Tetradecanoyl phorbol acetate suppresses follicle-stimulating hormone-induced synthesis of the cholesterol side-chain cleavage enzyme complex in rat ovarian granulosa cells

The effect of tetradecanoylphorbol acetate (TPA) on follicle-stimulating hormone (FSH)-induced synthesis of the cholesterol side-chain cleavage (SCC) enzyme complex was studied in rat ovarian granulosa cells cultured for 48 h in serum-free medium. Cell proteins were radiolabeled with [35S]methionine, followed by immunoprecipitation of cholesterol side-chain cleavage cytochrome P-450 (P-450SCC) as well as the iron-sulfur protein adrenodoxin. Polyacrylamide gel electrophoresis and fluorography of the immunoprecipitates showed that TPA, when added in combination with FSH (50 ng/ml) or dibutyryl cAMP (Bt2cAMP; 1 mM), suppressed the stimulatory effects of these compounds on the synthesis of the SCC components in a concentration-dependent fashion. The effect of TPA was accompanied by decreased progesterone formation and decreased cAMP accumulation. The structural analog of TPA, phorbol-4 alpha-didecanoate, which does not activate protein kinase C (Ca2+/phospholipid-dependent enzyme), had no effect on the FSH- or Bt2cAMP-stimulated synthesis of SCC and progesterone or on cAMP formation. In addition to inhibiting the synthesis of these proteins, TPA greatly reduced the FSH- and Bt2cAMP-induced increase in levels of mRNA encoding the precursor form of P-450SCC. It is concluded that the effect of the phorbol ester TPA to inhibit FSH-stimulated progesterone formation in cultured ovarian granulosa cells of the rat involves decreased synthesis of the components of the SCC enzyme complex due to reduced levels of mRNA encoding the precursor forms of these proteins. The results are indicative that TPA not only inhibits FSH-mediated stimulation of cAMP formation but also may block cAMP-mediated induction of SCC synthesis. It is postulated that the effects of TPA may reflect the physiological role of protein kinase C in the regulation of ovarian steroidogenesis.     

Androgen inhibition of FSH-stimulated progesterone production by granulosa cells of prepubertal pigs

Androgens have been reported to stimulate progesterone production by granulosa cells of several species, and to act synergistically with FSH in stimulation of progesterone accumulation by rat granulosa cells. Studies were undertaken to examine the effect of androgens on FSH-stimulated progesterone production in culture by granulosa cells derived from prepubertal pig ovaries. When included in 24-h culture with FSH, both androstenedione and testosterone caused a reduction in progesterone accumulation, but dihydrotestosterone and androsterone did not. Granulosa cells were cultured for 24 h with FSH and [14C]progesterone with or without testosterone; testosterone did not affect the rate of overall metabolism of [14C]progesterone and it was therefore concluded that testosterone inhibited progesterone synthesis, rather than enhancing its catabolism. 17 beta-Estradiol also inhibited FSH-stimulated progesterone accumulation. To determine whether the action of testosterone was mediated by conversion to estradiol, granulosa cells were cultured with FSH and testosterone with or without an aromatase inhibitor (4-acetoxy-androstenedione). The aromatase inhibitor failed to prevent the testosterone-induced reduction in progesterone accumulation, although it markedly inhibited estradiol accumulation. These results indicate that theca-derived androgens can inhibit FSH-stimulated progesterone production by granulosa cells in the prepubertal pig, independently of estradiol.     

Luteinization factor-stimulated steroidogenesis in porcine granulosa cells

Luteinization stimulator (LS), an intrafollicular compound of preovulatory (5-8 mm) follicles, increased both the basal and gonadotropins-stimulated production of progesterone by immature (1-3 mm) granulosa cells. The mechanism by which LS enhance steroidogenesis was investigated by studying the modulation of progesterone biosynthesis from exogenous cholesterol and pregnenolone in cultured porcine granulosa cells in serum-free medium. Progesterone production by cultured granulosa cells was stimulated by FSH, while treatment with 22-OH-cholesterol further enhanced the gonadotropin action. The activity of LS was found in cell conditioned media obtained after 3-day cultivation of preovulatory granulosa cells. Conversion of 22-OH-cholesterol into progesterone by granulosa cells isolated from small follicles was significantly stimulated in the presence LS in culture media. Also, progesterone production by granulosa cells in the presence of pregnenolone was increased considerably. Concomitant treatment with LS led to a further augmentation in progesterone synthesis. Endogenous formation of pregnenolone was inhibited by aminoglutethimide. Thus, LS enhancement of progesterone production in cultured porcine granulosa cells is associated with an increase in the activity of cytochrome P450 cholesterol side-chain cleavage and 3beta hydroxysteroid dehydrogenase enzymes.     

