Progesterone Production In Granulosa Cells Research Articles

Activin A, B and AB decrease progesterone production by down-regulating StAR in human granulosa cells

Activins are homo- or heterodimers of inhibin β subunits that play important roles in the reproductive system. Our previous work has shown that activins A (βAβA), B (βBβB) and AB (βAβB) induce aromatase/estradiol, but suppress StAR/progesterone production in human granulosa-lutein cells. However, the underlying molecular determinants of these effects have not been examined. In this continuing study, we used immortalized human granulosa cells (SVOG) to investigate the effects of activins in regulating StAR/progesterone and the potential mechanisms of action. In SVOG cells, activins A, B and AB produced comparable down-regulation of StAR expression and progesterone production. In addition, all three activin isoforms induced equivalent phosphorylation of both SMAD2 and SMAD3. Importantly, the activin-induced down-regulation of StAR, increase in SMAD2/3 phosphorylation, and decrease in progesterone were abolished by the TGF-β type I receptor inhibitor SB431542. Interestingly, the small interfering RNA-mediated knockdown of ALK4 but not ALK5 reversed the activin-induced suppression of StAR. Furthermore, the knockdown of SMAD4 or SMAD2 but not SMAD3 abolished the inhibitory effects of all three activin isoforms on StAR expression. These results provide evidence that activins A, B and AB down-regulate StAR expression and decrease progesterone production in human granulosa cells, likely via an ALK4-mediated SMAD2/SMAD4-dependent pathway. Our findings provide important insights into the molecular mechanisms underlying the regulatory effects of activins on human granulosa cell steroidogenesis.

Expression and effect of NAMPT (visfatin) on progesterone secretion in hen granulosa cells

In mammals, nicotinamide phosphoribosyltransferase (NAMPT) is an adipokine produced by adipose tissue that is found in intracellular and extracellular compartments. The intracellular form of NAMPT is a nicotinamide phosphoribosyltransferase, whereas the extracellular form is considered an adipokine. In humans, NAMPT regulates energy metabolism and reproductive functions, such as ovarian steroidogenesis. To date, no study has investigated the role of NAMPT in hen ovaries. We investigated whether NAMPT is present in hen ovarian follicles and its role in granulosa cells. Using RT-PCR, western blotting and immunocytochemistry, we detected mRNA transcripts and proteins related to NAMPT in theca and granulosa cells from pre-ovulatory follicles. Using RT-PCR, we demonstrated that mRNA NAMPT levels were higher in granulosa cells than they were in theca cells and that during follicle development, theca cell levels decreased, whereas levels remained unchanged in granulosa cells. NAMPT protein quantities were significantly higher in theca cells than they were in granulosa cells, but they were unchanged during follicular development. Plasma NAMPT levels, as determined by ELISA and immunoblotting, were significantly lower in adult hens than they were in juveniles. In vitro, treatment with human recombinant NAMPT (100 ng/ml, 48 h) halved basal and IGF1-induced progesterone secretion, and this was associated with a reduction in STAR and HSD3B protein levels and MAPK3/1 phosphorylation levels in granulosa cells. These effects were abolished by the addition of FK866, a specific inhibitor of NAMPT enzymatic activity. Moreover, NAMPT had no effect on granulosa cell proliferation. In conclusion, NAMPT is present in hen ovarian cells and inhibits progesterone production in granulosa cells.

