Inhibited Progesterone Production Research Articles

Daidzein does affect progesterone secretion by pig cumulus cells but it does not impair oocytes IVM

Daidzein, an isoflavone abundant in soybeans and other legumes, displays estrogen like properties. This study was aimed at evaluating the effect of daidzein (1 and 10 microM) on nuclear and cytoplasmic maturation of pig oocytes and on steroidogenic activity of cumulus cells. Daidzein supplementation during IVM had no effect on nuclear maturation and on fertilization traits. By contrast, both concentrations significantly (P < 0.05) inhibited progesterone production by cumulus cells after 24 and 48 h of culture while they did not induce any effect on estradiol production. Furthermore, daidzein did not exert any effect on the percentage of embryos that developed to blastocyst stage, on the number of blastomeres per blastocyst, or on the level of Hsp-70 and -90 gene transcript. Overall, our data demonstrate that daidzein added during oocyte maturation does not affect pig embryo development even if it markedly inhibits progesterone production by cumulus cells. Further studies are needed to evaluate the possible effect of daidzein during embryonic development.

Hypoxia is Important for Establishing Vascularization During Corpus Luteum Formation in Cattle

Hypoxia-inducible factor 1 (HIF1) has been demonstrated to have critical roles in angiogenesis via transcriptional regulation of angiogenic factors, such as vascular endothelial growth factor (VEGF). In the ovary, angiogenesis is known to occur after ovulation in the developing corpus luteum (CL) in mammals. To determine whether HIF1 participates in angiogenesis in bovine CL, the present study investigated the mRNA and protein expressions of the HIF1 alpha subunit (HIF1A) and VEGF in bovine CL during the estrous cycle. The effects of hypoxia on the expressions of HIF1A protein, VEGF mRNA and VEGF protein in bovine luteal cells were also examined by using a cell culture system. HIF1A mRNA expression was less at the regressed stage than at the other stages, whereas protein expression of HIF1A was highest at the early luteal stage and decreased thereafter. VEGF mRNA expression was highest at the developing luteal stage and decreased thereafter. VEGF protein expression was highest at the early luteal stage and decreased significantly at the regressed luteal stage. Hypoxia increased the amounts of HIF1A protein, VEGF mRNA and VEGF protein in cultured bovine luteal cells. Furthermore, we found that hypoxia inhibited progesterone production in the mid luteal cells, but not in the early luteal cells. The overall findings indicate that HIF1 is one of the factors promoting VEGF-induced angiogenesis during luteal development, and suggest that the hypoxic conditions formed after follicle rupture contribute to establishing luteal vascularization in cattle.

Effects of a trichothecene, T-2 toxin, on proliferation and steroid production by porcine granulosa cells

Fusarium mycotoxins, such as trichothecenes and zearalenone, are produced by molds and contaminate a large variety of grains and feedstuffs worldwide. Mycotoxins of Fusarium fungi include the trichothecenes, deoxynivalenol and T-2 toxin (T2), and zearalenone, and have been implicated in poor reproductive performance in pigs. However, direct ovarian effects of T2 toxin have not been reported. Therefore, porcine granulosa cells (GC) from small follicles (1-5mm) were cultured for 2 days in 5% fetal bovine serum and 5% porcine serum-containing medium followed by 2 days in serum-free medium containing various doses of FSH, insulin-like growth factor-I and T2 (at various doses/combinations) to evaluate the influence of T2 on steroid production and cell proliferation. T2 at 1, 3, 30 and 300 ng/mL completely inhibited FSH plus IGF-I-induced estradiol production, whereas 0.3 ng/mL of T2 inhibited estradiol production by 40%. Progesterone production was less sensitive to the inhibitory effects of T2 with 0.3 ng/mL having no effect and 1 ng/mL inhibiting progesterone production by only 30%. At 30 and 300 ng/mL, T2 completely inhibited FSH plus IGF-I-induced progesterone production. The impact of T2 on the dose-response to IGF-I (0, 3, 10 and 30 ng/mL) was also evaluated; T2 blunted the stimulatory effect of 3-30 ng/mL of IGF-I on steroid production and cell proliferation. Serum-induced granulosa cell proliferation was decreased (P<0.05) by 40% after 1 day and by 56% after 2 days of T2 treatment. The present studies indicate for the first time that T2 may be able to alter in growth of the granulosa layer within ovarian follicles in addition to their effect on steroidogenesis. In conclusion, T2 has potent direct dose-dependent effects on granulosa cell proliferation and steroidogenesis. These direct ovarian effects could be one mechanism whereby contaminating Fusarium mycotoxins in feedstuffs could impact reproductive performance in swine.