Inverse relationship between the expression of messenger ribonucleic acid for peroxisome proliferator-activated receptor gamma and P450 side chain cleavage in the rat ovary

Messenger RNA for peroxisome proliferator-activated receptor gamma (PPARgamma) has been found in granulosa cells, and its expression decreases after the LH surge. We determined which developmental stage of ovarian follicle expresses mRNA for PPARgamma and evaluated the impact of PPARgamma agonists on steroidogenesis. Ovaries were collected from immature eCG/hCG-treated rats at 0 (no eCG), 24, and 48 h post-eCG and 4 and 24 h post-hCG. Ovarian tissue was serially sectioned and processed for in situ hybridization to localize mRNA corresponding to PPARgamma, aromatase, and the LH receptor, and P450 side chain cleavage (P450SCC) and to determine whether apoptotic cells were present. During follicular development, there was no correlation between the expression of mRNAs for PPARgamma and aromatase or the presence of apoptotic cells, but a general inverse correlation was observed between the expression of PPARgamma mRNA and LH receptor mRNA. At 4 h post-hCG, follicles expressing P450SCC mRNA had lost expression of PPARgamma mRNA. This inverse pattern of expression between PPARgamma and P450SCC mRNAs was also observed 24 h post-hCG, with developing luteal tissue expressing high levels of P450SCC mRNA but little or no PPARgamma mRNA. To determine the impact of PPARgamma on steroidogenesis, granulosa cells were collected from ovaries 24 h post-eCG and cultured alone, with FSH alone, or with FSH in combination with the PPARgamma agonists ciglitazone or 15-deoxy-delta 12,14-prostaglandin J2 (PGJ2). Treatment of granulosa cells with PGJ2 stimulated basal progesterone secretion, whereas ciglitazone or PGJ2 had no significant effect on FSH-stimulated steroid production. These findings suggest that 1) PPARgamma may regulate genes involved with follicular differentiation and 2) the decline in PPARgamma in response to LH is important for ovulation and/or luteinization.     

Sandwich configuration of type I collagen suppresses progesterone production in primary cultured porcine granulosa cells by reducing gene expression of cytochrome P450 cholesterol side-chain cleavage enzyme

When porcine granulosa cells were cultured on type I collagen (TIC)-coated dishes, progesterone was continuously secreted in the culture medium. However, when cells were overlaid with a TIC gel, progesterone production was decreased to 34% (day 3) and 16% (day 4) of the value measured for cells without the overlay. The effect of TIC gel overlay on cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), which catalyzes the conversion of cholesterol to pregnenolone and is thought to be the rate-limiting enzyme in the conversion of cholesterol to progesterone, was examined. P450scc gene expression in cells overlaid with a TIC gel was decreased to 62% (day 3) and 36% (day 4) of the value measured for cells without the overlay. Amounts of P450scc were also reduced in the cells overlaid with a TIC gel. When pregnenolone, the direct precursor of progesterone, was added to the culture medium, the increase in progesterone production by cells overlaid with a TIC gel was much greater than that for cells without a TIC gel and a statistical difference in progesterone production was no longer observed between the two groups of cells. Treatment of the cells with human FSH (hFSH) enhanced progesterone production in a dose-dependent manner, irrespective of the presence of a TIC gel overlay. Moreover, hFSH induced P450scc gene expression in cells with and without a TIC gel overlay. These results indicate that a TIC gel overlay reduces progesterone production in granulosa cells via the suppression of P450scc gene expression. This supports the possibility that the existence of a TIC gel on the apical side of granulosa cells prevents the spontaneous luteinization of granulosa cells cultured on TIC-coated dishes. The fact that hFSH overcomes the suppressive effect of the TIC gel overlay on progesterone production may explain the mechanism for the subtle rise in serum progesterone concentration in the late follicle phase of the "in vitro fertilization" program.     