T-2 toxin regulates steroid hormone secretion of rat ovarian granulosa cells through cAMP-PKA pathway

T-2 toxin is a secondary metabolite produced by Fusarium genus and is a common contaminant in food and feedstuffs of cereal origin. In porcine granulosa cells(GC), T-2 toxin has been shown to inhibit the steroidogenesis; however, the mechanism has not been well understood. Gonadotropin-stimulated steroidogenesis is regulated by the cAMP-PKA pathway. In this study, we investigated potential mechanisms for T-2 toxin-induced reproductive toxicity focusing on the critical steps of the cAMP-PKA pathway affected by T-2 toxin. We first analyzed the effects of T-2 toxin on progesterone and estrogen production in rat granulosa cells. For this purpose the granulosa cells were cultured for 48h in 10% fetal bovine serum-containing medium followed by 24h in serum-free medium containing FSH (10ng/ml) and androstenedione (3ng/ml), both are required for normal steroidogenesis. Treatment of these cells with T-2 toxin dose-dependently inhibited the growth of cells and the steroid hormone production. Cellular cyclic AMP levels were dose-dependently inhibited by T-2 toxin (0, 1, 10 and 100nM, 24h). Furthermore, we found that although the induction of progesterone by 8-Br-cAMP (a FSH mimetic) and 22R-HC (substrate for progesterone) could both be inhibited by T-2 toxin treatment, the T-2-imposed inhibitory effects could be reversed by increasing doses of 22R-HC, while increasing 8-Br-cAMP had no effects, suggesting that T2 toxin targeted at distinct mechanisms. cAMP-stimulated steroidogenic acute regulatory protein (StAR) is a rate limiting protein in progesterone synthesis. Exposure to T2 toxin caused significant suppression of StAR expression as determined by Western blotting and semi-quantitative RT-PCR suggesting StAR is a sensitive target for T-2 toxin. Taken together, our results strongly suggest that T2 toxin inhibits steroidogenesis by suppressing cAMP-PKA pathway and StAR is a target for T-2-toxin. The antisteroidogenesis effects were observable at low T-2 dose (1ng/ml) suggesting T-2 toxin has an endocrine disruptive effect.

TGF-β1 downregulates StAR expression and decreases progesterone production through Smad3 and ERK1/2 signaling pathways in human granulosa cells.

Regulation of progesterone production in granulosa cells is important for normal reproductive functions. Steroidogenic acute regulatory protein (StAR) is recognized as the key regulatory protein involved in the rate-limiting step of steroidogenesis. TGF-β1 protein is detected in human follicular fluid, and TGF-β1 and its receptors are expressed in human granulosa cells. However, the functional role of TGF-β1 in the regulation of StAR expression and progesterone production in human granulosa cells remains unknown. Our objective was to investigate the effects of TGF-β1 on StAR expression and progesterone production in human granulosa cells. SVOG cells are human granulosa cells that were obtained from women undergoing in vitro fertilization and immortalized with SV40 large T antigen. SVOG cells were used to investigate the effects of TGF-β1 on StAR expression and progesterone production at an academic research center. Levels of mRNA and protein were examined by RT-qPCR and western blotting, respectively. The accumulation levels of progesterone were measured by enzyme-linked immunosorbent assay (ELISA). TGF-β1 treatment downregulated StAR expression and decreased progesterone production. The suppressive effects of TGF-β1 on StAR expression and progesterone production were abolished by the inhibition of TGF-β type I receptor. In addition, treatment with TGF-β1 activated the Smad2/3 and ERK1/2 signaling pathways. The inhibition of the Smad3 and ERK1/2 signaling pathways attenuated the TGF-β1-induced downregulation of StAR expression and progesterone production. TGF-β1 downregulated StAR expression and decreased progesterone production by activating the Smad3 and ERK1/2 signaling pathways in human granulosa cells.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and phytoestrogen genistein on the activity and the presence of steroidogenic enzyme proteins in cultured granulosa cells of pigs