Suppression of progesterone production by stresscopin/urocortin 3 in cultured human granulosa-lutein cells

Corticotropin-releasing hormone (CRH) and its receptors have been identified in female reproductive tissues. CRH regulates follicular maturation, ovulation, luteolysis and steroidgenesis. A CRH-related peptide stresscopin (SCP), or urocortin III (Ucn3), has recently been identified, but its functions in the ovary remain to be elucidated. In the present study, we investigated the effects of SCP/Ucn3 on progesterone production in cultured human granulosa-lutein cells. The presence of SCP/Ucn3 and CRH type-2 receptor (CRHR2) in cultured granulosa-lutein cells from 21 infertile women (aged 22-36 years) was examined by RT-PCR and immunocytochemistry. The concentration of SCP/Ucn3 in follicular fluid, human serum and culture medium was examined by radioimmunoassay. Progesterone production by cultured granulosa-lutein cells treated with SCP/Ucn3 was examined by enzyme-linked immunosorbent assay. SCP/Ucn3 and CRHR2 mRNAs and proteins were expressed in granulosa-lutein cells. SCP/Ucn3 was detected in culture media of granulosa-lutein cells and follicular fluid. Treatment of cultured granulosa-lutein cells with 0.1, 1.0 or 10 nM SCP/Ucn3 decreased progesterone secretion when compared with untreated control (all P < 0.05). Concomitant treatment with the CRHR2 antagonist antisauvagine-30 counteracted the inhibitory effects of SCP/Ucn3 on progesterone secretion, suggesting a mediatory role of CRHR2. The present results suggest that the SCP/CRHR2 system is present in human ovaries and treatment with SCP/Ucn3 inhibits progesterone production by cultured granulosa-lutein cells through interaction with CRHR2.

The Phytoestrogen Quercetin Impairs Steroidogenesis and Angiogenesis in Swine Granulosa Cells In Vitro

Experimental evidence documents that nutritional phytoestrogens may interact with reproductive functions but the exact mechanism of action is still controversial. Since quercetin is one of the main flavonoids in livestock nutrition, we evaluated its possible effects on cultured swine granulosa cell proliferation, steroidogenesis, and redox status. Moreover, since angiogenesis is essential for follicle development, the effect of the flavonoid on Vascular Endothelial Growth Factor output by granulosa cells was also taken into account. Our data evidence that quercetin does not affect granulosa cell growth while it inhibits progesterone production and modifies estradiol 17β production in a dose-related manner. Additionally, the flavonoid interferes with the angiogenic process by inhibiting VEGF production as well as by altering redox status. Since steroidogenesis and angiogenesis are strictly involved in follicular development, these findings appear particularly relevant, pointing out a possible negative influence of quercetin on ovarian physiology. Therefore, the possible reproductive impact of the flavonoid should be carefully considered in animal nutrition.

Defining the role of notch family members in the luteinization of murine granulosa cells in vitro