Inhibitory actions of adenosine on follicle-stimulating hormone-induced differentiation of cultured rat granulosa cells

To determine the effects of adenosine on follicle-stimulating hormone (FSH)-induced differentiation, granulosa cells isolated from the ovaries of diethylstilbestrol-treated immature rats were cultured with increasing concentrations of the nucleoside and modulators of adenosine action. Although adenosine had no effect on basal granulosa cell function during 48 h of culture, concentrations of the nucleoside from 10 microM to 1 mM progressively inhibited FSH-induced responses, including progesterone production and expression of FSH and luteinizing hormone (LH) receptors. Adenosine had biphasic effects on FSH-stimulated cAMP accumulation, causing inhibition of cAMP production at 10 to 100 microM and stimulation at higher concentrations. The enhancement of cAMP production by 1 mM adenosine occurred during the first 24 h of culture, while both 100 microM and 1 mM adenosine reduced FSH-stimulated cAMP production from 24 to 48 h. The inhibitory effects of adenosine were prevented by adenosine deaminase and dipyridamole, an inhibitor of adenosine transport, and were antagonized by 1-methyl-3-isobutylxanthine. The inhibition of cAMP and progesterone production by adenosine was partially overcome when cells were washed and reincubated with forskolin, but not with FSH. Adenine, guanosine, and inosine at concentrations of 100 microM did not modify FSH-induced cAMP formation or LH receptor induction. These results indicate that adenosine exerts predominantly inhibitory actions on hormone-induced granulosa cell differentiation, as manifested by prominent reductions in steroidogenesis and gonadotropin receptor expression.     

Dichlorodiphenyldichloroethylene potentiates the effect of protein kinase A pathway activators on progesterone synthesis in cultured porcine granulosa cells.

The insecticide dichlorodiphenyltrichloroethane (DDT) and its major metabolite p,p'-dichlorodiphenyldichloroethylene (DDE) have been implicated as endocrine-modulating chemicals. The DDT metabolite p, p'-DDE has been found contaminating human tissues and follicular fluid because of dietary exposure. We investigated the effects of DDE on progesterone synthesis in a stable porcine granulosa cell line, JC-410, and in primary cultures of porcine granulosa cells. Progesterone synthesis was not affected by 0.1-100 ng/ml DDE in the JC-410 cells. However, 10 ng/ml DDE increased 8-bromo-cAMP (8-Br-cAMP)-stimulated progesterone synthesis 0.4-fold (P < 0.05) over the levels observed with 1 mM 8-Br-cAMP alone. The effect of cholera toxin (CT) on progesterone synthesis was increased 0.7-fold (P < 0.05) by 10 ng/ml DDE over the value observed with 30 ng/ml CT alone. In primary cultures of porcine granulosa cells, 10 ng/ml DDE potentiated CT-stimulated progesterone synthesis 1.2-fold over the value observed with CT alone. In the JC-410 cells, 1 and 10 ng/ml DDE increased CT-stimulated cytochrome P450-cholesterol side-chain cleavage (P450(scc)) mRNA levels 0.3- and 0.4-fold, respectively, over the values obtained with CT alone. Neither basal nor CT-stimulated cAMP levels were changed by DDE. We conclude that DDE affects granulosa cell response to protein kinase A activators by altering the expression of the P450(scc) gene.     