Environmental estrogens such as dioxins (e.g. 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD) and phytoestrogens (e.g. genistein; G) are known to influence endocrine and reproductive processes in humans and animals. Because living organisms are usually exposed to small, non toxic, doses of dioxins and phytoestrogens, the aims of the study were to determine the effects of small, environmentally relevant doses of TCDD (100pM) and/or genistein (500nM) on: (1) the activity of steroidogenic enzymes (cholesterol side-chain cleavage enzyme, P450scc; 3β-hydroxysteroid dehydrogenase, 3β-HSD and aromatase, P450arom) and (2) amount of protein of the enzymes in granulosa cells isolated from medium and large ovarian follicles of pigs. To determine the activity of the enzymes, the incubation medium was supplemented with specific steroid substrates (25-hydroxycholesterol; pregnenolone; testosterone) of particular steroidogenic enzymes (P450scc, 3β-HSD and P450arom, respectively). Subsequently, the production of progesterone (P450scc and 3β-HSD) or estradiol (P450arom) was compared in the presence and absence of the appropriate steroid precursor. Neither genistein nor genistein combined with TCDD affected activity of P450arom and relative amounts of steroidogenic enzyme proteins in the examined granulosa cells of pigs. In contrast, genistein alone and in combination with TCDD decreased P450scc and 3β-HSD activity as well as progesterone production in granulosa cells isolated from medium and large follicles of pigs. Because TCDD alone did not affect steroid hormone production or enzyme activity, the above effects should be ascribed solely to genistein. It appears that the effects of the examined doses of TCDD and genistein on granulosal cell functions were not additive.

Activity of 3β-hydroxysteroid dehydrogenase associated with progesterone production in bovine granulosa cells cultured under different concentrations of serum, insulin-like growth factor I, and gonadotropin

Three-b-hydroxysteroid dehydrogenase (3b-HSD) is the enzyme responsible for progesterone production. This study aimed to determine whether 3b-HSD activity can be shown to reflect progesterone production by granulosa cells cultured under different serum conditions and follicle stimulating hormone (FSH), luteinising hormone (LH), and insulin-like growth factor I (IGF-I) concentrations. Large bovine follicles were dissected from abattoir ovaries to recover granulosa cells. Cells were washed, stained for viability, and plated for 48 h in basic medium with or without 5% foetal calf serum (FCS). Subsequently, cells were exposed to FSH (1 ng/mL), LH (10 or 100 ng/mL), or FSH (1 ng/mL) + IGF-I (1 or 10 ng/mL) in a serum-free medium for another 96 h to predict degree of luteinisation. Before and after incubation, granulosa cells were stained for 3b-HSD activity. The high dose of IGF-I (10 ng/mL) increased (P < 0.05) progesterone secretion over 2.5-fold compared with FSH alone or the low dose of IGF-I (1 ng/mL) in cells preincubated with FCS. This was clearly reflected by darker 3b-HSD staining than in cells exposed to FSH and low dose IGF-I.

BMP15 Suppresses Progesterone Production by Down-Regulating StAR via ALK3 in Human Granulosa Cells

In addition to somatic cell-derived growth factors, oocyte-derived growth differentiation factor (GDF)9 and bone morphogenetic protein (BMP)15 play essential roles in female fertility. However, few studies have investigated their effects on human ovarian steroidogenesis, and fewer still have examined their differential effects or underlying molecular determinants. In the present study, we used immortalized human granulosa cells (SVOG) and human granulosa cell tumor cells (KGN) to compare the effects of GDF9 and BMP15 on steroidogenic enzyme expression and investigate potential mechanisms of action. In SVOG cells, neither GDF9 nor BMP15 affects the mRNA levels of P450 side-chain cleavage enzyme or 3β-hydroxysteroid dehydrogenase. However, treatment with BMP15, but not GDF9, significantly decreases steroidogenic acute regulatory protein (StAR) mRNA and protein levels as well as progesterone production. These suppressive effects, along with the induction of Sma and Mad-related protein (SMAD)1/5/8 phosphorylation, are attenuated by cotreatment with 2 different BMP type I receptor inhibitors (dorsomorphin and DMH-1). Furthermore, depletion of activin receptor-like kinase (ALK)3 using small interfering RNA reverses the effects of BMP15 on SMAD1/5/8 phosphorylation and StAR expression. Similarly, knockdown of ALK3 abolishes BMP15-induced SMAD1/5/8 phosphorylation in KGN cells. These results provide evidence that oocyte-derived BMP15 down-regulates StAR expression and decreases progesterone production in human granulosa cells, likely via ALK3-mediated SMAD1/5/8 signaling. Our findings suggest that oocyte may play a critical role in the regulation of progesterone to prevent premature luteinization during the late stage of follicle development.