OBJECTIVE: Spontaneous luteinization of murine granulosa cells occurs in culture, and is characterized by increased production of progesterone, changes in cellular morphology, and biochemical markers. The Notch signaling pathway has been implicated in the processes of cellular proliferation and differentiation. Our goal was to investigate the significance of Notch signaling in the process of granulosa cell (GC) differentiation/luteinization.DESIGN: Analysis of protein expression and progesterone levels in murine GC cultures after inhibition of Notch protein activation or specific knockdown of Notch2.MATERIALS AND METHODS: Groups of C57BL/6 mice (6-9 weeks of age/6-14 per group) were injected (SQ) with Pregnant Mare Serum Gonadotropin (PMSG) to stimulate follicular development. Granulosa cells were obtained from the ovaries 40-44 hours later by follicular puncture. Cells were cultured in 12-well dishes, and the media and cell lysates were collected at 24 hour intervals for 96 hours. For the inhibition experiments, DAPT (γ-secretase inhibitor which prevents the activation of Notch proteins), was added to the media after 24 hours of culture. For the Notch2 knockdown experiments, siRNA transfection was performed after 24 hours of culture. Media and cell lysates were collected at 72 hours of culture. Media samples were tested for progesterone content using ELISA. Cell lysates were tested for expression of Notch2 protein by Western blot.RESULTS: Notch2 protein expression increased in concert with progesterone production in culture. When DAPT was used to inhibit the activation of Notch proteins, the resulting 20% decrease in Notch2 led to an 80% decrease in progesterone levels. Specific knock-down of Notch2 expression resulted in approximately 30% decrease in the protein concentration, and yielded a consistent 30-50% reduction in progesterone levels.CONCLUSIONS: The inhibition of progesterone production by DAPT indirectly suggests that members of the Notch family are involved in the differentiation/luteinization of granulosa cells. The siRNA knockdown experiments identify Notch2 as one of the potential specific players. OBJECTIVE: Spontaneous luteinization of murine granulosa cells occurs in culture, and is characterized by increased production of progesterone, changes in cellular morphology, and biochemical markers. The Notch signaling pathway has been implicated in the processes of cellular proliferation and differentiation. Our goal was to investigate the significance of Notch signaling in the process of granulosa cell (GC) differentiation/luteinization. DESIGN: Analysis of protein expression and progesterone levels in murine GC cultures after inhibition of Notch protein activation or specific knockdown of Notch2. MATERIALS AND METHODS: Groups of C57BL/6 mice (6-9 weeks of age/6-14 per group) were injected (SQ) with Pregnant Mare Serum Gonadotropin (PMSG) to stimulate follicular development. Granulosa cells were obtained from the ovaries 40-44 hours later by follicular puncture. Cells were cultured in 12-well dishes, and the media and cell lysates were collected at 24 hour intervals for 96 hours. For the inhibition experiments, DAPT (γ-secretase inhibitor which prevents the activation of Notch proteins), was added to the media after 24 hours of culture. For the Notch2 knockdown experiments, siRNA transfection was performed after 24 hours of culture. Media and cell lysates were collected at 72 hours of culture. Media samples were tested for progesterone content using ELISA. Cell lysates were tested for expression of Notch2 protein by Western blot. RESULTS: Notch2 protein expression increased in concert with progesterone production in culture. When DAPT was used to inhibit the activation of Notch proteins, the resulting 20% decrease in Notch2 led to an 80% decrease in progesterone levels. Specific knock-down of Notch2 expression resulted in approximately 30% decrease in the protein concentration, and yielded a consistent 30-50% reduction in progesterone levels. CONCLUSIONS: The inhibition of progesterone production by DAPT indirectly suggests that members of the Notch family are involved in the differentiation/luteinization of granulosa cells. The siRNA knockdown experiments identify Notch2 as one of the potential specific players.

Effects of blue-green algal toxin cylindrospermopsin (CYN) on human granulosa cells in vitro

The blue-green algal toxin cylindrospermopsin (CYN) occurs in public water supplies. CYN was hepatotoxic when administered orally to mice, and cytotoxic and genotoxic to human cell lines. To determine the effects of CYN on primary human IVF-derived granulosa cells, 0-1 microg/ml CYN was added to cells for 2, 4 or 6h+/-hCG (n=6), or for 24, 48 and 72 h (n=6). Cytotoxicity was evaluated by MTT assay, and secreted progesterone or estrogen quantified by radioimmunoassay. 24h exposure to 1 microg/ml CYN was cytotoxic (p<0.05), whereas 0.0625 microg/ml CYN did not cause cytotoxicity or affect estrogen production, but did inhibit basal progesterone production (p<0.01). Similarly, 6h exposure to 1 microg/ml CYN did not affect cytotoxicity or hCG-stimulated estrogen production, but did inhibit hCG-stimulated progesterone production (p<0.01). In this in vitro assay, CYN inhibited progesterone production and therefore has the potential to be an endocrine disrupter by changing the progesterone:estrogen ratio in women.