Induction of synthesis of cholesterol side chain cleavage cytochrome P-450 and adrenodoxin by follicle-stimulating hormone, 8-bromo-cyclic AMP, and low density lipoprotein in cultured bovine granulosa cells

The actions of follicle-stimulating hormone (FSH), 8-bromo-cyclic AMP (8-Br-cAMP), and low density lipoprotein (LDL) to stimulate the production of progesterone and the synthesis of cholesterol side chain cleavage cytochrome P-450 (cytochrome P-450ssc) and adrenodoxin were investigated in bovine granulosa cells maintained in primary monolayer culture. Treatment of granulosa cells in culture with FSH resulted in an increased incorporation of [35S]methionine into immunoprecipitable cytochrome P-450scc in a concentration-dependent fashion with a maximal effect being obtained at an FSH concentration of 500 ng/ml. Treatment of granulosa cells with FSH also resulted in the induction of synthesis of adrenodoxin. The cyclic AMP analog, 8-Br-cAMP, induced the synthesis of both cytochrome P-450scc and adrenodoxin to a greater extent than did FSH. LDL also stimulated the synthesis of both cytochrome P-450scc and adrenodoxin, when added to cells maintained in the presence of lipoprotein-poor serum. The presence of FSH or 8-Br-cAMP together with LDL resulted in a higher rate of enzyme synthesis than that observed with each effector alone. FSH, 8-Br-cAMP, and LDL also stimulated progesterone production by cultured granulosa cells. The results of this study offer a possible mechanism whereby granulosa cells undergo cytodifferentiation in vivo into luteal cells. The concentration of LDL in follicular fluid is very low. Following ovulation, vascularization of the follicle occurs and thus the granulosa cells are exposed to high levels of LDL, allowing for provision of substrate cholesterol, as well as stimulation of the synthesis of the enzymes involved in cholesterol side chain cleavage.     

Evidence for the presence of cholesterol side chain cleavage cytochrome P-450 and adrenodoxin in fresh granulosa cells. Effects of follicle-stimulating hormone and cyclic AMP on cholesterol side chain cleavage cytochrome P-450 synthesis and activity

The synthesis of cholesterol side chain cleavage cytochrome P-450 (cytochrome P-450scc) and adrenodoxin was studied both in freshly harvested bovine granulosa cells and in granulosa cells maintained in primary monolayer culture. In addition, the action of follicle-stimulating hormone (FSH) and cyclic AMP analogs to stimulate the synthesis of cytochrome P-450scc was investigated in cultured cells. Precursor forms of cytochrome P-450scc and adrenodoxin were immunoisolated from a cell-free translation system directed by RNA prepared from freshly obtained granulosa cells that were not luteinized. Furthermore, the presence of cytochrome P-450scc in lysates of granulosa cells freshly obtained from very small follicles (containing less than 0.1 ml of follicular fluid) and in mitochondria of freshly obtained granulosa cells was demonstrated by using an immunoblotting technique. Continuous treatment of cultured granulosa cells with FSH or with cyclic AMP analogs (dibutyryl cyclic AMP or 8-bromo cyclic AMP) for 72 h increased incorporation of [35S]methionine into immunoprecipitable cytochrome P-450scc. Moreover, FSH, dibutyryl cyclic AMP, and 8-bromo cyclic AMP stimulated pregnenolone production by cultured granulosa cells (2.3-, 4.0-, and 7.5-fold increase over control, respectively), indicative of an increase in cholesterol side chain cleavage activity. The results of this study demonstrate for the first time the presence of two components of the cholesterol side chain cleavage system in freshly obtained granulosa cells, and provide direct evidence for the trophic effect of FSH and its presumed mediator, cyclic AMP, on the synthesis of cytochrome P-450scc in granulosa cells.     

Follicle-stimulating hormone regulation of cytochrome P-450 side-chain cleavage messenger ribonucleic acid accumulation by porcine granulosa cells isolated from small and medium follicles

