant milk samples using an Image Master followed by MALDI TOF-MS. Analysis of the 2-DE gel image revealed a total of approximately 600 - 700 protein spots, of which 39 were differentially expressed in pregnant milk versus non-pregnant milk samples. Eight of these 39 spots corresponded to pregnancy-specific proteins; 11 other spots were down-regulated and 20 were up-regulated in the pregnant milk sample. Peptides were synthesized based on amino acid sequence information obtained from a proteomics analysis of pregnancy-specific milk whey proteins and then used to raise antibodies Western blotting. Antibodies to the following proteins were generated: lactoferrin, lactotransferrin, carrier organic anion transporter, similar to MAK31-like protein, alpha1G, cystatin C, AMP-activated protein kinase gamma subunit, transferrin, and conglutinin precursor protein. Western blot analyses showed that the expression of lactoferrin, lactotransferrin and alpha1G increased during pregnancy compared with non-pregnant samples. The remaining antibodies exhibited non-specific reactivity, possibly because the synthetic peptides used to raise them correspond to regions of the target protein that may not be accessible to antibodies in the mature folded protein, and are thus undetectable. The results of this study suggest that proteomic analyses could be systematically applied to the production of antibodies and confirmation of their potential applications. The proteins identified in pregnant samples and their antibodies could be good candidate starting points for the development of a pregnancy detection for cows.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
RNA ultra deep sequencing was used to strive for the generation of a complete catalog of transcriptome changes in bovine endometrium during the pre-implantation period. Endometrial tissue samples were collected from Simmental heifers after slaughter at days 15 (D15) and 18 (D18) of pregnancy and at the corresponding days of the estrous cycle (n=4 per group). RNAseq libraries starting from polyA RNA were prepared and analyzed on an Illumina Genome Analyzer IIx. For the D18 libraries 36 bp paired-end reads (sequences) were generated sequencing one sample per flow-cell lane resulting in more than 40 million uniquely mapped paired-end reads with 4 to 12 million reads per sample. These reads corresponded to approx. 15,000 annotated genes. For the D15 libraries 100 million uniquely mapped reads (54 bp paired-end) were produced ranging from 7 to 26 million reads per sample that corresponded to approx. 16,000 genes. Based on the results of the mapping to known genes normalized expression values (reads per kilobase mRNA and million reads, RPKM) were calculated for every sample and used for statistical analysis (SAM Excel Add-in). At D18 664 differentially expressed genes (DEGs) were identified (FDR 1%, at least 2-fold change), from which 414 were up-regulated and 250 down-regulated in pregnant endometrium. Analysis of the same RNA samples with Affymetrix Bovine Genome Arrays resulted in 336 DEGs (279 up- and 57 down-regulated). At D15, both RNAseq and microarray analysis revealed only up-regulated genes, namely 167 and 108 DEGs (FDR 5%, at least 2-fold change), respectively. Of the RNAseq DEGs, which were represented and detectable on the microarray, 90% (Day18) and 84% (D15) also showed differential expression in the microarray analysis indicating high consistency between RNAseq and Affymetrix microarray data. Comparison of RNAseq DEGs of D18 and D15 revealed that almost 90% of the genes up-regulated at D15 were also up-regulated at D18 of pregnancy. Direct comparison of RNAseq datasets from D15 and D18 pregnant endometrium showed that nearly all D15 DEGs had similar or higher expression levels in D18 pregnant endometrium. For selected genes differential expression was confirmed using quantitative real-time RT-PCR. Both, RNAseq and Affymetrix results showed good correlation to the qPCR results. Finally, analysis of differential splicing and 3' UTR variants based on RNAseq data is in progress to get further insights into the complex gene regulation networks in the bovine endometrium during early pregnancy. In conclusion, based on the higher sensitivity and detection of transcripts not represented on commercially available bovine microarrays, transcriptome analysis using RNAseq provides significant additional information on differential mRNA expression in bovine endometrium during early pregnancy. Furthermore, the expression values obtained by RNAseq have a more quantitative character than microarray results allowing direct comparison of different RNAseq datasets. Research supported by the German BMBF (FUGATOplus Compendium).
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Successful pregnancy is associated with an appropriate maternal immune response to the implanting embryo. Compared to the species forming invasive placenta, maternal immune response during pregnancy has not been much understood in the species forming noninvasive placenta including pigs. Previously, we have found expression of a MHC class II gene, swine leukocyte antigen (SLA)-DQA1, in the uterine endometrium on day (D) 12 of pregnancy in pigs. To further understand the role of SLA-DQA1 during pregnancy in pigs, we analyzed expression of SLA-DQA1 in the uterine endometrium during the estrous cycle and pregnancy in pigs. SLA-DQA1 mRNA was detectable in the endometrium during the estrous cycle and pregnancy and levels of SLA-DQA1 mRNA were changed during pregnancy. SLA-DQA1 mRNA and protein were localized mainly to subepithelial stroma and blood vessels. Treatment of endometrial explants from D12 of the estrous cycle with steroid hormones showed that progesterone induced SLA-DQA1 mRNA. Furthermore, we analyzed expression of SLA-DQA1 in the endometrium of the uterus carrying embryos from natural mating and embryos derived from somatic cell nuclear transfer (SCNT) on D12, D30 and at term (D114). Results showed that SLA-DQA1 expression was greatly decreased in the uterus with embryos derived from SCNT compared to those from natural mating at term, but not on D12 and D30 of pregnancy. Results of this study showed that SLA-DQA1 was expressed cell type- and pregnancy stage-specifically and its expression was greatly decreased in the uterus with cloned embryos, indicating a critical role of uterine SLA-DQA1 in the maintenance of pregnancy in pigs. This reseach was supported by the Yonsei University Research Fund of 2009, and by the BioGreen 21 Program (#20070301034040, #20080401034003), Rural Development Administration, Republic of Korea.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Plasma ATP elicits profound uterine vasodilation through activation of endothelial nitric oxide synthase (eNOS), an enzyme whose activity is regulated by its partitioning between caveolar and non-caveolar subcellular domains in uterine arterial endothelial cells (UAECs). Based on these profound effects that ATP has on caveolar eNOS, we hypothesized that UAEC caveolae contain specific protein machinery for ATP actions and that extracellular ATP will alter this protein profile with rapid trafficking of specific ATP-related proteins into or out of caveolae. Primary passage 4 cultured third trimester pregnant ovine UAECs (80% confluence) were treated with or without ATP (5 min, 100 µM). Sucrose gradient centrifugation followed by nanoLC-MS/MS was utilized to assess the contribution of the caveolar subcellular compartment in ATP-mediated uterine endothelial signaling. Proteome changes were validated by Western blotting. Proteomic classification of UAEC caveolar proteins demonstrated that 14 of the 100 most abundant proteins were related to ATP. These included ATPsynthases, ATPases, and ATP transporters. Treatment with ATP for 5 min rapidly increased the overall abundance of these ATP-related proteins by 41.6%. Interestingly, ATP treatment selectively produced a marked increase in the levels of caveolar ATP transporters alone. Notably, the adenine nucleotide translocator 2 (ANT2) increased substantially by 429%, whereas all voltage dependent anion channel (VDAC) proteins increased between 30 and 200%. Western blotting validated proteomics data; specifically ANT2 and VDACs were concentrated in the caveolar domain in response to ATP treatment and were elevated by similar magnitudes. The uterine endothelial caveolae are enriched with ATP-associated proteins and the caveolae appear to be dynamic "hubs" for ATP-actions. Furthermore, these data demonstrate that physiologic levels of ATP induce acute trafficking of ATP transporters within 5 min into the caveolae. Consistent with previous reports that ATP exhibits pronounced attraction to ATP flux-related ANT2 and VDAC channels, we observed that all these transporters are rapidly trafficked into the caveolae in response to acute ATP treatment. Further, we herein show definitive evidence for the presence of classical mitochondrial and endoplasmic reticulum proteins such as ATP synthases, ATPases, and ATP transporters in the plasmalemmal caveolar invaginations. In summary, this study demonstrates that the caveolae are enriched with ATP-related proteins and that acute extracellular ATP treatment rapidly induces trafficking of specific ATP transporters into the UAEC caveolae in late gestation. NIH HL49210, HD38843, HL87144, and HL70562.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
The HSD11B1 enzyme catalyzes the interconversion of inactive cortisone to active cortisol, which is a biologically active glucocorticoid, whereas HSD11B2 converts active cortisol to inactive cortisone. Recently, we found that endometrial epithelial HSD11B1 expression is induced by ovarian progesterone (P4) and further stimulated by interferon tau (IFNT) and that the glucocorticoid receptor is expressed in all cell types in the ovine endometrium. Available results support the working hypothesis that HSD11B1-regenerated cortisol and prostaglandin synthase two (PTGS2)-derived prostaglandins (PGs) from the endometrium and/or conceptus regulate trophoblast growth and development during conceptus elongation in sheep. In Study 1, ewes were hysterectomized on Days 12 to 16 of pregnancy (n=4-6 per day). In vitro endometrial explant cultures determined that HSD11B1 activity was highest (P<0.05) on Days 12 and 13 of pregnancy (9.6 and 10.4 pg/mg tissue/hour) and lowest in Day 16 pregnant ewes (3.4 pg/mg/hour). Interestingly, HSD11B1 activity was higher in the Day 14, 15 and 16 conceptuses (~75 pg/mg/hour). Neither HSD11B1 activity nor HSD11B1 and HSD11B2 mRNAs changed (P>0.10) in the conceptus between Days 14 and 16. Radioimmunoassay of uterine flushes from Day 12 to 16 pregnant ewes detected cortisol (~1 ng total). In Study 2, the ipsilateral uterine horn of Day 8 bred ewes was surgically ligated near the base and a catheter, connected to an implanted Alzet 2ML1 osmotic pump containing either vehicle (control) or meloxicam (2.5 mg; Boehringer Ingelheim Pharmaceuticals), was inserted into the uterine lumen. All ewes were hysterectomized on Day 15. Normal elongated, filamentous conceptuses were recovered from the uteri infused with the vehicle (n=4/5 ewes), whereas conceptuses from uteri infused with meloxicam were growth retarded and fragmented (n=3/5 ewes). Endometrial explants from meloxicam-infused ewes had 23% lower HSD11B1 activity (P<0.10) as compared to those from control ewes. Enzyme-linked immunoassay found that PGs were non-detectable in the uterine flushings from meloxicam as compared to control ewes. Further, meloxicam dose-dependently decreased (P<0.05) PG synthesis in endometrial explants cultures from Day 14 cyclic ewes. In Study 3, each uterine horn of Day 8 cyclic ewes was catheterized with an Alzet 2ML1 osmotic pump containing vehicle (control), meloxicam (2.5 mg), recombinant ovine IFNT (1 x 109 antiviral units), or meloxicam plus IFNT (n=4-5 ewes per treatment). All ewes were hysterectomized on Day 14. Endometrial explants from IFNT infused ewes had 1.8-fold higher (P<0.01) levels of HSD11B1 activity than control ewes. Further, HSD11B1 activity was 30% lower (P<0.05) in endometria from IFNT and meloxicam than IFNT infused ewes. Present and published results indicate that the elongating conceptus secretes more PGs and regenerates more cortisol from cortisone than the endometria during early pregnancy and that PTGS2-derived PGs and IFNT stimulate endometrial HSD11B1 activity. These results support the hypothesis that PTGS2-derived PGs, regenerated cortisol and IFNT from the conceptus and/or endometrium act in a paracrine and/or autocrine manner to stimulate endometrial and trophoblast functions important for conceptus elongation in sheep. Research supported by Agriculture and Food Research Initiative Competitive Grant No. 2009-01722 from the USDA National Institute of Food and Agriculture.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Recently we reported that physiologic levels of estradiol-17β (E2β) and the catecholestradiols 2-Hydroxyestradiol (2-OHE2) and 4-Hydroxyestradiol (4-OHE2) induce pregnancy-specific proliferation of uterine artery endothelial cells (UAECs). However, unlike E2β, the proliferative effects of its catechol metabolites were not mediated via estrogen receptors (ER) ERα or ERβ. Thus the exact mechanism of action of estrogen metabolites on UAECs mitogenesis remains to be determined. Due to the structural similarities between catecholestradiols and catecholamines and the fact that the catecholestradiols have affinity for adrenergic receptors (AR), we proposed the possibility that the catecholestradiols stimulate proliferation of UAECs via ARs and that catecholamines could also modulate these mitogenic effects. We hypothesized that catecholestradiols will partly mediate proliferation of UAECs via either α-AR or β-AR and that catecholamines will directly increase these proliferative effects. Identification of AR subtypes (α1, α2, β1, β2 and β3) in UAECs was performed by Western Blotting. Validated UAECs from late pregnant sheep (day 120-130; term = 147 d; n=5) were pretreated (10µM; 1 hr) in the absence or presence of the nonselective α-AR blocker phentolamine or nonselective β-AR blocker propanolol followed by either endothelial basal medium (EBM; vehicle), 0.1 nM or 100 nM of 2-Hydroxyestradiol (2-OHE2) and 4-Hydroxyestradiol (4-OHE2). AR activation alone or their additive effects with catecholestradiols were evaluated by combining treatments of 0.1 nM or 100 nM of epinephrine or norepinephrine together with 2-OHE2 or 4-OHE2. Western Blotting revealed the presence of α2, β2 and β3 AR in UAECS; however, α1 and β1 were not detectable. Both 2-OHE2 and 4-OHE2 treatment significantly increased (P<0.01) UAEC mitogenesis. Pretreatment with the alpha-AR antagonist phentolamine did not alter (P>0.05) the UAECs proliferation response to 2-OHE2 and 4-OHE2. By contrast the β-AR blocker propanolol pretreatment completely abrogated (P<0.01) the proliferative effects of the catecholestradiols. Both catecholamine agonists alone increased proliferation. Treatments of UAECs with combinations of epinephrine or norepinephrine with 2-OH E2 or 4-OHE2 significantly enhanced UAEC proliferation compared to either catecholamine agonists or catecholestradiols alone. These data demonstrate that catecholestradiols stimulate proliferation of UAECs via β2 and α3 AR and not α-ARs. Moreover, catecholamines that directly activate β-ARs can mimic these mitogenic actions. However combination treatments provide significantly greater stimulation than either catecholamines or catecholestradiols alone, suggesting additional recruitment of signaling pathways to achieve an additive effect. These data point to the potential relevance of the convergence of the sympathomimetic system and catecholestradiols in the regulation of pregnancy-induced angiogenesis. NIH HL49210, HD38843, HL87144 and R25GM083252.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
NOT PRESENTED.