Effects of epigallocatechin-3-gallate (EGCG) on in vitro maturation and fertilization of porcine oocytes

The beneficial properties of green tea and especially of its principal active polyphenol, epigallocatechin-3-gallate (EGCG), have led to an increased demand for dietary supplements with highly enriched EGCG concentrations. In order to investigate the possible reproductive-related consequence of EGCG supplementation, the effects of this catechin on in vitro maturation (IVM) and fertilization (IVF) of oocyte, using the pig as experimental model, were examined. In the first series of experiments EGCG, at concentrations ranging from 0 to 25 microg/ml, was added during in vitro maturation of pig oocytes. EGCG had no effect on nuclear maturation of pig oocytes and on fertilization traits considered after IVF at any of the doses tested. By contrast, a significant (p<0.05) decrease in the number of embryos that developed to blastocysts following parthenogenetic activation was recorded when 25 microg/ml EGCG was added to IVM medium; in addition this catechin concentration significantly (p<0.05) inhibited progesterone production by cumulus cells after 48 h of culture. When induction of sperm capacitation was performed in presence of EGCG, a significantly lower percentage of spermatozoa showing a Hsp70-capacitated pattern and a significant reduction of sperm H(2)O(2) production were evident at a concentration of 25 microg/ml EGCG (p<0.05). During gamete coincubation EGCG reduced, in a dose response manner, the number of reacted spermatozoa suspended in fertilization medium and increased the number of sperm bound to ZP. Supplementation of 10 microg/ml EGCG during IVF significantly increased the fertilization rate while higher EGCG concentrations (25 microg/ml) decreased the percentage of fertilized oocytes (p<0.05). In conclusion, our data suggest that high EGCG concentrations could affect in vitro maturation and fertilization in pig; it cannot be totally excluded that excessive EGCG concentrations could induce reproductive-related consequences also in vivo.

The Cooperative Effect of Growth and Differentiation Factor-9 and Bone Morphogenetic Protein (BMP)-15 on Granulosa Cell Function Is Modulated Primarily through BMP Receptor II

Growth and differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15, GDF9B) are oocyte-derived proteins essential for the growth and function of ovarian follicles. Moreover, ovine (o) GDF9 and oBMP15 cooperate to increase both (3)H-thymidine incorporation and alpha-inhibin production and to inhibit progesterone production by rat or ovine granulosa cells. Although the receptors through which these proteins act individually have been determined, the receptor(s) involved in mediating the cooperative effects of GDF9 and BMP15 is (are) unknown. In this study, the effects of the extracellular domains of the types I and II TGFbeta receptors on (3)H-thymidine incorporation by rat granulosa cells stimulated by oGDF9 and oBMP15 were investigated. Stimulation of (3)H-thymidine incorporation was completely blocked by the BMP receptor II (BMPRII) extracellular domain but unaffected by any other type II or any type I receptor. These results suggest that the initial interaction of oGDF9 and oBMP15 is with BMPRII and that a type I receptor is either recruited or already associated with BMPRII to mediate the cooperative effects of these growth factors.

Fenvalerate inhibits progesterone production through cAMP-dependent signal pathway

Fenvalerate is a widely used synthetic pyrethroid insecticide and is known to impede the male reproductive function. However, the mechanisms remain to be elucidated. In this study, mouse Leydig tumor cells (MLTC-1) were used to investigate the effects of fenvalerate on progesterone production. Fenvalerate treatment inhibited progesterone secretion induced by human chorionic gonadotropin (hCG), cholera toxin (CT) or forskolin and decreased cAMP levels induced by hCG, but not by CT or forskolin, which suggested a repaired site on the upstream components of G protein or G protein per se by fenvalerate in the cAMP-mediated signal pathway. Furthermore, the addition of cAMP analog, 8-Br-cAMP, could not reverse fenvalerate-suppressed progesterone synthesis, indicating that fenvalerate interfered with the downstream molecules of cAMP. In addition, fenvalerate decreased steroidogenic acute regulatory protein (StAR) mRNA and protein levels, and also profoundly inhibited the activity of P450 side chain cleavage enzyme (P450scc) which was consistent with the decreased expression of P450scc mRNA and protein in MLTC-1 cells. These results suggested that fenvalerate might inhibit progesterone production by attenuating cAMP generation and inhibiting StAR expression and P450scc activity.