The experiments described here were conducted to examine regulation of cytochrome P-450 side-chain cleavage (SCC) mRNA accumulation in porcine granulosa cells isolated from small (1-4-mm) and medium (5-6-mm) follicles. Granulosa cells were cultured under the following conditions: 1) for 48 h or 96 h with 0, 50, or 200 ng/ml porcine FSH; 2) for 96 h with 200 ng/ml FSH and aminoglutethimide (100 microM); and 3) for 96 h with forskolin (100 microM). Total RNA was extracted and examined by Northern and dot-blot hybridization analysis, and culture media were assayed for progesterone concentration. Northern blot analysis revealed a single band approximately 2.1 kb in size. Accumulation of SCC mRNA by granulosa cells was both FSH dose- and culture time-dependent (p less than 0.05) with maximal increases approximately 4.5 times control levels. Aminoglutethimide reduced progesterone production by about 80% while having no effect on granulosa cell accumulation of SCC mRNA compared to cells stimulated with 200 ng/ml of FSH. Forskolin-treated cells produced significantly more progesterone than did cells treated with FSH, but accumulation of SCC mRNA was similar. In response to FSH, concentration of SCC mRNA did not vary with follicle size, but granulosa cells from small follicles produced significantly more progesterone than did those from medium follicles. These results demonstrate that concentration of SCC mRNA in cultured porcine granulosa cells is FSH dose-dependent, does not vary significantly in cells from small- and medium-sized follicles, and is correlated with progesterone production, but may not parallel progesterone secretion. This last observation indicates that control at sites other than SCC mRNA can affect progesterone production.     

Effects of ovarian theca cells on granulosa cell differentiation during gonadotropin-independent follicular growth in cattle

We investigated the effects of theca cells or FSH on granulosa cell differentiation and steroid production during bovine early follicular growth, using a co-culture system in which granulosa and theca cells were cultured on opposite sides of a collagen membrane. Follicular cells were isolated from early antral follicles (2-4 mm) that were assumed to be in gonadotropin-independent phase and just before recruitment into a follicular wave. Granulosa cells were cultured under serum-free conditions with and without theca cells or recombinant human FSH to test their effects on granulosa cell differentiation. Messenger RNA levels for P450 aromatase (aromatase), P450 cholesterol side chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), LH receptor (LHr), and steroidogenic acute regulatory protein (StAR) in granulosa cells were measured by real-time quantitative RT-PCR analysis. FSH enhanced aromatase mRNA expression in granulosa cells, but did not alter estradiol production. FSH also enhanced mRNA expression for P450scc, LHr, and StAR in granulosa cells, resulting in an increase in progesterone production. In contrast, theca cells enhanced aromatase mRNA expression in granulosa cells resulting in an increase in estradiol production. Theca cells did not alter progesterone production and mRNA expression in granulosa cells for P450scc, 3beta-HSD, LHr, and StAR. The results of the present study indicate that theca cells are involved in both rate-limiting steps in estrogen production, i.e., androgen substrate production and aromatase regulation, and that theca cell-derived factors regulate estradiol and progesterone production in a way that reflects steroidogenesis during the follicular phase of the estrous cycle.     

Androgens augment the mitogenic effects of oocyte-secreted factors and growth differentiation factor 9 on porcine granulosa cells

In this study, we test the hypothesis that the growth-promoting action of androgens on granulosa cells requires paracrine signaling from the oocyte. Mural granulosa cells (MGCs) from small antral (1-3 mm) prepubertal pig follicles were cultured in the presence or absence of denuded oocytes (DO) from the same follicles to determine whether mitogenic and/or steroidogenic responses, to combinations of FSH, insulin-like growth factor 1 (IGF1), and dihydrotestosterone (DHT) were influenced by oocyte-secreted factors (OSFs). To further explore the identity of such factors we performed the same experiments, substituting growth differentiation factor 9 (GDF9), a known OSF, for the DO. OSFs and GDF9 both potently enhanced IGF1-stimulated proliferation, and inhibited FSH-stimulated progesterone secretion. Alone, DHT had little effect on DNA synthesis, but significantly enhanced the mitogenic effects of OSFs or GDF9 in the presence of IGF1. Denuded oocytes, GDF9, and DHT independently inhibited FSH-stimulated progesterone secretion, and androgen, together with DO or GDF9, caused the most potent steroidogenic inhibition. Focusing on mitogenic effects, we demonstrate that both natural androgen receptor (AR) agonists, testosterone and DHT, dose-dependently augmented the mitogenic activity of DO or GDF9. Antiandrogen (hydroxyflutamide) treatment, which is used to block androgen receptor activity, opposed the interaction between androgen and GDF9. In conclusion, androgens stimulate porcine MGC proliferation in vitro by potentiating the growth-promoting effects of oocytes or GDF9, via a mechanism that involves the AR. These signaling pathways are likely to be important regulators of folliculogenesis in vivo, and may contribute to the excess follicle growth that is observed in androgen-treated female animals.     