LH and hCG Action on the Same Receptor Results in Quantitatively and Qualitatively Different Intracellular Signalling

Human luteinizing hormone (hLH) and chorionic gonadotropin (hCG) act on the same receptor (LHCGR) but it is not known whether they elicit the same cellular and molecular response. This study compares for the first time the activation of cell-signalling pathways and gene expression in response to hLH and hCG. Using recombinant hLH and recombinant hCG we evaluated the kinetics of cAMP production in COS-7 and hGL5 cells permanently expressing LHCGR (COS-7/LHCGR, hGL5/LHCGR), as well as cAMP, ERK1/2, AKT activation and progesterone production in primary human granulosa cells (hGLC). The expression of selected target genes was measured in the presence or absence of ERK- or AKT-pathways inhibitors. In COS-7/LHCGR cells, hCG is 5-fold more potent than hLH (cAMP ED50: 107.1±14.3 pM and 530.0±51.2 pM, respectively). hLH maximal effect was significantly faster (10 minutes by hLH; 1 hour by hCG). In hGLC continuous exposure to equipotent doses of gonadotropins up to 36 hours revealed that intracellular cAMP production is oscillating and significantly higher by hCG versus hLH. Conversely, phospho-ERK1/2 and -AKT activation was more potent and sustained by hLH versus hCG. ERK1/2 and AKT inhibition removed the inhibitory effect on NRG1 (neuregulin) expression by hLH but not by hCG; ERK1/2 inhibition significantly increased hLH- but not hCG-stimulated CYP19A1 (aromatase) expression. We conclude that: i) hCG is more potent on cAMP production, while hLH is more potent on ERK and AKT activation; ii) hGLC respond to equipotent, constant hLH or hCG stimulation with a fluctuating cAMP production and progressive progesterone secretion; and iii) the expression of hLH and hCG target genes partly involves the activation of different pathways depending on the ligand. Therefore, the LHCGR is able to differentiate the activity of hLH and hCG.

Effects of testosterone, FSH, and LH on oestradiol and progesterone secretion by preovulatory cumulus oophorus complexes of the rat.

Female Wistar rats exhibiting a regular 4-day oestrous cycle were included in this study. They were killed in succession on the day of pro-oestrus at 11.00, 18.00, and 22.00 h. From ovarian preovulatory follicles cumulus oophorus complexes (COCs) were isolated and subsequently cultured with or without testosterone (T), T plus FSH, or T plus LH. In control cultures COCs isolated at all investigated hours released similar amounts of oestradiol. T stimulated this basal secretion and the effect was usually enhanced in the presence of FSH or LH. In control cultures the amount of released progesterone was greatest when expanded COCs were isolated (22.00 h). T present in culture media diminished the amount of secreted progesterone. However, when T was added with FSH or LH a distinct stimulatory effect was observed, except in cultures with T plus FSH set up at 22.00 h. Previously, gonadotrophins alone did not effect progesterone secretion. The results suggest that T can regulate steroid, and especially progesterone secretion by COCs. Until the preovulatory gonadotrophin surge T can inhibit luteinization of COCs, while afterwards, acting synergestically with gonadotrphins (especially with LH), T can stimulate progesterone production in the cumulus granulosa cells.

The Effects of Platelet-Derived Growth Factor BB on Ovine Granulosa Cell Function.