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Sperm motility is an important determinant of sperm quality. However, the mechanisms that mediate sperm motility are still poorly understood. Previously, we have shown that a teleost progestin hormone, 17,20beta,21-trihydroxyprogesterone (20beta-S) can mediate sperm hypermotility in Atlantic croaker, Micropogonias undulatus, by a nongenomic mechanism that is likely mediated by membrane progestin receptor-alpha (mPRalpha) coupled to a stimulatory olfactory G-protein, Golf. An earlier study has implicated a role of the alpha subunit of Golf in mediating sperm motility via increases in membrane-bound cyclic adenosine monophosphate (cAMP). There is evidence that the beta-gamma subunits of G-proteins may be involved in mediating sperm functions in various mammalian systems. A commonly studied intermediary of beta-gamma subunit function is the PI3K/Akt pathway. To the best of our knowledge, no reports of the PI3K/Akt pathway and its functions exist for fish sperm. The aim of this study was to investigate if the PI3K/Akt pathway exists in fish sperm, and whether the modulation of this pathway played a role in mPRalpha-mediated sperm hypermotility. The presence of Akt and its activated form (phosphorylated-Akt) in croaker sperm were confirmed by Western blots using commercially available antibodies. Recent experimental results further suggest that the beta-gamma subunits of Golf in croaker sperm are also involved in mediating the effects of 20beta-S on sperm activation, via activation of the PI3K/Akt pathway. Treatment of croaker sperm with 100nM 20beta-S and 100nM of the mPRalpha-specific agonist, Organon OD 02-0 caused activation of Akt. Specific inhibitors of the PI3K/Akt pathway (1nM Wortmannin, 25uM LY294002, or 50uM ML-9) significantly reduced the percentage of hypermotile croaker sperm in the presence of 20nM 20beta-S and 20nM OD 02-0. Treatment of croaker sperm with 10nM Wortmannin also prevented the activation of Akt, in the presence of 100nM 20beta-S or 02-0. Taken together, these results indicate an involvement of the PI3K/Akt pathway in mPRalpha mediated sperm hypermotility. To our knowledge, this is the first evidence that the PI3K/Akt pathway is involved in progestin-mediated sperm motility in a vertebrate species.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
In order to penetrate into the egg, the capacitated spermatozoon should bind to the zona pellucida (ZP) and undergo the acrosomal reaction. The acrosome reaction is mediated by receptor activation that leads to calcium influx into the sperm. In the present study we showed that isolated ZP activates sperm alpha7-nicotinic-achetylcholine receptor (á7nAChR) leading to the trans-activation of epidermal-growth-factor-receptor (EGFR), calcium influx and the acrosome reaction. The data revealed that alpha7nAChR agonist initiates the acrosome reaction which was inhibited by EGFR-antagonist, suggesting that EGFR mediates this reaction. Moreover, the ZP induced acrosome reaction was significantly inhibited by alpha-bungarotoxin (alpha7 antagonist) and by tyrphostin (EGFR antagonist), suggesting that EGFR and the alpha7 subunit are activated by ZP. Furthermore, ZP or EGF did not induce the acrosome reaction in sperm from alpha7 null mice, suggesting that ZP-induced acrosome raction depends on the existance of sperm alpha7 and mediated by EGFR. Inhibition of alpha7 in wild type sperm revealed only partial inhibition of the acrosome reaction induced by EGF, supporting the idea that the alpha7nAChR is localized up-stream to the EGFR. Recent studies suggest that Src kinase can activate EGFR in sperm. It was also shown that Src co-localized with the alpha7 in human sperm. We showed here that Src inhibitor blocked the alpha7-dependent acrosome reaction and EGFR-phosphorylation suggesting that SRC-family mediate these processes. Moreover, EGFR phosphorylation on tyrosine-845 induced by ZP is also inhibited by SRC-family inhibitor suggesting that this kinase mediate the ZP-induced EGFR phosphorylation. It was recently shown in our lab that EGFR activation leads to calcium influx. Here we showed that agonist of the alpha7, EGFR or ZP induced calcium influx that leads to the acrosome reaction. However, in alpha7 null mice, these agonists did not induce calcium influx or acrosome reaction, and this blockade could be bypassed using calcium ionophore supporting the role of alpha7 in calcium influx induced by ZP. In conclusion we suggest that activation of alpha7 by ZP leads to SRC-family dependent-EGFR activation, Ca2+ influx and the occurrence of the acrosome reaction.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Na+K+-ATPase acts as a signaling molecule when ouabain binds, inducing dose-dependent protein tyrosine phosphorylation (p-tyr) during sperm capacitation. Src is present in head membranes of bull sperm and its expression alters during ouabain-induced p-tyr, so this study investigated Src's role in capacitation. Fresh bull sperm (n=3-6 ejaculates per trial) were pre-incubated with or without Src inhibitors herbimycin A (inhibits kinase) or SU6656 (prevents ATP binding to Src), followed by incubation in the presence or absence of ouabain or heparin. Extracted proteins were assessed by western blotting, band intensity measured, and quantities of each protein band compared by ANOVA. Both protein p-tyr and LPC-induced acrosome reaction increased (p<0.05) upon exposure of sperm to ouabain (242, 176, 81, 49 kDa) or heparin (176kDa). Inhibition of Src activity by herbimycin or SU6656 antagonized (P<0.05) various ouabain- and heparin-induced p-tyr, and herbimycin inhibited the ouabain-induced acrosome reaction, confirming Src's integral role in capacitation. Herbimycin actually reduced some Src itself (p<0.05) during ouabain- or heparin-induced capacitation incubation. However, since Src was activated by auto-phosphorylation in the presence of ouabain or heparin, it was of greater interest that herbimycin significantly reduced ouabain-enhanced Src autophosphorylation of the 59kDa phospho-Src and the other ouabain- (166-31kDa) and heparin-induced phospho-Src (130, 40kDa) (P<0.05) implying that those are coupling proteins or substrates of Src. Src can also be activated by serine phosphorylation (p-ser) and both ouabain and heparin significantly stimulated p-ser of various sperm proteins (254-18kDa). SU6656 inhibited ouabain-induced p-ser at 40 and 32kDa, but heparin overcame this inhibition, implying that the Na+K+-ATPase pathway requires ATP binding to Src. In contrast, heparin is less ATP sensitive, perhaps primarily stimulating the cAMP/PKA pathway. Ouabain appears to induce capacitation through the ERK pathway, since phospho-ERK1/2 (Thr202/Tyr204, Thr185/Tyr187) during capacitation were ouabain-sensitive. SU6656 antagonized ouabain-enhanced phosphorylation of ERK2, and other ERKs (39-17kDa) at 2h and herbimycin inhibited ERK1 in control sperm incubated for 5h. Heparin overcame the Src inhibitors' effect on ERKs possibly through the heparin-induced cAMP/PKA pathway. In summary, this study demonstrates Src's activation and involvement in tyrosine phosphorylation-dependent signaling through involving and activating ERK1/2 (potentially ERK2) in ouabain-induced bull sperm capacitation or acrosome reaction. The ouabain-sensitive Na+K+-ATPase signaling cascade is unique and distinct from that of heparin, but this novel research does identify for first time that Src cross talks with the heparin signaling pathway to induce capacitation.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Although many events of developing spermatozoa occur within the testis, the maturation of sperm in the epididymis is a critical part of normal male fertility. The epididymis is not only where sperm are stored but also where spermatozoa gain their motility as well as their ability to fertilize an egg. As the spermatozoa traverses from the caput to the cauda many different proteins are added or removed from the spermatozoa. Many studies have investigated the epididymal transcriptome there have been few studies investigating the proteomics of spermatozoa maturation. We have begun a high throughput proteomic approach to catalog of all proteins associated with murine sperm in both the caput and the cauda epididymis. Sperm were purified from the caput or cauda and proteomic analysis was performed as follows: 1) proteins were extracted and digested into tryptic peptides, 2) the resulting peptides were separated into approximately 30 fractions, 3) each fraction was analyzed by high sensitivity liquid chromatography coupled to tandem mass spectrometry. Preliminary results have identified over 300 unique peptides in the cauda sperm when compared to the caput sperm and over 500 unique peptides in the caput when compared to cauda sperm. We hope that this combined global analysis will allow us to gain new insights into the sperm maturization process.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Storage of fresh liquid boar semen for artificial insemination is associated with decreased sperm fertility, viability and motility over time. Boar sperm plasma membranes contain approximately 60% long chain poly-unsaturated fatty acids (PUFA) and the omega-3 (n-3) and omega-6 (-6) fatty acids are essential PUFA which cannot be synthesized by the animal. It has been proposed that mammals evolved on a dietary n-3 to n-6 ratio of 1:1, but the ratio in typical boar diets is 1:6, risking sub-optimal supply of long chain n-3 fatty acids for spermatogenesis. This study evaluated dietary flaxseed as a source of n-3 fatty acids on fresh and stored boar sperm. Three boars were fed for 3wk according to NRC (1998) recommendations for breeding boars, followed by an iso-caloric and iso-nitrogenous diet containing 15% flaxseed for 9 weeks. Semen was collected 2× per week and analyzed for concentration, volume, and function (motility, viability, morphology, membrane fluidity and capacitation status) during weeks 1-3 (W1/3), weeks 6 and 7 (W6/7), and weeks 10 and 11 (W10/11). Sperm function was also assessed after 5 and 7 days of storage (BTS, 17°C) at weeks 3, 7 and 11. Ejaculate volume increased through the 12 wks for each boar while concentration increased for only one boar; total sperm per ejaculate differed among periods. Total motility increased from W6/7 to W10/11 for one boar, but not the others. The percent motile sperm declined from day 0 to 5 of storage at all times, but there was significantly less loss of motility at W10/11 (p < 0.05). Mean number of viable sperm was greater at W6/7 and W10/11 than W1/3, and viability after 5 and 7 days of storage also was significantly better at W6/7 and W10/11 than W1/3 (p < 0.06 and p < 0.05 respectively). Dietary flaxseed increased the percent of morphologically normal sperm, and decreased of percent of sperm with tail abnormalities. Fluidity of the sperm plasma membrane as assessed by flow cytometry increased with the flax diet. Diet did not affect the acrosome reaction in fresh or stored sperm. These results indicate that the magnitude of the loss in sperm motility and viability following storage are reduced following dietary flax supplementation and the fluidity of the sperm plasma membrane is increased.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Cyclin Dependant Kinase 1 (CDK1) is a protein kinase that plays a critical role in the G2/M progression in mitotic cell divisions and in the prophase to metaphase I progression during meiosis in oocytes. In these cells, CDK1 heterodimerizes with Cyclin B1 (CCNB1) to form the M-phase Promoting Factor (MPF) complex and the CCNB1 subunit regulates the kinase activity of CDK1. We showed earlier that the HSPA2 chaperone was required for MPF assembly. However, others reported that disruption of the gene for cyclin-dependent kinase 2 (Cdk2) resulted in the arrest in prophase I of meiosis in mid-pachytene spermatocytes and that CDK2 can interact with Cyclin B. In addition, disruption of the gene for testis-specific Cyclin A1 (Ccna1) resulted in the arrest in prophase I of meiosis in late spermatocytes and a lack of MPF activation. It was not clear from these results if CDK1 serves an essential role in the meiotic divisions of spermatocytes. We employed the conditional gene targeting approach to disrupt Cdk1 during spermatogenesis to address this issue. Mice in which LoxP sites flanked exon 3 of Cdk1 were produced and mated to Hspa2-cre transgenic mice that expressed cre recombinase specifically in pachytene spermatocytes. By mating the offspring of these mice, we generated mice in which both alleles of Cdk1 were disrupted in pachytene spermatocytes (PS Cdk1-/-). Western blotting indicated that the level of CDK1 in the testes in PS Cdk1-/- mice was reduced, but not eliminated. However, immunohistochemistry showed that CDK1 was present in spermatogonia and pachytene spermatocytes in wild type mice, but only in spermatogonia of PS Cdk1-/- mice. Male PS Cdk1-/- mice mated with wild type females failed to sire offspring and when the testes from PS Cdk1-/- mice were examined histologically, they were found to lack post-meiotic germ cells. These data indicate that Cdk1 is essential for meiotic progression in the male. In addition, they also indicate that CDK2 cannot heterodimerize with CCNB1 or CCNA1 to form an active MPF in pachytene spermatocytes in the absence of CDK1. This research was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
NOT PRESENTED.
Biol Reprod. 2010 Nov;83(1 Suppl):3.
The gonadotropins, luteinizing hormone (LH) and follicle stimulating hormone (FSH), play a critical role in the development of a reproductively competent male. Postnatally, the actions of LH mediated through its receptor (LHR) is critical for testicular steroidogenesis and testosterone produced by the Leydig cells is absolutely required for complete spermatogenesis. FSH, acting on the Sertoli cells, stimulates early events in spermatogenesis and is also necessary for the maintenance of spermatogenesis. In an effort to understand how chronic ligand mediated activation of LHR affects gonadal development and function, transgenic mice expressing a constitutively active yoked hormone-receptor complex (YHR), achieved by covalently linking human chorionic gonadotropin to LHR were generated. Prepubertally elevated testosterone levels, decreased gonadotropin levels, smaller testis, and smaller average seminiferous tubule diameters were detected in male mice expressing YHR (YHR+) when compared to wild type (WT) mice. It was also observed that YHR+ mice had significantly fewer total and motile epididymal sperm compared to WT animals, and breeding studies indicated that YHR+ mice were sub-fertile. The objective of the current study is to determine if the decrease in the testis size and epididymal sperm are a result of changes in the testicular germ cell population. Analysis of testicular germ cell populations in WT and YHR+ mice from 10 days to 24 weeks of age revealed that there are significantly fewer cells in the YHR+ animals starting at 5 weeks of age, suggesting that the decrease in average seminiferous tubule diameter may in part be a result of fewer germ cells. To further evaluate if the reduction in germ cell numbers is a result of a disruption at a specific stage in spermatogenesis, flow cytometric analysis of testicular spermatogenic cells was performed. The analysis showed a significant decrease in the number of spermatogonia, spermatocytes, and round spermatids at 5, 8, and 12 weeks of age. These data suggest that spermatogenesis is impaired in YHR+ males, most likely at the spermatogonial stage, resulting in a decrease in the subsequent germ cell populations. These results also suggest that premature elevation in testosterone levels may directly or indirectly affect germ cell development. Further studies are warranted in order to determine if a decrease in proliferation and/or differentiation of spermatogonia, or an increase in apoptosis, is responsible for the decreased number of germ cells. Supported by NIH HD 044119
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Semen quality of bull is very important both in natural service condition and artificial insemination program. Some bulls produce less or substandard quality of spermatozoa, which makes less number of inseminates and also impairs fertility post insemination or post service. Such bulls mostly have some testicular pathology such as testicular mixed atrophy, which may be irreversible. However, it is difficult to understand their testicular physiology precisely and determine their future utilization. The present study was designed to investigate the difference between poor and good quality semen donating Holstein bulls on GnRH induced testosterone response (GITR). The semen quality parameters of semen volume, spermatozoa concentration, progressive motility of spermatozoa after collection and after freezing and thawing were evaluated by using routine evaluation techniques in the semen collection centers for at least 4 months, and bulls (n=5) having poor semen quality parameters were selected as experimental bulls (experimental group). Similarly, bulls (n=4) having good semen quality parameters were used as control bulls (control group). Both groups were administered once intramuscularly with GnRH (250 μg of fertirelin acetate). Blood samples were taken at -1d, -30 min and 0h (time of treatment) followed by every 30 min for 5h and 1d, 3d and 5d post treatment, and assayed by enzyme immunoassay to measure testosterone and Estradiol (E2) concentrations. The pre-treatment concentrations were used to establish the basal levels of these hormones. The percentage increments based on 0h level were calculated for individual bull for each sampling time during first five hour. The average increments were compared between two groups. Although the result indicated high bull to bull variation in the basal levels of these hormones, no difference was observed in the basal levels between two groups. The first five hour increment pattern of testosterone was quite similar in all bulls and the average levels in both groups indicated the maximum rise of testosterone at 3h post treatment. Four bulls out of five (80%) in the experimental group never reached 60% testosterone increment and remaining one bull also had <80% peak increment. The lowest testosterone increment of only 20.4% was observed in one experimental bull. However, all bulls in control group had >80% testosterone increment with the highest of 102.2% increment during this time. The average testosterone increment in experimental group was lower (P<0.05) from 1h onward until 5h. There was no such difference in E2 increments. The concentrations of both hormones in all bulls almost returned to the basal level on 1d post treatment and no considerable changes were observed until 5d. In conclusion, it can be suggested that GITR may be considered as an indicator of semen quality and less than 60% GITR may be a tool to predict poor semen quality in Holstein bulls.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Glucose tolerance decline and insulin resistance often accompanied functional decline in multiple organs. The present study was to examine whether administration of Danseng (Codonopsis pilosula) root extract to adult male rats will reverse glucose tolerance impairment induced sperm dysfunction. The adult Sprague Dawley male rats were divided into three groups (n=6): control group, fructose alone group, fructose plus Danseng group. In the fructose group, the tap drinking water was replaced by a 10% fructose solution for 8 weeks. All the rats administered with fructose got glucose tolerance impairment. In the fructose plus Danseng group, after the adult male rats got glucose tolerance impairment, Danseng were added by 1 mg/kg body weight in their drinking water for another 4 weeks. Control rats were having standard chow and tap drinking water ad libitum. The rats were anaesthetized and the epididymis was excised and weighed, and used for biochemical analysis. The epididymis was disected and incubated in Locke solution containing 0.18% glucose for obtaining the sperm suspension. The morphology of sperm will be observed under light microscope. Sperm concentration and motility will be determined on counting chamber. Samples were kept frozen (-20°C) until assay. Activities of antioxidant enzymes in testis were measured by assay kit. The combination of Danseng reversed the effect of fructose. The protein expression of antioxidant enzymes, eNOS, and MAPK were detected by Western blot. There were found no significant changes in weight of epididymis and the ratio of epididymis and body weight. The consumption of fructose decreased motility and total count of sperm by 24% and 7% significantly. No significant changes in epidydimis thiobarbituric acid reactive substance (TBARS), superoxide dismutase (SOD), catalase and glutathione peroxidase, glutathione reducatse, glutathione (GSH), and GSSG were observed between the three groups. The administration of fructose resulted in decreased the ratio of GSH and GSSG compared to control group, combination of fructose and Danseng significantly enhanced the ratio of GSH and GSSG compared to fructose group (p<0.05). A correspondent higher increase of epididymis Mn-SOD expression was also observed in the fructose plus Danseng group. The expression of eNOS was also increased significantly in the fructose plus Danseng group compared with the fructose group (p<0.05). The consumption of fructose decreased the phosphorylation of Akt by 32%, combination of Danseng restored phosphorylated Akt expression near the expression of control group. There were no significant change in phosphorylation of p44/42 MAPK, and JNK/SAPK were observed between the three groups. In conclusion, the obtained results strongly suggest that the consumption of fructose decreased the sperm motility and total sperm counts due to the declined ratio of GSH and GSSG, and the decreased Mn-SOD expression. The combination of Danseng has protective effects on epididymis tissues by increased the expression of Mn-SOD, eNOS, and phosphorylation of Akt.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Until recently, insulin production was thought to be restricted almost entirely to the beta cells of the pancreas. However, recent studies have revealed insulin mRNA expression in both the testes and the sperm. This discovery has important implications for the impact of diabetes on male fertility. Both Type I and Type II diabetic men are known to experience fertility problems, although the root of these problems remains unclear. We showed that Type I diabetic mice have smaller testes as well as abnormal testes histology. This phenotype was rescued with the administration of exogenous insulin. Previous data from our lab shows that Type I diabetic mice have lower fertility levels and produce lower quality sperm. Here we show that the lack of insulin in these mice is directly responsible for lower sperm quality by preventing proper testes development and subsequent sperm production. We also sought to characterize transcriptional control of insulin production in the testes of diabetic mice to determine whether testicular insulin production responds to diabetic conditions similarly to the beta cells of the pancreas. The pancreas produces insulin for all the cells in the body to regulate glucose metabolism, yet it remains unclear why the reproductive organs require their own source of insulin. Using quantitative real time PCR, we showed that insulin mRNA expression is regulated independent of the pancreas. In the type I diabetic mouse, we showed that insulin mRNA expression decreases in the pancreas. Conversely, insulin mRNA levels significantly increased in the testes of Type I diabetic mice. The type II diabetic mouse model displays a similar disparity in testes versus pancreas insulin mRNA levels. While pancreatic insulin transcription increases as males become hyperinsulinemic, testes insulin production remains stable or even slightly decreased. This suggests that the testes may be able to sense disturbances in systemic insulin levels induced by diabetes. This work provides insight into the potential functions of insulin in testes development and spermatogenesis. Determining the mechanism by which diabetes affects reproductive tissues may identify novel targets for fertility treatments for both Type I and Type II diabetic males. Travel for this meeting was supported by the Burroughs Welcome Fund.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Spermatogonial stem cells (SSCs) are at the foundation of spermatogenesis. SSC can both self-renew and produce daughter cells that under go differentiation in seminiferous tubules of testis. Therefore, transplantation of genetically modified SSCs can achieve SSC-mediated transgenesis. A number of new techniques have been devised recently to introduce foreign genes into SSCs using viral vector. Yet the most commonly used techniques are still based on the use of high molecular weight polycations such as polybrein. In this study, we describe a transduction method based on polybrein and compacted lipopolyamine(DOGS)-coated lentivirus. Donor cells were collected from B6.129S7-Gtrosa26(designated ROSA) and C57BL/6 pup testes. After enzymetic digestion with 0.25 % trypsin and 7 mg/ml DNase I, SSCs were enriched by MACS with anti-Thy-1 antibody. Enriched SSCs were cultivated consisted of serum-free medium and mitotically inactivated STO cell feeders. Established SSCs were infected with letivirus MOI of 5 for 6 hours with 4, 8 µg/ml of polybrein, 2, 4 µg/ml of DOGS and without any polycationic agents. The infected SSCs were washed 3 times with culture medium and analyzed under fluorescence microscope after 3 weeks of culture. The average transduction efficiency of infection with 4, 8 µg/ml of polybrein, 2, 4 µg/ml of DOGS were 47.0 ± 1.73, 45.7 ± 2.89, 53.7 ± 1.53 and 60.7 ± 2.08 respectively . Significant increase of transduction efficiency could be seen for those cells that were infected with 4 µg/ml of DOGS. And 1.56 fold increased transduced cells could be collected for those cells that were infected with DOGS when compared with the infected with polybrein. In this study we report a highly efficient method for transduction of mouse spermatogonial stem cells using DOGS in vitro. Finally, based on our results in this, the use of lipopolyaimne on lentiviral transduction will provide efficient system to generate trangenic animals using spermatogonial stem cells. This study was supported by Grant from Stem Cell Research Center(SC-5140), the Korea Healthcare technology R&D Project(A083418), 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):3.
