Progesterone Synthesis In Granulosa Cells Research Articles

MiRNA novel-216 regulated fatty acid composition and progesterone synthesis in goat granulosa cells by targeting TPD52.

Granulosa cells in the ovaries of livestock are crucial for secreting steroid hormones that regulate follicular development, with lipid synthesis and metabolism playing key roles in this process. The molecular mechanisms behind steroid hormone secretion regulated by fatty acid metabolism in goat granulosa cells have been unclear. Our previous transcriptome analysis of Yunshang black goat ovaries revealed that miR-novel-216, which had lower expression in high-fertility goats, might regulate granulosa cell function. We further investigated the role of miR-novel-216 by isolating and culturing goat granulosa cells in vitro, and found that it inhibits cell proliferation, lipid accumulation, and progesterone synthesis in goat granulosa cells. The qTar and miRanda analyses predicted TPD52 as a target of miR-novel-216, confirmed by dual luciferase and transfection assays. Previous studies have shown that progesterone synthesis in granulosa cells is closely related to free fatty acid composition. We investigated the effect of in vitro construction of TPD52 overexpression and interference plasmids on the free fatty acid content of goat granulosa cells using mass spectrometry sequencing. The results showed that overexpression of TPD52 in goat granulosa cells significantly increased free fatty acid content and promoted granulosa cell proliferation, lipid accumulation, and progesterone synthesis, whereas the opposite was true for inhibition of TPD52. It was shown that miR-novel-216 affects granulosa cell proliferation and free fatty acid levels by regulating the expression of TPD52, which increases reproductive hormone secretion and promotes polytocous trait in goats. This provides a foundation for developing breeding strategies to improve goat fertility.

Corticosterone stage-dependently inhibits progesterone production presumably via impeding the cAMP-StAR cascade in granulosa cells of chicken preovulatory follicles

Stress can suppress reproduction capacity in either wild or domestic animals, but the exact mechanism behind it, especially in terms of steroidogenesis, remains under-investigated so far. Considering the important roles of progesterone in avian breeding, we investigated the modulation of corticosterone on progesterone production in cultured granulosa cells of chicken follicles at different developmental stages. Using enzyme immunoassays, our study showed that corticosterone could only inhibit progesterone synthesis in granulosa cells from F5-6, F4, and F3 follicles, but not F2 and F1 follicles. Coincidentally, both quantitative real-time PCR and western blotting revealed that corticosterone could downregulate steroidogenic acute regulatory protein (StAR) expression, suggesting the importance of StAR in corticosterone-related actions. Using the dual-luciferase reporter system, we found that corticosterone can potentially enhance, rather than inhibit, the activity of StAR promoter. Of note, combining high-throughput transcriptomic analysis and quantitative real-time PCR, phosphodiesterase 10A (PDE10A), protein kinase cAMP-dependent type II regulatory subunit alpha (PRKAR2A) and cAMP responsive element modulator (CREM) were identified to exhibit the differential expression patterns consistent with cAMP blocking in granulosa cells from F5-6, F4, and F3, but not F2 and F1 follicles. Afterward, the expression profiles of these genes in granulosa cells of distinct developmental-stage follicles were examined by quantitative real-time PCR, in which all of them expressed correspondingly with progesterone levels of granulosa cells during development. Collectively, these findings indicate that corticosterone can stage-dependently inhibit progesterone production in granulosa cells of chicken preovulatory follicles, through impeding cAMP-induced StAR activity presumptively.

MiR-99a-5p inhibits target gene FZD5 expression and steroid hormone secretion from goat ovarian granulosa cells

MicroRNA (miRNA) has vital regulatory effects on the proliferation, differentiation and secretion of ovarian granulosa cells, but the role of miR-99a-5p in goat ovarian granulosa cells (GCs) is unclear. Both miR-99a-5p and Frizzled-5 (FZD5) were found to be expressed in GCs in goat ovaries via fluorescence in situ hybridization and immunohistochemistry, respectively, and FZD5 was verified (P<0.001) as a target gene of miR-99a-5p by double luciferase reporter gene experiments. Furthermore, FZD5 mRNA and protein expression were both found to be regulated (P<0.05) by miR-99a-5p in GCs. Moreover, the overexpression of miR-99a-5p or knockdown of FZD5 suppressed (P<0.05) estradiol and progesterone secretion from the GCs, as determined by ELISA. In summary, miR-99a-5p inhibits target gene FZD5 expression and estradiol and progesterone synthesis in GCs. Our study thus provides seminal data and new insights into the regulatory mechanisms of follicular development in the goat and other animals.

IL-1β Upregulates StAR and Progesterone Production Through the ERK1/2- and p38-Mediated CREB Signaling Pathways in Human Granulosa-Lutein Cells.

The proinflammatory cytokine interleukin-1β (IL-1β) may be involved in several ovulation-associated events, such as protease synthesis, prostaglandin production, and steroidogenesis in granulosa cells. However, the exact effect of IL-1β on progesterone synthesis in granulosa cells and the underlying mechanism remain unclear. By using cultured granulosa-lutein cells collected from women undergoing in vitro fertilization or intracytoplasmic sperm injection, we found that IL-1β upregulated steroidogenic acute regulatory protein (StAR) expression and progesterone synthesis in granulosa-lutein cells, which was comparable with luteinizing hormone effect and could be abolished by an IL-1 receptor antagonist. Moreover, IL-1β activated the phosphorylation of cyclic adenosine monophosphate response element-binding protein (CREB), and knockdown of CREB attenuated the induction of StAR expression and progesterone synthesis by IL-1β in granulosa-lutein cells. Furthermore, IL-1β activated the extracellular signal-regulated kinase (ERK)1/2 and p38 pathways and inhibition of the ERK1/2 and p38 pathways attenuated the IL-1β-induced phosphorylation of CREB, StAR expression, and progesterone synthesis in granulosa-lutein cells. In conclusion, IL-1β could upregulate StAR expression and stimulate progesterone biosynthesis through increase in CREB phosphorylation via activating the ERK1/2 and p38 pathways in human granulosa-lutein cells.

