NOT PRESENTED.
. 2010 Nov;83(1 Suppl):5.
Biol Reprod. 2010 Nov;83(1 Suppl):5.
Biol Reprod. 2010 Nov;83(1 Suppl):5.
Numerous studies have demonstrated progesterone's inhibitory effect on granulosa cell proliferation. Although the mechanisms involved are poorly understood, P4's anti-mitotic action in granulosa cells could be mediated by Progesterone Receptor Component 1 (PGRMC1). Therefore the present studies were designed to assess PGRMC1's role in regulating mitosis in spontaneously immortalized granulosa cells (SIGCs). First the cellular localization of PGRMC1 in SIGCs at different stages of mitosis was examined by co-immunostaining with PGRMC1 and β-tubulin antibodies. During interphase, PGRMC1 was localized in both nuclear and cytoplasmic compartments, where co-localization with the microtubules (β-tubulin) was not evident. On the contrary, at each stage of mitosis from prophase to cytokinesis PGRMC1 was found distributed to the spindle apparatus as indicated by its colocalization with β-tubulin. During Anaphase, Telophase, and Cytokinesis, PGRMC1 also associated with the midzone and the midbody. Given its specific cellular localization, we hypothesized that PGRMC1 plays an important role in regulating mitosis by affecting the mitotic spindle. To test this possibility, we have delivered the PGRMC1 antibody into SIGCs using the Chariot transfection reagent. Transfection efficiency was tested using fluorescent-labeled IgG and was estimated to be nearly 100%. After 18 hours of treatment with PGRMC1 antibody, cell proliferation was significantly reduced by 48% when compared with IgG control group (unpaired t-test, p<0.05). In contrast, the mitotc index (% of mitotic figures) was 61% higher in PGRMC1 antibody-treated group (unpaired t-test, p<0.05). These data indicate that the presence of the PGRMC1 antibody specifically delays the mitotic progression without affecting spindle morphology. Although the PGRMC1 antibody did not alter spindle morphology, it may influence mitosis by regulating microtubule dynamics. Therefore we tested the effect of PGRMC1 antibody on the rate at which β-tubulin depolymerized in response to cooling (0°C) and reassembled after warming to 37°C. SIGCs transfected with either PGRMC1 antibody or IgG were incubated on ice for 60 minutes, then the medium was replaced and the cells were incubated for an additional 7 minutes at 37°C. Cells were then fixed and immunostained for β-tubulin after 0, 3, 7, 15, 30 and 60 min of cooling and after 7 min of re-warming. The fluorescence intensity (FI) of the metaphase spindle was quantified and used as an indicator of the quantity of polymerized β-tubulin in each spindle. The presence of the PGRMC1 antibody did not influence the rate of β-tubulin depolymerization in response to cooling. However after re-warming, the metaphase spindles in the PGRMC1 antibody treated group reassembled to 94% of the FI of the metaphase spindle at time 0. In the IgG control group the rate of metaphase spindle reassembly was significantly slower returning to only 73% of the time 0 value (t test, p<0.05). This study indicates that, while the presence of the PGRMC1 antibody did not affect the rate of cold-induced depolymerization, it promoted a faster microtubule reassembly when compared with the control group. This suggests that PGRMC1-tubulin interaction can alters microtubule dynamics. Taken together our results suggest a crucial role of PGRMC1 in granulosa cells mitosis, possibly mediated through the regulation of a microtubule-dependent process. (Work supported in part by NIH grant 5R01HD052740)
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Biol Reprod. 2010 Nov;83(1 Suppl):5.
Follistatin is an antagonist of activin and related TGFβ superfamily ligands. It is synthesized as three protein isoforms. The longest FST315 isoform is found in the circulation while the shortest FST288 isoform is typically found in or on cells and tissues and the intermediate FST303 isoform is found in gonads, especially follicular fluid. We recently demonstrated that the FST isoforms have distinct biological actions in vitro which, taken together with their differential distribution, suggests they may also have different roles in vivo. To explore the specific role of individual FST isoforms, the Fst gene was altered so that only the FST288 isoform was produced (FST288-only). We previously reported that adult female FST288-only mice have fertility defects including reduced litter size and longer intervals between litters. Superovulation revealed that FST288-only females have fewer ovulated eggs and embryos as young adults - indicative of impaired follicular development. Consistent with this reduced fertility, FST288-only ovaries had fewer healthy antral follicles at 100-250 days of age but significantly more secondary, primary, and primordial follicles at 8.5 days of age. Interestingly, superovulation of 1-yr-old FST288-only females produced no normal eggs compared to 15 normal eggs in WT females, indicating premature cessation of ovarian activity. We have now examined the mechanisms behind the initially enlarged primordial follicle pool in neonatal FST288-only females. The number of germ cells as well as whether they were still in germ cell nests or had formed primordial follicles were counted in 0.5, 3.5, 5.5 and 8.5 day ovaries. The total numbers of germ cells was greater at all time points in FST288-only females. Double immunofluorescence staining for germ cell markers (GCNA-1 or MVH/Vasa) combined with markers of proliferation (BrdU or Ki67) failed to identify proliferating germ cells in WT or FST288-only ovaries at any age tested, although pre-granulosa and granulosa cells were vigorously proliferating in both genotypes. On the other hand, apoptosis markers (Tunel or cleaved PARP-1) revealed that germ cell apoptosis was essentially complete by days 5.5 in WT ovaries but continued until day 8.5 in FST288-only ovaries. In fact, germ cell nest breakdown itself was delayed in FST288-only ovaries such that the percentages of oocytes remaining in nests was greater at days 3.5-8.5 in FST288-only ovaries compared to WT ovaries. These results indicate that deletion of the FST315 and 303 isoforms, along with reduced expression (50% of WT) of the FST288 isoform leads to delayed germ cell nest breakdown and the accompanying germ cell apoptosis while having no effect on proliferation after birth. This altered primordial follicle formation is likely responsible for the increased number of primordial follicles observed at day 8.5 in FST288-only ovaries and perhaps also for the more rapid decline in this oocyte pool, thereby leading to premature loss of fertility in FST288-only females. Since neutralization of activin and related ligands is the only known activity of FST, these results suggest that increased activin bioactivity is at least partly responsible for these alterations in follicle development. Moreover, some of the observed fertility defects in FST288-only females resemble human premature ovarian failure (POF) so that they may be a useful model for elucidating mechanisms that lead to this syndrome.
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Biol Reprod. 2010 Nov;83(1 Suppl):5.
Androgens have well defined roles in male reproduction and prostate cancer. In contrast, other than the obligatory role of testosterone as an estradiol precursor in steroidogenesis, little is known about androgens and androgen receptor (AR) actions in the female. Interestingly, female mice with global knockout of AR expression were found to have reduced fertility with abnormal ovarian function. However, since ARs are expressed in a myriad of reproductive tissues, including the hypothalamus, pituitary, and various ovarian cells, the role of tissue-specific ARs in regulating female fertility remained unknown. To examine the importance of ovarian ARs in female reproduction, we generated granulosa cell (GC)- and oocyte-specific AR null mice by crossing AR-flox mice with MisRIIcre (granulosa cell-specific) or GDF9cre (oocyte-specific) mice. Relative to wild-type (WT) and heterozygous (AR+/-) mice, GC-specific AR-/- mice had sub-fertility and premature ovarian failure. This sub-fertility was manifested by smaller litter size and fewer litter numbers at 8-9 weeks of age, with disrupted estrous cycling and even more dramatic reduction in fertility by 24-25 weeks. Interestingly, GC-specific AR-/- mice ovulated significantly lower number of oocytes relative to AR+/- mice, possibly explaining the observed reduced litter size and numbers. Furthermore, corresponding with the reduced ovulation, the ovaries of the GC-specific AR-/- mice contained more pre-antral and atretic follicles, with fewer antral follicles and corpora lutea, relative to AR+/- mice. This accumulation of small pre-antral follicles was not due to increased recruitment of primordial follicles, as the percentage of primordial and primary follicles in GC-specific AR-/- and AR+/- mice were similar. These observations suggest that a sub-set of follicles in GC-specific AR-/- mice may be stalled in the pre-antral phase of development, thereby subjecting them to become atretic rather than develop into antral follicles, followed by ovulation and corpora lutea production. In fact, consistent with this observation that pre-antral follicles have difficulty growing and progressing through normal folliculogenesis, in-vitro growth of pre-antral follicles from GC-specific AR-/- mice was significantly slower than follicles from WT animals. Moreover, under macroscopic examination, the uteri of GC-specific AR-/- animals appeared to be normal, suggesting that the observed reproductive defects and sub-fertility are not due to changes in the uterus, but owing exclusively to lack of ARs in the GCs. In contrast to GC-specific AR-/- mice, fertility, estrous cycle, and ovarian morphology of oocyte-specific AR-/- mice were normal, although androgens no longer promoted oocyte maturation in these animals. Since follicular development and overall fertility of the oocyte-specific AR-/- mice were normal, AR expression in oocytes does not appear to be essential for reproduction. In summary, our data show that ARs in GCs, but not oocytes, regulate female fertility, perhaps by promoting pre-antral follicle growth and development, while preventing follicular atresia. In fact, nearly all the reproductive phenotypes observed in the global AR knockout mice can be explained by the lack of AR expression in GCs, suggesting that ovarian actions of androgens can have significant effects on the entire hypothalamic-pituitary-gonadal axis.
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Biol Reprod. 2010 Nov;83(1 Suppl):5.
The Forkhead boxO (Foxo) family of transcription factors is conserved from C.elegans to man and controls a wide range of cellular functions, including metabolism, cell survival and apoptosis in a cell and tissue specific manner. We have shown previously that Foxo1, Foxo3 and Foxo4 are differentially expressed in the various cell types of the mouse ovary. Foxo1 is highly expressed in granulosa cells and also oocytes (but not luteal cells) whereas Foxo3 is most abundant in oocytes but is also expressed in granulosa and luteal cells. Although these patterns of expression suggest that the FOXO factors serve important physiologic roles in granulosa cells, the critical role of Foxo3 in the maintenance of germ cell quiescence in primordial follicles, and the essential role of Foxo1 in embryologic development has significantly hindered comprehensive genetic analyses of these factors in somatic cells of the adult ovary. To overcome these limitations and gain new insights into the physiologic roles of the FOXO factors in granulosa cells, we generated Foxo1/3/4 tKO mice in which we conditionally inactivated all three Foxo genes in granulosa cells by mating mice expressing Foxo1/3/4 floxed alleles with mice expressing Amhr2-Cre. These matings have also generated the Foxo1/4 double KO and Foxo3 single KO mice. We document in Foxo1/3/4 tKO mice (by real-time RT-PCR and immuno-staining) that expression of each Foxo factor is reduced in granulosa cells whereas expression of Foxo1 and Foxo3 in oocytes remains intact. The Foxo1/3/4 tKO mice appear to be infertile based on preliminary breeding results of adult tKO females mated to fertile wild-type male mice and on the dramatic, consistent ovarian phenotype of tKO mice. Specifically, histological sections of ovaries of 2 month old tKO mice show that follicular growth and apoptosis are impaired and corpora lutea are absent. Granulosa cells within growing follicles exhibit abnormal detachment from the basal lamina but are not apoptotic. Immunohistochemical, real-time RT-PCR and in situ hybridization analyses of granulosa cell and luteal cell marker genes document that major changes in gene expression profiles occur in the Foxo mutant cells. Expression of the granulosa cell genes Inhba (activin), Cyp19a1 (aromatase), Bmp2 (BMP2), Nr5a1 (LRH1), Lhcgr (LH receptor) and the luteal cell gene Cyp11a1 (cholesterol side chain cleavage cytochrome P450) are altered dramatically both in vivo and in culture whereas expression of Fshr (FSH receptor) and Esr2 (estrogen receptor beta) remains unchanged. The Foxo1/4 and Foxo3 cKO mice are fertile indicating that there are overlapping and/or stage-specific functions of the Foxo factors in the ovary. These data indicate that FOXO factors impact granulosa cell fate decisions by regulating not only apoptosis but also other growth regulatory pathways. NIH-HD-16272 and -16229.
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Biol Reprod. 2010 Nov;83(1 Suppl):5.
A three-dimensional (3D) culture system supports development of primate preantral follicles to the antral stage with appreciable steroid production. The present study assessed: (a) whether in vitro developmental competence of follicles is age-dependent, and (b) the role of insulin in supporting folliculogenesis. Ovaries were obtained from prepubertal (n=3, 1-3 years, noncycling), young (n=6; 4-11 years), or older (n=3, 13-16 years) adult, rhesus macaques during the early follicular phase (day 1-3 of the cycle). Ovary strips were incubated in 0.1 mg/ml collagenase-DNAase-PBS at 37°C for 30 min. Secondary follicles (diameter = 120-220 μm) were isolated, encapsulated into 0.25% (w/v) alginate beads, and cultured individually for 40 days in 48-well plates containing 300 μl αMEM supplemented with 138 ng/ml recombinant human (rh) FSH, 3% human serum protein supplement, 1 mg/ml bovine fetuin, 5 μg/ml transferrin, and 5 ng/ml sodium selenite. The effects of high or low concentrations of insulin (5 or 0.05 μg/ml, respectively) were examined. Each culture condition had 12-24 follicles from each monkey. Follicles that reached antral stage were treated with 100 ng/ml rhCG for 34 h to determine competency for oocyte meiotic maturation. For follicles cultured with high-dose insulin, survival was lower (P<0.05) in follicles from older animals (15±5%) than those from prepubertal (59±8%) and young adult monkeys (55±11%). The surviving follicles were categorized as no-grow (NG, <250 μm), slow-grow (SG, 250-500 μm), and fast-grow (FG, >500 μm) according to their diameters on day 40. FG follicles were only collected from young adults which represented 43% of the total cohort; whereas 9% were NG and 48% were SG follicles. The percentage of NG and SG follicles were 14% and 86%, respectively, for the prepubertal group. There was a greater proportion of NG (71%) and a smaller proportion of SG (29%) follicles from the older animals. The SG follicles from young adults reached larger (P<0.05) diameters compared to the prepubertal group, and produced more (P<0.05) ovarian steroids than those from prepubertal and older animals. In young adults, FG follicles achieved larger (P<0.05) diameters in the presence of high-dose insulin compared to low-dose insulin. Ovarian steroid production was greater (P<0.05) in SG follicles cultured with high-dose relative to low-dose insulin. Despite hCG exposure, healthy oocytes remained at the germinal vesicle (GV) stage. More GV oocytes with larger (P<0.05) diameters were retrieved from young adults whose follicles were cultured with high-dose insulin. The results indicate that developmental competence of secondary follicles during encapsulated 3D culture is age-dependent in macaques. Follicles from prepubertal or young adults survive, grow faster, produce more ovarian steroids, and yield more healthy GV oocytes. Furthermore, insulin did not affect follicle survival, but at a high dose promoted growth of FG follicles, steroidogenesis of SG follicles, and production of healthy GV oocytes from young adults. This culture system offers an opportunity to characterize the endocrine and paracrine functions of primate follicles that influence follicle growth and oocyte maturation, with relevance to translational efforts to grow human follicles in vitro for fertility preservation in cancer survivors.