WITHDRAWN.
Biol Reprod. 2010 Nov;83(1 Suppl):3.
From previous experiments, our laboratory has determined that Vascular Endothelial Growth Factor (VEGF) pro-angiogenic isoforms increase germ cell numbers and anti-angiogenic isoforms reduce the seminiferous cord area and increase the interstitial area in vivo in perinatal male mice. Therefore, our hypothesis was that VEGF pro-angiogenic and anti-angiogenic isoforms altered germ cell apoptosis in testes of adult mice. Adult mice (60-90 days old) expressing LacZ driven by the Kdr promoter (KDR-lacZ) were injected (IP) with recombinant anti-angiogenic isoform VEGF165b (1µg; n=8), PBS Control (1µg; n=8), an antibody which neutralizes anti-angiogenic isoforms of VEGF (AntiVEGFxxxb, 1μg; n=9) or IgG control (1µg; n=8) and blood samples and testes were collected, at 3 or 9 hours following injection. Blood samples were analyzed for testosterone. There were no differences in testosterone among treatments (P > 0.05). Testes were embedded, sectioned and the number of apoptotic cells was quantified on testis sections with a TUNEL assay. At 3 hours, the VEGF165b treatment group tended (P < 0.06) to have increased number of apoptotic germ cells (16.5±4.2) compared to its PBS control (8.0±1.5). At 9 hours after injection, the VEGF165b treatment group had an increased number of TUNEL positive germ cells (20.0±3.0; P < 0.05) compared to PBS Controls (10.1±1.7). Taken together these data indicate that VEGF165b is detrimental to germ cell survival and may initiate the apoptosis pathway. Further experiments are underway to elucidate the VEGF165b mediated mechanisms of apoptosis in germ cells. This research was supported by NIH/NICHD RO1-HD051979.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Ethylene Dimethane Sulfate (EDS) is an alkylating agent that selectively destroys adult Leydig cells in rat testes. Mature Leydig cells produce testosterone (T) the vital steroid hormone for maintaining healthy spermatogenesis. EDS depletion of mature Leydig cells is a model system to study effects of T withdrawal on spermatogenesis and T dependent gene expression in testes. Testosterone depletion after EDS treatment is reported to induce germ cell apoptosis. However, only a few studies focus on genes that regulate germ cell apoptosis in the absence of T. Here, we report that EDS treatment induces changes in gene expression for granzyme (GzmA, GzmB, GzmK, GzmM and GzmN) variants reported to be present in testes. For comparison, control groups (N=6) of untreated (Unt), vehicle treated (Veh) and vehicle+T (Veh+T) were maintained and assayed simultaneously. For EDS studies, male rats (>90 days of age) were given a single intraperitoneal injection (75 mg of EDS in 25% dimethyl-sulfoxide (DMSO)/75% water/kg of body weight), to kill Leydig cells. As a Leydig cell (-) androgen control, T was replaced on days 0, 2 and 4 for 5 days post-EDS and on days 0, 2, 4, and 6 for 7 days post-EDS by subcutaneous injection of testosterone propionate (TP) at 10 mg/kg of body weight, (20 mg TP/ml of sesame seed oil). At 5 and 7 days post-EDS, rats were euthanized for serum and tissue collection. From trunk blood, serum T levels were quantified by radioimmunoassay (RIA). From testicular tissue, gene expression was quantified from total RNA by reverse transcription and real time PCR. The presence and abundance of tissue proteins expressed were evaluated by western blot. For cellular localizations immuno-histochemistry on testicular tissues was performed. Testicular weights were significantly reduced in the EDS treated rats in both 5 day (13%) and 7 day (18%) EDS groups compared to controls - Unt, Veh, Veh+T, and the Leydig cell (-) control (EDS+TP). Serum T was below detection limits of the assay (<0.05 ng/ml) in both 5 and 7 days EDS treated rats. Loss of Leydig cells in EDS treated rats was quantified by analyzing the loss of luteinizing hormone receptor (LHR) mRNA. In both 5 and 7 days EDS and EDS+TP groups ≈90% LHR expression was lost. Quantification of germ cell apoptosis was performed by Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) assay. Five days after EDS treatment there was a significant increase in the germ cell apoptosis and the rate of apoptosis increased further in 7 days EDS treatment group. T replacement after EDS significantly improved the survival rate of the germ cells. In both 5 and 7 days EDS treated groups GzmK, GzmM and GzmN mRNA expression were increased over controls. T replacement brought the level of mRNA expression for GzmK, GzmM and GzmN down. Collectively, these data indicate that germ cell survival is dependent on T and depletion of T results increased germ cell apoptosis. Thus, GzmK, GzmM and GzmN may be involved in germ cell apoptosis following testosterone depletion in testes. Support: Texas Woman's University 2008-2009 Research Enhancement Program.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Testosterone is a steroid hormone known to be critically involved in the initiation and maintenance of spermatogenesis. For this reason, the objective of this study was to investigate the concentration of testosterone in blood and seminal plasma and their relationship to the sperm count in the domestic cat. Six adult mixed-breed male cats, exposed to more than 12h of natural light per day were used as semen donors. Semen collection was performed during breeding season (October to December) with at least 2-d interval and all tomcats were submitted to semen collection on the same day. Using an artificial vagina, sperm samples were collected two consecutive times with a rest period of 5-10 min. After sperm collection, samples were centrifuged for 5 min at 700 × g and seminal plasma was separated from spermatozoa, re-centrifuged at 1500 × g during 15 min and stored at -80°C. Pellet was resuspended in phosphate buffered saline and total sperm number was determined. Before testosterone measurement, seminal plasma samples from the same tomcat were thawed, pooled and centrifuged at 3000 × g for 10 min. For each male, blood samples were collected in two different occasions, one in November and other in December. Total testosterone level in serum and seminal plasma were measured by a commercial solid-phase radioimmunoassay kit (Coat-A-Count kit, Diagnostics Products Corporation, Los Angeles, CA, USA) with an intra-assay coefficient of variation of 4.8%. The average concentration of testosterone in seminal plasma (11.2 ± 6.3 ng/dL) was approximately seventeen times lower than the concentration found in blood (193.2 ± 63.8 ng/dL) resulting in a seminal plasma/ blood testosterone level ratio of 0.055 ± 0.017. Mean value for total sperm was 64.7 ± 26.4 million. A positive and significant correlation was found between seminal plasma and blood levels of testosterone (r = 0.85, p < 0.05), whilst the correlation between both blood and seminal plasma testosterone level with sperm count was statistically non-significant (r = 0.27 and 0.16, respectively). These results indicate that testosterone level in seminal plasma is lower than and positively correlated with blood testosterone level and that both hormone levels are not correlated with the sperm production. Considering these findings, we can affirm that the testosterone concentration in blood is a good predictor of the seminal plasma testosterone level in domestic cat.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Immature Sertoli cells cease to proliferate, and differentiate into their adult form during the first wave of spermatogenesis. Since Sertoli cell number determines the number of germ cells that can be supported, the rate and duration of Sertoli cell proliferation during postnatal development determines adult sperm production capacity. In the past, the testis was considered to be unresponsive to thyroid hormones, but more recent studies in rats and humans have shown that hyperthyroidism causes Sertoli cells to cease proliferation and hypothyroidism prolongs proliferation. This implies that T3 or T4 may affect the duration of proliferation of Sertoli cells in euthryoid animals during postnatal development, but this question has not been directly tested. The objectives of this study are to determine serum levels of free T3 and T4 in normal Rideau Arcott ram lambs, and to compare this to postnatal Sertoli cell proliferation and differentiation. We designed our experiments to accommodate the age discrepancy in onset of testicular maturation that we see in our flock. Serum was collected twice weekly from the jugular vein of 12 Rideau Arcott ram lambs between 10 and 19 weeks of age, and testicular biopsies were taken once a week from the left testis of these same animals over the same time span. Testicular biopsies were processed for histology and immunohistochemistry, and free T3 and T4 were determined by radioimmunoassay using Coat-A-Count kits (Siemens Med. Solu. Diag. Los Angeles, CA). Serum free T3 levels increased between 12 and 13 wk of age and then decreased, but when free T3 was compared to lumen formation (indication of tight junction formation by Sertoli cells), T3 remained at about 4.5 pmol/l until 1 wk after initiation of lumen formation and then decreased during the first wave of spermatogenesis and leveled off at about 2 pmol/l by the end of first wave of spermatogenesis. Free serum T4 levels decreased at 14 wk of age, but when compared to lumen formation, the decrease was observed 1 wk before initiation of lumen formation. Decrease in neither free T3 nor free T4 correlates with the onset of lumen formation in ram lamb seminiferous tubules. We have developed an immunohistochemistry technique for the sheep biopsies that identifies cells that are both proliferating (anti-PCNA positive) and are Sertoli cells (anti UCHL1 negative) for similar comparison to serum TH levels.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
The development of the male reproductive tract (MRT) is highly dependent on androgens. However, it is not clear as to whether there is a critical developmental period when androgen exposure can adversely affect this development. Estrogens have also been shown to play an important role in MRT development. Information on the effects of neonatal exposure to androgens, with or without estrogens, on the developing MRT is limited. The current objective was to determine the effects of dihydrotestosterone (DHT) and diethylstilbestrol (DES) on MRT development. Seventy-seven male pups (n=8-10/group) were randomly assigned to one of the following groups and treated by injection: 1) Control, 2) DES 1-6 days, 3) DES and DHT 1-6 Days, 4) DES 1-6 days and DHT 7-12 days, 5) DES 1-6 days and DHT 13-18 days, 6) DHT 1-6 days, 7) DHT 7-12 days, and 8) DHT 13-18 days. DES resulted in delayed testicular descent (Groups 2-5). Similarly, preputial sheath development (PSD) was delayed (P<0.001) in Groups 2, 4, and 5; however treatment with DHT up to day 6 (Group 3) reversed the effects of DES. DHT alone on d1-6 resulted in delayed PSD, suggesting that there is a critical period when androgen exposure can adversely affect the MRT development. Males were mated with post-pubertal females on d142 for up to nine days and evaluated for vaginal plugs as an indication of fertility. Vaginal plugs were significantly decreased (P< 0.05) in Groups 2, 4, 5, and 8 compared to control, suggesting that adverse affects by DES could be reversed by DHT from d1-6. However, DHT alone, administered after d13, adversely affect fertility. Based on current findings, we conclude that there is a critical developmental period when androgen exposure can adversely affect MRT development.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
In many vertebrate species, sex is determined at fertilization of zygotes by sex chromosome composition. But in some species - fish, amphibians and reptiles - sex is determined by environmental factors such as temperature and sex steroids. We previously showed that female-to-male sex-reversal could be induced when R. rugosa tadpoles were treated with testosterone. In this case, CYP17, which is responsible for production of androstenedione in the sterodogenic pathway, was upregulated in the sex-reversed gonad. However, little is known about the mechanism involved in the sex-reversal. To clarify this, we cloned and characterized two types of cDNAs, CYP17 and CYP17-pseudo, from R. rugosa. The nucleotide sequence of CYP17 cDNA is highly similar to that of CYP17-pseudo. However, a 2-bp insertion into exon 1 of CYP17-pseudo resulted in a frameshift mutation that generated a premature stop codon. CYP17 is composed of 8 exons, but CYP17-pseudo has only one. Fluorescence in situ hybridization (FISH) analysis mapped the R. rugosa CYP17 to chromosome 9, whereas CYP17-pseudo was mapped to chromosome 4. In addition, RT-PCR analysis showed that the CYP17 expression was much higher in the gonad of males during and after sex determination than in females. By contrast, CYP17-pseudo expression was generally very low and displayed no sexual dimorphism. Finally, we examined the nucleotide sequence of promoter regions of CYP17 and CYP17-pseudo. Interestingly, there was little similarity between the promoter regions of the two genes, indicating that expression of each gene is probably regulated by different transcription factors. Taken together, our results indicate that CYP17 is implicated in testicular differentiation of the gonad in R. rugosa, although the factor(s) controlling CYP17 expression still remain to be identified.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Reproductive strategies of seasonal breeders are adaptations to annual changes in the environment and they minimize the animals' energetic efforts for reproduction. Mature males show synchronized cycles of testicular growth and involution between the breeding and non-breeding periods. The wild male ground squirrel (Citellus dauricus Brandt) is such a typical seasonal breeder, which has a breeding season from April to May after hibernation and then goes through a long period of sexual dormancy from June to March. It provides us a useful animal model to study the testicular growth and involution. In the aspect of morphology, the testicular weight and size of wild ground squirrels decreased significantly from the breeding season to the non-breeding season. And the histological results of testes in the breeding and non-breeding seasons exhibited the seasonal changes in cells' types and number. Leydig cells around serminiferous tubules and spermatogonia, primary spermatocytes, second spermatocytes and spermatozoa in serminiferous tubules were identified in testes during the breeding season, however, Leydig cells around serminiferous tubules and spermatogonia and primary spermatocytes in serminiferous tubules were observed in testes during the non-breeding season. To clarify the mechanism about the seasonal changes in testes of wild animals, we hypothesized that the change patterns of testicular weight and size were consistent with germ cells' proliferation and apoptosis according to the histological observation. Firstly, the immunohistochemistry of bcl-2 and bax indicated that the positive staining of bcl-2 was showed in spermatocytes and round spermatozoa in the breeding season and bax was mainly expressed in spermatocytes in the non-breeding season. Also, the positive immunoreactivities of bcl-2 in the breeding season and bax in the non-breeding season were confirmed by Western blotting analysis. The results of bcl-2 and bax suggested that apoptosis may be one reason for the seasonal changes in testicular weight and size of wild ground squirrels. And then, the immunohistochemistry and western blotting analysis for PCNA were used to detect the difference of cell proliferation between the breeding and non-breeding seasons. The positive immunoreactivity of PCNA was observed in spermatogonia and spermatocytes during the breeding season, and weakly in spermatocytes during the non-breeding season. The results of PCNA suggested that the proliferation of germ cells may be another reason for the increased weight and size of testes in the breeding season. Finally, the testosterone concentrations in serum were assayed by ELISA and this result has positive correlation with the weight and size of testes from the breeding season to the non-breeding season. These results suggested that the proliferation and apoptosis of germ cells may be an important reason for testicular recrudescence and regression from the breeding season to the non-breeding season in wild ground squirrels and this cycling expression of bcl-2, bax and PCNA in testis would underlie the reproductive physiological adaption for natural environment. The future study will focus on other factors that affect the cells proliferation and apoptosis and thus help to elucidate the mechanism of the wild animals' testicular recrudescence and regression. This study is supported by Program for Changjiang Scholars and Innovative Research Team in Universities (IRT0607) from China.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
NOT PRESENTED.