Platelet derived growth factors (PDGF) have been shown to modulate ovarian follicular development in some species. However, relatively little is known regarding the potential role of PDGF in regulating granulosa cell function in ruminants. Oocytes and granulosa cells in growing ovine follicles express mRNA encoding both PDGFA and PDGFB and follicular atresia appeared to cause decreased expression in the granulosa cells. However, with in situ hybridization, mRNAs for PDGF receptors (PDGFRA and PDGFRB) were not detectible in the granulosa cells. Thus, it is not clear whether PDGF can regulate granulosa cell function in vitro. Therefore, our aim was to determine the effects of PDGF on thymidine incorporation and progesterone production in sheep granulosa cells. Granulosa cells were collected from ovine follicles 1-2 mm in diameter. The incorporation of 3H-thymidine was determined during a 24 hour culture (20,000 viable cells per well) with M199 media. Secretion of progesterone was measured following culture of granulosa cells (100,000 viable cells per well) in an enriched McCoys 5a media with 30 ng/ml androstenedione, 3 ng/ml ovine FSH and 1 ng/ml IGF-1. Cells were cultured for 6 days with media being replaced every 48 hrs. The concentration of progesterone was measured by RIA in the media collected at the end of the culture period. The amount of DNA at the end of the culture period was also measured using binding of Hoechst 33258 dye. Triplicate wells were included in each assay and a minimum of 3 independent granulosa cell cultures were examined. The effects of human PDGFBB (10 ng/ml) was compared to vehicle treated control cells using paired t-tests. Addition of PDGFBB increased (P<0.05) 3H-thymidine incorporation 4.33 ± 1.10 fold compared to control cultures. Progesterone concentrations in PDGFBB treated granulosa cells decreased (P<0.01) to 0.33 ± 0.03 of those observed in control cultures with no effect on DNA content (1.06 ± 0.09 fold relative to control cultures). Given that PDGFBB regulated ovine granulosa cell function even though mRNA encoding PDGF receptors could not be localized to granulosa cells by in situ hybridization, RT-PCR was used to determine if culture of the granulosa cells might induce expression of PDGF receptors. Total cellular mRNA was extracted from freshly isolated granulosa cells or those cultured for 2 days (2 pools each). One pool of freshly isolated granulosa cells and both pools of cultured granulosa cells contained both PDGFRA and PDGFRB mRNA. Therefore, PDGFs are likely regulators of granulosa cell function in sheep although differences in receptor expression requires further exploration.

Progesterone production requires activation of caspase-3 in preovulatory granulosa cells in a serum starvation model

Granulosa cells proliferate, differentiate, and undergo apoptosis throughout follicular development. Previous studies have demonstrated that stimulation of progesterone production is accompanied by caspase-3 activation. Moreover, we previously reported that arsenic enhanced caspase-3 activity coupled with progesterone production. Inhibition of caspase-3 activity can significantly inhibit progesterone production induced by arsenic or follicle-stimulating hormone (FSH). Here, we report that serum starvation induces caspase-3 activation coupled with augmentation of progesterone production. Serum starvation also increased the levels of cytochrome P450 cholesterol side chain cleavage enzyme (P450scc) and steroidogenic acute regulatory (StAR) protein, both of which may contribute to progesterone synthesis in preovulatory granulosa cells. Inhibition of caspase-3 activity resulted in a decrease in progesterone production. Deactivation of caspase-3 activity by caspase-3 specific inhibitor also resulted in decreases in P450scc and StAR expression, which may partly contribute to the observed decrease in progesterone production.Our study demonstrates for the first time that progesterone production in preovulatory granulosa cells is required for caspase-3 activation in a serum starvation model. Inhibition of caspase-3 activity can result in decreased expression of the steroidogenic proteins P450scc and StAR. Our work provides further details on the relationship between caspase-3 activation and steroidogenesis and indicates that caspase-3 plays a critical role in progesterone production by granulosa cells.