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Biol Reprod. 2010 Nov;83(1 Suppl):5.
Uterine leiomyomas are the most common pelvic neoplasms in women and are diagnosed in 25-60% of women depending on age. Available treatments for these benign tumors are limited to surgical procedures or hormonal therapy, which are associated with high costs and significant side effects. Since one of the main characteristics of these tumors is excessive collagen deposition, we decided to examine the effects of anti-fibrotic drugs as well as the use of collagen siRNAs as alternative therapeutic approaches. Primary leiomyoma smooth muscle cells (LSMCs) from patients undergoing hysterectomy were cultured on monomeric and fibrillar collagen as well as plastic dishes. While cells grown on plastic and monomeric collagen had a spindle-like morphology, cells on fibrillar collagen had extended surface areas with numerous projections. Using thymidine incorporation assays, we measured proliferation of these cells in the absence or presence of platelet derived growth factor (PDGF). In comparison to the rate of growth on plastic, monomeric collagen induced up to a 45% increase in LSMC proliferation while fibrillar collagen acted as a non-conducive matrix decreasing the rate of proliferation by as much as 75%. Treatment of LSMCs cultured on monomeric collagen with 10 ng/ml PDGF for 24 hours increased proliferation by up to 60% over similarly treated cells cultured on plastic, suggesting a synergistic effect between extracellular matrix and growth factors. We next investigated the effect of two major anti-fibrotic drugs, trichostatin A (TSA) and halofuginone (HF), on expression of collagens I and III and proliferation of LSMCs cultured on different collagen matrices. Both of these pharmacological agents were able to down-regulate the expression of collagens I and III by 95% within 6 hours. Dose response studies on these two drugs, showed that 50 nM HF and 100 nM TSA decreased the rate of proliferation by 50% in cells cultured on plastic whereas in cells cultured on monomeric collagen higher concentrations of these drugs were required to achieve 50% inhibition. Proliferation of LSMCs cultured on plastic and treated with PDGF plus one of these anti-fibrotic drugs was reduced to a greater extent than when the cells were cultured on monomeric collagen. These results indicate that the monomeric collagen matrix can partially compensate for the inhibitory effects of TSA and HF on cellular proliferation. To validate that the inhibitory effects of TSA and HF on the proliferation of LSMCs occur through specific down-regulation of collagens, we tested the effects of silencing expression of collagens I and III on LSMC proliferation. Treatment of cells with 60 nM siRNA against collagen I or III for 24 hours resulted in almost complete loss of expression of their proteins. Treatment with collagen type I siRNA reduced the rate of proliferation of cells treated with PDGF by about 20% regardless of the matrix. In contrast, treatment with collagen type III siRNA inhibited proliferation of LSMCs by 40%. These results confirm the extracellular matrix-mediated regulation of proliferation by anti-fibrotic drugs, TSA and HF. Taken together our findings demonstrate an important role for different forms of collagen in the morphology and proliferation of LSMCs and how antifibrotic agents might be used to overcome the proliferative effects of specific matrix proteins. (Supported by NICHD #PO1HD057877)
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Biol Reprod. 2010 Nov;83(1 Suppl):5.
The mouse uterus is a sensitive biological system in which to assess the mechanisms of estrogen response. Biological, biochemical and genomic events initiated by estrogens have been well-characterized and described, allowing assessment of chemicals with suspected estrogenic activity by comparison to the signature responses of proven estrogens. Some of these responses include binding to the estrogen receptor alpha (ERα), which is abundant in uterine cells, interaction with estrogen responsive enhancer DNA sequences (EREs) resulting in regulation of estrogen responsive transcripts, proliferation of the uterine epithelial cells, and increased tissue weight. Uterine tissue is comprised of several cell types, including myometrial cells on the outer margins, epithelial cells lining the inner lumen (luminal epithelium) and glands (glandular epithelium), with stromal cells in the middle compartment. All of these cell types express ERα; earlier studies using tissue recombination have suggested that estrogen-dependent epithelial cell proliferation relies on estrogen-induced stromal growth factors, such as IGF1 or EGF, which then impact the epithelial cells. We have developed a conditional knockout of ERα selective to the uterine epithelial cells (UtEpiαERKO) by intercrossing Wnt7acre and floxed ERα lines. Resulting females have uteri with all cell types present, but are infertile. Mammary glands are fully developed, reflecting normal ERα function in mammary tissue. Examination of the uteri by immunohistochemistry indicates selective ablation of ERα protein only in luminal and glandular epithelia. The administration of estrogen for 24 hours results in epithelial proliferation, with increased markers for S phase as well as M phase. Estrogen also increased the uterine weight after 3 days, but the increase was diminished in comparison to the increase seen in WT animals (4.3-fold for WT, 3.0-fold for the conditional knockout), and was inhibited by co-administration of the ER antagonist, ICI 182,780, indicating the effect is ER-mediated. Interestingly, after 3 days of estrogen treatment epithelial cells of the UtEpiαERKO had gross indications of apoptosis (blebbing) that was confirmed by increased terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) levels. The apoptosis inhibitory protein transcript, Birc1, is increased by E2 in WT but not UtEpiαERKO uteri. No increase in the epithelial-specific estrogen-responsive lactoferrin transcript was seen in the UtEpiαERKO, whereas this transcript was increased 17-fold in WT animals, supporting the epithelial specific ablation of hormone-dependent gene responses. These results demonstrate that in adult animals ERα remaining in other cells is sufficient to initiate the epithelial proliferation. Additionally, epithelial ERα is necessary to elicit a full uterine physiological response to estrogen, including water imbibition, uterine weight increase, gene regulation, inhibition of apoptosis and the hyperplasia of the endometrial tissues.
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Biol Reprod. 2010 Nov;83(1 Suppl):5.
Endometriosis is a debilitating disease where endometrium-like tissue forms lesions outside the uterus that result in chronic pelvic pain and infertility in 10-20% of women of reproductive age. Endometriosis is an estrogen-dependent disease and current medical therapies for the disease include oral contraceptives, aromatase inhibitors, and gonadotropin releasing hormone analogues that act by suppressing estrogen action. These therapies are not ideal in that they frequently produce undesirable menopause-like side effects such as loss of bone density and hot flushes and fail to restore fertility in women of reproductive age. Moreover, cessation of medical therapy results in reestablishment of the disease within a year in ~50-60% of patients. Our broad goal is to investigate new therapies for endometriotic disease. There is growing evidence that prostaglandin E2 (PGE2) can act to mediate the actions of steroid hormones to promote growth and development of endometriotic lesions. PGE2 biosynthesis is limited by cyclooxygenase-2 (COX-2) and prostaglandin E-synthase-1 (PGES-1) and its actions are mediated through G-protein coupled membrane receptors (EP1, EP2, EP3, and EP4). Rhesus macaques can develop endometriosis and provide a useful animal model for testing novel therapies for the disease. However, little is known regarding the PGE2 paracrine system in rhesus macaque endometrium. This study had two objectives. Our first objective was to characterize tissue specific expression on steroid hormone regulation of COX-2, PGES-1, and the EP receptors in eutopic and ectopic endometria from rhesus macaques. Our second objective was to assess the effect of COX-2, EP2 and EP4 on cell proliferation in rhesus macaque endometrial epithelial and stromal cells by pharmacological blockade. Samples were obtained from animals assigned to studies at the Oregon National Primate Research Center (ONPRC). Animal use was reviewed and approved by the ONPRC Institutional Animal Care and Use Committee. The samples included formaldehyde-fixed paraffin-embedded endometrium, spontaneous endometriosis and ectopically grafted endometrium (induced endometriosis) from castrated monkeys under estrogen treatment (n=3 each). Fresh samples of endometrium from spontaneous endometriosis were also used to prepare isolated epithelial and stromal cells cultures for in vitro study. We found that COX-2 and PGES-1 were expressed in glandular epithelia (GLE) and stroma (STR) and were significantly up regulated in ectopic compared to eutopic endometria. EP1 protein was present at similar levels in the GLE and STR of both ectopic and eutopic endometria. However, EP2 and EP4 proteins were expressed at greater levels in GLE and STR of ectopic compared to eutopic endometria. Expression of EP3 protein was very low or undetectable in both eutopic and ectopic macaque endometrium. Functional studies revealed that progesterone down-regulated EP4 but not EP2, COX-2 or PGES-1 in ectopic and eutopic endometria in vivo. Pharmacological blockade of COX-2 or EP2 and EP4 decreased proliferation of primary cultured endometrial GLE and STR. In conclusion, our data suggest that PGE2 may play an important role in the pathogenesis of endometriosis and targeting COX-2 or EP2 and EP4 could develop into novel non-estrogen or non-steroidal therapies for treatment of endometriosis in women. Supported by NIH grants HD18185 and RR000163 to ONPRC.
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Biol Reprod. 2010 Nov;83(1 Suppl):5.
Matrix metalloproteinases (MMPs) digest the extracellular matrix to facilitate cellular proliferation and tissue growth. The gelatinase MMP9, expressed in the neonatal and prepubertal porcine uterus, has been linked to porcine uterine growth and development. Colostrum (first milk) is rich in bioactive factors that include relaxin and estradiol, both of which can induce uterine MMP9 expression. Lactocrine signaling occurs when bioactive factors are transferred from mother to offspring as a consequence of nursing. Thus, lactocrine-acting factors are likely to affect critical developmental events in neonatal tissues. For example, ingestion of colostrum during the first 48 h of life is necessary to support normal estrogen receptor alpha (ESR1) expression in the neonatal porcine uterus, a requirement for normal uterine development. Whether nursing affects uterine MMP9 expression during this period is unknown. Moreover, effects of nursing after colostrum deprivation for 48 h from birth on the expression of specific markers and mediators of uterine development remain undefined. Here, objectives were to determine effects of: (1) age and nursing on porcine uterine MMP9 expression at birth [postnatal day (PND) 0] and on PND 2 and; (2) the age at first nursing (PND 0 or PND 2) and duration of nursing on expression of porcine uterine MMP9 and ESR1 at PND 14. For objective 1, neonatal gilts (n=4-5/group) were assigned randomly at birth to either nurse ad libitum or to receive hormone-free milk replacer via gavage (50 ml/2 h for 48 h). Uteri were collected on PND 2. In addition, uteri were obtained from gilts at birth (PND 0), prior to their consumption of colostrum. For objective 2, neonatal gilts (n=4-5/group) were: (a) allowed to nurse ad libitum; (b) nursed for 2 d from birth and switched to milk replacer; (c) fed milk replacer ad libitum; or (d) fed milk replacer for 2 d from birth and switched to nursing. Uterine tissues were collected on PND 14. MMP9 and ESR1 proteins were detected by immunoblotting using actin as a loading control. Uterine MMP9 activity was evaluated by gelatin zymography. For objective 1, uterine MMP9 protein was undetectable at birth and induced at PND 2 in nursing, but not replacer-fed animals. For objective 2, uterine MMP9 and ESR1 protein levels were similar at PND 14 in gilts that nursed for either two weeks or for only 48 h from birth. In gilts fed milk replacer for two weeks, uterine MMP9 was reduced (P<0.05) and ESR1 protein was undetectable on PND 14 in comparison to colostrum-fed gilts. Return to nursing after 48 h from birth did not restore uterine MMP9 or ESR1 signals at PND 14 to levels observed for colostrum-fed gilts. Zymographic analyses indicated an increase (P<0.05) in uterine MMP9 gelatinolytic activity in animals that nursed from birth to PND 14 in comparison to gilts that were fed replacer over the same period. Overall, results indicate that normal induction of porcine uterine MMP9 and ESR1 requires ingestion of colostrum during the first 48 h of life. Data support the idea that maternally-driven lactocrine signaling for two days from birth may be essential to establish an optimal developmental program in neonatal uterine tissues. Depriving neonates of such lactocrine-acting factors could alter the developmental trajectory of uterine tissues with negative reproductive consequences in adulthood. (Support USDA-NRI 2003-35203-1357 and 2007-35203-18098; NSF-EPS 0814103)
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Biol Reprod. 2010 Nov;83(1 Suppl):5.