Effects of Fusarium mycotoxins on steroid production by porcine granulosa cells

Fusarium mycotoxins, such as trichothecenes and zearalenone, are common grain and foodstuffs contaminants. Some of these like deoxynivalenol (DON) can negatively impact pregnancy success in swine, but evidence for direct ovarian effects of DON, zearalenone, and its major metabolite, alpha-zearalenol (ZEA) is meager. To evaluate the effects of two mycotoxins, DON and ZEA on porcine granulosa cell(s) (GC) proliferation, steroidogenesis and gene expression, pig GC from small follicles (1-5mm) were cultured for 2 days in 5% fetal bovine serum and 5% porcine serum-containing medium followed by 2 days in serum-free medium containing control (no mycotoxins) or mycotoxins (at various doses/combinations). Both DON and ZEA had biphasic effects on IGF-I-induced estradiol production, increasing estradiol production at smaller doses and inhibiting at larger doses. ZEA at 3,000 ng/mL (9.37 microM) increased IGF-I-induced progesterone production and at 30 ng/mL (0.0937 microM) and 300 ng/mL (0.937 microM) were without effect, but these doses of ZEA increased FSH-induced progesterone production. ZEA at 3,000 ng/mL inhibited FSH plus IGF-I-induced CYP19A1 and CYP11A1 mRNA abundance. DON inhibited progesterone production at 100 ng/mL (0.337 microM) and 1,000 ng/mL (3.37 microM) but at 10 ng/mL (0.0337 microM) was without effect. DON at 1,000 ng/mL (but not at 10 ng/mL) completely inhibited FSH plus IGF-I-induced CYP19A1 and CYP11A1 mRNA abundance. The concomitant treatment of ZEA had little effect on the dose response to DON. DON increased IGF-I-induced cell numbers at 10 and 100 ng/mL and inhibited cell numbers at 1,000 ng/mL, whereas ZEA had no effect on GC numbers. Only a combined treatment of DON and ZEA increased serum-induced cell proliferation. In conclusion, mycotoxins have direct dose-dependent effects on GC proliferation, steroidogenesis and gene expression. These direct ovarian effects could be one mechanism whereby contaminating Fusarium mycotoxins in feedstuffs could impact reproductive performance in swine.

Nitric oxide in bovine corpus luteum: Possible mechanisms of action in luteolysis

ABSTRACTAlthough prostaglandin (PG) F2α is considered as the principal luteolytic factor, its action on the bovine corpus luteum (CL) is mediated by other intraovarian factors. Among them, nitric oxide (NO) seems to play a mandatory role in luteolysis. In this article we review the background and current status of work on possible roles of NO in the CL function, based on available information and our own experimental data. NO is produced in all three main types of bovine CL cells: steroidogenic, endothelial and immune cells. PGF2α and some luteolytic cytokines (tumor necrosis factor, interferon) increase NO production and stimulate NO synthase expression in the bovine CL. NO inhibits progesterone production, stimulates the secretion of PGF2α and leukotriene C4, reduces the number of viable luteal cells and, finally, participates in functional luteolysis. NO induces the apoptotic death of CL cells by the modulation of bcl‐2 family gene expression and the stimulation of caspase‐3 gene expression and activity. However, this simple molecule shows both luteolytic and luteotropic actions during the estrous cycle in ruminants. The aim of this overview is to present and discuss the recent findings crucial for understanding NO role in the process of CL regression in cattle.

Inhibition of progesterone production in human luteinized granulosa cells treated with LXR agonists

Progesterone production by luteal cells is dependent on the supply of cholesterol by lipoproteins. The aim of this study was to determine whether the liver X receptors (LXRs) contribute to cholesterol homeostasis and progesterone secretion in human luteinized granulosa cells. Cells were isolated from follicular aspirates of patients undergoing in vitro fertilization. Luteinization was induced by a 7-day treatment with human chorionic gonadotrophin. LXR beta was expressed at higher levels than LXR alpha in granulosa cells and its expression was increased during luteinization. Treatment of luteinized granulosa cells by LXR agonists induced a significant time- and dose-dependent reduction in progesterone secretion (50% reductions after a 7-day treatment with 1-microM of either GW3965 or T0901317). mRNA levels of steroidogenic genes including steroidogenic acute regulatory protein and P450 side-chain cleavage were only moderately affected by LXR activation, with a significant reduction that was observed at 10 microM agonist concentration. Cellular cholesterol was markedly reduced after treatment with LXR agonists as a result of an increased cholesterol efflux that was related to the induction of LXR target genes (ABCA1, ABCG1, apo E, PLTP). Our study identifies LXRs as new, key actors contributing to regulation of cholesterol metabolism and steroidogenesis in luteinized granulosa cells.