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Survival of each mammalian species depends on fertilization, and control of reproduction remains a significant global human health issue. Although progress has been made, much remains unknown about the molecular events that ensure that a single sperm fertilizes a single egg. Failure to do so results in polyspermy and embryonic death. Sperm bind to eggs, but not to two-cell embryos and a major defense against post-fertilization polyspermy is provided by the zona pellucida. The mouse zona pellucida is composed of three glycoproteins, ZP1, ZP2 and ZP3. Following fertilization, ZP2 is cleaved by a protease thought to reside in cortical granules (CGs) located at the periphery of growing oocytes. Upon egg activation initiated by gamete fusion, cortical granules migrate to the periphery and exocytose their contents which presumably diffuse through the zona pellucida. Having biochemically characterized the proteolytic cleavage site of mouse ZP2, we have recently identified a candidate CG-specific metalloprotease (CGM) in mice. The CGM has been fluorescently tagged with GFP and mCherry, transiently expressed in CHO cells and analyzed on immunoblots with polyclonal antibody to CGM as well as to GFP/mCherry. Expression vectors encoding these CGM-fluorescent fusion constructs have been microinjected into growing oocytes and preliminary results using confocal microscopy demonstrate successful targeting to cortical granules. Using site-directed mutagenesis of these expression vectors, we are in the process of identifying protein motifs essential for intracellular trafficking to cortical granules which will be confirmed with mouse transgenesis. In addition, we have established a mouse line lacking CGM using DNA recombineering and embryonic stem cell technology to successfully target the single-copy gene encoding CGM. We are in the process of evaluating the phenotype of this mutant mouse line and are seeking to rescue it by establishing a second transgenic mouse line expressing CGM fluorescently tagged with mCherry. Both the CGM null mutant and rescue mouse lines will be used for in vivo dissection of the intracellular trafficking of this metalloprotease and its role in the post-fertilization block to polyspermy. By characterizing these events in vivo and in vitro, these investigations will provide insight into the molecular mechanisms of fertilization with translational implications for reproductive medicine.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
During fertilization, the sperm delivers the enzyme phospholipase C-zeta to the oocyte's cytoplasm that leads to the release of calcium from the intracellular stores. The sperm-induced calcium signal consists of an initial calcium increase followed by a series of transients that results from the cyclic release and subsequent re-uptake of calcium by the endoplasmic reticulum. The train of calcium spikes is maintained by an influx of calcium across the oocyte's plasma membrane. Despite their importance in oocyte activation, little is known about the identity and regulation of such calcium influx channels. Our previous study indicates that Orai1, a recently identified store-operated calcium entry (SOCE) channel is localized in the plasma membrane of pig oocytes and may be responsible for the maintenance of the long-lasting calcium signal during fertilization. Here, we investigated the function of Orai1 protein in SOCE in porcine oocytes. The oocytes were collected from ovaries of prepubertal gilts and matured in TCM 199 medium supplemented with 0.07 mg/ml cysteine, 10 ng/ml EGF, 0.5 IU/ml LH and 0.5 IU/ml FSH at 39 °C in 5% CO2 in air for 36 hours. Messenger RNA encoding for Orai1 tagged with the green fluorescent protein on its N-terminus (GFP-Orai1) was generated by in vitro transcription and microinjected into the cytoplasm of in vitro matured oocytes. After injection the oocytes were cultured for an additional 8 hours to allow for translation to take place. Overexpression of GFP-Orai1 was verified in the injected oocytes by detecting the fusion protein with laser-scanning confocal microscopy and western-blot using anti-Orai1 and anti-GFP antibodies. Injected and control (carrier medium-injected) oocytes were placed into calcium-free medium containing 10 uM CPA (cyclopiazonic acid) for 2 hours to deplete the calcium stores. Oocytes were then incubated in calcium-free TL-HEPES medium containing the calcium indicator dye fura-2 AM for 45 minutes. Calcium-containing medium was then added to the oocytes and the changes in the intracellular free calcium concentration in these oocytes were monitored using an InCyt Im2 fluorescence imaging system. Data were analyzed using student's T-test. Results show that the addition of calcium after store depletion induced an elevation in the cytosolic calcium concentration in both Orai1-overexpressing and control oocytes through SOCE. The increases in the intracellular free calcium concentrations were significantly lower in Orai1-overexpressing oocytes compared to control oocytes; the folds change in the fluorescence ratios (that indicate calcium levels) were 3.5±0.33 and 5.4±0.46, respectively (p=0.015). The calcium influx was also significantly slower in GFP-Orai1-injected oocytes compared to that in the control group; the time between calcium add-back and the peak values were 827.5±122.8 and 97.5±67.5 seconds, respectively (p=0.002). These findings indicate that elevated levels of Orai1 in oocytes have a negative effect on SOCE activity. This is in accordance with other studies showing that in somatic cell types Orai1 was activated by Stim1, a sensor of calcium levels in the endoplasmic reticulum, and the expression ratio between these two interacting proteins had a profound effect on the calcium influx generated. Further studies have been designed to determine the effects of Orai1 down regulation on SOCE and characterize the communication between Orai1 and Stim1 in oocytes.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Dysregulation of metabolic pathways in the oocyte is associated with reduced competence. Glucose is metabolized primarily by glycolysis and the pentose phosphate pathway (PPP), and these pathways are important to both cytoplasmic and nuclear events during the maturation process. However, an excess of glucose, as in conditions of diabetes and obesity, can have deleterious effects on the oocyte. Leptin, which is associated with insulin sensitivity in several tissues, is important for correct ovarian function. The objective of these experiments was to determine the effect of leptin under in vitro maturation (IVM) conditions of physiological (Experiment 1) or high (Experiment 2) glucose concentration on porcine oocytes. We hypothesize that addition of leptin during maturation will impact oocyte quality via changes in glucose metabolism. This in vitro system could be a good model in which to study the effects of obesity and diabetes on the oocyte. Cumulus-oocyte complexes isolated from abattoir derived sow ovaries were cultured in a chemically defined medium, Purdue Porcine Medium (PPM) for 44h, in 7% CO2 in air at 38.7°C. In Experiment 1, medium was supplemented with 4 different concentrations (0, 10, 100, 1000 ng/mL) of leptin (LEP), in the presence of 2mM glucose (GLUC). Mature oocytes were collected and lysed for quantitative real time PCR (qPCR) analysis in three groups of 20 oocytes. Data were analyzed using the REST software. ADIPONECTIN and ADIPOR1 transcripts were not expressed in oocyte samples. IRS1, IGF1, 3βHSD, PPARG, and CYP19 were not differentially expressed between oocytes matured with or without LEP at any concentration. ADIPOR2 transcripts significantly decrease with the addition of 100 compared to 10 ng/ml LEP, and then significantly increase when oocytes were supplemented with 1000 compared to 100 ng/ml, suggestive of a biphasic effect. In experiment 2, medium was supplemented with 5mM GLUC in the absence of LEP (control) or 50mM GLUC in the presence of increasing concentrations of LEP (0, 10, 100, 1000ng/mL). For assessment of nuclear maturation, oocytes were fixed and stained after IVM. Nuclear maturation was scored as 1 (mature; anaphase I, telophase I or metaphase II) or 0 (not mature). Data were analyzed by PROC GENMOD. There was an effect of treatment on nuclear maturation (P < 0.01). In the absence of LEP, 50mM GLUC had a deleterious effect on oocyte maturation compared to control (71.0 vs. 86.5%, respectively). Addition of 10ng/ml of LEP improved oocyte maturation (85.6%) to levels similar to control (86.5%). In contrast, higher concentrations of LEP failed to improve nuclear maturation (100 ng/mL, 76.0; 1000 ng/mL, 67.6 %). To determine the effect of LEP on GLUC metabolism, glycolysis and PPP were simultaneously measured in mature oocytes using [5-3H] and [1-14C] glucose. High GLUC concentrations reduced glycolysis independent of LEP (1.07, 1.05, 1.18, 0.89 pmol/oocyte/3h, for 0, 10, 100 and 1000 ng/mL LEP respectively) compared to control (1.46 pmol/oocyte/3h). There was no difference in PPP activity between treatments (P>0.05). This data suggests that LEP and GLUC interact to regulate oocyte nuclear and cytoplasmic maturation. Leptin, in high concentrations, is able to down regulate adiponectin receptor in the oocyte. In hyperglycemic conditions, leptin was not able to improve glycolysis or PPP activity. However, leptin (10ng/ml) diminished the negative effect of high glucose on nuclear maturation.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
NOT PRESENTED.
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Luteinizing hormone (LH) is a heterodimeric protein hormone that is produced in the anterior pituitary gland as two separate subunits (α and β) that are non-covalently associated. The α subunit is highly conserved among other pituitary hormones, as well as among mammalian species, while the β subunit is distinct. In females, LH is responsible for ovulation of the dominant follicle; in males, LH triggers Leydig cells to produce testosterone within the testes. LH is also thought to facilitate the final maturation of the dominant follicle as indicated by the presence of LH receptors expressed on the granulosa cells directly following deviation. LH is under the direct control of gonadotropin releasing hormone (GnRH) from the hypothalamus, causing an LH pulsatile surge about 12 hours after GnRH is released. GnRH is used in superovulatory protocols as an LH agonist because a purified source of LH is not commercially available for superovulatory purposes; therefore, a recombinant source of highly purified, bioactive LH would be an important contribution to superovulatory protocols, particularly because GnRH is thought to elicit the release of other pituitary hormones to a degree (namely follicle stimulating hormone). Further, the amount of LH released in direct proportion to GnRH may be enough for a single ovulation, but superovulation may require a higher amount of LH to complete maturation of every follicle that will be ovulated. A single polypeptide harboring the β subunit directly upstream of the α subunit was generated by PCR using individual subunits derived from bovine anterior pituitary cDNA; this gene fusion product was ligated into a shuttle vector and cloned into pGEX4T-1 for expression as a fusion to GST using novel restriction enzyme sites. The construct was transformed into BL21-DE3 bacterial cells for protein expression; cultures were induced and protein was expressed for 12 hours prior to harvesting by centrifugation. Following lysis, protein in the soluble fraction was purified by affinity chromatography, taking advantage of the GST tag. BLHβα was further purified using hydrophobic interaction chromatography, ion exchange chromatography, and size exclusion chromatography to net a purity of about 80%.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Polycystic ovary syndrome (PCOS) is one of the most common endocrine diseases among reproductive women and usually associated with obesity and insulin resistance. However, the etiology of PCOS is still unknown. A rat PCOS model is described for investigating possible pathogenesis. Female prepubertal rats were randomly divided into three experimental groups treated with vehicle (sesame oil, 1ml/kg/day, n=18), testosterone propionate (TP, 10mg/kg/day, subcutaneously, n=17), or dihydrotestosterone (DHT, 10mg/kg/day, subcutaneously, n=16) for 56 days. Vaginal smears of both TP and DHT groups showed continuous diestrus phase rather than normal 4~5-day estrous cycle in vehicle group. DHT treated female rats were significantly heavier and their ratios of parametrial fat weight to total body weight were also higher. Oral glucose tolerance test performed at 12-week-old revealed insulin resistance phenomenon in DHT group but not in the other two groups. Ovarian histology showed polycystic morphology in DHT treated group but not in vehicle and TP-treated groups. There were no significant differences in serum follicle-stimulating hormone (FSH) levels, but in vitro culture of anterior pituitaries demonstrated that DHT group produced significantly more FSH but less luteinizing hormone (LH). Anterior pituitaries of TP group also produced less LH but even more FSH than those in DHT group. Abnormally low LH/FSH ratio might result in estrous cycle disruption, but was not correlated with the severity of metabolic disturbance. This research was supported by a grant (V98B1-008) from Taipei Veterans General Hospital.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Pulsatile release of gonadotropin-releasing hormone (GnRH) is indispensable to maintain normal gonadotropin release from the pituitary and hence gonadal activities. Previous studies with several types of the hypothalamic deafferentation or lesion suggest that the GnRH pulse generator consists of non-GnRH neurons and is located in the mediobasal hypothalamus. The cellular source of this ultradian rhythm in GnRH activity remains to be determined. Inputs from kisspeptin neurons in the posterior part of the arcuate nucleus to GnRH neuronal terminals in the median eminence could be a candidate for an intrinsic source to drive the GnRH pulse generator in rats. To explore the possibility whether the arcuate kisspeptin signaling is involved in producing pulsatile GnRH release, pulsatile luteinizing hormone (LH) release was determined in ovariectomized rats bearing hypothalamic deafferentation, which cuts off the posterior part of the arcuate nucleus from the median eminence. One weak after the brain surgery, blood samples were taken from freely moving conscious rat though an indwelling atrial cannula every 6 min for 3 h. After blood sampling, coronal sections of the hypothalamus were strained with thionin to determine the placement of the deafferentation. Pulsatile LH release was obvious with high frequent and amplitude in ovariectomized rats with sham operation. On the other hand, an irregular fluctuating pattern in plasma LH levels was found in rats bearing the deafferentation. Apparent LH pulses were found in rats with incomplete deafferentation. The mean LH level and the frequency and amplitude of LH pulses during the 3-h sampling period were lower in rats bearing the deafferentation compared with sham-operated controls or animals with incomplete deafferentation. The present result indicates that pulsatile LH release is maintained by neural inputs from the posterior part of the arcuate nucleus. Thus, it is possible that the arcuate kisspeptin neuron is a component of GnRH pulse generating mechanism in female rats. This work was supported in part by PROBRAIN of Japan.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
It is generally assumed that the preovulatory LH surge of mammals is triggered by estradiol (E2), acting within the hypothalamus to enhance the synthesis and release of gonadotropin-releasing hormone (GnRH). However, it is unclear how E2 inhibits GnRH neuronal activity for most of the menstrual cycle, but then stimulates it during the late follicular phase. Using in situ hybridization histochemistry, RT-PCR, gene microarray analysis, and real-time PCR, we show that the hypothalamus of female rhesus macaques (Macaca mulatta) contains two distinct GnRH neuronal populations. One of these populations expresses the traditional mammalian form of GnRH (GnRH-I) while the other expresses the chicken-II form (GnRH-II); both of these neuropeptides are highly effective at stimulating LH release in vivo. Importantly, only the GnRH-II neuronal population was found to respond positively to E2, by showing enhanced GnRH-II gene expression during the late follicular phase of the menstrual cycle or after administration of exogenous estrogen (either E2 or estradiol benzoate). In contrast, the responses of the GnRH-I neurons consistently reflected only a negative influence of estrogen, suggesting that the primary role of these neurons is to mediate negative E2 feedback and to play a relatively minor role in the generation of the preovulatory LH surge. Because humans also express two forms of GnRH (GnRH-I and GnRH-II), it is plausible that fertility in women is regulated by the coordinated action of two distinct GnRH neuronal populations, which are differentially affected by the sex-steroid environment. Supported by NIH Grants: HD-29186 & RR-00163.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Metabolic stress, as in individuals with low body fat, promotes hypothalamic amenorrhea. Leptin, a hormone produced by adipocytes in proportion to fat/energy stores, is a humoral factor that signals the status of body fat (energy) stores to the brain. Leptin acts via the leptin receptor (LepRb) on the surface of specialized populations of neurons in the hypothalamus and elsewhere to permit energy expenditure on processes such as reproduction. Thus, animals deficient in leptin (e.g., ob/ob mice) or LepRb (db/db) exhibit a phenotype similar to the neuroendocrine starvation response, including hypothalamic infertility, despite their obesity and large energy stores. Indeed, leptin treatment restores the function of the neuroendocrine reproductive axis in animals and humans with low body fat, as well as in ob/ob mice. The neural mechanism(s) by which leptin regulates the neuroendocrine reproductive axis remain poorly understood, however, since GnRH neurons do not express LepRb. The potential interaction of leptin with the two populations (ARC and AVPV) of kisspeptin neurons implicated in reproductive control has been incompletely studied. To examine the neural mechanisms by which leptin acts to control the neuroendocrine reproductive axis, we thus generated a number of mouse models that permit the visualization of LepRb neurons and their downstream contacts. We utilized these models to examine the potential for GnRH and Kisspeptin neurons to express LepRb or to lie in synaptic contact with LepRb neurons. This analysis demonstrated no LepRb expression in GnRH and kisspeptin neurons in the hypothalamus. In addition to revealing the innervation of GnRH neurons by ARC and AVPV kisspeptin neurons, however, this analysis demonstrated that GnRH and kisspeptin neurons (both ARC and AVPV) are directly innervated by LepRb neurons. The soma of GnRH-projecting LepRb neurons lie predominantly in the ARC, DMH/VMH, and PMv nuclei of the hypothalamus. Defining these LepRb-expressing neuronal circuits that are positioned to control the reproductive axis will provide insight into mechanisms that may link changes in nutritional status to alterations in reproductive function.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
NOT PRESENTED.
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Iron deficiency (ID), a common nutrient deficiency, impairs brain development. Newborn screening for ID has advantages, as earlier recognition of at-risk children could prevent long-term neurocognitive morbitity. ZnPP/H is an available, cost effective and sensitive biomarker of incomplete iron incorporation into erythrocytes. ZnPP/H, a potential candidate for newborn screening for iron deficiency, is measurable on whole cord blood or filter paper spots. Previous studies rinsed cord blood samples to remove pigments including bilirubin, that interfere with readings. Filter paper newborn screen samples cannot be rinsed and are obtained at 24-48 hrs after birth, when bilirubin levels may be elevated. Our aims were first, to examine the reproducibility of filter paper ZnPP/H measurements and second, to examine methods to limit bilirubin interference with ZnPP/H. Methods: We measured reproducibility of cord blood ZnPP/H eluted as hemolyzed blood from filter paper blood spots and whether eluted values correlated to the same whole blood samples. We measured the degree of bilirubin interference with ZnPP/H readings. We evaluated potential candidates to remove bilirubin interference with ZnPP/H, including detergents, albumin, and bilirubin oxidase. Results: Filter paper ZnPP/H was reproducible. Filter ZnPP/H correlated to whole or rinsed cord blood, P<0.0001, but lines of identity did not pass through zero. Although 5 mg/dL levels of bilirubin were similar to ZnPP/H without bilirubin, 10, 15 and 20 mg/dL increased the levels of ZnPP/H in a dose response fashion (P<0.0001). Tween 20 and Triton X-100 increased ZnPP/H baseline variability and did not remove bilirubin interference. Albumin decreased baseline ZnPP/H values to those of rinsed whole blood, but did not remove bilirubin interference. Bilirubin oxidase reagent is inexpensive and stable when refrigerated or frozen in water. Although not returned to baseline, bilirubin oxidase removes bilirubin interference by 70% (p<0.0001). Conclusion: Filter paper ZnPP/H correlated to intact whole and rinsed ZnPP/H, but bilirubin levels of 10 mg/dL interfere with readings. Bilirubin oxidase removes most bilirubin interference, improving the accuracy of filter paper ZnPP/H. Further studies are needed to validate the use of ZnPP/H from blood on filter paper.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
As women begin to enter menopause, the risks of developing cardiovascular diseases and type 2 diabetes appears to climb. This cluster of risk factors, including obesity, insulin resistance, dyslipidemia, hypertension and decreasing of high density lipoprotein-cholesterol, were called metabolic syndrome. Although there were reports about adverse effects, hormone replacement therapy (HRT) was used to relief menopausal symptoms and treat post-menopausal osteoporosis clinically. Besides estrogen supplement, some healthy food like soy isoflavone, has high affinity to estrogen receptor and it has been reported to improve lipid profiles in post-menopausal women. However, the beneficial effects of HRT and isoflavone on menopausal-associated metabolic syndrome have not been fully verified yet. In our study, we have established a screening platform to evaluate the protective effects of estrogen and isoflavone on developing menopause-associated metabolic syndrome in women. First, we established an animal model of menopause-associated metabolic syndrome. Female rats were ovariectomized to create a menopause status, and then fed with high-fructose diet to induce development of metabolic syndrome. Body weight, adiposity, insulin sensitivity, plasma lipid profiles, and blood pressure were measured. Second, we investigated the protecting effects of hormone replacement therapy on menopause-associated metabolic syndrome in this model. Finally, we evaluated the protecting effects isoflavone on menopause-associated metabolic syndrome in this model. Our results showed that high-fructose feeding induced obesity, insulin resistance, hyperlipidemia, and hypertension in ovariectomized rats. Treatment with estrogen (17£-estradiol, 0.5 mg/pellet, 60-day release) reduced body weight and fat mass and improved insulin sensitivity. Besides, oral administration of soy isoflavone extract (450 mg/day) lowered body weigh, fat mass, improved insulin sensitivity and plasma lipid profiles in high-fructose fed ovariectomized rats. Our results demonstrated that estrogen supplement improved metabolic parameters in ovariectomized rats. Isoflavone also had the similar protective effects, and there was no adverse effect such as hypertriglyceridemia, which presence in rats received estrogen supplement. In conclusion, HRT and isoflavone have significant protective effects on prevention of menopausal-associated metabolic syndrome. This research is supported by DOH098-TD-F-113-098004, Department of Health, Taiwan.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
NOT PRESENTED.