The transforming growth factor β (TGFβ) superfamily, the largest family of growth factors in mammals, plays key roles in numerous biological processes. TGFβ ligands bind to their type 1 and type 2 receptors and activate intracellular SMAD transducers to initiate signal transduction. Although recent studies have achieved tremendous insights into this growth factor family in female reproduction, the functions of the receptors in vivo remain poorly defined, partially due to receptor redundancy or lethal phenotypes of genetically engineered ubiquitous null mouse models. TGFβ type 1 receptor (TGFBR1), also known as activin receptor-like kinase 5 (ALK5), is the type 1 receptor for TGFβ ligands. Tgfbr1 null mice die embryonically, precluding functional characterization of TGFBR1 postnatally. To study TGFBR1-mediated signaling in female reproduction, we generated a mouse model with conditional knockout (cKO) of the TGFBR1 in the female reproductive tract using anti-Mullerian hormone receptor type 2 (Amhr2) promoter-driven Cre-recombinase. We found that Tgfbr1 cKO females are sterile during a 6-month fertility test. To uncover the causes of the sterility, we performed histological, functional, and molecular analyses of the female reproductive tract. Strikingly, we discovered that the Tgfbr1 cKO females developed oviductal diverticuli which phenocopy the cKO of DICER1, the RNase III that processes microRNAs in the cytoplasm. These findings suggest that the microRNA pathway is derailed in the oviduct in the absence of TGFBR1. Further studies demonstrated that embryo development and transit to the uterus are severely compromised in the Tgfbr1 cKO mice due to the formation of the oviductal diverticuli. Thus, we identified essential roles of TGFβ family signaling through TGFBR1 in female reproductive tract development and function. Further understanding of the regulatory significance of TGFBR1-mediated signaling in the female reproductive tract may help to discover novel therapeutic approaches for infertility treatment. Supported National Institutes of Health Grant HD33438.
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Biol Reprod. 2010 Nov;83(1 Suppl):5.
In mice, the coordinated actions of estrogen (E) and progesterone (P) via their respective nuclear receptors lead to successful establishment of embryo implantation. It is known that ovarian E stimulates the P-primed uterus to undergo receptive transformation to initiate the attachment of blastocyst to luminal epithelium. Subsequently, uterine stromal cells undergo a unique differentiation process, known as decidualization. However, the molecular mechanisms underlying the actions of E during implantation remain unclear. In this study, we sought to identify the E-induced gene networks that operate in uterine stromal cells during implantation. Ovariectomized mice were treated with a well-established regimen of E and P that mimics the hormonal sequence prior to implantation and creates a receptive uterus. The global gene expression profiling was performed using stromal cells isolated from P-primed uterus treated with or without E. We identified several hundred genes whose expression was significantly altered in response to E. We grouped differentially expressed genes into biological pathways using Ingenuity Pathway Analysis. Strikingly, we found that the expression of 18 genes associated with cholesterol biosynthesis was significantly up regulated in stromal cells upon administration of E to P-primed uterus. These genes included several key enzymes that participate in the cholesterol biosynthesis as well as the sterol regulatory element-binding protein 2 (Srebp2), which is the master regulator that controls the expression of these enzymes. To explore the potential role of Srebp2 in implantation, we monitored its uterine expression during early pregnancy using immunohistochemistry. The Srebp2 protein was induced in uterine stromal cells on day 4 of pregnancy, and its expression was further enhanced in differentiating stromal cells at days 5-7 of gestation, suggesting that regulation of cholesterol biosynthesis is potentially critical for the decidualization process. To investigate this possibility, we utilized an in vitro decidulization system in which primary undifferentiated stromal cells, isolated from mouse uterus prior to implantation, were allowed to undergo differentiation in the presence of E and P. We observed that the expression of mRNAs encoding Srebp2 and several cholesterol biosynthesis-related enzymes was markedly elevated during the in vitro differentiation. Importantly, the level of endogenous cholesterol was markedly increased in stromal cells undergoing decidualization. Furthermore, the treatment of these cells with inhibitors of cholesterol biosynthesis resulted in the down-regulation of decidual biomarkers, such as decidual prolactin-related protein and connexin 43, indicating impairment in uterine stromal cell differentiation. Collectively, these studies uncover a unique pathway in which E stimulates the de novo biosynthesis of cholesterol by promoting Srebp2 expression. It is conceivable that the cholesterol synthesized within the pregnant uterus acts as the precursor of local production of hormonal signals critical for stromal decidualization during early pregnancy.
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Biol Reprod. 2010 Nov;83(1 Suppl):5.
During mammalian sperm capacitation, an increase in protein phosphorylation in tyrosine and serine/threonine residues has been described. The role of protein kinases, including tyrosine kinases and PKA, in this process is well documented. However, little is known about the role of protein phosphatases in this event. PP2A is serine threonine phosphatase whose activity has been detected in human and primate sperm extracts. Its role during sperm capacitation is not known. The aim of this work was to study the involvement of PP2A in the regulation of human sperm capacitation. To accomplish this, human sperm samples, obtained from normal donors according to the WHO guidelines, were selected by a Percoll gradient and then resuspended in a non-capacitating medium (modified Tyrode medium without BSA and bicarbonate). The pH was 7.4 and the osmolarity was adjusted between 280 and 300 mmol/kg. Immediately thereafter, some sperm aliquots were incubated at 37°C and 5% CO2 as follow: a) in non-capacitating medium plus 90 nM endothal; b) in capacitating medium (2.6% BSA and 25 mM bicarbonate); c) in capacitating medium plus 90 nM endothal; d) capacitating medium plus inhibitor solvent. At different periods (0, 15, 30, 60 and 300 min), sperm aliquots were withdrawn to evaluate the percent of capacitated sperm using the chlortetracycline fluorescence assay. The results indicate that incubation with endothal very rapidly increased the percent of capacitated sperm. Similar results were observed using 1 nM okadaic acid. The effect of the inhibitors took place only when the sperm were incubated in capacitating medium. The increase in sperm capacitation was especially impressive during the first half hour of incubation; thereafter, there was no difference between sperm incubated in capacitating medium plus the inhibitors versus sperm incubated in capacitating medium without the inhibitors. In addition, the effect of endothal was evidenced by an increase in protein phosphorylation in threonine residues by mean of western blot. Using a polyclonal rabbit antibody the presence of PP2A was confirmed in human sperm. These results suggest that PP2A may have an important role in regulating the initial events of the human sperm capacitation process. This research was supported by Fondecyt Project 1080028.
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Biol Reprod. 2010 Nov;83(1 Suppl):5.
Spermatozoa interact with different isoforms of glycodelin during their passage towards the oocytes in the female reproductive tract. The only known action of glycodelin-A, one of the glycodelin isoforms, is inhibition of spermatozoa-zona pellucida binding, an action paradoxical to general belief that the maternal body should promote fertilization. Therefore, we studied other actions of glycodelin-A on spermatozoa. The objective of this report was to investigate the mechanism of action of glycodelin-A on zona pellucida-induced acrosome reaction, a crucial step in the fertilization process. Glycodelin-A did not affect spontaneous acrosome reaction. Pre-treatment of human spermatozoa with glycodelin-A enhanced zona pellucida-induced acrosome reaction, i.e. the increase in the percentage of acrosome-reacted spermatozoa after sequential glycodelin-A and zona pellucida treatment was significantly higher than the sum of treatments with glycodelin-A and zona pellucida alone. Native human zona pellucida protein-3 (ZP3) and ZP4 were purified. Glycodelin-A primed ZP3-, but not ZP4-induced acrosome reaction. Other glycodelin isoforms and deglycosylated glycodelin-A did not have such priming activity. Glycodelin-A treatment increased cAMP level and protein kinase-A (PKA) activity, but decreased that of extracellular signal-regulated kinase (ERK). Treatment with inhibitors of ERK simulated the priming activity of glycodelin-A. Calcium influx is an important event in zona pellucida-induced acrosome reaction. Therefore, the action of glycodelin-A on calcium influx was studied by fluorescence imaging of individual spermatozoon using Fluo-4AM dye. Compared with the untreated control, glycodelin-A induced a stronger and more rapid calcium influx in spermatozoa upon exposure to solubilized zona pellucida. In conclusion, glycodelin-A sensitizes human spermatozoa for zona pellucida-induced acrosome reaction through activation of the cAMP/PKA-ERK signaling leading to enhanced zona pellucida-induced calcium influx. (This work is supported in part by a research grant from the University of Hong Kong.)
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Biol Reprod. 2010 Nov;83(1 Suppl):5.
The oscillation in the intracellular free Ca2+ and the depletion of the intracellular stores stimulates an influx of extracellular Ca2+. This Ca2+ influx is responsible for sustaining the long-lasting Ca2+ oscillation. A previous report from our lab indicates that STIM1, a known Ca2+ sensor in somatic cells is expressed in porcine oocytes and is essential for store-operated Ca2+ entry. Because a Ca2+ entry through the plasma membrane is critical for the maintenance of Ca2+ oscillation during fertilization, we hypothesized that STIM1 function has implications for subsequent embryo development. In this study, the inactivation of STIM1 in oocytes and its effect on early embryo development after fertilization was investigated using the pig as a model. Gilt ovaries were obtained at a local abattoir and immature oocytes were collected by aspirating mid-size follicles. The oocytes were matured in vitro in a TCM199-based medium. First, matured oocytes (34 hours after the beginning of maturation) were denuded, allocated into three groups and injected with either physiological saline (control), non-functional control siRNA, or siRNA against STIM1. Approximately 10 h later, injected oocytes were fertilized and cultured in Porcine Zygote Medium 3 (PZM-3) for 7 days. Frequencies of cleaved embryos and blastocyst formation were recorded and analyzed by ANOVA following with acrsin transformation. For the second experiment, oocytes were fertilized at 42-44 hours after the beginning of maturation, and then injected with saline or siRNA against STIM1 at 5 hours post fertilization. These embryos were also cultured for 7 days and the frequency of cleaved embryos was compared by chi-square analysis. Down regulation of STIM1 prior to fertilization was detrimental to embryo development. Frequencies of cleaved embryos after injections with saline, control siRNA, and siRNA against STIM1 were 67.8%, 72.5%, and 25.8%, respectively. Cleavage frequency in embryos injected with STIM1 siRNA was lower compared to that of the other groups (p<0.01). Similar tendencies were observed in blastocyst formation. Only 0.8% of embryos formed blastocyst after STIM1 siRNA injection, whereas blastocyst formation in the saline- and control siRNA-injected groups were 14.9% and 14.2%, respectively (p<0.01). When siRNA against STIM1 was delivered into oocytes 5 hours after fertilization, embryo cleavage was not affected: the proportions of 2-cell embryos after injection of saline or STIM1 siRNA were 66.0% and 72.9%, respectively. Our results indicate that STIM1 plays a critical role during fertilization and normal STIM1 function has an effect on subsequent embryo development. The fact that the absence of STIM1 affects embryo development only when inactivation takes place prior to fertilization indicates that STIM1 is essential during fertilization. On the other hand, STIM1 inactivation 5 h post fertilization had no detrimental effect on embryo development. These findings support our hypothesis that STIM1 is involved in the maintenance of Ca2+ oscillation during fertilization. The exact role of STIM1 in Ca2+ signaling during fertilization is under investigation.
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Biol Reprod. 2010 Nov;83(1 Suppl):5.
Recently it was shown that actin polymerization occurs during sperm capacitation. On the other hand, fast disassembly of actin filament is necessary for the acrosome reaction to occur. In the present study we demonstrate a role for gelsolin in the regulation of actin polymerization/depolymerization in human spermatozoa during capacitation and the acrosome reaction. We found that at the beginning of the capacitation, gelsolin is colocalized with G-actin in the Triton-X-100 soluble fraction, whereas at the end of capacitation most of the geloslin is colocalized with F-actin in the insoluble fraction. Furthermore, we showed that gelsolin translocates from the sperm tail to the head during capacitation, according to the increase in F-actin in the head. Interestingly, this increase of gelsolin in the head cannot be seen when intracellular calcium was celated by BAPTA/AM, indicating the importance of calcium ions for this translocation. The release of gelsolin bound to PIP2 by PBP10, a peptide containing PIP2-binding domain of gelsolin, caused fast calcium-dependent F-actin depolymerization as well as enhanced acrosome reaction rate. These effects could also be seen by increasing phospholipase C activity which hydrolyzes PIP2 resulting in the release of bound gelsolin to the cytosol. Using the immunoprecipitation method we showed that the tyrosine kinase SRC and gelsolin form a complex in human sperm. Moreover, activating SRC by adding 8-Br-cAMP revealed an increase in the complex level. Addition of 8-Br-cAMP also increased F-actin formation that was inhibited by the SRC inhibitor PP1, suggesting that the formation of this complex leads to the inhibition of gelsolin. This suggestion is supported by showing that PBP10 enabls to cause complete F-actin breakdown in the presence of 8-Br-cAMP or vanadate. Thus, the data suggest that activation of SRC causes inhibition of gelsolin probably due tyrosine phosphorylation of this protein. In conclusion, we suggest that actin polymerization during capacitation can occur due to the inactivation of gelsolin by its tyrosine phosphorylation and binding to PIP2. Prior to the acrosome reaction, gelsolin is activated by its dephosphorylation and release from its binding to PIP2. This activation of gelsolin is important step for Acrosome reaction to occur in the end of capasitetion.
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Biol Reprod. 2010 Nov;83(1 Suppl):5.