Evidence for a functionally active inhibitory guanine nucleotide-binding regulatory protein in the swine ovary

Incubation of particulate fractions of swine granulosa cells or luteal minces with purified pertussis toxin (islet-activating protein) and [32P]-NAD catalyzed the (32P)-ADP ribosylation of a 41,000 dalton membrane protein. ADP-ribosylation was markedly reduced by prior incubation of intact cells with toxin. The functional relevance of this presumptive inhibitory guanine nucleotide-binding protein in pig granulosa cells was indicated by the ability of prior treatment with pertussis toxin to increase cyclic AMP generation and progesterone production significantly in response to follicle stimulating hormone. Prior cellular intoxication also enhanced cyclic AMP production stimulated by luteinizing hormone and choleratoxin, but not basally or after forskolin. These results demonstrate the presence of an inhibitory guanine nucleotide-binding protein in both the follicular (granulosa cell) and luteal compartments of the mammalian ovary, and indicate its functional relevance in cyclic AMP generation and progesterone secretion.     

Decreased intracellular potassium levels underlie increased progesterone synthesis during ovarian follicular atresia

More than 99% of ovarian follicles are lost by a degenerative process known as atresia, a phenomenon characterized by apoptosis of granulosa cells. Uniquely, dying granulosa cells also greatly increase their progesterone biosynthesis while reducing estrogen production. Recent studies have documented a dramatic decrease in intracellular K+ concentration during apoptosis that plays an important role in regulating apoptotic enzymes. However, it is unclear whether this ionic change affects related processes such as the change in steroidogenesis in dying granulosa cells. To explore this question, granulosa cells were cultured in hypotonic medium, which initially swells the cells. The cells respond by extruding K+, which we have documented by fluorescence spectrophotometry. The K+ efflux osmotically draws water out the cell, returning it to a near normal volume (as measured by flow cytometry). The result is a cell of normal size with a decreased intracellular K+ concentration. FSH stimulation of these cells caused an increase in progesterone biosynthesis. This response was enhanced at higher doses of FSH, although basal progesterone production was not affected, suggesting that K+ levels may affect the gonadotropin-signaling pathway. No increase in steroidogenic acute regulatory or cholesterol side-chain cleavage cytochrome P450 mRNA was detected, although cAMP production was enhanced. These results suggest that the loss of intracellular K+ by apoptotic granulosa cells greatly facilitates FSH-stimulated progesterone production.     

三氯生对原代大鼠卵巢颗粒细胞孕酮分泌影响的实验研究

目的:研究三氯生对原代大鼠卵巢颗粒细胞孕酮(P4)分泌功能的影响。方法:原代大鼠卵巢颗粒细胞培养备用。取备用的卵巢颗粒细胞采用不同浓度的三氯生(0、0.01、0.1、1μM)染毒。24 h后分别采用MTT法检测颗粒细胞的相对活力、酶联免疫法(ELISA法)检测颗粒细胞P4分泌水平、实时荧光定量PCR法(q RT-PCR)及western blot法检测类固醇激素合成急性调节蛋白(St AR)、胆固醇侧链裂解酶(P450scc)以及3β-羟基类固醇脱氢酶(3β-HSD)的基因及蛋白表达水平。结果:三氯生在本研究所采用的浓度范围内对颗粒细胞的活性并没有影响(P0.05);三氯生(0.1、1μM)可抑制颗粒细胞P4的分泌,且呈现剂量依赖性下降(P0.05)。三氯生(0.1、1μM)可使St AR的基因表达水平显著增高、P450scc的基因表达水平下降(P0.05)。1μM三氯生可使St AR及P450scc的蛋白表达水平明显降低(P0.05)。三氯生对3β-HSD的基因及蛋白表达水平皆没有影响(P0.05)。结论:三氯生可抑制原代大鼠卵巢颗粒细胞的P4分泌,对类固醇激素合成关键分子的影响可能是其作用机制之一。     

Hormone stimulated steroid biosynthesis in granulosa cells studied with a fluorogenic probe for cytochrome P-450SCC.