Circadian Clock genes Per2 and clock regulate steroid production, cell proliferation, and luteinizing hormone receptor transcription in ovarian granulosa cells

Circadian Clock genes are associated with the estrous cycle in female animals. Treatment with Per2 and Clock siRNAs decreased the number of granulosa cells and LHr expression in follicle-stimulating hormone FSH-treated granulosa cells. Per2 siRNA treatment did not stimulate the production of estradiol and expression of P450arom, whereas Clock siRNA treatment inhibited the production of estradiol and expression of P450arom mRNA. Per2 and Clock siRNA treatment increased and unchanged, respectively, progesterone production in FSH-treated granulosa cells. Similarly, expression of StAR mRNA was increased by Per2 siRNA and unchanged by Clock siRNA. Our data provide a new insight that Per2 and Clock have different action on ovarian granulosa cell functions.

Effect of resistin on granulosa and theca cell function in cattle

Resistin is an adipokine that has not been extensively studied in cattle but is produced by adipocytes in greater amounts in lactating versus non-lactating cattle. Seven experiments were conducted to determine the effect of resistin on proliferation, steroidogenesis, and gene expression of theca and granulosa cells from small (1–5 mm) and/or large (8–22 mm) cattle follicles. Resistin had no effect on IGF-I-induced proliferation of large-follicle theca cells or small-follicle granulosa cells, but decreased IGF-I-induced proliferation of large-follicle granulosa cells. Resistin weakly stimulated FSH plus IGF-I-induced estradiol production by large-follicle granulosa cells, but had no effect on IGF-I- or insulin-induced progesterone and androstenedione production by theca cells or progesterone production by granulosa cells of large follicles. In small-follicle granulosa cells, resistin attenuated the stimulatory effect of IGF-I on progesterone and estradiol production of small-follicle granulosa cells. RT-PCR measuring abundance of side-chain cleavage enzyme ( CYP11A1), aromatase ( CYP19A1), FSH receptor ( FSHR) and LH receptor ( LHCGR) mRNA in large- and small-follicle granulosa cells indicated that resistin reduced the stimulatory effect of IGF-I on CPY11A1 mRNA abundance in large-follicle granulosa cells but had no effect on CYP19A1, FSHR or LHCGR mRNA abundance in large- or small-follicle granulosa cells. Resistin had no effect on CYP11A1, CYP17A1 or LHCGR mRNA abundance in theca cells. These results indicate that resistin preferentially inhibits steroidogenesis of undifferentiated (small follicle) granulosa cells and inhibits proliferation of differentiated (large follicle) granulosa cells, indicating that the ovarian response to resistin is altered during follicular development.

Inhibitory effects of epidermal growth factor on progesterone production of ovarian granulosa cells in Tsaiya duck (Anas platyrhynchos var. domestica)

1. The purpose of this study was to investigate the effects of epidermal growth factor (EGF) on progesterone (P4) secretion from ovarian granulosa cells in Tsaiya ducks (Anas platyrhynchos var. domestica). 2. We obtained the largest (F1) follicle from Tsaiya duck, the granulosa layer was separated and the cells were isolated according to their proximity to the germinal disc. 3. The granulosa cells were cultured in vitro, the culture media and the cells were used to determine P4 and steroidogenic enzyme concentrations, respectively. 4. P4 concentrations were decreased in cultured granulosa cells taken proximal to the germinal disc (GD) compared to those distal to the germinal disc (NGD). 5. EGF inhibited both basal and ovine luteinising hormone (oLH)-induced P4 concentrations. It also inhibited the P4 secretion via protein kinase A (PKA) pathway when cultured with GD and NGD granulose cells (mixed together) in vitro. 6. Western blot results showed decreased concentrations of cytochrome P450 side-chain cleavage (P450scc) enzyme, 3β-hydroxysteroid dehydrogenase (3β-HSD) enzyme and steroid acute regulatory (StAR) protein expression, when the cells were co-treated with EGF and oLH. 7. The inhibitory effect of oLH-induced P4 production was attenuated by EGF by the addition of MAP-erk kinase (MEK) inhibitor (PD98059) suggests EGF may inhibit P4 production by affecting via the mitogen-activated protein kinase (MAPK) pathway.