The Ubiquitin-Proteasome Pathway (UPP) is considered vital to mammalian fertilization as it participates in sperm capacitation, acrosomal exocytosis (AE), and sperm-zona pellucida (ZP) penetration. Ubiquitin binds in tandem to a variety of other proteins and targets them for proteolysis by a multi-subunit protease, the 26S proteasome. Proteasome inhibition stops fertilization. When presented with a proteasomal inhibitor cocktail (PIC), solubilized zona pellucida proteins (ZPP) are protected from proteasomal degradation by capacitated boar spermatozoa or by the proteasomes isolated from boar sperm acrosomes. To further understand the participation of sperm proteasomes in fertilization, we designed an experimentally consistent in vitro system in which 10,000 live, freshly collected (never cryospreserved), capacitated boar spermatozoa are co-incubated with ZPP solubilized by non-degrading/non-reducing methods from 100 meiotically mature porcine oocytes. Upon co-incubation, the soluble ZPP bind to sperm acrosomes as they would during fertilization and induce the process of acrosomal exocytosis that results in the formation of the membrane vesicle-composed "acrosomal shroud" this enables the exposure of acrosomal enzymes to ZPP. The capacitated boar spermatozoa were incubated with solubilized porcine ZP proteins with or without PIC composed of MG 132, CLβL and epoxomicin. After the two-hour coincubation, the entire reaction volume was incubated with a fluorescent marker of the exposed/remodeled outer acrosomal membrane, lectin PNA. Spermatozoa coincubated with ZPP under a variety of conditions, as well as those labeled with PNA before and after capacitation, were analyzed for intensity and number of intact, detached acrosomal shrouds using the Guava EasyCyte Plus flow cytometer (IMV Technologies). Acrosomal membrane remodeling was monitored by epifluoresce microscopy, and by conventional and environmental scanning electron microscopy (SEM & ESEM, respectively). Proteasomal degradation of acrosomal shroud proteins was examined by tandem mass spectrometry (LC-MS/MS). Results showed that when in the presence of the PIC, significantly more spermatozoa retained their acrosomal shrouds. Those acrosomal shrouds that detached from spermatozoa after sperm-ZPP coincubation seemed to disintegrate in vehicle control. In contrast, the number of intact, detached shrouds was significantly higher in the presence of PIC (ANOVA; p<0.05). Rippling and vesiculation of the OAM, as induced by sperm-ZPP coincubation and visualized by SEM/ESEM, was similar to that observed during IVF. Several acrosomal shroud proteins were protected from proteasomal degradation by specific proteasomal inhibitors, including SPAM1, MFGE8 (aliases lactadherin, SED1, SP47), ZPBP2 (alias IAM38) and ACRBP (alias SP32); the Angiotensin I Converting Enzyme 1 Isoform 2/ACE2 was more abundant in the vehicle group. Our results indicate that proteasomal inhibitors block fertilization by: 1) preventing the ZP-induced acrosome remodeling dependent on acrosome-borne proteasomes, and 2) directly inhibiting ZPP degradation by acrosomal proteasomes during sperm-ZP penetration. This work provides tools to modulate fertilization in vitro and points to a new direction in which to contemplate mammalian fertilization and non-hormonal contraceptive design. Supported by USDA-NRI grant #2007-01319 and by seed funding from the F21C Program of UM (PS).
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Biol Reprod. 2010 Nov;83(1 Suppl):5.
KLHL10 is a testis-specific protein exclusively expressed in haploid germ cells (spermatids) during spermatogenesis. Inactivation of Klhl10 leads to disruption of spermiogenesis and complete male infertility in mice. Our previous investigation has discovered that CULLIN3 (CUL3), a well-known ubiquitin-conjugating enzyme (E2)-recruiting protein, interacts with the BTB domain of KLHL10, suggesting a potential role of KLHL10 as a scaffold protein in this spermatid-specific CUL3-based E3 ligase complex. In most of the CUL3-BTB/Kelch E3 ligase complexes, kelch domain usually functions as a substrate recruiting domain. We therefore used yeast 2-hybrid assays and screened an adult mouse testis library to identify testicular proteins that interact with the kelch domain of KLHL10. We identified numerous Kelch repeats-interacting proteins, and spermatogenesis-associated proteins 3 (SPATA3) and 6 (SPATA6) were two among those proteins. Here we report our preliminary characterization of these two KLHL10-interacting proteins in the mouse testis. In vitro co-immunoprecipitation confirmed their interactions, and domain deletion studies further revealed six Kelch repeats are the only required domain for their interactions. Multi-tissue RT-PCR analyses showed that Spata3 was exclusively expressed in the testis, whereas Spata6 displayed a ubiquitous expression pattern. During mouse testicular development, the onset of Spata3 mRNA expression was at ~ postnatal day 28 (P28), while Spata6 mRNAs were detected at all ages examined. Using purified testicular cell types, we also investigated the cellular origin of Spata3 and Spata6 transcripts. Spata3 mRNA was detected at lower levels in pachytene spermatocytes and its levels peaked in spermatids. Spata3 mRNA was not detected in any other cell types within the testis. In contrast, Spata6 mRNA was detected in all cell types within the testis and the highest levels were detected in spermatids. Protein expression and localization studies are underway and the effects of KLHL10 inactivation on the turnover of these two proteins are also being investigated. Our data start to shed light on the KLHL10-mediated protein ubiquitination pathway during spermiogenesis. This work is supported by a grant from the NICHD (HD050281).
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Biol Reprod. 2010 Nov;83(1 Suppl):5.
Betaglycan (TGFBR3) is an accessory receptor that modulates the activities of members of the TGFβ superfamily of growth factors which regulate many aspects of reproductive biology. We have previously reported the presence of betaglycan on somatic and germ cells in fetal murine ovary from 12.5 dpc. However, the role of this receptor in early ovarian development is poorly understood. We hypothesised that, given its expression pattern, betaglycan could serve as a TGFβ accessory receptor on both oogonia and somatic cells during early ovarian development. In the current study, we examined the Tgfbr3 (betaglycan) null and wildtype ovaries from 11.5-14.5 dpc. During this window of development, no overt morphological phenotype was detected, and cell proliferation (PCNA immunostaining) and apoptosis (active caspase 3 immunostaining) were unchanged in the null ovary. To determine whether somatic and/or germ cell markers were altered in the absence of betaglycan, quantitative real time PCR analysis was conducted using total RNA derived from 11.5-14.5 dpc betaglycan wildtype, heterozygous, and null ovaries (n=3 at each age/genotype). At the time of sex determination 12.5 dpc, a (20%-30%) transient decrease of the mRNA expression of the germ cell markers Oct4 and Mvh was observed (P<0.05). After 12.5 dpc, genes associated with the differentiation of the female somatic lineages Wnt4, Fst, Bmp2 were significantly decreased in the null ovary by 30%-50% (P<0.05) while germ cell markers (Oct4, Mvh) showed no significant changes compared to wildtype ovary. These data were verified at the protein level by immunohistochemistry. By 14.5 dpc, mRNA and protein expression of the meiotic chromosome synapsis marker (Sycp3) was reduced in the null ovary (70%, P<0.01) consistent with a delay in entry into meiosis, a later block of meiosis or a reduction in the numbers of germ cells. Genes associated with the testis-differentiation pathway (e.g. Sox9, Cyp11a1, Cyp17a1, Hsd3b, Insl3, Dhh) were not aberrantly expressed in betaglycan null or heterozygous ovaries, with expression levels very low to undetectable. Collectively, these data suggest that loss of the receptor betaglycan results in a disruption to the differentiation of ovarian somatic cells and germ cell entry into meiosis.
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Biol Reprod. 2010 Nov;83(1 Suppl):5.
Any perturbation affecting normal development of ovaries during embryogenesis can lead to reproductive disorders such as infertility, premature ovarian failure or ovarian cancer. Recent studies have implicated signaling through Rspo1, Wnt4, and β-catenin as a critical event necessary for normal fetal ovarian development. Unfortunately, compared to fetal testis development, still little is known about molecular factors necessary for fetal ovarian development. To obtain better insight into this important process, whole genome gene expression profiling experiments have been conducted using whole ovaries and ovarian somatic cells. We recently reported on the use of a unique transgenic mouse strain that enabled us to isolate and perform a genome expression profiling experiment specifically on the precursor granulosa cell population. Data analysis from this experiment revealed that several guanine nucleotide exchange factors called dedicator of cytokinesis (Dock) genes are up-regulated in ovarian precursor granulosa cells compared to fetal testicular Sertoli cells. Of particular interest is the recent observation that DOCK4 is associated with the Wnt/β-catenin signaling pathway, and localizes to an eQTL involved in mammalian fetal ovarian development. We tested the hypothesis that Dock4 and Dock9 are expressed and necessary for proper fetal ovarian development. Real time RT-PCR analysis revealed that Dock4 is expressed during fetal ovarian development. Furthermore, whole mount immunohistochemistry revealed a center-to-pole pattern of expression for Dock4 in fetal ovaries between embryonic day (E) 11.5 and E13.5. Finally, both microarray and real time RT-PCR analysis identified Dock9 as a second Dock gene highly expressed during fetal ovarian development. We conclude that Dock4 and Dock9 are potential regulators of fetal granulosa cell differentiation and ovarian development. Funded by Colorado State University start-up funds.
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Biol Reprod. 2010 Nov;83(1 Suppl):5.
Cell fate specification begins in preimplantation embryo with the emergence of trophectoderm (TE). Primitive endoderm (PE) is the second extraembryonic lineage to form from cells within the inner cell mass (ICM) during embryogenesis. Ruminant conceptuses elongate dramatically prior to uterine attachment and extensive migration and proliferation of TE and PE is crucial for normal conceptus development and elongation. Work described here provides evidence that FGF2 and potentially other FGFs impact PE development during bovine embryonic development. Bovine blastocyst outgrowths were generated from in vitro-produced embryos using a feeder-layer free culture system containing a Matrigel-coated surface. Individually cultured bovine blastocysts were supplemented with FGF2 at the beginning of culture (day 8 post-IVF) and identification of PE-like cells was evaluated microscopically on days 13 and 15. In controls, only a single blastocyst outgrowth contained what appeared morphologically as PE cells (1.3% of all outgrowths). By contrast, 10.5±6.7%, 19.7±0.28% and 22.4±0.26% of outgrowths contained PE colonies on day 15 when embryos were exposed to 0.5, 5 and 50ng/ml FGF2, respectively. A subset of outgrowths (n=6 TE only and 6 PE-containing) were selected and cultured to day 20, then lineage-specific markers were evaluated using qRT-PCR. Transcripts for IFNT and CDX2 (TE markers) were present in TE outgrowths. Trace amounts of IFNT and CDX2 mRNA were detected in PE colonies (P<0.05) likely because residual TE cells still existed in some of these outgrowths. PE-containing outgrowths contained ample quantities of GATA4 and GATA6 mRNA (putative PE markers) whereas TE outgrowths contained no GATA4 and low amounts of GATA6 mRNA. None of the outgrowths contained NANOG mRNA whereas all TE and PE outgrowths contained OCT4 mRNA. Western blot analysis determined PE-containing outgrowths produced transferrin, a PE product. Moreover, immunoreactive GATA4 localized to nuclei of PE cells but was not detected in TE cells. By contrast, CDX2 localized to TE nuclei but was not detected in PE cells. Continued cultivation and selection for TE and PE yielded pure cell populations (n=6 of each). Differences in relative abundance of FGF receptors (FGFRs) and selective alternatively spliced receptor isoforms were evident between the cell types. Specifically, transcripts for FGFR2b and FGFR3 predominated in TE cells while FGFR1b represented the primary transcript in PE cells. TE and PE cells responded differently to FGF2 treatment. FGF2 supplementation did not affect the mitotic index of TE cells whereas the mitotic index of PE cells increased (P<0.05) following exposure to 5 or 50ng/ml FGF2 (35.1±0.09% and 34.7±0.11%, respectively versus 19.9±0.06% for controls). These observations indicate that the non-TE cells formed with blastocyst exposure to FGF2 are PE, and these cells respond differently to FGF2 than TE. Collectively, this work provides new insight into another vital activity for FGF2 and potentially other FGFs during early embryonic development in bovine. 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):5.
Studies using embryonic stem (ES) cells and gene targeting have identified Sox2 as one of the key genes controlling the transcriptional networks required for pluripotency. Expression of Sox2 is tightly controlled and small changes in Sox2 expression leads to differentiation of ES cells. Forced expression of Sox2 with three other transcription factors--Pou5f1, c-Myc, and Klf4--can convert highly differentiated somatic cells to a pluripotent state, although at a poor efficiency. To date, the role of Sox2 on development of a differentiated oocyte to totipotent blastomeres of a preimplantation embryo is unknown. Sox2, which is highly localized in the nucleus, was first zygotically expressed during the 2-cell stage but its expression dramatically increased between the morula and blastocyst stages. Injecting a cRNA encoding Sox2 into 1-cell embryos resulted in over-expression of SOX2 and developmental arrest at the 2-cell stage, whereas injecting cRNAs encoding Pou5f1, c-Myc and Klf4 had little effect on development. Injecting a 2-cell blastomere with a cRNA encoding Sox2 inhibited cleavage of the injected blastomere, but had no effect on cleavage of the uninjected blastomere. Global transcription, as assessed by BrUTP incorporation was reduced by ~25% in 2-cell embryos over-expressing SOX2, and transcript profiling of 2-cell embryos revealed that about one third of the genes zygotically expressed in 2-cell embryos were inhibited when SOX2 was over-expressed. These results suggest that SOX2 functions mainly as a transcriptional repressor during the course of genome activation. Analysis of the genes whose expression was perturbed by over-expressing SOX2 revealed a subset of genes known to be directly regulated by SOX2 in ES cells. Results of these experiments implicate Sox2 in conversion of a highly differentiated oocyte into totipotent blastomeres and that the amount of Sox2 is critical for this transition.
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Biol Reprod. 2010 Nov;83(1 Suppl):5.