Corticotropin-releasing hormone inhibits progesterone production in cultured human placental trophoblasts

Placental-derived corticotropin-releasing hormone (CRH) seems to play a major role in the mechanisms controlling human pregnancy and parturition. It has been suggested that CRH directly modulates the endocrine function of placental trophoblasts, including the production of estrogen, ACTH, and prostaglandin. In this study, we sought to investigate the effect of CRH, locally produced by placenta, on progesterone production. Percoll-purified placental trophoblasts were obtained from uncomplicated term pregnancies and cultured for 72 h. Progesterone concentration in culture media was measured by RIA. The mRNA transcripts encoding CYP11A1 and HSD3B1, the enzymes for progesterone synthesis, were determined by quantitative real-time reverse transcription (RT)-PCR. Results showed that CRH (10(-8)-10(-6) mol/l) caused a significant decrease in progesterone levels in a dose-dependent manner. The CRH antagonist, alpha-helical CRH 9-41, at 10(-7)-10(-5) mol/l stimulated progesterone secretion. Consistent with this thesis, CRH decreased, whereas alpha-helical CRH increased, the mRNA levels of CYP11A1 and HSD3B1. Since CRH has been shown to activate the phospholipase C-protein kinase C (PKC) signal pathway in placenta, we examined whether the effect of CRH on progesterone synthesis was dependent on PKC signal pathway. Treatment of cells with PKC inhibitor, Gö6976, resulted in a significant increase in progesterone production, and exogenous CRH restored progesterone production. In conclusion, placental CRH exhibits a tonic inhibitory effect on progesterone production in a PKC-dependent fashion.

Effects of Phytoestrogen Extracts Isolated from Rye, Green and Yellow Pea Seeds on Hormone Production and Proliferation of Trophoblast Tumor Cells Jeg3

Background: Phytoestrogens are a diverse group of non-steroidal plant compounds. Because they have chemical structures similar to estrogens they are able to bind on estrogen receptors in humans. Objectives: In this study, we tested the effects of crude phytoestrogen extracts from rye (Secale cereale), green pea (Pisum sativum) and yellow pea seeds (Pisum sativum cv.) on cell proliferation and the production of progesterone in trophoblast tumor cells of the cell line Jeg3. Methods: Isoflavone extracts from green and yellow pea seeds and lignan extracts from rye seeds were obtained, using different extraction methods. Isolated extracts were incubated in different concentrations with trophoblast tumor cells. Untreated cells were used as controls. At designated times, aliquots were removed and tested for estradiol and progesterone production. In addition, we tested the effects of the phytoestrogen extracts on cell proliferation. Results: Cell proliferation is significantly inhibited by potential phytoestrogens isolated from rye, green and yellow pea seeds in trophoblast tumor cells of the cell line Jeg3. We found a correlation between the effects of proliferation and production of estradiol in isoflavone extracts from green and yellow pea seeds in Jeg3 cells. In addition, higher concentrations of isoflavones isolated from green pea seeds and lignans from rye showed also a inhibition of progesterone production whereas higher concentrations of rye lignans elevated estradiol production in Jeg3 cells. Conclusion: A useful indicator test system for potential phytoestrogens could be established. Based on the obtained results it is proposed that green and yellow pea seeds contain measurable concentrations of isoflavones and rye seeds contain lignans which can be isolated and used for special human diet programs.