Biol Reprod. 2010 Nov;83(1 Suppl):3.
NOT PRESENTED.
Biol Reprod. 2010 Nov;83(1 Suppl):3.
NOT PRESENTED.
Biol Reprod. 2010 Nov;83(1 Suppl):3.
In mammals, the pool of primordial follicles present at birth represents the total population of germ cells available to females during their entire reproductive life. Basic mechanisms underlying primordial follicle formation as well as how these normal processes are disrupted in reproductive disorders are not well understood. During mouse embryonic development, oogonia undergo several rounds of mitotic division followed by incomplete cytokinesis to form germ cell cysts and at approximately 13.5 days post coitum (dpc) they enter meiosis becoming oocytes. Oocytes progress through the stages of prophase I of meiosis and arrest in the diplotene stage beginning at 17.5 dpc. After birth, the germ cell cysts break apart into single oocytes, some cells in each cyst die by programmed cell death and the remaining become enclosed by granulosa cells to form primordial follicles. The processes of diplotene arrest, cyst breakdown and primordial follicle assembly occur at approximately the same time but their relationship has not been established. By histological staining of ovaries at various time-points and analyzing their oocyte nuclear morphology, a correlation has been drawn regarding the progress of meiosis and the age of ovaries. Previous work demonstrated that treatment of neonatal mice with estrogenic compounds results in delayed cyst breakdown and primordial follicle formation suggesting a role for estrogen signaling in oocyte development. Our model of the effects of estrogen on cyst breakdown is that before birth maternal estrogen levels are high which prevents the cysts from breaking apart and immediately after birth there is a drop in estrogen levels which triggers cyst breakdown and primordial follicle assembly. To test this, levels of estradiol were measured using ELISA or RIA. Maternal estradiol levels measured so far indicate a drop in serum estradiol levels corresponding to the time of cyst breakdown. Alternatively, developing ovaries themselves could produce estradiol and this could affect cyst breakdown. To test this, fetal and neonatal mice ovaries were grown in organ culture and estradiol levels in the media measured by radioimmunoassay.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
NOT PRESENTED.
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Nuclear factor-κB (NF-κB) designates a family of transcription factors that have been shown to modulate antiviral, inflammatory and immune responses. Activation of NF-κB is dependent on IKKβ a component of the IκB kinase (IKK) complex which promotes degradation of IκB inhibitory proteins and allows nuclear translocation of NF-κB. Our studies in ovarian granulosa cell tumor cell lines (COV434 and KGN) indicate that NF-κB signalling is constitutively activated. Follicle stimulating hormone (FSH) has been reported to increase X-linked inhibitor of apoptosis (XIAP) expression through NFκB activity in granulosa cells, but beyond that a role for NFκB signalling in folliculogenesis has not been elucidated. To establish the significance of NF-κB signalling in the ovary we have created a gonadal specific IKKβ conditional knockout mouse. A transgenic mouse line containing floxed IKKβ alleles (gift of M Karin, UCSD) was crossed with a transgenic line expressing cre-recombinase under the control of the anti-Mullerian hormone receptor (AMHR) promoter (gift of M Matzuk, BCM). Female mice resulting will not express IKKβ in granulosa cells and thus cannot activate NFκB signalling. Mice were assessed at 7 and 15 weeks of age for body, uterine horn and ovarian weights. A histological assessment of the ovaries was undertaken, serum follicle stimulating hormone (FSH) and luteinising hormone (LH) levels were measured and vaginal smears were performed to evaluate estrus cyclicity. Body weight and uterine horn weights were not different from control mice however ovarian weights were significantly reduced. Estrus cycles were not significantly different to control counterparts. The ovaries of 7 week old mice contained follicles of all developmental stages but corpora lutea were notably absent indicating that these mice were infertile. The theca cell layer of secondary follicles was indistinct and there was a suggestion that numbers of each follicle type may be altered with an increase in the number of apoptotic follicles. Serum FSH and LH levels were elevated from week 7. By 15 weeks of age corpora lutea were present; the fertility of these mice as determined by breeding studies has yet to be assessed. In summary, the IKKβ conditional knockout mice exhibit a reproductive phenotype which includes delayed ovulation. These results validate the hypothesis that ovulation is an inflammatory-like response. This novel model will be a valuable tool for reproductive research; the subtlety of the phenotype allowing us to tease out the underlying mechanisms and role of NFκB signalling in reproductive function. Research supported by the NHMRC of Australia Regkey 494809.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
The LH surge induces dramatic increases in periovulatory gene expression to bring about ovulation. Interestingly, the expression of many of these LH-induced genes rapidly declines prior to or immediately after ovulation, but nothing is known about the mechanisms involved in the rapid down-regulation of these genes. In the present study, we investigated whether nuclear factor interleukin-3 (NFIL3), a transcriptional repressor, is expressed in the periovulatory follicle and if so, what is the specific function of NFIL3 in the rat ovary during the periovulatory period. First, to examine the spatiotemporal expression pattern of NFIL3, ovaries or granulosa cells were collected at various times after hCG administration from eCG/hCG-stimulated immature rats (n=3/time point). Real-time PCR analysis revealed that Nfil3 mRNA expression was highly induced both in intact ovaries and granulosa cells after hCG treatment. In whole ovaries, Nfil3 mRNA levels were increased approximately 8-fold at 4 h after hCG and remained elevated through 24 h. In granulosa cells, Nfil3 mRNA expression was highest at 12 h after hCG treatment. In situ hybridization analysis demonstrated that Nfil3 mRNA expression was highly induced in theca-interstitial cells at 4 and 8 h after hCG, localized to granulosa cells at 12 h, and decreased at 24 h in both theca-interstitial and granulosa cells. Secondly, to determine the functional impact of NFIL3 expression, granulosa cells or theca cells were infected with an adenoviral NFIL3 over-expression vector. Next day, the cells were treated with forskolin plus PMA to induce periovulatory gene expression. As screened using a DNA microarray and confirmed by real time PCR analyses, over-expression of NFIL3 inhibited the hCG induction of prostaglandin-endoperoxide synthase 2 (Ptgs2), progesterone receptor (Pgr), epiregulin (Ereg) and amphiregulin (Areg) in granulosa cells. NFIL3 over-expression in cultured granulosa cells also decreased the levels of prostaglandin E2 in the conditioned media. In theca-interstitial cells, the expression of 15-hydroxyprostaglandin dehydrogenase (Hpgd), an enzyme involved in prostaglandin metabolism, was also inhibited by NFIL3 over-expression. Lastly, to begin to unveil the molecular mechanism of NFIL3's action, NFIL3 interaction with the Ptgs2 promoter was investigated using granulosa cells. Data from luciferase assays demonstrated that NFIL3 overexpression decreased the induction of the Ptgs2 promoter activity while ChIP analyses indicated that NFIL3 binds to the promoter region containing the DNA binding sites of CREB and C/EBPβ. In summary, hCG induced NFIL3 expression in theca-interstitial and granulosa cells prior to the expected time of ovulation. Our findings suggest a novel role for NFIL3 in the process of ovulation and/or differentiation of the theca-interstitial and granulosa cells by regulating the expression of Ptgs2, Pgr, Ereg and Areg, all genes known to be critical in the ovulatory process. For example, the down-regulation of Ptgs2 and Hpgd would impact the synthesis and turnover of prostaglandins (PGs), resulting in alterations in levels of PGs. As NFIL3 is known to compete with CREB or C/EBPβ, it is possible that the rapid down-regulation of periovulatory gene expression may occur through the action of NFIL3 as a transcriptional repressor and competitor of transcriptional activators such as CREB or C/EBPβ. Supported by NCRR P20 RR15592 and HD057446.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Ovarian folliculogenesis is regulated by many endocrine, paracrine and autocrine factors, and as follicular development progresses, increased angiogenesis is essential to sustain development of the rapidly expanding follicle. Angiogenesis is tightly regulated by a variety of pro- and anti-angiogenic factors, including vascular endothelial growth factor (VEGF) and thrombospondin-1 (TSP-1). Aberrant angiogenesis occurs in a number of pathological conditions, such as polycystic ovary syndrome (PCOS) and endometriosis, and novel ways of targeting this could lead to new therapeutics. TSP-1 is an anti-angiogenic factor in many physiological systems and has been shown to have anti-angiogenic effects on follicles in vitro. ABT-428898 is an octpeptide mimetic of TSP-1 and we investigated the effect of ABT-898 on follicular angiogenesis using an in vitro angiogenesis assay, prior to studying its effects in vivo in the common marmoset (Callithrix jacchus). Intact preantral/early antral follicles from 21-day-old rat ovaries were cultured for six days in the presence of ABT-898 (0, 0.1, 1, 10, 100 and 1000nM). At the end of the culture period, angiogenic sprouting from the follicles was quantified using image analysis. In a subsequent experiment, the potential of this peptide to inhibit follicular angiogenesis was investigated in vivo, in marmoset monkeys. The marmosets (n=5) were treated with 2.5mg/kg ABT-898 twice daily throughout the follicular phase of the cycle and control marmosets (n=5) were treated with vehicle. On d10 of the follicular phase, which corresponds to the peri-ovulatory period in controls, all animals were injected with 20mg bromodeoxyuridine (BrdU) one hour before removal of ovaries which were fixed, sectioned and stained for haematoxylin and eosin, CD31, BrdU, CD31/BrdU and caspase-3. Treatment with ABT-898 resulted in the suppression of follicular angiogenesis, a reduction in endothelial cell proliferation at the pre- and early antral stages of follicular development and the suppression of granulosa cell proliferation at early preantral, preantral and early antral stages of follicular development. In addition to the inhibition of angiogenesis, ABT-898 treatment also resulted in a significant increase in the number of large preantral and early antral follicles undergoing apoptosis. However, follicle selection and ovulation were not inhibited by treatment, suggesting that the effects of ABT-898 are stage-specific. Taken together, these results indicate that the TSP peptide is capable of having a dual effect by inhibiting follicular angiogenesis and promoting atresia of antral follicles in vivo, but does not prevent ovulation, as has been observed with direct VEGF inhibitors. These results suggest that the TSP peptide could be a novel therapeutic to inhibit abnormal angiogenesis and induce atresia of accumulated follicles in polycystic ovary syndrome. This research was supported by a Medical Research Council Ph.D. studentship and a core grant to Medical Research Council Reproductive Sciences Unit (U.1276.00.002.00006.01).
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Anti-Mullerian hormone (AMH) is involved in the regression of the Mullerian ducts in mammalian and avian male embryos as well as the right oviduct in avian female embryos. AMH expression by granulosa cells has also been demonstrated in adult hens and mammals and is thought to be involved in the recruitment of follicles from the primordial pool as well as in regulating FSH sensitivity. We have shown that AMH expression by the granulosa layer of hens is high in the small growing follicles, but decreased in the larger hierarchical follicles. The decline in expression of AMH with increasing follicle size is associated with an increase in expression of the receptor for FSH (FSH-R) in the granulosa layer, although the mechanism is not known. In this experiment, our goal was to determine if vitamin D3 (1, 25-dihydroxyvitamin D3) is involved in regulating expression of AMH in granulosa cells of the hen. We collected granulosa cells from follicles 3-5 mm and 6-8 mm in size and cultured them for 24 hours in Medium 199 + 5% fetal bovine serum (n=7). The medium was removed and Medium 199 + 0.1% bovine serum albumin and vitamin D3 (at doses of 0, 10, 100 nM) were added. The cells were cultured for another 24 hours, harvested and RNA was extracted for use in quantitative PCR. AMH and FSH-R mRNA expression were evaluated and all values were standardized to 18S reactions. There was a significant (p<0.05) dose-related decrease in the expression of AMH in granulosa cells from 3- to 5-mm and 6- to 8-mm follicles. Additionally, FSH-R mRNA and cell proliferation were significantly (p<0.05) increased by vitamin D3 in both groups. Western blot analysis for the vitamin D3 receptor (VDR) showed doublet bands at the expected size (58 and 60 kDa) in protein isolated from chicken granulosa layer and intestine. We used this same antibody to visualize the location of VDR within the follicle with immunohistochemistry and found it predominantly localized to the nucleus of granulosa cells. VDR mRNA expression in the granulosa layer was also evaluated using quantitative PCR (n=4). We found an increased expression of VDR (p<0.05) with increasing follicle size with the highest expression in the F1 follicle. To determine if vitamin D3 is having a direct effect on AMH expression we searched for possible vitamin D response elements (VDRE) in the sequence upstream from the AMH gene. We found several putative VDREs based on their similarity to known VDREs. In conclusion, our data show that vitamin D3 decreased AMH expression in a dose-related way, concomitant with an increase of FSH-R. These results suggest that vitamin D3 may have a role in regulation of follicle selection in the hen. Supported by USDA-NRI-2008-35203-19097.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Mares are useful comparative research models for studying ovarian dynamics in women because of similarities in the number and nature of follicular waves and a constant relative diameter of the largest follicle between the two species at definable events throughout the ovulatory wave. Recent (2005 to 2009) temporal and mechanistic studies have contributed to the understanding of the hormonal changes during the periovulatory period in mares. However, regulatory mechanisms of ovulation in mares are not well known, especially, the role of the long secretory pattern of luteinizing hormone (LH) during the periovulatory period. Progesterone (P4) treatment for several days during the estrous cycle may depress gonadotropins concentrations, block follicular development, and induce persistent anovulatory follicles in several species. The goals of this study were: (1) to test if a high dose of exogenous progesterone (P4) given during the preovulatory period reduces the LH surge concentrations and blocks ovulation in mares with single or double dominant follicles; and (2) to study the effect of P4 treatment on follicle blood flow. Mares with a growing ≥25-mm follicle 15 days after ovulation were scanned daily by ultrasonography. When the largest follicle reached ≥30 mm and the endometrial echotexture score was indicative of preovulatory period, mares were randomly assigned to one of the following four groups (n=8-10 mares/group): single dominant control (SD); single dominant+progesterone (SD+P4); double dominant control (DD); and double dominant+progesterone (DD+P4). The treatments involved the vehicle (3 ml of safflower oil) for the control groups and P4 (150 mg in 3 ml of safflower oil) for the P4-treated groups. Treatment was given every 12 h for 7 days. Ovulation of the largest follicle occurred in 100, 38, 100, and 56% of the mares in the groups SD, SD+P4, DD, and DD+P4, respectively. Ovulation of the second largest follicle occurred in 44 and 11% in the groups DD and DD+P4. Follicle atresia or formation of a hemorrhagic anovulatory follicle was the outcome for follicles that did not ovulate. Plasma LH concentrations but not FSH were significantly (P<0.0001) inhibited by P4 treatment. Mares in the P4-treated groups ovulated in presence of very low (7-fold less) concentrations of LH when compared with the controls. Diameter of the largest ovulatory follicle and estradiol concentration did not differ among groups. Blood flow in the wall of the largest follicle was less (P<0.04) in P4-treated groups 3 days after beginning of treatment. In conclusion, the preovulatory LH surge does not seem to be crucial for ovulation to occur in mares. From a clinical standpoint, attention should be given to progesterone-based synchronization or protocols for inhibition of estrous behavior since progesterone-treated mares may ovulate with very low systemic concentrations of LH. The present findings may also have implications in human reproductive medicine due to the striking similarities between mares and women in several aspects of folliculogenesis.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
The Kit system, composed of Kit ligand (KL) and its cognate tyrosine kinase receptor, cKit, has been well studied in mammals, but its functions in reproduction of non-mammalian vertebrates remain largely unknown. Studies have shown that the Kit system is involved in signaling between the oocyte and somatic cells during the process of follicle maturation in mammals. In mammalian ovaries, KL is known to be present solely in the granulosa layer of ovarian follicles; cKit is present in both the theca layer and the oocyte. Although the pattern of KL expression during follicle development has been studied in mammals, this has not been examined in the hen. The unique hierarchical arrangement of follicles in the hen ovary makes it a good model for the study of folliculogenesis. The objective of our study was to characterize the expression pattern of KL during follicle maturation and to examine regulation of KL. The ovarian stroma and the granulosa layer from follicles of various sizes (1, 3, 5, 6-12 mm, F1) were collected from white Leghorn hens (n=4 replicates), and KL mRNA expression was assessed using quantitative PCR. All values were normalized to 18S values. We found that granulosa mRNA expression for KL decreased significantly (P <0.01) with increasing follicle development, with the highest expression found in 1 mm follicles and the lowest amount seen in the granulosa layer from 6-12 mm and F1 follicles. Additionally, western blot analysis confirmed the presence of cKit (at the expected size of 120 kDa) in the theca layer of 3-5 mm follicles and in a lysate of whole <1 mm follicles, suggesting oocyte expression of cKit. We also examined whether the oocyte (or oocyte factors) affected KL mRNA expression. In mammals, the oocyte-derived factors BMP15 and GDF9 have been found to influence KL expression. As a source of oocyte-derived factors, we used oocyte conditioned medium (OCM). OCM was produced by culturing small (<1 mm) follicles in M199 plus 0.1% BSA for three days, then collecting and filter sterilizing the supernatant. For these experiments, granulosa cells were collected from 6-8 mm follicles (n=4 replicates) and the cells were dispersed and plated in M199 plus 5% serum for 24 h. Medium was then replaced with M199 plus 0.1% BSA in the presence and absence of OCM (at 25% and 50% of volume). After another 24 h, the medium was removed, cells collected and RNA extracted. Quantitative real-time PCR was used to evaluate expression of KL mRNA, with all values normalized to 18S expression. OCM caused a dose-related increase (P < 0.05) in expression of KL. Western blot confirmed the presence of BMP15 and GDF9 in OCM, suggesting that one or both of these factors, possibly by signaling through cKit, may play a role in regulating KL expression in the hen. Supported by USDA-NRI-2008-35203-19097.