Gestational administration of testosterone increases ovarian androgen receptor protein expression in the fetus and culminates in hypersecretion of luteinizing hormone (LH), hyperestrogenism, persistent ovarian follicles, and oligo-/anovulation in adults. Since steroidogenic potential of the ovary gets established during fetal life, we hypothesized that maternal testosterone excess also alters steroidogenic enzyme expression within the fetal ovine ovary. Pregnant Suffolk ewes were treated with testosterone proprionate (T, 100 mg, twice weekly) or T + the androgen receptor antagonist, flutamide (15 mg/kg BW daily; T+F,) from days 30 to 90 of gestation. Fetal ovaries were collected on d65 (n=4, 5, and 5 for T, T+F, and control [C] groups, respectively) and d90 (n=5, 7, 4) of gestation and snap frozen. Ovarian RNA was subjected to quantitative RT-PCR to measure expression of steroidogenic acute regulatory protein (StAR), cytochrome P450 side chain cleavage (Cyp11A1), cytochrome P450 17α-hydroxylase (Cyp17), cytochrome P450 aromatase (Cyp19), 3-β-hydroxysteroid dehydrogenase (3βHSD), 5α-reductase, androgen receptor (AR), estrogen receptors (ER) α and β, and progesterone receptor (PR). Data were analyzed by 2-way ANOVA followed by Bonferroni post-hoc statistical tests. Maternal T had no effect on AR, PR, ERα and ERβ mRNA expression but levels of ERα and AR mRNA were higher in d90 than d65 ovaries. There was no age effect on steroidogenic enzyme expression in control ovaries. Gestational T excess increased (P<0.05) Cyp19 (3.9-fold) and 5α-reductase (1.8-fold) expression in fetal d65 ovaries but decreased (P<0.05) Cyp11A1 expression (3.7 fold) in d90 ovaries. Maternal T excess had no effect on mRNA expression of other steroidogenic enzymes. Co-treatment with the androgen antagonist, flutamide, prevented the T-induced decrease in Cyp11A1 mRNA seen at d90, but not the increase in Cyp19 and 5α-reductase seen in d65 ovaries. Co-treatment with T+F also increased (P<0.05) StAR (2.0-fold) and Cyp11A1 (3.0-fold) expression in d65 ovaries, compared to C and T-treated ovaries. Our findings suggest that the effects of gestational T treatment on Cyp11A1 is mediated by androgenic actions of T and the effects on Cyp19 and 5α-reductase likely via estrogenic actions stemming from aromatization of T to estrogen and that these changes are fetal age dependent. Increased StAR and Cyp11A1 mRNA expression at d65 in the T+F ovaries, but not the T-alone ovaries, indicates that AR is primarily repressing expression of these two transcripts. Additionally, the increased potential for estrogen metabolism (i.e., increased Cyp19) in the d65 T-alone but not T+F ovaries indicates that estrogenic effects can only be manifest if the AR is blocked. Evidence for estrogenic effect is consistent with our recent finding of increased estradiol levels in the fetal blood of day 65 and 90 T-treated fetuses. These studies demonstrate that maternal T treatment alters fetal ovarian steroidogenic gene expression and implicate direct actions of estrogens in addition to androgens in the reprogramming of fetal developmental trajectory leading up to adult reproductive pathologies. Supported by NIH P01 HD44232 (VP).
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Biol Reprod. 2010 Nov;83(1 Suppl):5.
It is widely accepted that maternal dietary alterations before conception and during gestation can result in reduced fecundity, compromised embryo and fetal development, as well as potential adult health concerns. One of the first and crucial requirements for viable development is the acquisition of oocyte competence. Indeed, oocyte quality is a primary contributor to human infertility. This study investigated the impact of maternal protein diet directly on mammalian oogenesis over three increasing time points. Six week old outbred female mice were randomly allocated to three maternal diet groups: control diet for breeding and reproduction (20% protein), 14% low protein diet (LPD) or 25% high protein diet (HPD) for 2, 6 and 10 weeks prior to oocyte collection or mating. Oocyte maturation rates showed a significant increase in the number of immature and degenerate oocytes compared to controls after more than 2 weeks on HPD (2 wk control = 5.5% vs 2 wk HPD = 6% NS; 6 wk control = 11.3% vs 6 wk HPD = 21.2% P<0.05; 10 wk control = 8.4% vs 10 wk HPD = 19.6% P<0.05). Quantitative real-time PCR was performed on individual MII oocytes from each group relative to an internal house keeping gene, Gapdh for the following key oocyte genes: Bmp15, Gdf9, Mos, Npm2, Zfp36l2 and Zp2. Differential MII oocyte gene expression profiles were observed following consumption of HPD at each time point compared to controls. All genes except Gdf9 were upregulated following 2 wk and 6 wk HPD (P<0.05), while at the 10 wk HPD interval all key oocyte genes were upregulated compared to controls (P<0.05). There were no differences observed in oocyte maturity or gene expression levels with LPD at any time point. Female CF1 mice that were mated following preconception maternal protein diet displayed similar plugging and absorption rates across all groups. The Codelink Whole Genome Mouse Bioarray (Applied Microarrays) was chosen for placental transcriptome analysis with data normalization and statistical analysis performed by Genespring software (Agilent Technologies). Unsupervised hierarchical clustering of individual placental samples completely separated the control and test groups with three distinct branches. However, significant differences in gene expression profiles were only observed following the 10 wk preconception maternal protein diet. Transcriptome analysis of biopsied placenta revealed 2,678 differentially expressed transcripts following 10 wk LPD and 1,366 following 10 wk HPD, compared to 10 wk controls (P<0.05). A total of 2,305 transcripts were differentially expressed between the diet test groups (P<0.05). Gene ontology annotation revealed predominant biological processes to include protein modification, cell proliferation, development, DNA and RNA metabolism, and response to stress. Additionally, 10% of the altered transcripts were identified with an unknown biological process or function. Our results demonstrate that the maternal protein diet during the time of oogenesis can directly influence oocyte quality. The consumption of a HPD had a direct effect on oocyte maturation, as well as oocyte and placental gene expression, while the consumption of a LPD prior to conception appeared to impact development at later stages with alterations observed in the placental transcriptome. Further understanding of maternal dietary impacts during oogenesis, including the underlying mechanisms, have the potential to improve reproductive success.
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Biol Reprod. 2010 Nov;83(1 Suppl):5.
Female obesity is associated with decreased fecundity with increased time-to-pregnancy and decreased chance of pregnancy. In obesity, accumulation of lipid in non-adipose tissues, or lipotoxicity, is associated with endoplasmic reticulum (ER) stress, mitochondrial dysfunction and ultimately apoptosis. We have previously shown that obese women have increased triglycerides in follicular fluid; thus, the present study examined whether diet-induced obesity causes lipotoxicity in granulosa cells and the cumulus oocyte complex (COC). COCs and granulosa cells were collected from ovaries of adult mice fed high fat or control diet for 4 weeks. COCs and oocytes were assessed for 1) lipid content by neutral lipid stain BODIPY 493/503; 2) ER homeostasis by measuring expression of ER stress marker genes; 3) Mitochondrial function by measuring membrane potential; and 4) apoptosis by DNA laddering and TUNEL assays. Quantification of lipid levels showed that in immature COCs lipid content was significantly higher in both cumulus cells and oocytes of mice fed high fat diet compared to control. Expressed as total lipid, oocytes from mice fed high fat diet had markedly increased lipid content compared to oocytes of mice fed control diet both before and following ovulation. The presence of ER stress was determined by measuring the expression of ER stress marker genes (ATF4, GRP78, CHOP10 and Hsp70) in COCs isolated from preovulatory follicles of eCG-treated mice. There were significantly higher levels of ATF4 and GRP78 expression in COCs from high fat diet fed mice relative to control. Mitochondrial damage initially manifests as a decrease in the mitochondrial membrane potential (MMP), determined by the ratio of red to green fluorescence using JC-1 probe. In mice fed high fat diet, the mean ratio of red/green fluorescence was significantly reduced in both immature, preovulatory, and ovulated oocytes compared to oocytes from control diet mice, demonstrating that oocytes from high fat diet fed mice have reduced MMP, indicative of mitochondrial damage. The incidence of apoptosis in granulosa cells and COCs of preovulatory follicles was investigated. DNA laddering assay showed that granulosa cells from high fat diet fed mice have greater 180bp and 360bp band intensities compared to cells from control diet fed mice, indicating increased DNA fragmentation. TUNEL assay on ovarian sections confirmed this result by demonstrating more TUNEL-positive cells within the ovarian follicles of mice fed high fat diet compared to control diet. The apoptotic index reached 44.03% in COCs and 31.83% in granulosa cells from high fat diet fed mice compared to 7.6% and 7.65%, respectively, in control diet fed mice. Indicators of lipotoxicity were analyzed in ovarian cells of women of varying BMI in order to determine whether lipotoxicity pathways may be activated in the human ovary in response to obesity. ATF4 expression was significantly increased in granulosa cells from obese women compared to granulosa cells of moderate or overweight women, suggesting that ER stress pathways are activated in granulosa cells of obese women. Thus, lipid accumulation, ER stress, mitochondrial dysfunction and apoptosis are markedly increased in ovarian cells of mice fed HFD. ER stress is also increased in granulosa cells from obese women. These results indicate that lipotoxicity may contribute to the reduced pregnancy rates observed in response to obesity.
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Biol Reprod. 2010 Nov;83(1 Suppl):5.
Assisted reproductive technologies are now routinely employed for laboratory animals, livestock, and in humans. Concerns have been raised, however, that transfer of blastocysts after culture could lead to an unintentional bias in the sex of the resulting offspring. In cattle, for example, culture of zygotes in presence of glucose at concentrations >2.5 mM leads to pronounced skewing of sex ratio towards males among resulting blastocysts. In addition, most successful human IVF programs now utilize blastocyst stage embryos to ensure that the embryos are developmentally competent through the cleavage stages, but there have been several reports of a distinct male bias in babies born after selection of such advanced embryos for uterine transfer. Finally, there have been numerous reports that male embryos have a faster growth rate during pre-implantation than females in a wide range of species, although contradictory data have also appeared. In litter-bearing species, such as the mouse or pig, where more eggs are fertilized than develop to term, uterine conditions that enhance sexually dimorphic differences in growth could provide a competitive advantage to one sex over the other in their ability to establish adequate placentation. Here, we have compared the effects of culture in presence of high (5.6 mM) and low (0.2 mM) D-glucose concentrations (in KSOM medium with amino acids) on sex ratio and cell numbers among blastocysts derived from CD1 mice, and the subsequent effects of embryo transfer on numbers of male and female pups born. In Experiment 1, CD1 females on a Purina 5015 Chow diet were bred to CD1 males and 1-cell embryos collected on the morning of the coital plug. Embryos from different dams were randomized and placed in culture under the two glucose conditions. Those embryos that had reached the blastocyst stage after 96 h were collected. Embryo sex was determined by XY fluorescence in situ hybridization and cell numbers assessed based on DAPI nuclear staining. No differences were observed in sex ratio for embryos cultured under either low (92 females and 113 males) or high glucose (111 females and 108 males). However, males cultured under both low and high glucose had higher cell numbers than females under the same conditions (Low: males 53.0 ± 1.3, females 48.3 ± 1.4; High: males 57.5 ± 1.3, females 51.2 ± 1.3; P<0.05). In Experiment 2, we determined whether this increased cell number provided male conceptuses an advantage over females. CD1 zygotes were cultured to blastocyst under high glucose conditions and transferred to the uteri of surrogate CD1 females "bred" 2.5 days earlier to vasectomized males. To replicate crowded uterine conditions, ~10 embryos were transferred into each uterine horn. The sex of resulting pups was determined at day 2 and 21 days of age. A total of 55 male and 34 female (62% males/38% females; P<0.02) pups were born from 14 litters, clearly indicating that males were at an advantage under these experimental conditions. However, it remains unclear whether the skewed sex ratio resulted from the more rapid pre-implantation growth of males, the intense competition for space resulting from embryo transfer to create crowded conditions, or to both. Experiments are underway to distinguish these possibilities. However, the experiments suggest that, in mice, sexually dimorphic responses to nutrient availability could skew sex ratios of offspring born. Research supported by NIH Grants HD 44042.
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Biol Reprod. 2010 Nov;83(1 Suppl):5.
The extent to which maternal nutrition alters placental and fetal development through epigenetic mechanisms, is still uncertain. In an established experimental model using the catabolic primiparous sow, feed restriction during lactation affects embryonic survival and development during the subsequent gestation. In a continuation of these studies, primiparous sows (n=34) were either fully fed (Control:C) or restricted to 60% of voluntary feed intake (Restrict:R) during the third week of a 21-d lactation and re-bred after weaning. Sows were euthanized at d 30 of gestation and reproductive and litter characteristics were recorded. Embryos and placentae (n=488 of each) were collected for each sow, and a sample of endometrium (n=68) was taken from the left and right horn from each sow, frozen immediately in liquid nitrogen and stored at -80°C. Total RNA was extracted from placental, endometrial and embryonic samples collected, DNAse-treated, reverse transcribed, and real-time PCR was used to determine transcript abundance of candidate genes involved in regulating endometrial and placental vascularity and embryonic muscle development. The relative deltaCt values were calculated using cyclophilin as an internal control, and data were analyzed using mixed models analysis with treatment and sex of embryo as independent variables and sow as a random variable. There were no differences in ovulation rate, embryonic survival or number of embryos in utero between C and R sows. However, litter sex ratio was different with more males in the R sows (P<0.05) and embryo weight was reduced (P<0.05) in R compared to C sows. Embryo weight and litter sex ratio were also related to sow weight loss in the previous lactation. When genes regulating vascular development were considered, FGF2 and VEGFA mRNA expression was lower in the endometrium, while ANGPT2 was lower (P<0.05) in the placenta, recovered from R sows. Expression of myogenic regulatory factors analyzed (MYF5, MYF6, MYOG, MYOD1, PAX3, GLI1 and WNT10b) was not different between C and R sows at d 30 of gestation. In addition, the expression of some imprinted genes were examined, and IGF2 expression tended to be higher (P=0.059) and INSR expression was higher (P=0.049) in the R embryos, and IGF2R demonstrated a tendency for a sex by treatment interaction (P=0.066) in the placenta. These results confirm that maternal metabolic state prior to conception can adversely affect embryonic size and sex ratio in the next litter. These effects involved expression of genes related to vascular development in the uterus and placenta, and were consistent with the concept of epigenetic regulation of developmentally important genes.
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Biol Reprod. 2010 Nov;83(1 Suppl):5.