The role of bone morphogenetic proteins 2, 4, 6 and 7 during ovarian follicular development in sheep: contrast to rat

The intraovarian roles of BMP family members such as BMP2, 4, 6 and 7 are not well understood, particularly in species with low ovulation rates such as sheep. Therefore, the objectives of these experiments were to determine the expression patterns of mRNAs encoding BMP2, 4, 6 and 7 during ovarian follicular development in sheep, and to determine the effects of these growth factors on ovine granulosa cell functions in vitro. For comparative purposes, the effects of these BMPs were also determined in rat granulosa cells since these factors have been most widely studied in this poly-ovulatory species. As assessed by in situ hybridization, non-atretic ovine follicles expressed mRNA for BMP6 but not 2, 4 or 7. Furthermore, expression of BMP6 was limited to the oocyte of primordial as well as primary, pre-antral and antral follicles. Reverse transcription-PCR of granulosa cell mRNA detected low levels of all the BMPs in some pools of cells. BMP2, 4, 6 and 7 each inhibited progesterone production from ovine granulosa cells without affecting cellular proliferation/survival. Similarly, these BMPs inhibited progesterone production from rat granulosa cells. However, they also stimulated cellular proliferation/survival of the rat granulosa cells highlighting a species-specific difference for these growth factors. In conclusion, in sheep, BMP2, 4, 6 and 7 inhibit granulosa cell differentiation without affecting proliferation. However, as BMP2, 4 and 7 were not detectable by in situ hybridization in any cells of non-atretic ovarian follicles, it seems unlikely that these proteins would have an important intra-ovarian role in regulating follicular development in sheep. In contrast, localization of BMP6 mRNA in the oocyte suggests that this BMP family member may have a paracrine and/or autocrine role in regulating follicular growth in sheep, as has been shown for two other oocyte derived from members of the transforming growth factor superfamily, BMP15 and growth differentiation factor 9.

Extra‐pituitary function of GnRH‐I and GnRH‐II in human reproduction

Recent studies have indicated that both the classical form of mammalian GnRH (GnRH-I) and its novel isoform (GnRH-II) are potent autocrine regulators in several non-pituitary tissues, including the ovary, endometrium and placenta. In human granulosa-luteal cells, we have demonstrated the expression of GnRH-I and GnRH-II, as well as GnRH receptor (GnRH-R). GnRH-II, like GnRH-I, directly inhibits progesterone production in human granulosa-luteal cells. In normal ovarian surface epithelium (OSE) and in 80% of human ovarian epithelial tumors, GnRHR is expressed. A direct anti-proliferative effect has been observed following treatment with GnRH-I or GnRH-II in different ovarian cancer cell lines, presumably via a common GnRHR (the type I GnRH-R). In human endometrial stromal cells and in the first-trimester trophoblast, GnRH-I and GnRH-II are expressed. These peptides differentially regulate the balance between the urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor (PAI-1) levels in the human decidua. GnRH-I and GnRH-II are potent regulators of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) that are important for the overall proteolytic activity of trophoblasts during human implantation. Together, these findings strongly support a multi-faceted role of the GnRH/GnRH-R system in the control of reproduction. [This research was supported by the Canadian Institutes of Health Research].

The regulatory mechanism of Tremella mesenterica on steroidogenesis in MA-10 mouse Leydig tumor cells

Tremella mesenterica (TM), a yellow jelly mushroom, has been traditionally used as tonic food to improve body condition in Chinese society for a long time. We have previously demonstrated that TM reduced in vitro hCG-treated steroidogenesis in MA-10 mouse Leydig tumor cells without any toxicity effect. In the present study, the mechanism how TM suppressed hCG-treated steroidogenesis in MA-10 cells was investigated. MA-10 cells were treated with vehicle, human chorionic gonadotropin (hCG, 50 ng/ml), or different reagents with or without TM to clarify the effects. TM significantly suppressed progesterone production with the presences of forskolin (10 and 100 microM) or dbcAMP (0.5 and 1mM), respectively, in MA-10 cells (p<0.05), which indicated that TM suppressed steroidogenesis after PKA activation along the signal pathway. Beyond our expectation, TM induced the expression of steroidogenic acute regulatory (StAR) protein with or without hCG treatments. However, TM profoundly decreased P450 side chain cleavage (P450scc) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) enzyme activities without any influences on the expression of both enzymes. These inhibitions on steroidogenic enzyme activities might counteract the stimulation of StAR protein expression. In conclusion, results suggest that TM suppressed hCG-treated steroidogenesis in MA-10 cells by inhibiting PKA signal pathway and steroidogenic enzyme activities.