LH-Induced Neuregulin 1 (NRG1) Type III Transcripts Control Granulosa Cell Differentiation and Oocyte Maturation

Epidermal growth factor (EGF)-like factors [amphiregulin (AREG), betacellulin, and epiregulin] are induced by LH and activate the EGF receptor (ERBB1)/ERK1/2 pathway in granulosa cells and cumulus cells of preovulatory follicles to impact ovulation. However, the expression and roles of other ERBB family members and their ligands have not been explored in detail. Herein, we document that two transcripts of the neuregulin (Nrg1) gene are expressed in granulosa cells, and that the type III Nrg1 is induced during ovulation in an ERK1/2 and C/EBPβ-dependent manner. Western blotting shows that intact (75 kDa) and secreted (45 kDa) forms of neuregulin 1 (NRG1) are present in the ovary. NRG1 likely binds to ERBB3/ERBB2 complexes that are expressed in granulosa cells and cumulus cells. In cultured granulosa cells, NRG1 selectively stimulates the phosphorylation of AKT/PKB compared to ERK1/2. However, when granulosa cells were cultured with NRG1 and AREG, the phosphorylation of ERK1/2 was markedly enhanced as compared with that by AREG alone. Cotreatment with NRG1 and AREG also increased progesterone production. When cumulus-oocyte complexes (COCs) were cultured with both NRG1 and AREG, the matured oocytes exhibited significantly higher developmental competence as compared with that of oocytes cultured with AREG alone. Collectively, these results document that the expression of type III NRG1 is induced in granulosa cells during ovulation and that NRG1 enhances AREG-induced ERK1/2 phosphorylation in both granulosa cells and cumulus cells. The NRG1 pathway has two roles: one is to enhance AREG-induced progesterone production in granulosa cells, and the other is to regulate oocyte maturation by a cumulus cell-dependent mechanism.

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

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

Effects of Nonylphenol on the Production of Progesterone by Rat Granulosa Cells.

Nonylphenol (NP) is a metabolite of detergent. The estrogenic effects and acute toxicity of NP causes disturbance in reproductive system. It has been reported that NP inhibits testosterone synthesis in male gonads, while the effects on progesterone release from female ovarian cells are unclear. In the present study, the effects of NP on steroidogenesis of progesterone in rat granulosa cells were examined both in vitro and in vivo. Immature (3-week old) female rats were intraperitoneally injected with pregnant mare serum gonadotropin (PMSG, 20 IU). After 48 hrs, the PMSG-treated rats were sacrificed and granulosa cells were collected from rat ovaries. The granulosa cells were cultured in 24-well plates (105 cells/ 0.5 ml) for 48 hrs, then treated with NP (4.3 µM, 12.8 µM, 43 µM ), in the presence or absence of human chorionic gonadotropin (hCG, 0.05 IU/ml) for 2 to 24 hrs to evaluate the NP effects on progesterone steroidogenesis. The levels of progesterone in medium were measured by radioimmunoassay, and the protein expressions were detected by western blotting. The data indicated that NP stimulated production of progesterone via a dose-dependent manner, especially for 2, 4 and 8 hrs, while treatment of NP for 24 hrs decreased progesterone release. Treatment of NP for 16 and 24 hrs prevented the stimulatory effects of hCG. We also gave the immature female rats gavage of NP with daily dose of 0, 50, 100 and 200 µg per kg body weight of NP for 7 days. The progesterone levels were raised following administration of 100 µg NP. Stimulation with hCG and 8-bromo-adenosine cyclic monophosphate (8-Br-cAMP) showed similar trends. MAPK/ERK kinase (MEK) inhibitor PD98059 did not affect progesterone as compared with vehicle treatment. Expressions of steroidogenic acute regulatory (StAR) protein were enhanced with 100 µg of NP. Moreover, gavage of NP (100 µg /kg/day) during 21-day pregnancy increased progesterone production of the female puppies' granulosa cells compared with those treatment without NP. These data suggested that NP directly stimulated StAR protein expression and affected the synthesis and production of progesterone in rat granulosa cells via activation of cAMP pathway. (poster)