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Several members of the TGF-beta family of secreted products, including GDF9, BMPs, Activin, and AMH, are critical regulators of ovarian function. These proteins bind to cell-surface receptor and activate SMAD signaling pathways. GDF9 and Activin signal through the SMAD2/3 pathway, while BMPs and AMH signal through the SMAD1/5/9 pathway. Both pathways require cooperative binding to SMAD4, a central signaling molecule for all members of the TGFβ superfamily. SMAD signaling is critical for follicular development and function and effects on granulosa cells are well accepted, but direct effects on oocytes have not been described in the literature. Recently, we have shown that activated (phosphorylated) SMAD1/5/9 and SMAD2/3 proteins are present in the nucleus of mouse oocytes from primordial to antral follicles. To investigate further the possible role of SMAD signaling in oocytes, we have generated mice with oocyte-specific deletion of the Smad4 gene by crossing mice with a floxed Smad4 allele to mice expressing Cre-recombinase driven by the Zp3 promoter. Histological analysis revealed that follicles of all developmental stages were present on the ovaries of Smad4 mutant mice. Surprisingly, mutant ovaries had greater number of follicles compared to control littermates at 2 and 4 months of age. Consistent with an observed increase in follicle number, we observed a 3- to 4-fold increase in oocyte transcripts that drive follicular development in Smad4 mutant oocytes collected from 3-week-old mice. These transcripts include Gdf9, Bmp15, Figla, and Zp3. Thus, SMAD4 signaling in oocytes suppresses positive regulators of follicular development. In a second experiment examining the role of SMAD signaling during follicle assembly and activation, newborn ovaries were treated with an inhibitor of SMAD2/3 signaling (SB431542) for 2 days. Consistent with our results in the Smad4 mutant animals, ovarian expression of Gdf9, Bmp15, Figla, and Zp3 mRNA were markedly increased (2- to 3-fold) in ovaries treated with the SMAD2/3 inhibitor compared to control ovaries. We then treated newborn ovaries for 6 days with A-83, a more potent and specific inhibitor of SMAD2/3 signaling than SB431542. Normal primary and early secondary follicles were present in control ovaries cultured in medium only. However, the morphology of ovaries treated with the inhibitor was striking. Many large apparently activated oocytes were observed in treated ovaries. However, these large oocytes were surrounded by few granulosa cells, suggesting a deficiency of granulosa cell proliferation. In summary, loss of Smad4 in oocytes leads to increased follicular development co-incident with upregulation of specific oocyte transcripts that promote granulosa cell function, particularly Gdf9 and Bmp15. Inhibition of SMAD2/3 signaling in newborn ovaries causes upregulation of oocyte transcripts and oocyte activation, but without an increase in granulosa cell proliferation. Based on these findings, our working model is that SMAD signaling acts differently in oocytes versus granulosa cells. In oocytes SMAD signals suppress oocyte-stimulated follicular development, while in granulosa cells SMAD activation stimulates proliferation and consequently follicular growth.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Feeding fat supplements high in omega-3 long-chain polyunsaturated fatty acids (PUFAs) have been to shown to increase the reproductive performance of dairy cows. The objective of the current study was to evaluate the effect of two rumen-protected n-3 PUFA (fish oil and marine microalgae) supplements on ovarian function of lactating cows on a pasture-based feeding system during Ovsynch. Eighteen Holstein cows were blocked for parity and expected date of calving. Cows were assigned randomly within block to one of three isolipidic dietary treatments, fed from 30 d before to 100 d after calving. Six cows were assigned to the control supplement (hydrogenated plant oil; HPO), 6 to the marine microalgae (MA) supplement, and 6 to the fish oil (FO) supplement. The cows were synchronized for estrus and ovulation during Days 35-68 post-partum. Daily and twice-daily blood sampling and transrectal ovarian ultrasonography were conducted during the Ovsynch period. A validated enzyme and radioimmunoassay procedure were used to determine plasma progesterone and estradiol concentrations, respectively. Cows that calved early in the pasture season had a longer heat after the 1st Factrel injection than the cows that calved later in the pasture season (P<0.05). Heat was significantly longer in FO-treated cows compared to HPO-treated cows after the Lutalyse injection (3.0±0.5 d and 0.8±0.1 d, respectively; P<0.05); however, MA-treated cows did not significantly differ from HPO- or FO-treated cows. Ovulation was delayed in MA-treated cows compared to HPO-treated cows after the 2nd Factrel injection (1.6±0.1 d and 1.3±0.1 d, respectively; P<0.05); however, FO-treated cows did not significantly differ from HPO- or MA-treated cows. No significant differences were found among treatments for maximum preovulatory follicular size after the 1st or 2nd Factrel injection. The number of CL in MA-treated cows was higher than in HPO-treated cows at the time of the Lutalyse (2.2±0.3 and 0.8±0.2, respectively; P<0.05) and 2nd Factrel (2.0±0.3 and 0.7±0.2, respectively; P<0.05) injection; however, cows fed FO had a similar number of CL as the cows fed MA and HPO (P>0.05). Total luteal volume was not different among the treatments (P>0.05). We concluded that supplementation with n-3 PUFAs affects ovarian function in lactating dairy cows during Ovsynch.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
The mechanisms that regulate variation in ovarian function, which may contribute to infertility, are poorly understood. Previous results from our laboratory demonstrate that cattle can be phenotyped reliably based on peak number of follicles ≥ 3 mm in diameter growing during follicular waves, and that young adult cattle of similar ages with consistently low versus high peak number of follicles during follicular waves (hereafter called antral follicle count, AFC) have much lower circulating AMH concentrations and a markedly diminished ovarian reserve (total numbers of healthy follicles/oocytes in ovaries), but higher circulating FSH concentrations, greater intrafollicular estradiol concentrations, and greater abundance of aromatase mRNA in granulosa cells of early growing antral follicles during follicular waves. Based on these observations, it is hypothesized that the FSH-induced capacity of granulosa cells to produce estradiol is greater while AMH production is lower in cattle with low vs a high AFC. To test this hypothesis, granulosa cells from small antral follicles (3-5 mm follicles) were collected from pairs of ovaries with low (<15 &ge 3 mm in diameter) versus high (>25 follicles) numbers of follicles and were cultured in serum-free media containing 10-6M androstenedione, 2 ng/ml LR3-IGF-1 and 1 ng/ml insulin with increasing doses of oFSH (0, 0.01, 0.05, 0.1, and 0.5 ng/ml). Media was collected and replaced every 48 h. After 6 d of culture, media was harvested, granulosal cell numbers determined, and cells collected for RNA isolation. Estradiol and AMH concentrations in media were determined by immunoassays, and abundance of aromatase and AMH mRNA determined by real-time PCR. Results demonstrated that granulosa cells from cattle with low follicle numbers had >80% decrease in estradiol and AMH production and abundance of mRNAs for aromatase and AMH compared to cattle with high follicle numbers. Based on these results, it is concluded that FSH action on granulosal cells is diminished in cattle with a relatively low versus a high number of follicles growing during follicular waves. Further, the diminished responsiveness of granulosa cells to FSH in cattle with low follicle numbers may partially explain why circulating AMH concentrations are also much lower in these animals despite much higher circulating FSH concentrations during follicular waves. Research supported by USDA-NRI 2004-01697, 2007-01289 to JJI.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
The mammalian ovulatory process is accompanied by a gonadotropin-dependent regulation of several genes. Little is known about ovarian regulation of epidermal growth factor (EGF)-like growth factors in non-primate species, including epiregulin (EREG). The objective of the study is to characterize the regulation of EREG during hCG-induced ovulation in mares. Follicles during estrus between 0 and 39 h post-hCG and corpora lutea on day 8 of the estrous cycle were collected, and RNA and proteins were extracted. Results from semi-quantitative RT-PCR/Southern blot analyses showed that levels of EREG mRNA were very low in follicles obtained at 0 h but markedly increased thereafter (P < 0.05). Tissue blot analyses revealed that levels of EREG transcripts were relatively high in muscle but low or undetectable in corpora lutea and other non-ovarian tissues tested. Analyses performed with individual preparations of theca and granulosa cells indicated that EREG was regulated significantly in both cell layers (P < 0.05), with a maximal induction obtained 33-39 h post-hCG. Immunohistochemistry and immunoblotting confirmed regulated-expression of EREG protein in both cell types after hCG. Results from primary granulosa cultures isolated from dominant follicles revealed that levels of ADAMTS1, PGR, and CTSL2 mRNA were initially low but markedly increased in response to the treatment with EGF. This treatment had no effect on PTGS2 and PTGER2 transcript expression. Thus, this study reports the gonadotropin-dependent regulation of EREG during ovulation in mares and shows for the first time the stimulation of genes required for the rupture of follicles after the treatment with EGF.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Anti-Mullerian hormone (AMH) produced by granulosa cells is proposed as a predictor of fertility in several mammalian species. AMH production is positively associated with the number of healthy growing follicles in ovaries of women, while recent studies in cattle have demonstrated that serum AMH concentrations are greater in animals with high, compared to low, antral follicle counts. Additionally, AMH may be involved in granulosa cell differentiation and follicle selection. For instance, in rodents, AMH inhibits FSH-induced aromatase activity and luteinizing hormone receptor expression in granulosa cells. Dimerization of the type II (AMHR2) receptor with a type I receptor initiates AMH signaling via SMAD1,-5,-8. To date, regulation of AMHR2 expression in granulosa cells and its role in follicle selection is poorly understood. We hypothesized that the expression of AMHR2 mRNA in bovine granulosa cells (bGC) is regulated during follicle development and that AMH can regulate bGC function. The objectives of the present study were to: 1) compare AMHR2 expression with other TGFbeta super family type I and type II receptors, plus related transcription factors, in female reproductive tissues; 2) characterize AMHR2 expression in bGC during follicle development; and 3) examine a role for AMH signaling in bGC differentiation and proliferation. bGC were isolated from small (5-8 mm), medium (9-12 mm), and large (13-24 mm) antral follicles obtained from dairy cattle collected at local abattoirs. Following isolation, some bGC were frozen for RNA analysis. Expression of ACVR1, ACTR2, BMPR1A, BMPR1B, BMPR2, TGFBR2, WT1, and SF1 was evident in bGC, oviduct, uterus (both fetal and adult) and liver, whereas AMH and AMHR2 were restricted to bGC and fetal oviduct, while AMHR2 was also present in fetal uterus. Further analyses revealed that expression of AMHR2 and its requisite transcription factor WT1 (but not SF1) were greater in small antral follicles compared to larger antral follicles (p<0.05). In addition, bGC from 5-8 mm follicles were cultured for 24 h in media containing 2.5% FCS with 0, 10, 25, or 100 ng/ml rhAMH and/or a maximally effective dose of FSH (100 ng/ml) to evaluate a role for AMH in bGC differentiation. There was no difference in bGC AMHR2 expression among treatments (p>0.05); thus, it was concluded that neither AMH nor FSH directly regulate AMHR2 expression. To evaluate cell proliferation, bGC were labeled with 2 μM carboxyfluorescein succinimidyl ester and treated 24 h later with 0, 10, 25, or 100 ng/ml rhAMH, betacellulin (20 ng/ml), FSH (100 ng/ml), or LH (100 ng/ml). Cell proliferation was evaluated at 24, 48 and 72 h post treatment. No differences in bGC proliferation were observed at 24 and 48 h; however rhAMH inhibited bGC proliferation following 72 h in vitro (p<0.05). It is concluded that bGC express AMHR2, and that AMH can inhibit the proliferation of granulosa cells from small antral follicles. We propose AMH signaling is abated through the gradual loss of AMHR2 expression during follicular development, and that the loss of AMH signaling enhances FSH-responsiveness and the differentiation of bGC. These data contribute to our understanding the cellular and molecular actions of AMH signaling, and its predicted role in regulating the ovulation rate in a monovular species. Supported by NSF IOS-0968784 to ALJ.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Theca cells are critical components of ovarian steroidogenesis as they synthesize androgens, which are subsequently converted to estrogens by the granulosa cells. In addition to their roles in normal ovary development, theca cells are implicated in pathological conditions of the ovary such as polycystic ovary syndrome. Despite their importance in the ovarian development, the source of theca cells and the mechanism by which they are specified remain a mystery in ovarian biology. By studying the differentiation of mouse Leydig cells, the male counterparts of theca cells, we have obtained information that could be applied to theca cells. Specification of the Leydig cells in fetal testes requires activation of the Hedgehog (Hh) pathway that consists of transcription factors Gli1 and Gli2. We first investigated whether Gli1 and Gli2 mark the potential theca cell precursors in the ovary. At birth, the expression of Gli1 was present only in the mesonephric tubules that connect to the rete ovarii of the ovary whereas the ovary was devoid of any Gli1-positive cells. Between birth and postnatal day 3 (PD3), Gli1 expressing cells gradually spread from mesonephric tubules into the interstitium of the ovary. By PD6, when the putative theca cells appear, Gli1-positive cells were found to surround the developing follicles outside of the granulosa cell layer. In the adult ovary, Gli1 expression was detected specifically in the theca layer. These findings suggest Gli1-positive cells from the mesonephros could be precursors of theca cells. To test this possibility, we cultured newborn ovaries with or without mesonephros attached. We found that, in the absence of the mesonephros, the Gli1 positive cells in the ovary were significantly decreased. Gli2, also a component of the Hh pathway, often displays an expression pattern overlapping with Gli1 in many tissues including the theca layer of adult ovaries. However, when we performed a time course analysis of Gli2 expression in the ovary, we found a pattern completely different from that of Gli1. Gli1 expression was never detected in the fetal ovaries whereas Gli2 expression was found in the fetal ovary as early as embryonic day 14.5. The expression of Gli2 exhibited a network-like pattern surrounding the germ cell nests. In the adult ovaries, Gli2 became positive in the theca layers, overlapping with Gli1. In summary, based on the interstitial pattern of expression, we speculate that Gli1 and Gli2 mark different populations of theca cells progenitors. Different time frame of expression and cellular distribution between Gli1 and Gli2 further suggest the presence of complicate mechanisms in theca cells specification and differentiation. The importance of the mesonephros to Gli1 expression in the ovary reveals an unexpected role of the mesonephros in theca cell development. We are currently investigating whether Gli1- and Gli2-positive cells represent two different populations of theca cells and their functional significance in theca cell development. This study was supported by NIH-HD046861 and HD059961.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Nonylphenol (NP) is a metabolite of detergent. The estrogenic effects and acute toxicity of NP causes disturbance in reproductive system. It has been reported that NP inhibits testosterone synthesis in male gonads, while the effects on progesterone release from female ovarian cells are unclear. In the present study, the effects of NP on steroidogenesis of progesterone in rat granulosa cells were examined both in vitro and in vivo. Immature (3-week old) female rats were intraperitoneally injected with pregnant mare serum gonadotropin (PMSG, 20 IU). After 48 hrs, the PMSG-treated rats were sacrificed and granulosa cells were collected from rat ovaries. The granulosa cells were cultured in 24-well plates (105 cells/ 0.5 ml) for 48 hrs, then treated with NP (4.3 µM, 12.8 µM, 43 µM ), in the presence or absence of human chorionic gonadotropin (hCG, 0.05 IU/ml) for 2 to 24 hrs to evaluate the NP effects on progesterone steroidogenesis. The levels of progesterone in medium were measured by radioimmunoassay, and the protein expressions were detected by western blotting. The data indicated that NP stimulated production of progesterone via a dose-dependent manner, especially for 2, 4 and 8 hrs, while treatment of NP for 24 hrs decreased progesterone release. Treatment of NP for 16 and 24 hrs prevented the stimulatory effects of hCG. We also gave the immature female rats gavage of NP with daily dose of 0, 50, 100 and 200 µg per kg body weight of NP for 7 days. The progesterone levels were raised following administration of 100 µg NP. Stimulation with hCG and 8-bromo-adenosine cyclic monophosphate (8-Br-cAMP) showed similar trends. MAPK/ERK kinase (MEK) inhibitor PD98059 did not affect progesterone as compared with vehicle treatment. Expressions of steroidogenic acute regulatory (StAR) protein were enhanced with 100 µg of NP. Moreover, gavage of NP (100 µg /kg/day) during 21-day pregnancy increased progesterone production of the female puppies' granulosa cells compared with those treatment without NP. These data suggested that NP directly stimulated StAR protein expression and affected the synthesis and production of progesterone in rat granulosa cells via activation of cAMP pathway.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
During the mono-ovulatory estrus cycle, FSH and LH play an important role for growth, selection and dominance of the preovulatory follicle. The dominance of a follicle is characterized by the acquisition of luteinizing hormone (LH) receptors on granulosa cells (GC) and the transfer of its dependency for follicle-stimulating hormone (FSH) to LH. The LH surge induces profound changes in GC functions, which must be modified in a precise chronological order. These changes ultimately lead to the release of the matured oocyte during the follicular rupture at ovulation. On a transcriptional level, the gene expression cascades induced by this hormone, which lead to ovulation, are complex and still poorly understood. The objective of this study is to determine how the gene expression profile of GCs is modulated by the LH surge. The estrus of thirty animals were synchronized, they were then stimulated with FSH and finally the LH surge was induced by gonadotropin releasing hormone injection on day 14.5. Three different groups of GCs were collected by ovariectomy; (1) 2 h before the induction of the LH surge, (2) 6 h and (3) 22 h after the LH surge. The RNA of the different GC groups was extracted, amplified, labeled and hybridized on a microarray slide. A bovine oligonucleotide-spotted microarray (24K) was used to profile all known protein-encoding transcripts. The results obtained from the microarray data analysis showed that most transcripts were present in every type of GC studied. However, candidates were found to be preferentially expressed in only one of the GC states. Moreover, an unsupervised hierarchal clustering indicates that the overall GC expression of 2 h pre-LH and 6 h post LH correlate more closely than with the 22 h post LH, which is consistent with the endogenous ovarian physiological functions as GCs undergo extreme changes in order to achieve their luteal role. To understand the effect of LH on granulosa cell gene expression, transcripts fluctuating at the 6h post-LH stage were analyzed using pathway analysis software. Globally, the representation at 6 h post LH indicates that the follicles expresses genes involved in cell-to-cell interactions (like PTP4A3, THBS1, PPAP2B), in the inflammatory response (like B4GALT1, THBS1, TRAF3IP2) and in lipid metabolism modifications (like ACSL3, CYP51A1, FADS2). In conclusion, our goal is to put these follicular events in the context of oocyte developmental competence as it is gradually acquired during follicular growth and maximized during the last stages of follicular maturation.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
WITHDRAWN.