Feeding a low-protein, casein-based, methionine (MET) supplemented diet to pregnant rats is a well-established model to study developmental programming. Currently, the mechanisms leading to these reprogramming events are unresolved; one possible explanation is that the higher supplemental MET in the low-protein diet could disrupt one-carbon (1-C) metabolism at the level of the mother, the fetus or both. Since 1-C metabolism, involving such events as nucleoside synthesis and epigenetic transfers, is especially critical during the embryonic/ fetal period of life, it seems plausible that disruption of these processes could result in the long-term reprogramming of offspring observed. We propose that a low-protein high-MET diet will disrupt 1-C metabolism in dams and/or fetuses. Phase one of this study presents plasma amino acid profiles of dams at day 20 of gestation. Wistar rats were fed ad libitum, commencing on day zero of pregnancy, one of three diets: 18% casein with 0.5% supplemental MET (18-0.5, "control"; n=6), 9% casein with either 0.5% or 1.0% supplemental MET (9-0.5; n=6 and 9-1.0; n=6, respectively). Food consumption and maternal weight gain were monitored throughout the study. On day 20 of gestation, subsequent to maternal euthanasia, fetuses were excised and weighed and litter size was assessed. Maternal and fetal plasma, livers and kidneys were collected. Plasma free amino acid profiles were determined by HPLC using the Pico-Tag method (Waters). No significant differences were observed in food consumption, maternal weight gain or litter sizes (18-0.5: 14.3±0.7; 9-0.5: 14.5±0.9; 9-1.0: 12.4±1.6 fetuses). Fetal weights were unaffected by diet. Surprisingly, levels of the toxic, non-protein, sulfur-containing amino acid homocysteine (HCY), which serves to connect the folate and MET cycles in 1-C metabolism, did not differ significantly (18-0.5: 30±8; 9-0.5: 43±11; 9-1.0: 52±6 μM). However, maternal plasma MET levels were elevated by 42% in both 9-0.5 and 9-1.0 (respectively 148±23; 149±10 μM; p<0.05) compared to the control diet (86±12). Threonine (THR), the catabolism of which can be used to increase folate supply, was decreased in 9-0.5 by 47% and by 57% in 9-1.0 (respectively 186±24; 151±19 μM; p<0.001) compared to controls (18-0.5: 355±24), but levels were not significantly different between the low-protein diets. Relative to the control group, serine (SER) and glycine (GLY), two amino acids which are catabolised in the regeneration of methylated folates, tended to be reduced in 9-1.0 (respectively p=0.06; p=0.08), with GLY tending to be further reduced in 9-1.0 relative to 9-0.5 (p=0.08). Together, these results indicate a possible disruption of 1-C metabolism, specifically at the intersection of the MET and folate cycles. Decreases in plasma THR, SER and GLY, in combination with the increased plasma MET and unaltered plasma HCY suggests usage of 5-methyl-tetrahydrofolate for HCY re-methylation. Thus, regulation of plasma levels of HCY may be achieved at the expense of creating a functional folate deficiency within dams of the 9-0.5 and 9-1.0 treatment groups. Reprogramming could result from the impaired folate status, or through interactive effects of depleted folates and reduced dietary protein. Further assessment of 1-C metabolism disruption at the fetal and maternal level is currently underway.
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Biol Reprod. 2010 Nov;83(1 Suppl):5.
Ovarian cancer is a deadly disease. It is referred to as the "silent killer" due to the late stage at which the disease is usually diagnosed, by which time the survival rate is very low and conventional treatment modalities such as surgery and chemotherapy are often ineffective. Progress to devising effective therapies and dietary interventions for the treatment and prevention of ovarian cancer has been hampered by the lack of suitable animal models. With the exception of the laying hen, there are no other animal models of spontaneous epithelial ovarian cancer that replicate the human disease. We have shown that, analogous to the human cancer, a dramatic up-regulation of E-cadherin (CDH1) is an early defining event in ovarian cancer in the laying hen. A diet which is high in meats and low in vegetables may be corellated with increased ovarian cancer incidence. An imbalance of omega 3 (OM-3FA) and omega 6 (OM-6FA) fatty acids contributes to excess cancer risk. Increasing the consumption of OM-3FA may be a nontoxic way to prevent ovarian cancer, augment cancer therapy and to significantly increase life span. An effective treatment or dietary intervention that significantly reduces the progression of ovarian cancer has the potential for turning ovarian cancer into a disease that women die WITH not from. We have shown that supplementing hen's rations with flaxseed, a rich source of OM-3FA, results in an amelioration of the severity of ovarian cancer. The objective of this study was to assess the effect of a flaxseed enriched diet on E-cadherin expression and to determine the correlation of E-cadherin expression with ovarian cancer progression. Old laying hens which had completed their 2nd year of lay were fed a 10% flaxseed-enriched or standard diet for one year. The incidence and severity of ovarian cancer was determined on necropsy and normal and cancerous ovarian tissues were collected and subjected to immunohistochemical staining, western blot, and qPCR to assess changes in E-cadherin. In addition, DNA microarray analysis was conducted to identify other cancer associated genes that were affected by flaxseed dietary intervention. The results showed that flaxseed diet decreased the severity of ovarian cancer and decreased morbidity of hens. Microarray analysis revealed that E-cadherin was the mostly highly up-regulated gene in ovarian cancer and its expression was significantly decreased by the flaxseed diet. The decrease in E-cadherin in ovarian cancers from flaxseed-fed hens was verified by qPCR. Immunohistochemistry and western blot confirmed that there was a significant decrease in E-cadherin expression in cancerous ovaries from flaxseed compared to control fed hens. In parallel, in vitro studies showed that the addition of OM-3FA decreased the viability, rate of proliferation and increased the rate of apoptosis in human ovarian cancer cell lines. The addition of OM-3FA resulted in a concomitant decrease E-cadherin expression epithelial cancer cells. These results define E-cadherin as an important molecular target for treatment of primary ovarian cancer. [Supported by: NIH/NCCAM AT004085, American Institute for Cancer Research AICR06043, NIH Training Grant T32 HL007692]
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Biol Reprod. 2010 Nov;83(1 Suppl):5.
Regulation of uterine angiogenesis during pregnancy is mediated partly by estrogens, which are significantly elevated during gestation. We have recently demonstrated that estradiol-17β (E2β) and its cytochrome P450- (CYP450) and catechol-O-methyltransferase (COMT)-derived metabolites induce pregnancy-specific proliferation of uterine artery endothelial cells (UAECs). Moreover, E2β-induced proliferation of UAECs was ERβ-mediated, whereas its metabolites stimulate proliferation via ER-independent mechanisms. Despite the absence of common receptors that mediate the proliferative effects of E2β and its metabolites, we hypothesize that it is still possible that the pathways that mediate UAEC proliferation converge at the level of mitogen-activated protein kinases (MAPK) signaling cascade. Validated UAECs from late pregnant ewes (day 120-130; term = 147 days; n=5) were serum starved (24 hrs) and pretreated (1 hr) with or without 2.5 µM/5 µM of PD98059 (ERK-1/2 MAPK inhibitor) or 2.5 µM/5 µM of LY294002 (PI3K inhibitor) followed by treatment with endothelial basal media (EBM; vehicle) or EBM with 0.1 nM of E2β or 2-Hydroxyestradiol (2-OHE2), 4-Hydroxyestradiol (4-OHE2), 2-Methoxyestradiol (2-ME2) or 4-Methoxyestradiol (4-ME2). An in vitro index of proliferation was evaluated utilizing the Brdu Proliferation Assay technique following 24 hrs of estrogens' treatment. Western blotting was utilized for evaluation of phosphorylated and total ERK-1/2, p38 MAPK and PI3K. Activation of native p38 MAPK protein was further validated utilizing a cell-based ELISA immunoassay kit. Pretreatment with PD98059 at all doses significantly abrogated (P<0.01) UAECs proliferation in response to E2beta, 2-OHE2, 4-OHE2 or 4-ME2. However, LY294002 pretreatment did not inhibit (P>0.05) UAECs proliferation induced by E2β, 2-OHE2, 4-OHE2 or 4-ME2. Western Blotting revealed a proliferation-specific activation of phosphorylated ERK-1/2 MAPK and p38 MAPK but not phosphorylated PI3K in response to E2β, 2-OHE2, 4-OHE2 and 4-ME2. Total ERK-1/2, p38 MAPK and PI3K were not significantly changed among the different treatments of estradiol or its metabolites. P38 MAPK ELISA immunoassay revealed proliferation-specific changes in native phosphorylated p38 MAPK activity in response to treatments with estradiol or its metabolites. 2-ME2 treatment did not stimulate proliferation or significantly stimulate any of the protein kinases. These data demonstrate that despite the absence of common receptors, E2β and its CYP450- and COMT-derived (4-ME2) metabolites activate signaling pathways that converge at ERK-1/2 and p38 MAPK signaling to trigger proliferation which is independent of PI3K. Our findings indicate that physiological and pharmacological approaches to investigating endothelial proliferation and pregnancy-induced angiogenesis requires specific appreciation of both ERK-1/2 MAPK and p38 MAPK signaling pathways that may be dysfunctional in gestational vascular diseases. NIH HL49210, HD38843, HL87144 and R25GM083252.
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Biol Reprod. 2010 Nov;83(1 Suppl):5.
Four mammalian sprouty (Spry) proteins have been identified to function as endogenous inhibitors for FGF signaling. Protein kinase B (Akt) dependent ser1179 phosphorylation of endothelial nitric oxide (NO) synthase (eNOS) is a key mechanism for FGF2 stimulation of endothelial NO production. Concomitant increases in placental FGF2 expression and local NO production are pivotal for maternal-fetal interface vasodilation and angiogenesis during pregnancy. However, it is unknown if sprout proteins are expressed in the placenta and whether they regulate FGF2 signaling to nitric oxide (NO) production in placental endothelial cells. Hypotheses: Sprouty proteins are expressed in the placenta and they negatively regulate FGF2 activation of eNOS via the PI3K-Akt pathway primary placental endothelial cells. Methods: Immunofluorescence microscopy was used to detect sprout proteins in paraffin embedded sections of fetal placental arteries from pregnant (d120-130) ewes. Immunoblotting was used to detect sprout proteins in primary ovine fetoplacental artery endothelial (oFPAE) cells. Cells were transfected with or without pcDNA3-flag-Spry plasmids and then treated with or without increasing doses of FGF2 for up to 60 min. Spry protein overexpession was monitored by immunoblotting with an anti-flag antibody. Activation of Akt and eNOS was determined by immunoblotting with specific phosphor-Akt and eNOS antibodies. Results: Spry proteins 1-4 were expressed in placental artery endothelial cells in vivo and in vitro. Treatment with FGF2 stimulated Akt and eNOS activation in a time- and dose-dependent manner. Wortmannin (20 nM) effectively blocked FGF2 stimulation of Akt and eNOS phosphorylation. Overexpression of flag-Spry2 or Spry4 significantly inhibited FGF2-induced Akt and eNOS activation. Conclusion: Sprouty proteins are expressed in placental endothelial cells and functions endogenous antagonists of FGF2 signaling to eNOS activation through the PI3K-Akt pathway (HL74947 & HL70562).
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Biol Reprod. 2010 Nov;83(1 Suppl):5.
Insulin-like growth factor-I (IGF1) is a growth factor that increases the proportion of embryos becoming blastocysts in many species. The mechanism is unknown but could involve, among other possibilities, stimulation of blastomere proliferation, inhibition of apoptosis or cell-cycle arrest and stimulation of expression of genes involved in compaction and blastocyst formation. The overall objective of the present study was to determine the mechanism by which IGF1 increases the percent of oocytes becoming a blastocyst. Specifically, it was tested whether IGF1 acts before or after embryonic genome activation, stimulates development through the MAPK pathway, and increases transcript abundance for genes controlling compaction and blastocyst formation. Experiments were performed with embryos produced in vitro and cultured in a modified Synthetic Oviductal Fluid. In Exp. 1, it was demonstrated that recombinant human IGF1 could increase the proportion of oocytes becoming blastocysts and that concentrations of 10, 100 and 200 ng/mL were equally effective. In Exp. 2 it was tested whether IGF1 acts to increase blastocyst yield before or after day 4 of development. Embryos were cultured with the following treatments: 1) medium alone from day 0-8 post insemination (pi), 2) medium alone from day 0-4 and medium with 100 ng/mL IGF1 from day 4-8, 3) medium with 100 ng/mL IGF1 from day 0-4 and medium alone from day 4-8, or 4) medium plus 100 ng/mL IGF1 from day 0-8. As compared to controls (30.4% ± 1.89), IGF1 increased (P<0.01) the percent of oocytes that were blastocysts at day 8 pi when added from day 4-8 (42% ± 1.89) or day 0-8 (40% ± 1.89) but not from day 0-4 (33.6 ± 1.89). For Exp. 3, it was tested whether IGF1 increases blastocyst development by acting through MAPK signaling. At day 4, embryos were placed in fresh medium to achieve the following treatments: 1) medium + 0.1% DMSO (vehicle); 2) medium + 0.1% DMSO and 100 ng/mL IGF1; 3) medium + 0.1% DMSO (vehicle) + 100 µM PD 98059 (MAPK inhibitor); and 4) medium +DMSO + 100 ng/mL IGF1 + 100 μM PD 98059. In the absence of IGF1, PD 98059 did not decrease the percent of oocytes that became blastocysts at day 8 (21% vs 24% for control and PD 98059, pooled SEM=0.02). IGF1 increased blastocyst yield (P<0.001) and PD 98059 decreased this effect (37% vs 29% for IGF1 and IGF1 + PD 98059; P<0.05). In Exp. 4 quantitative real time PCR was used to analyze genes involved in compaction and blastocyst formation. At day 4, embryos received IGF1 (100 ng/mL) or vehicle. Embryos were harvested on day 5 (those ≥ 16 cells) or 6 (morulae and early blastocysts) and analyzed for expression of CDX2, CDH1, ATP1A1, OCLN and Hist2h2aa2. IGF1 tended to increase ATP1A1 at day 5 (1.3 fold; P=0.09) and increased the expression of ATP1A1 day 6 (1.68 fold; P<0.04), but there was no effect on IGF-1 on the other genes. In conclusion, IGF1 promotes development to the blastocyst stage by regulating MAPK-dependent events at day 4 or later. Among these events is upregulation of expression of ATP1A1, which is required for blastocoel formation (Support: USDA NRI Grants 2006-55203-17390 and 2007-35203-18070).