The regulation of steroidogenesis by luteinizing hormone (LH) was studied in granulosa cells during follicular development using a fluorescent reporter assay based on the metabolism of a fluorescent probe specific for cytochrome P-450SCC (cholesterol side-chain cleavage enzyme). Intact granulosa cells or mitochondria were obtained from the first (F1) second (F2) and third (F3) largest preovulatory follicles of the hen ovary and incubated with the fluorogenic substrate. Metabolism of this substrate by cytochrome P-450SCC generates the highly fluorescent resorufin anion (the fluorescent reporter). In both mitochondria and intact granulosa cells, incubated with the fluorescent substrate, an increase in resorufin fluorescence was observed and the increase was greater in samples derived from F1 than in samples from F2 or F3. In cells, LH added simultaneously with the P-450SCC substrate significantly increased resorufin fluorescence above control values in a time- and dose-dependent manner up to 2-3 h after the incubation was initiated. Forskolin and 8-bromo-cAMP also stimulated metabolism of the P-450SCC substrate significantly by 15 min. When granulosa cells were preincubated with LH before exposure to the P-450SCC substrate resorufin fluorescence was significantly attenuated compared to controls (not exposed to LH in the preincubation period). The decrease in resorufin fluorescence observed when cells were pretreated with LH, may be due to the release of cholesterol from endogenous pools and its competition with the exogenous fluorogenic for the substrate P-450SCC enzyme. In granulosa cells that were preloaded with the P-450SCC substrate, the stimulatory effect of LH treatment remained constant from 30 min to 2 h after hormone addition. The results show that this fluorescent probe can be used in a rapid assay for the continuous measurement of the acute effects of hormone agonists on cholesterol conversion to pregnenolone in steroidogenic cells.     

FSH and growth factors affect the growth and endocrine function in vitro of granulosa cells of bovine preantral follicles

The hypothesis was tested that bovine preantral follicles can be stimulated to grow in vitro by FSH and by the mitogens, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), but not by transforming growth factor-beta (TGFbeta), which generally inhibits EGF and bFGF action. Preantral follicles, 60 to 179 mum in diameter, were isolated from fetal ovaries by treatment with collagenase and DNase and cultured for 6 d in serum-free medium, with or without FSH and growth factors. Basic FGF (50 ng/ml), and to a lesser extent FSH (100 ng/ml) and EGF (50 ng/ml), stimulated thymidine incorporation by granulosa cells in bovine preantral follicles compared to control cultures (8-, 4- and 2.5-fold the labeling index of the controls; P < 0.05). Alone TGFbeta (10 ng/ml) had no effect on (3)H-thymidine incorporation, but it completely inhibited the bFGF- but not the FSH-stimulated increase in the labeling index and mean follicular diameter of preantral follicles (P < 0.05). By the end of the culture period oocytes in most treatments had degenerated, and the few surviving oocytes were in preantral follicles cultured with FSH or bFGF. Progesterone accumulation was greater (P < 0.05) in the presence of FSH (100 ng/ml) or EGF (50 ng/ml) than with bFGF, TGFbeta or control medium. Basic FGF strongly inhibited the effect of FSH on progesterone secretion (P < 0.05). Only FSH stimulated the conversion of exogenous testosterone to estradiol and both bFGF and TGFbeta markedly inhibited FSH-stimulated estradiol accumulation. These results indicate that proliferation of granulosa cells of bovine preantral follicles can be stimulated by bFGF, FSH and EGF, whereas TGFbeta inhibits growth, and that they are steroidogenically active in culture. Basic FGF and TGFbeta antagonize FSH-stimulated steroid production by granulosa cells of cultured bovine preantral follicles.     

Effect of cannabinoids on progesterone production by ovarian granulosa cells of pig and rat

Various cannabinoids inhibited both basal and FSH-stimulated progesterone synthesis by pig and rat granulosa cells $\text{in vitro}$ in a dose- (0.09 – 16 μM) and time- (4 – 24 h) dependent manner. The data present further evidence for a possible direct interference of cannabinoids with ovarian functions.