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Adiponectin and resistin are part of the adipokine family of hormones produced and secreted by adipose tissue. Circulating adiponectin is associated with increased adiposity and can promote glucose uptake, possibly via increased expression and/or activation of glucose transporters. In addition, a role for adiponectin in reproductive function has been put forth to include effects on ovulation and steroidogenesis. Resistin, in contrast, may be more closely linked to inflammation, although there is some evidence that resistin is associated with insulin resistance. The goal of this study was to examine intra-follicular and serum concentrations of adiponectin and resistin during the periovulatory interval in rhesus monkeys undergoing controlled ovarian stimulation (COS), as well as determine the expression of adiponectin receptors during granulosa cell luteinization. Adult females were treated with recombinant human follicle-stimulating hormone for 7 days and follicles were aspirated before (0 hr), 3, 6, 12, and 24 hr after ovulatory bolus of hCG. Follicular fluid and serum levels of adiponectin and resistin were measured by ELISA before (0 hr) and 24 hr after hCG. Adiponectin decreased (p<0.05) 24 hr after hCG treatment while serum levels remained unchanged. In contrast, follicular fluid levels of resistin were unchanged after hCG, while serum concentrations increased 24 hr post-hCG. The mRNA expression of adiponectin and adiponectin receptors (AdipoR) I and II were analyzed using RT-PCR. Adiponectin mRNA was not detectable in granulosa cells at any time point. However, AdipoRI expression was increased transiently 3 hrs after hCG (5.5-fold, p<0.05), while AdipoRII mRNA was elevated 3 and 6 hrs after hCG treatment before returning to 0 hr levels (6- and 23-fold, respectively; p<0.05). Granulosa cells isolated prior to hCG in vivo were cultured with FSH or FSH+hCG +/- adiponectin (0-25 µg/ml). In the presence of FSH only, adiponectin increased progesterone levels (0 µg/ml adiponectin: 1.7 ng/ml ±0.2; 5 µg/ml: 2.0 ± 0.3; 25 µg/ml: 8.9 ± 0.7). Similarly, hCG markedly increase progesterone over FSH alone (7.7-fold; p<0.05), although adiponectin did not further increase progesterone. Adiponectin did not alter the mRNA expression of glucose transporters (GLUT) 1, 2, or 4, but in the presence of hCG, dose-dependently increased GLUT3 mRNA (p<0.05). These data confirm and quantify the presence of adiponectin and resistin in the luteinizing primate follicle, as well as the dynamic expression of adiponectin receptors. Importantly, the lack of granulosa cell expression of adiponectin mRNA indicates a complex origin for follicular adiponectin. A limited local role for adiponectin through its increased receptors may facilitate glucose uptake by luteinizing granulosa cells. Supported in part by NIH HD043358 (CLC) RR00169 (CNPRC) and RR13439 (CAV).
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
WITHDRAWN.
Biol Reprod. 2010 Nov;83(1 Suppl):3.
NOT PRESENTED.
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Orchitis is characterized by inflammatory infiltrates in the testicular interstitium and is associated with focal or total impairment of spermatogenesis eventually leading to male infertility. Cytokines are considered to play a major role in the maintenance of normal testicular function. Under normal conditions, members of the Interleukin-1 (IL-1) family are expressed at basal levels in different types of testicular cells. During inflammation several cytokines are increased in the testis potentially leading to disturbed testicular function. IL-1Receptor antagonist knock out (IL-1Ra K/O) mice show elevation of IL-1alpha and IL-1beta levels in testicular tissue and impaired male fertility. The aim of this study is to investigate the expression levels of IL-1 family members (IL-1alpha, IL-1beta and IL-1Ra) in the testis during the progression of experimental autoimmune orchitis (EAO) in mice and to localize their production. A model for murine EAO was established. In brief, testes of adult wild type (WT) BALB/c mice (12 weeks old) were homogenized, mixed with complete Freund's adjuvant and injected subcutaneously in tail, neck and foot pad of Balb/c wildtype mice. Pertussis toxin (20µg/ml) was used as a co-adjuvant. In adjuvant control group testis homogenate was replaced with saline. Sham-treated animals were injected with saline only. At day 0 (untreated group), 20, 30, 40, 50 and 70 days after the first immunization mice were sacrificed, testes were weighted and either fixed in Bouin solution for histology assessment or homogenized for cytokine measurements. A scoring system reflecting the severity of EAO was developed based on examinations of hematoxylin-eosin stained paraffin sections. IL-1alpha, IL-1beta and IL-1Ra levels in the testes were measured using specific ELISA for each cytokine. The cellular localization of IL-1 in the testis of the EAO and control groups was determined by immunofluorescence staining. In the EAO model, testicular weight of the WT-EAO-mice was significantly decreased after 50 days compared to control group (p<0.001). Testes showed severe morphological changes such as germ cell sloughing, disruption of spermatogenesis and Sertoli-cell-only-syndrome as well as massive interstitial cell infiltration. Based on the histoscore, the disease progression was subdivided into five stages and the animals were grouped accordingly. ELISA measurements revealed higher levels of IL-1alpha, IL-1beta and IL-1Ra in EAO mice starting with gradual increase from day 30 after the first immunization compared to control groups. At day 70 differences between EAO and adjuvant control culminated and EAO mice displayed ca. 3-fold higher levels of the respective cytokines. EAO histology scores tended to coincide with the increased cytokines levels. Compared to the adjuvant control groups, testis sections of EAO-mice showed increased expression levels of IL-1alpha, IL-1beta, IL-1Ra and IL-1 receptor 1 (IL-1 R1) using immunofluorescence, which was particularly evident in the interstitial area. Increased staining of IL-1 R1 was also detected in the seminiferous epithelium. Members of IL-1 family are involved in the development of experimental autoimmune orchitis and may play a crucial role in the onset and progression of the disease.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Environmental factors including nutritional availability are well understood to control oocyte production in Drosophila. Based on reports in the literature of deleterious effects on ovary function during periods of viral infection, we hypothesized that infection with Flock House Virus (FHV) would result in altered egg chamber development, and possibly degradation. We have shown that when wild-type Canton-S flies are injected with FHV, mid-stage egg chambers (stage 8/9) are destroyed due to rapid tissue remodeling. This effect was found to be dependent upon the concentration (established as plaque forming units using S2 cells) of virus injected. As early as 12 hours post-infection, posterior egg chamber follicle cells (PFC) invade and phagocytose enclosed oocytes in a process that we have termed Virus-Induced Somatic Oocyte Destruction, or VISOD. Using whole-mount fluorescence immunostaining of FHV particles in situ, we determined that FHV is present at high levels in cells throughout the ovary, including follicle cells engaged in oocyte phagocytosis. Mated infected flies thus lay significantly fewer embryos than PBS-injected controls. We are now engaged in a screen for genes required for VISOD induction. As seen in a model of chemical insult (Rapamycin ingestion), flies heterozygous for the epithelial polarity genes discs large and merlin fail to activate oocyte destruction as seen in wild-type flies, suggesting that epithelial remodeling is crucial for the process to begin. Similarly, genes involved in innate immunity and autophagy were found to rescue oogenesis during infection. We hypothesize that the induction of oocyte loss in response to viral infection is conserved in mammals, potentially in women.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
NOT PRESENTED.
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Cryopreservation of canine spermatozoa offers potential exchange of genetic material, and thus may lead to improvement in the breeding management programs used to produce working dogs. In particular, in guide dog colonies, application of transcervical artificial insemination using frozen canine semen is anticipated to assist with meeting the demand for adequate supply of guide dogs for the blind. Egg yolk is the most commonly used compound in canine semen extenders for protection of spermatozoa from cold shock and disruption during the freezing and thawing process. However, due to a recent outbreak of avian influenza and its triggering of growing concern throughout the world, transportation of frozen or chilled semen exposed to egg yolk has become extremely difficult. Thus, it is an urgent matter to develop a novel semen extender without egg yolk for use in freezing of canine spermatozoa. As an alterative compound to egg yolk, skim milk seems to be especially suitable as a semen extender in canine species, since a skim milk extender is the most commonly used extender for mouse and goat sperm. We preveously reported that the skim milk/glucose-based extender without egg yolk was effective on the motility and fertilizing capacity of canine spermatozoa frozen-thawed. In this study, we further examined the optimal concentration of glucose, trehalose and raffinose in skim milk extenders to improve the sperm motility after frozen and thawed. The optimal concentration of glucose, trehalose and raffinose were 0.3 M, 0.2 M and 0.2 M respectively, and the mean percentages of motile frozen-thawed spermatozoa in skim milk/trehalose-based extender (74.3% ± 7.04 %) or skim milk/raffinose-based extender (70.9% ± 10.2 %) were significantly higher than that of skim milk/glucose-based extender (59.6% ± 8.63 %) or egg yolk extender (53.7% ± 9.28 %). To examine the fertilizing capacity, frozen spermatozoa with skim milk/raffionse-based extender was transcervically inseminated into a recipient bitch and its conception was admitted by echography and radiography. These data indicate that canain frozen spermatozoa using raffinose in skim milk based extender may contribute to exchange of genetic material and efficient production of companion and working dogs, such as guide dogs for the blind.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
The endangered black-footed ferret (Mustela nigripes; BFF) is a species that was rescued from the brink of extinction when the last 18 individuals remaining were brought into captivity to begin a breeding program. Over 20 years later, more than 6,500 individuals have been produced. This population, however, has experienced a recent decline in normal sperm (NS; 50% to 25%), which may be linked to inbreeding depression and/or a change from a manually prepared rabbitmeat diet to a commercially available horsemeat diet. Because nutrition can be a limiting factor for reproductive success, we investigated its potential role in the observed fitness decline in the BFF population. Here we examined the role of vitamin (vit) E and selenium (Se), which are antioxidants that can promote sperm viability by eliminating reactive oxygen species. We also investigated vit A, which can decrease the absorption of vit E from the intestine. Our specific objective was to determine the effects of two commercially available diets (horse- and beef-based) and of dietary vit E and whole carcass supplementation on serum vit E, A and Se, dental health and semen quality. Black-footed ferrets (n=55 males) were randomly assigned to one of five diet treatments (n=11/treatment): 1) horsemeat diet (control); 2) horsemeat diet + vit E (400 IU/kg DM) daily; 3) horsemeat diet + whole carcass (2 hamsters/week); 4) horsemeat diet + vit E daily + whole carcass; and 5) beef diet. Blood was taken prior to and 6 months after the diet change and both blood and diets were analyzed for vit E, A and Se concentrations. Diets were also analyzed for proximate analysis and fatty acid profiles. Electroejaculates were collected monthly from males throughout the breeding season (Feb-May) and evaluated for for sperm concentration, sperm motility index (SMI; includes % motile and forward progression) and NS. Additionally, males were paired with females during the breeding season to assess the effect of diet on fecundity. The beef and horsemeat diets had comparable vit E and Se concentrations and all diets met most nutrient requirements for small carnivores; however, the horsemeat diet was excessive in vit A and the beef diet was deficient in vit A. Blood Se and vit A concentrations were comparable among treatments. Vit E supplementation increased serum vit E; serum vit E was lower (P<0.001) in BFFs fed the horsemeat diet without vit E supplementation as compared to the other treatments. The addition of whole carcass reduced tartar and gingivitis. Seminal results demonstrated: 1) no effect of diet on sperm concentration; 2) beef diet and both carcass treatments had improved (P<0.001) SMI over the horsemeat control group; and 3) horsemeat diet supplemented with vit E had the lowest (P<0.001) NS. No significant difference in successful pairing and/or in the number kits/litter was determined among diet treatments. In conclusion, the high concentrations of vit A in the horsemeat diet could compete with vit E which may elicit pro-oxidative effects and reduce reproductive success in this endangered species. The addition of whole carcass and vit E may improve overall BFF health. Additionally, increased inbreeding over time may also be attributing to the declining seminal quality and should be further investigated. Successful reproduction is important to BFF survival; therefore, it is important to monitor and adapt management practices.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
NOT PRESENTED.