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Biol Reprod. 2010 Nov;83(1 Suppl):5.
Follicle-stimulating hormone mediates cyclic follicle growth and is widely used clinically for controlled ovarian stimulation. The ovarian response to FSH is variable ranging from low to high and has been partly attributed to two common variants of the FSH receptor (FSHR). In addition to these, we have recently identified four abnormal FSHR splicing products (three exon deletions and one intron insertion) in the cumulus cells of 37% (13 of 35) of infertile women tested. All alterations affected the extracellular part of the receptor. All patients with variant receptors also expressed normal FSHR. We focused on the splice variant lacking exon 2 because it was only detected in young women (<35) with low ovarian response (≤4 oocytes) when treated with FSH. In vitro analyses indicated that when the normal and deleted variants were co-expressed, as found in patients, the response of the normal receptor was significantly reduced. Our aim in this study was to determine the molecular mechanism of action of the mutant receptor. We established stable inducible HEK293 cells expressing wild-type [FSHR(wt)] and splice-variant receptor [FSHR(del ex2)]. FSHR is a G-protein coupled receptor and upon FSH stimulation the level of intracellular cAMP is increased in a dose-response manner. To measure cAMP in our system, the cells were transfected with a cAMP-responsive luciferase reporter plasmid, stimulated with 0-1000 mIU/ml FSH for 12 h. The cells were lysed and luciferase amount was measured. In vitro expression of FSHR(del ex2) alone did not elicit a response to FSH, while co-expression of FSHR(del ex2) with FSHR(wt) resulted in a significant reduction of the wt activity when stimulated with FSH, indicating that it can form functional heterodimers with the wt receptor. Co-immunoprecipitation confirmed the presence of such heterodimers. Immunostaining and surface biotinylation showed that, unlike the FSHR(wt), less than half of the total amount of FSHR(del ex 2) protein is present on the cell surface, and a large amount of protein remains trapped in intracellular compartments. Subcellular localization was determined using fractionation in sucrose gradients. The separation of plasma membrane, Golgi, and endoplasmic reticulum was verified by Western blot with antibodies against Na-K-ATPase, GM130, and Calnexin, respectively. Subcellular fractionation identified FSHR(del ex2) protein in Golgi and Endoplasmic reticulum. Our findings indicate severe impairment of the cellular mechanism that processes and traffics the variant receptor to the plasma membrane. The variant receptor requires the presence of wt but can significantly alter its activity, resulting in less than optimal ovarian stimulation. Therefore, FSHR variants can contribute to abnormal response to stimulation in certain women undergoing infertility treatment. Further elucidation of the signaling cascades downstream of FSHR, in combination with newly developed fertility drugs, could in the future lead to personalized treatments for a subgroup of patients that can bypass the FSHR and target downstream effectors of folliculogenesis. This research was supported by NIH HD055274 (MDL) and partly by NCRR/NIH RR024138 (MDL).
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Biol Reprod. 2010 Nov;83(1 Suppl):5.
Inhibin is the only TGFβ superfamily ligand assembled from two dissimilar subunits; a β-subunit it shares with activin and an α-subunit. The N-terminal extension (1-32 aa) of the mature vertebrate inhibin α-subunit is unique within the superfamily. In the presence of the ancillary binding protein betaglycan, inhibin competes with activin to bind the activin type II receptors (ActRIIA/B) to block activation of downstream signaling. The conventional thinking is that inhibin binding to ActRII (alone or with betaglycan) is sufficient to inhibit activin. However, despite its high affinity for ActRII, the concentration of circulating inhibin is too low to achieve the profound antagonism of constitutive activin signaling that occurs in the pituitary. We hypothesized that inhibin interacts with ALK4 to achieve full antagonism of activin signaling. Conditioned media containing the human or chicken inhibin free α-subunit inhibited activin signaling in LβT2 cells in a dose-dependent manner. In contrast, conditioned media containing an N-terminal extension deletion mutant of the inhibin free α-subunit (αHext-) failed to inhibit activin signaling. An inhibin a-subunit N-terminal peptide (WPWSPSALRLLGRPPE) and wrist region peptide (PPNLSLPVPGAPPTP) were added to media with activin A; only the N-terminal peptide demonstrated inhibitor function at a concentration of 2.5 mM. Biotinylated inhibin A, but not activin A, was found to bind ALK4, though the affinity of inhibin A was weaker for ALK4 compared with ActRIIB. A chimera mutant containing the inhibin α-subunit N-terminal extension region swapped into βA-subunits (βAext+/ βAext+), as well as an N-terminal extension deletion inhibin A mutant (αHext-/βA), were produced to test their binding affinity to ALK4 compared with wild type human biotinylated inhibin A and activin A by competitive binding ELISA. Wild type human inhibin A competed well with inhibin A for ALK4 binding. Whereas wild type human activin A did not bind ALK4, the N-terminal extension region chimera mutant acquired ALK4 binding capacity. By contrast, the N-terminal deletion inhibin A mutant showed decreased binding affinity for ALK4 compared with wild type inhibin A. The N-terminal peptide can compete with inhibin A for ALK4 compared with the wrist region peptide. Native gels confirmed the formation of complexes containing inhibin A and ALK4, though complex formation was not as robust as that seen with inhibin A with ActRIIB. The N-terminal extension region of the inhibin α-subunit directly associates with ALK4, thereby disrupting the activin/ActRIIB/ALK4 complex. Thus, the N-terminal extension region imparts inhibin with its antagonistic function.
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Biol Reprod. 2010 Nov;83(1 Suppl):5.
Previous studies indicate that local factors (paracrine and/or autocrine factors) modulate LH-induced Leydig cell steroidogenesis. The insulin-like growth factor (IGF) system (IGF-I, IGF-II, their receptors, and binding proteins) has been proven to be a modulator of reproductive function. Previous studies indicate that the action of IGF-I on basal and/or hCG-induced testosterone production varies depending on the type of species, developmental stage and/or the experimental conditions. Recently, localization of IGF-I and IGF-IR were observed in equine Leydig cells using immunohistochemistry (IHC), suggesting IGF-I may play a role in equine Leydig cell steroidogenesis. In contrast, a previous experiment showed that treatment with increasing doses of IGF-I did not increase testosterone production in cultured equine Leydig cells. The objectives of this study were to test the synergistic effect of IGF-I on eLH-induced testosterone production in cultured equine Leydig cells and a potential age-effect difference. Testes were collected from 5 pubertal (12-18 mo) and 5 post-pubertal stallions (2- 4 y) during routine castrations at the UC Davis Veterinary Hospital. Equine Leydig cells were isolated using validated enzymatic and mechanical procedures. Leydig cells were treated with purified eLH (0,1,5 or 10 ng/ml; E263B, Papkoff) and/or recombinant human IGF-I (0, 1, 5, 10, or 50 ng/ml, rhIGF-1; Parlow AF., National Hormone and Peptide Program, Harbor-UCLA Medical Center) and incubated in 95% air: 5% C02 at 34°C for 24 h. At 24 h, culture media was collected and frozen at -20°C until analyzed for testosterone by a validated RIA. In response to eLH, in both pubertal and post-pubertal stallions, the level of testosterone from cultured equine Leydig cells significantly increased in a dose-dependent manner compared with 0 controls. In contrast, treatment with IGF-I only (1 to 50 ng/ml) failed to increase levels of testosterone in either age group. In pubertal stallions, treatment with both IGF-I (1 to 50 ng/ml) and 5 ng eLH failed to show a significant difference in testosterone production compared with 5 ng eLH only. However, a significant increase in the level of testosterone was observed from Leydig cells treated with both 50 ng/ml of IGF-I and 5 ng/ml of eLH compared with 5 ng/ml eLH only in post-pubertal stallions. In summary, IGF-I did not increase basal production of testosterone from cultured equine Leydig cells from pubertal or post-pubertal stallions, but a synergistic effect of IGF-I on eLH-stimulated testosterone production was observed in cultured equine Leydig cells from post-pubertal stallions. This is consistent with the recent IHC study which demonstrated relatively lighter IGF-I staining in Leydig cells from pubertal stallions than from post-pubertal stallions. In conclusion, the results of this study suggest that IGF-I has a paracrine and/or autocrine action on Leydig cell steroidogenesis in post-pubertal stallions.
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Biol Reprod. 2010 Nov;83(1 Suppl):5.
Obesity is a major risk factor in the development of insulin resistance and type II diabetes. The adipocyte-derived hormone leptin maintains energy balance by acting on hypothalamic leptin receptors (LRbs) that trigger activation of the JAK2/STAT3 pathway. Although disruption of LRb-STAT3 signaling promotes obesity in mice, other neuroendocrine features of LRb function such as fertility appear normal, suggesting that other effectors are required for leptin signaling. Here we describe a novel role for the cAMP and calcium-responsive CREB coactivator Crtc1 in energy balance and reproduction. Crtc1 -/- mice are hyperphagic, obese, and infertile. While Crtc1 -/- mice are sensitive to the anorexigenic effects of the alpha-MSH analog, MTII, Crtc1 -/- mice display persistent weight gain during chronic leptin infusion, implicating Crtc1 as a downstream effector of leptin signaling. Moreover, in leptin deficient ob/ob mice, hypothalamic Crtc1 is highly phosphorylated and inactive. Conversely, leptin treatment triggers dephosphorylation and activation of Crtc1 in ob/ob mice. Notably, Crtc1 regulates the expression of Cartpt and Kiss1 genes, which encode hypothalamic neuropeptides that mediate leptin's effects on satiety and fertility, respectively. Crtc1 transcriptional activity over the Cartpt and Kiss1 promoters is maximally activated through synergistic leptin- and cAMP-dependent signaling. Crtc1 over-expression in cultured cells increases the transcriptional activity of Cartpt and Kiss1 promoters, while Crtc1 depletion by RNAi in vitro or by targeted gene disruption in vivo, decreases the expression of Cartpt and Kiss1 genes. Leptin administration also increases recruitment of hypothalamic Crtc1 to Cartpt and Kiss1 promoters in vivo. Thus, our results indicate that Crtc1 is a key effector of leptin signaling that links energy balance and fertility.
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Biol Reprod. 2010 Nov;83(1 Suppl):5.
Reproductive maturation (i.e., puberty onset) and function (i.e., fertility) are sensitive to the state of body energy reserves, as evidenced by the fact that conditions of disturbed energy homeostasis are commonly linked to different degrees of reproductive disruption. The neuroendocrine basis for such a metabolic control of reproduction remains partially unknown, although much has been learnt in recent years about the factors and circuits involved. In this context, compelling evidence, gathered mostly in laboratory rodents, has demonstrated that kisspeptins, the products of the Kiss1 gene that act via the G protein-coupled receptor GPR54 (or Kiss1R), are essential signals for the central regulation of reproduction by nutritional and metabolic cues. Thus, kisspeptin neurons in the forebrain appear to operate as sensors of energy stores and the metabolic state of the organism, since conditions of negative energy balance (e.g., fasting) or metabolic stress (e.g., experimental diabetes, acute inflammation) that inhibit the reproductive axis are coupled to decreased Kiss1/kisspeptin tone at the hypothalamus. In turn, exogenous kisspeptin administration rescues the state of hypogonadotropism associated to energy deficit. The adipose hormone, leptin, seems to play an important role in the metabolic control of the Kiss1 system, as leptin stimulates Kiss1 mRNA expression at the hypothalamus, thus defining a putative leptin-kisspeptin-GnRH pathway responsible for the metabolic regulation of puberty onset and fertility. Recent studies have identified different molecular mediators of this pathway, such as the sensor of cellular energy status, mammalian target of rapamycin (mTOR), and Crtc1. In addition, several reports have pointed out the potential involvement of other neuropeptide and hormonal regulators (e.g., ghrelin, NPY, melanocortins, MCH) in the metabolic control of the Kiss1 system. Altogether, the available experimental data strongly suggest that Kiss neurons play a prominent integratory role in the hypothalamic circuitry conveying metabolic information onto the reproductive centers, thus serving a key function in the physiological coupling of body energy status, puberty onset and fertility.
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Biol Reprod. 2010 Nov;83(1 Suppl):5.
Early reports gave rise to the concept that energy reserves must exceed critical threshold to allow the onset of puberty and for normal sexual maturation. It is now well established that the adipocyte hormone leptin plays an essential role linking metabolism and reproduction. Humans and mice lacking leptin (ob/ob) or leptin receptors (LepR, db/db) are infertile. Leptin administration to leptin-deficient subjects and ob/ob mice induces puberty and restores fertility. However, the exact site/s of leptin action has been difficult to determine. In this presentation, we will show that bilateral excitotoxic lesion of the hypothalamic ventral premammillary nucleus (PMV) of ob/ob mice blunts the ability of leptin to induce sexual maturation and preclude leptin stimulatory effects on LH secretion. Moreover, unilateral re-expression of endogenous LepR selectively in PMV neurons is sufficient to induce sexual maturation and improve fertility in female LepR null mice. These effects are independent on chances in Kiss1 gene expression. Leptin responsive PMV neurons coexpress glutamate and innervate GnRH terminals in the mediobasal hypothalamus. Notably, leptin action on PMV neurons normalized the increased GnRH content characteristic of leptin signaling deficiency. Collectively, these findings establish the PMV as a key site for leptin action on reproductive control.