Identification of genes targeted by FSH and oocytes in porcine granulosa cells

In the mammalian ovarian follicle maturing oocytes are nurtured and supported by surrounding somatic cells, the mural granulosa cells and the cumulus cells. These cells are regulated by follicle-stimulating hormone (FSH), originating from the pituitary, and paracrine factors derived from the oocyte. To gain insight into the mechanisms involved in the regulation of granulosa cell function, this study aimed to identify genes in mural granulosa cells that are regulated by FSH and oocyte secreted factors using the pig as a model organism. Mural granulosa cells were collected from 3–6 mm follicles from sow ovaries and cultured in serum free medium in the presence or absence of FSH and/or isolated cumulus oocyte complexes (COCs). FSH significantly increased both the metabolic activity and progesterone production of granulosa cells, while the presence of COCs reversed these FSH effects. Expression levels of mRNA in the absence/presence of FSH and COCs were analyzed on porcine specific microarrays representing 11,300 genes. Both previously identified and novel FSH target genes as well as some oocyte affected genes were found. Expression of inhibitor of DNA binding protein 2 and 3, ID2 and ID3, was decreased by FSH but increased by COCs, as validated by quantitative PCR. These proteins function as dominant negative basic helix loop helix (bHLH) transcription factors and since all regulated genes contain the consensus E-box sequence that can bind bHLH factors, our data suggest that FSH and COCs may regulate granulosa cell function by tuning the activity of bHLH factors, through ID2 and ID3.

Stimulatory Effects of Propylthiouracil on Pregnenolone Production through Upregulation of Steroidogenic Acute Regulatory Protein Expression in Rat Granulosa Cells

Propylthiouracil (PTU) is a common and effective clinical medicine for the treatment of hyperthyroidism. Our previous study demonstrated that short-term treatment with PTU inhibits progesterone production in rat granulosa cells. However, our present results indicate that a 16-h treatment with PTU was able to stimulate pregnenolone production in rat granulosa cells, although progesterone production was diminished by PTU through inhibition of 3β-hydroxysteroid dehydrogenase. Notably, we found that PTU treatment enhanced the conversion of cholesterol into pregnenolone, whereas the protein level of the cytochrome P450 side-chain cleavage enzyme (P450scc, which is the enzyme responding to this conversion) was not affected. Interestingly, the levels of steroidogenic acute regulatory protein (StAR) in both total cell lysate and the mitochondrial fraction were significantly increased by PTU treatment. Furthermore, the binding of steroidogenic factor-1 (SF-1) to the StAR promoter region was also enhanced by PTU treatment, which suggests that PTU could upregulate StAR gene expression. In addition to SF-1 regulation, we found that mitogen-activated protein (MAP) kinase kinase activation is an important regulator of PTU-stimulated StAR protein expression, based on the effects of the MEK inhibitor PD98059. In conclusion, these results indicate that PTU plays opposite roles in the production of progesterone and its precursor, pregnenolone. The regulation of negative feedback on speeding the cholesterol transportation and pregnenolone conversion after a 16-h PTU treatment may be the mechanism explaining PTU's inhibition of progesterone production in rat granulosa cells.

Peroxisome proliferator-activated receptor gamma and early folliculogenesis during an acute hyperandrogenism condition

Acute hyperandrogenism decreases serum P levels and induces early apoptosis of antral follicles by a mechanism mediated by the peroxisome proliferator-activated receptor gamma system and independent of the steroidogenic acute regulator protein.