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Cryotolerance of in vitro produced embryos can be improved by reduction of lipid droplet accumulation by microsurgery or chemical delipidation. We previously determined that forskolin caused delipidation during culture of bovine embryos; however, pregnancy rates were not evaluated; also, we hypothesized that thawing embryos and culturing them in vitro for 10 h in order to transfer only the expanded blastocysts could result in improved pregnancy rates. Recent research has shown that synchronization with a 5 day CO-synch plus progestagen protocol results in higher pregnancy rates after artificial insemination than a 7 day protocol; however, these protocols have not yet been compared for embryo transfer. The aim of this experiment was to evaluate pregnancy outcomes of delipidated, frozen-thawed-cultured in vitro produced embryos vs. in vitro and in vivo controls, as well as the two estrus synchronization protocols mentioned above. Oocytes were collected from slaughterhouse ovaries, matured, fertilized, and the resulting embryos cultured in vitro by standard procedures in a chemically defined medium. Day 0 of culture was ~18 h after the onset of IVF. At 6.0 days of culture, half of morulae were exposed to 10 μM forskolin, and the rest served as controls. At 7.5 days after fertilization, embryos were conventionally frozen with ethylene glycol. Also, in vivo produced embryos were collected nonsurgically from superovulated cows, and cryopreserved conventionally. Recipients were assigned to two synchronization protocols: 5 (n=59) or 7 day (n=56) CO-synch plus progestagen plus 400 IU equine chorionic gonadotrophin on the day of CIDR removal; embryos were transferred nonsurgically 7 days after the last GnRH injection. Recipients were assigned to 4 embryo cryopreservation treatments: 1) in vivo produced control (n=29), 2) in vitro produced control (n=21), 3) in vitro produced plus forskolin (n=36), 4) in vitro produced plus forskolin, plus thawed in the laboratory and cultured for 10 h, and only transferring expanded blastocysts (n=29; ~70% expanded). Pregnancies were evaluated at 41 days post GnRH by ultrasonography by detecting an amniotic vesicle or live fetus. Data was analyzed by Proc GenMod, using 1 for pregnant and 0 for nonpregnant. Pregnancy rate with the 5 day synchronization protocol was not different (P > 0.05) from that obtained with the 7 day protocol (27.1 vs. 23.2%, P > 0.1). There was an effect (P < 0.05) of cryopreservation treatment on pregnancy rates; in vivo control 41.4%a, in vitro plus forskolin plus 10 h culture 31.0a,b in vitro plus forskolin 16.7%b,c, and in vitro control 9.3%c (means without common superscripts differ, P < 0.05). There was no interaction (P > 0.1) among treatments. In conclusion, 5 day CO-Synch + progestagen treatment was similar to 7 day, and in vitro treatment of embryos to delipidate them with forskolin plus 10 h of culture post-thaw was similar to the in vivo control, and could be an alternative to improve pregnancy rates. Research supported by PROMEP, SEP, Grant UACH-PTC-087.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Superovulation is defined as the induction of multiple ovulation products (oocytes or embryos) and is an invaluable tool in animal breeding and assisted reproduction. This technique works efficiently in domestic and rodent models, however, it has not been successful in primate species. The rhesus macaque is an excellent translational model for human reproductive issues. Like the human, the rhesus monkey is monovular, which translates into the collection of perhaps 1 embryo after mating or A.I. in every 3 natural cycles. It would facilitate research in early embryo development if rhesus monkeys could be induced to provide multiple embryos after superovulation, mating and uterine flushing. Females, which had been previously tested for ease of cervical cannulation, underwent superstimulation and superovulation (rhFSH, LH, hCG) and natural mating. On Days 4 and 5 following presumed conception, flushing of the uterus was performed to recover ovulation products (unfertilized oocytes or developing embryos). To date, 56% of attempted flush cycles (10/18) resulted in the collection of ovulatory products for a total of 10 unfertilized oocytes and 16 embryos in varying degrees of development (4-cell to blastocyst). Of these 10 successful cycles, 2 cycles only produced 1 ovulation product (1 blastocyst found in each) and 8 resulted in multiple ovulation products corresponding to multiple sites of ovulation on the ovaries examined through laparoscopy. One particular cycle resulted in the collected of 1 unfertilized oocyte, 1 8-cell embryo and 5 blastocysts. Another cycle resulted in the collection of 4 blastocysts. This report provides the first substantial evidence that it is possible to superovulate non human primates for the collection of multiple in vivo produced embryos for research. However, at present, success is sporadic and further work is needed, including hormone profiling to assess individual female response to exogenous hormones, to improve rates of embryo recovery. Nevertheless, this result offers proof of principle that rhesus monkeys can produce multiple ovulations in each cycle. Embryos resulting from these cycles will provide an excellent model for establishing normal primate embryo characteristics, as well as providing the basis for embryonic stem cell lines from gold standard in vivo produced embryos compared to their in vitro produced counterparts.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
The Arabian Tahr is one of the endangered species found entirely in the mountains of United Arab Emirates and Oman. Assisted reproductive technologies (ARTs) such as in vitro fertilization and embryo transfer could play essential role in saving the population of endangered species. Identifying suitable surrogate mothers will be one means of increasing the population of the Arabian Tahr through assisted reproduction. The objective of the present study was to investigate the ability of Tahr sperm to fertilize goat oocytes in vitro, and the developmental potential of the resulted hybrid embryos. Oocytes were obtained from ovaries of slaughtered domestic goats and in vitro matured in TCM (with fetal calf serum, FSH, LH, Na-pyruvate, estradiol and cysteamine) in a humidified atmosphere of 5% CO2 and 39°C. Matured oocytes were in vitro fertilized with frozen-thawed or fresh Tahr sperms collected by electro-ejaculation at a concentration of 1-2 x 106 sperm/ml. Fertilized oocytes were cultured in SOF medium and cleavage was checked 44 h post insemination. Over 70% and 55% cleavage rates were obtained with fresh and frozen sperms, respectively. When the hybrid embryos (2 to 4-cell stages) were surgically transferred into 4 domestic goats, pregnancy was established in 3 goats as indicated by progestin level in fecal samples and by ultrasound examination at 2 months of pregnancy. Our results confirmed that Arabian Tahr's sperms are capable of fertilizing the domestic goat's oocytes in vitro, and that pregnancy could be achieved when the resulted hybrid embryos are transferred into domestic goats. This finding is crucial for us in our effort to produce a hybrid offspring that can be used as surrogate mothers to propagate the Arabian Tahr.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
The mammalian ovarian lifespan is determined at the time of birth through a delicate balance of oocyte survival and apoptosis, and the mechanism by which these germ cells die is not understood. It is our hypothesis that c-kit signaling regulates Bcl2 family proteins to effect oocyte death or survival. In order to test this, we began by determining if the Bcl2 protein was important for germ cell survival. Previous studies have demonstrated that overexpression and deletion of Bcl2 altered the primordial follicle pool at postnatal days (PND) 9 and 42, however no studies have been conducted to identify the effect of Bcl2 on the pool of follicles at earlier time points. To elucidate the effects of Bcl2 on the number of oocytes and formation of primordial follicles, we examined oocyte specific Bcl2 over-expressing mice with an insertion of the human bcl-2 gene fused to the c-kit promoter. Wild-type FVB mice without the transgene were used as the control strain. Eight ovaries from the c-kit/bcl2 overexpressing mice, and eight ovaries from wild-type control mice were harvested and fixed overnight at PND 1, 4 ,7 and 9. All ovaries were stained with the oocyte marker, STAT 3, and propidium iodide to visualize germ and somatic cells within the ovary. Ovarian whole mounts were analyzed via confocal microscopy for number of germ cells, number of oocytes in cysts and number of oocytes in primordial, primary and secondary follicles. Overall, no significant differences were found for cyst breakdown, oocyte numbers or follicle development at PND 1, 4, 7 or 9. It does not appear that the overexpression of Bcl-2 has any affect on cyst breakdown, follicle development or cell death. This may be due to a redundancy with other anti-apoptotic Bcl2 family members such as Bcl-x, which has been shown to be expressed in the neonatal ovary by immunohistochemistry and RT-PCR. Moreover, overexpression of the protein may not be able to rescue the dying oocytes, producing no new phenotype in the ovary. It is also possible that cell death is not dependent on the Bcl2 intrinsic apoptotic pathway. To further clarify the role of Bcl2 protein in oocyte death, bcl2 knockout models were used to test the effects of abrogation of the bcl2 gene on oocyte death and follicle development. Ovaries were stained with STAT-3 and propidium iodide and labeled with their genotype for analysis. Eight wild-type, heterozygous and homozygous null mice were obtained at PND 1, 4 and 7 and the ovaries were analyzed by confocal microscopy. As in the overexpressing models, knockout of bcl2 did not affect cyst breakdown, follicle development or oocyte numbers. This data, like the overexpression data, shows that Bcl2 may not have a role in oocyte cell death or in follicle formation. We hypothesize that in the absence of Bcl2, other proteins in this family may substitute for its function in preventing cell death. More work is needed on the inhibition of Bcl2 in conjunction with inhibition of other family members to see if this is the case. Future work will examine the importance other Bcl2 family proteins and the effect of c-kit stimulation on their activity.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Ovarian cancer is the most lethal genealogical malignancy due to the fact that is undetectable in early stages and is only caught in women after metastasis has occurred. Eighty percent of ovarian cancers have p53 mutations, which are also associated with primary tumor size, serous stage III cancers, and abnormal accumulation of p53 protein, suggesting that p53 mutant cancers gain metastatic properties. Additionally there is interplay between the transforming growth factor beta (TGFbeta) pathway and p53 such that TGFbeta affects cell proliferation, apoptosis, and metastasis. Cooperation between Smads, the downstream transcription factors of the TGFbeta pathway, and tumor suppressor p53 has been documented. Mutations in both TGFbeta signaling and p53 cause aberrant cell behavior, disease, and cancer, making their interaction critical to the understanding of cancer progression. The role of TGFbeta in cancer changes as the disease progresses; in the early stages TGFbeta suppresses tumor growth; however, after tumor formation TGFbeta promotes metastasis. Our hypothesis is that TGFbeta signaling prevents tumor progression in ovarian cancer p53 wild-type cell lines, while p53 mutants exhibit TGFbeta-dependent migration, proliferation, and tumorogenesis. To determine the role of p53 in the TGFbeta pathway, a panel of seven ovarian cancer cell lines was chosen based on p53 status (OVCA420, HEYC2, OVCA433 - p53 wild-type; OVCA432, OVCAR3 - p53 mutant; SKOV3, OVCAR5 - p53 null). Proliferation, apoptosis, and protein expression were measured in these cell lines after stimulation with TGFbeta (10ng/ml) and Activin (20ng/ml). Luciferase assays were performed to determine the ability of the cells to elicit a Smad-dependent transcriptional response after TGFbeta and Activin ligand stimulation. All cell lines responded to TGFbeta portraying that all elements of the pathway are present including an activated Smad2/3 and Smad 4. In response to TGFbeta, p53 wild-type ovarian cancer cells, unlike p53 mutants and nulls, are arrested in G1, suggesting coregulation between p53 and p21 expression. p15 acted as a negative control for cyclin-dependent kinase inhibitors as it was not regulated in a TGFbeta/p53-dependent manner. Known tumor suppressor and anti-invasion protein Maspin, shown to mediate BRAt in breast cancer, was absent in mutant p53 cell lines suggesting a gain-of-function that could account for increased metastatic properties from mutant p53 in the presence of TGFbeta. MapK/Erk expression was also upregulated only in p53 wild-type cell lines indicating that MapK pathway might encourage p53 phosphorylation and promote p53/Smad interaction. Re-expression of p53 in the null SKOV 3 cell line reinstated that both p21 and MapK/Erk expression are regulated by p53. We have identified p53 as a modifier of Smad signaling that encourages cell cycle arrest in its wild-type form as opposed to the mutant and null forms. Mutant p53 cell lines have shown a gain of function in the loss of tumor suppressor Maspin, which is also known to be anti-invasive in breast cancer. Further studies will help determine whether the loss of Maspin accounts for increased metastasis in p53 mutants and whether Mapsin expression is TGFbeta dependent. Eventually this research will reveal whether p53 is the molecular switch that triggers TGFbeta-induced invasion and tumor progression in ovarian cancer.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
MicroRNAs are small, single-stranded RNA molecules that regulate translational repression. Our group and others have shown that microRNAs play important functional roles in epithelial ovarian cancer, specifically clear cell and serous histotypes. To explore the role of microRNAs in endometrioid ovarian cancer, we preformed deep sequencing in primary tumors. Our microRNA and gene expression profiling show that endometrioid tumors cluster separately from serous tumors. When we compared the expression of these differentially expressed microRNAs in endometrioid tumors with short-term cultures of normal ovarian surface epithelium, we discovered 81 microRNAs were significantly differentially expressed with a fold change of 2.0. Using genome wide gene expression data from the controls and the endometrioid tumors, we performed in silico microRNA-mRNA targeting analysis. Pathway analysis revealed that a significant number of our targeted genes were involved in Ephrin receptor signaling. Ephrin receptors are receptor tyrosine kinases. Numerous studies have shown that increased EphA2 expression correlates with angiogenesis and poor prognostic outcomes in patients with ovarian cancer. We have found that a small number of microRNAs in endometrioid ovarian cancer diversely target a pathway in which several elements are clinically important in ovarian cancer. The project described was supported by Award Number R01CA060651 from the National Cancer Institute, National Institutes of Health 5K12HD050128 Women's Reproductive Health Research Program, and the Ovarian Cancer Research Fund.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
NOT PRESENTED.
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Embryonic stem (ES) cells are pluripotent cell lines derived from the blastocyst-stage of mammalian embryos. These unique cells are characterized by their capacity for prolonged undifferentiated proliferation in culture, with the potential to differentiate into derivatives of all three germ layers. Additionally, these cells closely resemble theirs in vivo counterparts, providing a stable in vitro model of embryo growth and development that functions as a tool by which specific cell signaling systems can be investigated. Therefore, mouse ES cells constitute a versatile biological system, which has facilitated major advances in the fields of cell and developmental biology. Though melatonin has a wide variety of biological functions, its effects on the ES cells is still unknown. In this study, we examined the effects of melatonin pharmacological concentrations (100 or 200 µM) on the proliferation and differentiation of ES cells (ES-E14TG2a cell line) using an in vitro culture system. We found that melatonin at pharmacological concentrations affected morphology and expressions of phospho-ERK in 200 µM melatonin and phosphor-Akt in 100 µM of melatonin. Melatonin treatment (100 or 200 µM) increased Bcl-2 expression and suppressed Bax expression as well as phospho-GSK alpha and beta. The POU (N-terminal to homeobox) domain containing the transcription factor Oct4, HMG (high mobility group) domain containing the transcription factor Sox2, the zinc finger transcription factor Zfp206 and the zinc finger gene REX-1 (Znf42) are all important for cellular pluripotency and pre-implantation development. Their expression regulates ES cell differentiation that control a set of genes expressed very early in development. In this study, melatonin (100 µM) treatment influenced the Oct4 and REX-1 expressions in 1 day, but did not significantly in 2 and 3 days. Also, Sox2 and Zfp206 expressions did not change in 1 to 3 day of melatonin treatment. Taken together, melatonin at pharmacological concentrations may affect morphology and expressions of phospho-ERK and phosphor-Akt, but not influence the expressions of Oct4, REX-1, Sox2, and Zfp206.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
The miniature pig is regarded as a better organ donor breed for xenotransplantation than other commercial pig breeds because the size of their organs is similar to that of humans. The cell-mediated xenograft rejection, including NK cells and CD8+ CTL, is a major obstacle for successful pig to human xenotransplantation. The cytotoxicity of human CD8+ CTL and NK cells against pig cells is highly detrimental and mediated at least in part by the Fas/FasL pathway. To prevent this cell-mediated xenocytotoxicity, we generated a membrane-bound form of human FasL (mFasL) as an inhibitor of CTL and NK cells cytotoxicity that cannot be cleaved by metalloproteinase producing a putative soluble form FasL. To improve efficiency of transgenic miniature pig production, we analysed the effect of breed difference between donor cell-breed and oocyte-breed on pregnancy and delivery rate, respectively. Cloned porcine embryos derived from transgenic miniature pig donor cells containing transgene were transferred to domestic or miniature recipient pigs. Pregnancy rate was higher in miniature pig recipients compared with domestic pig recipients. However, delivery rates were higher in domestic pig recipients than in miniature recipients. The domestic and miniature recipients produced one healthy transgenic pig, respectively. In cytotoxicity assay using transgenic clonal cell lines and transgenic pig's ear cells, the rate of CD8+ CTL-mediated cytotoxicity was significantly reduced in transgenic pig's ear cells (31.2 ± 11.59%) compared to normal minipig fetal fibroblasts (72.5 ± 8.42%). This result indicated that grafts of transgenic pigs expressing membrane-bound human FasL could control the cellular immune response to xenografts, and create a window of opportunity to facilitate xenograft survival. This research was supported by the BioGreen 21 Program (#PJ004200201003), Rural Development Administration, Republic of Korea.
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
NOT PRESENTED.
Biol Reprod. 2010 Nov;83(1 Suppl):3.
NOT PRESENTED.
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Despite evidence indicating that differentiating mouse embryonic stem cells (mESC) can generate oocytes and follicle-like structures in vitro, fertilizable oocytes have not been produced. Unlike endogenous primordial germ cells (PGC), which have the innate ability to form oocytes that initiate and progress through meiosis, mESC-derived germ cells exhibit many meiotic abnormalities and fail to progress through prophase. As such, isolation of early stage (pre-meiotic) ESC-derived germ cells may prove useful for identifying key processes that lead to meiotic commitment and progression. Although the timing of meiotic entry in embryonic ovaries in vivo has been well characterized, the molecular events associated with irreversible meiotic commitment are not well defined. Induction of stimulated by retinoic acid gene 8 (Stra8) is associated with the early onset of meiotic commitment, and has been previously used to isolate male germ cells from mESC cultures. In the present study, we combined a dual reporter system with germline specification by mESC to identify two distinct populations of Stra8-positive germ cells that differ in their degree of commitment to meiosis. To track germ cells, we used a karyotypic XX mESC line derived in our lab from transgenic mice expressing green fluorescent protein (GFP) driven by a fragment of the mouse Oct4 promoter in which the epiblast-specific proximal enhancer has been inactivated (TgOG2 or ΔPE-Oct4-Gfp). When undifferentiated, TgOG2 mESC are GFP positive (GFP+). Following initiation of differentiation by removal of leukemia inhibitory factor (LIF) and mouse embryonic fibroblast (MEF) feeder cells, TgOG2 mESC were transiently transfected with a DsRed2 expression vector under control of the murine Stra8 promoter. In the continued absence of LIF and MEF, the level of GFP gradually decreased such that comparatively few GFP+ cells remained detectable after 5 days; however, DsRed2-expressing cells became identifiable by fluorescence microscopy 5 days after the induction of differentiation. Subsequent isolation by fluorescence-activated cell sorting (FACS) revealed two distinct DsRed2-expressing cell populations: one that was GFP+ and one that was GFP-negative (GFP-). The sorted populations were re-plated separately and while the DsRed+/GFP- population remained quiescent, DsRed+/GFP+ germ cells actively proliferated and maintained high levels of GFP expression. These findings indicate that mESC-derived Stra8+/Oct4- germ cells commit to meiosis and lose replicative capacity; however, there is a subset of Stra8- expressing germ cells that continue to express Oct4 and retain high proliferative potential. In light of recent evidence indicating that Stra8 binds to DNA and exhibits transcriptional activity, we hypothesize that irreversible meiotic entry is a transcriptionally defined event regulated by Stra8 in those germ cells that repress Oct4 expression. (Supported by Ruth L. Kirschstein National Research Service Award F32AG034809 to D.C.W, NIH MERIT Award R37-AG012279 to J.L.T, and Vincent Memorial Research Funds).
(poster)
Biol Reprod. 2010 Nov;83(1 Suppl):3.
Conservation of germplasm from endangered species is a priority for all classes of animals. However, the large avian oocyte and the inability to consistently superovulate birds make techniques such as cloning and oocyte cryopreservation unlikely tools for avian conservation. Instead, the use of domestic birds as hosts to produce sperm of exotic species for use in artificial insemination may be a practical approach to conserve avian germplasm. During early embryonic development (stages 14-17) the primordial germ stem cells circulate in the blood prior to colonization of the gonadal ridge. We propose that transfer of adult exotic bird germ stem cells (GSCs, collected at necropsy) to host embryos of non-seasonal domestic species can provide year-round production of sperm from genetically valuable individuals. Following the death of a donor, developing host embryos to stages 14-17 requires 2.5 days. Therefore, viable donor cells must be maintained in culture until host embryos are ready for transfer. Testes from three quail (10-12 wks, young adult), one lesser flamingo (26 yrs, mid life) and one gold-breasted starling (21 yrs, geriatric) were mechanically dispersed prior to 10-day culture. Cells were cultured in DMEM/F12 medium supplemented with 15% FBS and FGF and LIF at 40°C with 6% CO2 in air. The testicular cells were stained with stem cell-specific antibodies SSEA-1, SSEA-3, SSEA-4, EMA-1, CD9, integrin (int) beta1 and int alpha6 at days 0, 2, 4, 6, 8 and 10 followed by flow cytometry to determine if stem cell-specific antigen expression changed throughout the culture period. Immunohistochemistry established location of antibody positive cells within quail and lesser flamingo testes. The patterns of antigen expression varied in a species-specific manner over time in culture. However, because the flamingo and starling cells were collected opportunistically from birds of different ages, it was not possible to exclude age as a variable in this study. In quail cultures the percentage of cells positive for most stem cell markers increased over the course of the culture: SSEA-3 (0.4-0.8), SSEA-4 (undetectable (UD)-59), EMA1 (UD-2), CD9 (UD-0.2), int alpha6 (UD-0.4) and int beta1 (UD-0.2). SSEA-1 expression was consistently high throughout culture at 87-93%. No EdU incorporation was detected by flow cytometry, indicating that cells did not proliferate in quail cultures. Although total cell numbers decreased by 58% by day 10, the number of antibody positive stem cells increased by 2 (CD9) to 60-fold (SSEA-4). In flamingo cultures the percentage of cells positive for some stem cell markers increased over the course of the culture: SSEA-4 (UD-12), int alpha6 (UD-0.7) and int beta1 (1.8-2.2). SSEA-1 decreased (16%-10%) and SSEA-3 (20%-2%). EMA1 and CD9 remained UD throughout culture. In gold-breasted starling cultures the percentage of cells positive for some stem cell markers increased over the course of the culture: SSEA-4 (UD-3), int alpha6 (UD-0.3) and int beta1 (UD-0.09). SSEA-1, EMA1 and CD9 remained UD throughout culture. SSEA-3 decreased (0.7-0.4). These preliminary data suggest that the survival of testicular stem cells may be prolonged by in vitro culture. In addition, in vitro culture may be a useful adjunct to xenotransfer studies by increasing the number of stem cells available for transfer. This culture system will provide the foundation for developing techniques to preserve the genetic material from endangered or genetically valuable specimens.
(poster)