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Biol Reprod. 2010 Nov;83(1 Suppl):5.
Vascular endothelial growth factor (VEGF) is a potent inducer of vascular hyperpermeability and the key mediator of angiogenesis, both of which are involved in normal development and reproduction, as well as cancer and other diseases. VEGF is rapidly and strongly induced in endometrial epithelial cells by 17beta-estradiol (E2). We have proposed that this "immediate-early" response is the key to E2-induced cell proliferation. Recently, we linked E2-induced VEGF expression in the endometrium to the transcription factor hypoxia-inducible factor 1alpha (HIF-1alpha), which regulates over 100 genes essential for cellular adaptations to low oxygen and other stresses. E2, via the PI3K/Akt pathway, induces the rapid recruitment of both HIF-1alpha and ERalpha to the VEGF gene promoter. In contrast to the endometrium, however, E2 has little or no effect on VEGF expression in breast and endometrial cancer cells in vitro. We recently resolved this inconsistency by showing that the lack of E2 induction in vitro is due to a deficiency of HIF-1alpha caused by the high oxygen level to which cultured cells are exposed. Restoration of HIF-1alpha in endometrial and breast cancer cells restores rapid and strong VEGF expression by E2. Thus, VEGF-induced vascular hyperpermeability, which floods tissues with plasma proteins, should now be considered a potential mediator of E2-induced cell proliferation in both normal target tissues and E2-dependent tumors. In summary, we have for the first time linked E2 action to HIF-1alpha activation and VEGF expression, thereby providing new insight into E2's effects in target tissues, and into possible treatments for E2-associated cancers.
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Biol Reprod. 2010 Nov;83(1 Suppl):5.
Human placental development starts in a low oxygen environment because the cytotrophoblast plug the spiral arterioles. This situation changes at about 11 weeks of gestation by the extravillous trophoblast which invade into the spiral arteries and remodel the vessel walls to increase placental blood perfusion. These events are accompanied by a switch in oxygen concentration from 2% to 8% and correlate to important steps in placental development. The exact mechanism, however, by which trophoblast cells sense oxygen tension remains still elusive. The hypoxia-inducible factor-1α (HIF-1α) tightly coordinates the expression of several target genes responsible for trophoblast invasion and function. We focused on key molecules in the invasion and differentiation process, CCN1 (CYR61) and CCN3 (NOV) and the gap junction proteins Cx43 and Cx40, which are all upregulated in the invasive trophoblast of placental explants and trophoblast cell lines by low oxygen. We identified a direct role of HIF-1α for their elevation under low oxygen. Increase in CYR61 and NOV increases invasion properties of JEG3 trophoblast cells combined with an elevated MMP-2 and MMP-9 activity. The effect of hypoxia on Cx43 and Cx40 in the placental cell line JAr expressing both connexins was associated with a translocation of Cx43 from the cell membrane to the cytoplasm resulting in a reduction of channel function analysed by calcein transfer. Our data show that important genes involved in trophoblast invasion and differentiation are regulated via HIF-1α and that their regulation is associated with changes in function which could contribute to the pathophysiology in preeclampsia.
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Biol Reprod. 2010 Nov;83(1 Suppl):5.
Fertility in females requires exquisite regulation of the ovarian follicle that houses the oocyte, granulosa, and theca cells. Follicles do not progress beyond the preantral stage in the absence of FSH. FSH signals through its G protein-coupled receptor on granulosa cells (GCs) to activate genes that promote maturation of the follicle to a preovulatory phenotype that can respond to the surge of LH and produce a viable oocyte. A key pathway by which FSH signals to regulate expression of genes critical for GC maturation is the phosphatidylinositol 3-kinase (PI3K) pathway. We have shown that FSH via PI3K/AKT and downstream mammalian target of rapamycin/raptor promotes enhanced translation of HIF1α. HIF1αis a ubiquituous transcriptional activator absent from most cells under normoxia, due to oxygen-dependent, post-translational modifications that target it for proteosomal degradation. Thus, HIF1α accumulates only under hypoxia in most cells. HIF1α together with constitutively expressed HIF1β form the heterodimeric transcriptional factor HIF1 that activates a hierarchy of target genes, including vascular endothelial growth factor (Vegf) and various glycolytic enzymes that permit cells to survive under reduced oxygen concentrations. Our results show under normoxic conditions that HIF1α accumulates in GCs in response to FSH and that HIF1 appears to be necessary for FSH regulation of at least three target genes, namely inhibin-α(Inha), luteinizing hormone/choriogonadotropin receptor (Lhcgr), and Vegf. Accumulation of HIF1α also requires AKT-stimulated phosphorylation/inactivation of forkhead box O factor 1. Taken together, these results suggest that HIF1 regulates a unique set of target genes in GCs. Supported by NIH RO1HD62503.
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Biol Reprod. 2010 Nov;83(1 Suppl):5.
The generation of pluripotent cell lines in livestock, including cattle, has been inefficient with putative stem cells undergoing rapid spontaneous differentiation. Direct reprogramming of mouse fibroblasts to an ES-like state, is possible simply by forcing expression of a handful of transcription factors. The cells, termed induced pluripotent stem cells (iPSCs), can exhibit most of the characteristics of ES cells, including contribution to the germ-line in mouse. We hypothesized that a similar approach can result in the generation of pluripotent cells from cattle. Anticipated hurdles to translating iPSC technology to cattle included- 1) Efficient transfection methods, 2) Virus packaging cell type, 3) Whether to transduce cells with mouse, human or bovine pluripotency genes and 4) Conditions for maintaining BiPSCs in an undifferentiated state. We used a GFP vector to quantify viral transduction efficiency of bovine adult fibroblasts (BAFs). Putative BiPSCs were cultured in alpha-MEM medium supplemented with 20% FCS. Colonies, resembling mouse ESC colonies, with multi-layered growth and high nucleus to cytoplasm ration, were observed. Putative BiPSCs were initially manually passaged and then dissociated enzymatically for >30 passages. All colonies stain brightly for alkaline phosphatase. The iPS cells have a normal karyotype and express Oct4, Sox2, Rex1, SSEA1, and alkaline phosphatase by RT-PCR analysis. And SSEA1, SSEA4, and Oct4 by immunocytochemistry. The cells form embryoid bodies containing cells from the three germ layers, and formed teratomas in SCID mice. We conclude that pluripotency can be induced in bovine somatic cells, which will be useful for a range of biotechnology applications.
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Biol Reprod. 2010 Nov;83(1 Suppl):5.
Embryonic stem (ES) cells represent permanent cell lines that can be maintained in an undifferentiated state and induced to form derivatives of the three embryonic germ layers. These characteristics give ES cells great potential for both basic research and clinical applications in regenerative medicine. The establishment of ES cells from large animals that model human diseases is of significant importance. We derived permanent canine cell lines from preimplantation stage embryos. These cells expressed the core pluripotency transcription factors OCT3/4, NANOG, SOX2 and, similar to human ES cells, they expressed SSEA-3, SSEA-4, TRA-1-60, TRA-1-81. Canine ES cells maintained a normal karyotype and morphology after multiple in vitro passages and rounds of cryopreservation. Plating cells in the absence of a feeder layer resulted in their in vitro differentiation to multiple cell types. In vivo, canine ES cells gave rise to teratomas comprising cell types of the three embryonic germ layers. In an attempt to define the signaling cascades regulating cES cell self-renewal and pluripotency, we assessed the utility of the small molecule inhibitors of Mek/Erk and Gsk3 in combinations with LIF, BMP4 and bFGF for the derivation and maintenance of canine ES cells. Preliminary results suggested that although morula-stage embryos initially underwent ex vivo maturation with expansion of the inner cell mass, embryos arrested at the expanded blastocyst stage regardless of the combination of small molecules and growth factors used. Moreover, supplementation of early passage cultures with inhibitors induced proliferative arrest and cell death. Thus, the signaling pathways regulating the self-renewal of canine ES cells remain unclear.
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Biol Reprod. 2010 Nov;83(1 Suppl):5.
Authentic embryonic stem cells (ESC) have probably never been derived from the inner cell mass (ICM) of pig, despite over 25 years of effort. Recently, several groups, including ours, have reported the induced pluripotent stem cells (iPSC) from swine by reprogramming somatic cells with a combination of four factors (OCT4, SOX2, KLF4, C-MYC) delivered by retroviral transduction. The piPSC resembled FGF2-dependent human (h) ESC and are likely to advance swine as a model in biomedical research, since grafts could potentially be matched to the animal that donated the cells for re-programming. The dependence of piPSC on FGF2 also draws parallels to murine pluripotent stem cells derived from advanced epiblast, so-called ‘epiblast stem cells’. Indeed, an emerging concept is that there are two kinds of ESC, one dependent on FGF2 and not competent to contribute to germ-line chimeras, the other upon LIF and germ-line competent. The objective of our recent investigations has been to develop LIF-dependent piPSC by using the same reprogramming factorsbut selecting the colonies on a modified LIF- medium supplemented with two kinase inhibitors, CHIR99021, which inhibits GSK-3beta, and kenpaullone, which inhibits both GSK-3beta and CDK1. The LIF-dependent piPSC, derived here from outgrowths of umbilical cord mesenchyme, expressed markers consistent with pluripotency and bore a striking resemblance to ICM-derived murine ESC in colony morphology, culture characteristics, and short cell cycle time. Currently, the ability of LIF-piPSC to give rise to teratomas and chimeras is under investigation. Supported by Missouri Life Sciences Board Grant 00022147 and NIH grant HD21896.
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Biol Reprod. 2010 Nov;83(1 Suppl):5.
Abstract not available.
Biol Reprod. 2010 Nov;83(1 Suppl):5.
Abstract not available.
Biol Reprod. 2010 Nov;83(1 Suppl):5.
Steroidogenic cells such as cortical cells in the adrenal, Leydig cells in the testis, and granulosa and theca cells in the ovaries, are derived from a common primordium that consists of Steroidogenic factor 1 (SF1)-positive precursor cells. SF1 defines the steroidogenic lineages in these organs and at the same time, induces expression of steroidogenic enzymes and production of steroids. The Hedgehog (Hh) signaling pathway, which is known to play roles in organogenesis and cell fate determination, has been shown to be a downstream regulator of SF1 in the appearance of steroidogenic cells in these organs. We will present results demonstrating that the Hh signaling pathway serves as a common crosstalk component in these organs and yet evolves diverse functions in the expansion and differentiation of the steroidogenic cells in a tissue-specific manner (supported by NICHD-059961).
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Biol Reprod. 2010 Nov;83(1 Suppl):5.
Exposure to naturally-occurring and synthetic chemicals in the environment with estrogenic activity is a known risk factor for adverse effects on reproductive structure and function; these adverse effects include altered puberty, subfertility/infertility, menstrual cycle irregularities and even neoplasia later in life. These affects were previously thought to occur only at high levels of exposure. However, concern with endocrine disrupting chemicals now focuses on the potential for adverse effects from low levels exposures especially when exposure occurs during critical developmental windows. Thus to test the hypothesis that low levels of estrogen exposure may have adverse consequences, we tested several estrogenic compounds including the pharmaceutical diethylstilbestrol (DES) and the phytoestrogen genistein found in soy products in a neonatal CD-1 mouse model where treatment was on Days 1-5 of life. Subsequent changes in ovarian structure and function, and fertility were evaluated. Mice were exposed either subcutaneously or orally to the compounds. Alterations in reproductive tract development and function will be described and the molecular mechanisms involved these changes will be discussed.
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Biol Reprod. 2010 Nov;83(1 Suppl):5.
Dioxins are environmental contaminants of great concern because of their persistence and toxicity. These compounds act via a unique signaling pathway to elicit a variety of undesirable effects including immunosuppression, teratogenesis, carcinogenesis, endocrine disruption and infertility. Prepubertal or developmental exposure to dioxin shortens the reproductive lifespan by delaying the onset of puberty and hastening reproductive aging in female rats. We have also shown that the hormonal mechanisms controlling ovulation and subsequent fertility in the female are disrupted by low concentrations of dioxins. The ovary, pituitary gland and hypothalamus all appear to be targets of dioxin action. The mechanisms of disruption of female reproductive endocrinology by dioxin appear to involve antagonism of ovarian development and estrogen action and vary with dose and age at exposure. Efforts are currently underway to further disclose the effects of dioxins on the developing ovary and embryo.
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Biol Reprod. 2010 Nov;83(1 Suppl):5.
Women are exposed to endocrine disrupting chemicals (EDCs) on a daily basis. This is of concern because many EDCs cause infertility and premature ovarian failure. Infertility is a public health concern because it occurs in 5-6 million women in the United States alone, making it a fairly prevalent condition. Premature ovarian failure poses health risks because it is associated with an increased risk of cardiovascular disease, osteoporosis, and premature death. Our laboratory is interested in the effects of the EDC methoxychlor (MXC) on fertility and ovarian function. MXC is a pesticide which is used against insects that attack produce and home gardens. MXC exposure causes infertility and ovarian failure in a wide variety of species. Our studies show that MXC may cause infertility and ovarian failure by inhibiting growth and inducing atresia of antral follicles. Further, our studies indicate that MXC inhibits production of androgens and estradiol. MXC also impairs mitochondrial respiration, increases production of reactive oxygen species, causes oxidative DNA and protein damage, and decreases expression and activity of selected anti-oxidants. In addition, MXC-induced atresia is prevented by estradiol and anti-oxidant treatment. Taken together, these data indicate that MXC causes atresia via mechanisms that involve inhibition of sex steroid biosynthesis and induction of oxidative stress. These findings have increased our understanding of the mechanisms by which a selected EDC causes ovotoxicity. This information may lead to the development of novel targets for the treatment of infertility and premature ovarian failure induced by EDCs. Supported by NIH R01 ES012893.
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