Localized accumulation of angiotensin II and production of angiotensin-(1–7) in rat luteal cells and effects on steroidogenesis

These studies aim to investigate subcellular distribution of angiotensin II (ANG II) in rat luteal cells, identify other bioactive angiotensin peptides, and investigate a role for angiotensin peptides in luteal steroidogenesis. Confocal microscopy showed ANG II distributed within the cytoplasm and nuclei of luteal cells. HPLC analysis showed peaks that eluted with the same retention times as ANG-(1-7), ANG II, and ANG III. Their relative concentrations were ANG II >or= ANG-(1-7) > ANG III, and accumulation was modulated by quinapril, an inhibitor of angiotensin-converting enzyme (ACE), Z-proprolinal (ZPP), an inhibitor of prolyl endopeptidase (PEP), and parachloromercurylsulfonic acid (PCMS), an inhibitor of sulfhydryl protease. Phenylmethylsulfonyl fluoride (PMSF), a serine protease inhibitor, did not affect peptide accumulation. Quinapril, ZPP, PCMS, and PMSF, as well as losartan and PD-123319, the angiotensin receptor type 1 (AT1) and type 2 (AT2) receptor antagonists, were used in progesterone production studies. ZPP significantly reduced luteinizing hormone (LH)-dependent progesterone production (P < 0.05). Quinapril plus ZPP had a greater inhibitory effect on LH-stimulated progesterone than either inhibitor alone, but this was not reversed by exogenous ANG II or ANG-(1-7). Both PCMS and PMSF acutely blocked LH-stimulated progesterone, and PCMS blocked LH-sensitive cAMP accumulation. Losartan inhibited progesterone production in permeabilized but not intact luteal cells and was reversed by ANG II. PD-123319 had no significant effect on luteal progesterone production in either intact or permeabilized cells. These data suggest that steroidogenesis may be modulated by angiotensin peptides that act in part through intracellular AT1 receptors.

Meat and Livestock Association Plenary Lecture 2005. Oocyte signalling molecules and their effects on reproduction in ruminants

Sheep (Ovis aries) are a highly diverse species, with more than 900 different breeds that vary significantly in their physiological characteristics, including ovulation rate and fecundity. From examination of inherited patterns of ovulation rate, several breeds have been identified with point mutations in two growth factor genes that are expressed in oocytes. Currently, five different point mutations have been identified in the BMP15 (GDF9b) gene and one in GDF9. Animals heterozygous for the GDF9 and/or the BMP15 mutations have higher ovulation rates than their wild-type counterparts. In contrast, those homozygous for any of the aforementioned BMP15 or GDF9 mutations are sterile owing to arrested follicular development. In bovine and ovine ovaries, GDF9 was expressed exclusively in oocytes throughout follicular growth from the primordial stage of development, whereas in sheep BMP15 was expressed exclusively in oocytes from the primary stage: no data for the ontogeny of BMP15 expression are currently available for cattle. In vitro, ovine growth differentiation factor 9 (oGDF9) has no effect on (3)H-thymidine incorporation by either bovine or ovine granulosa cells, whereas ovine bone morphogenetic protein 15 (oBMP15) has modest (1.2- to 1.6-fold; P < 0.05) stimulatory effects. Ovine GDF9 or oBMP15 alone inhibited progesterone production by bovine granulosa cells, whereas in ovine cells only oGDF9 was inhibitory. The effects of oGDF9 and oBMP15 together were often cooperative and not always the same as those observed for each factor alone. Active immunisation of ewes with BMP15 and/or GDF9 peptides affected ovarian follicular development and ovulation rate. Depending on the GDF9 and/or BMP15 vaccine formulation, ovulation rate was either increased or suppressed. A primary and single booster immunisation of ewes with a BMP15 peptide in a water-based adjuvant has led to 19-40% increases in lambs born per ewe lambing. Collectively, the evidence suggests that oocyte signalling molecules have profound effects on reproduction in mammals, including rodents, humans and ruminants. Moreover, in vivo manipulation of these oocyte signalling molecules provides new opportunities for the management of the fertility of ruminants.