BMP-4 suppresses progesterone production by inhibiting histone H3 acetylation of StAR in bovine granulosa cells in vitro

This study examined the molecular mechanism by which BMP-4 inhibits progesterone production and the expression of genes involved in steroidogenesis. Granulosa cells were cultured in medium with or without BMP-4 for 0-96 h. BMP-4 inhibited progesterone secretion in granulosa cells and suppressed the expression of steroidogenic acute regulatory protein (StAR) at the mRNA and protein levels, whereas BMP-4 did not affect the proliferation of granulosa cells. In addition, we found that BMP-4 affected the expression of SR-B1 mRNA but not LDL-R in granulosa cells. To examine the protein-DNA interaction at specific sites within the StAR gene promoter, we used the quantitative real-time PCR and the ChIP technique. We demonstrated that BMP-4 suppresses the acetylation of histone H3 associated with the StAR promoter region at 48 and 72 h of culture in bovine granulosa cells. Our results showed for the first time that BMP-4 inhibited the acetylation of histone H3 associated with the StAR promoter region in bovine granulosa cells. Taken together, we propose that the inhibition of the acetylation of histone H3 associated with the StAR promoter region by BMP-4 may be one of the inhibitory molecular mechanisms of progesterone synthesis in granulosa cells. Our data suggested that theca cell-derived BMP-4 is important as a regulator of steroid hormone synthesis in granulosa cells during follicular development in the mammalian ovary.

In vitro exposure of porcine granulosa cells to the phytoestrogens genistein and daidzein: Effects on the biosynthesis of reproductive steroid hormones

Since a discrepancy concerning the effects of phytoestrogens on steroidogenesis exists in the literature we investigated the effects of genistein and daidzein on progesterone and estradiol synthesis in cultured primary granulosa cells derived from follicles of porcine ovaries. In this context, the investigation was performed to test the hypothesis that isoflavones can reduce hydroxysteroid dehydrogenase/isomerase (3β-HSD) activity by down-regulation of its transcription. We found that daidzein did not impair the viability of cultured granulosa cells in the concentration range from 0.1 to 100 μM, but genistein inhibited the cell viability at 50 μM compared to the unexposed controls. Forskolin (10 μM) and pregnenolone (2.5 μM) enhanced the basal progesterone secretion in the absence of both phytoestrogens. Daidzein or genistein at non-toxic concentrations alone or combined with forskolin or pregnenolone significantly reduced progesterone synthesis. This reduction was not due to changes of the abundance of P450scc protein, but the gene hydroxysteroid dehydrogenase/isomerase ( 3β-HSD) was significantly decreased at a non-toxic concentration of daidzein (50 μM) in non-stimulated and pregnenolone-stimulated cells. Moreover, genistein (1, 10 μM) significantly inhibited the 3β-HSD-mRNA only in pregnenolone-stimulated granulosa cells. It can be suggested that the effect of genistein on steroidogenesis only partly results from the impairment of 3β-HSD gene expression. In non-toxic concentrations daidzein and genistein did not change the androstenedione- or testosterone-stimulated estradiol-17β synthesis. In summary, genistein and daidzein have direct effects on porcine granulosa cell progesterone synthesis which involve the inhibition of 3β-HSD enzyme activity across the post-cyclic AMP pathway.

201.Regulation of mouse mural granulosa cell progesterone synthesis by oocyte paracrine factors

Oocytes secrete soluble paracrine factors that play an important role in the growth and development of surrounding follicular cells. It is known that oocytes suppress FSH-induced progesterone production by mural granulosa cells (MGC), however it is unclear which growth factor(s) are involved. Some candidate molecules include growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15) and BMP6. The aim of this study was to examine the role of these factors in the regulation of FSH-induced MGC progesterone production. Prepubertal 129/SV mice were primed with eCG and ovaries were collected ~46�h later. MGC from large antral follicles were cultured with either denuded oocytes (DO; 0.25/μl), GDF9 or BMP15 (0.25-4% v/v) or BMP6 (10-200ng/ml), in the absence or presence of FSH. Cells were cultured for 18�h followed by a further 6�h pulse of 3H-thymidine. After 24�h cells and media were harvested for assessment of MGC DNA and progesterone synthesis, respectively. Treatment with FSH increased MGC progesterone production 9-fold, which, as expected, was antagonised by coculture with DO (by 73%). GDF9 and BMP15 both decreased FSH-induced MGC progesterone in a dose dependent manner, significantly reducing control levels (100%) to 17% and 30%, respectively, at doses of 2%v/v. All doses of BMP6 abolished FSH-stimulated progesterone. Even though all treatments inhibited progesterone production, only two of these, GDF9 (0.25%v/v) and DO (0.25DO/μl), stimulated MGC DNA synthesis (2.1 and 3.3 fold above controls, respectively). The BMP receptor type-II (BMPR-II) is a known receptor of several oocyte factors. Treatment with a BMPR-II ectodomain completely antagonised DO- and GDF9-stimualted MGC DNA synthesis, but progesterone levels were only partially restored (by 50%). These data indicate that BMP15 and BMP6 mimic the progesterone-regulating, but not the growth-promoting, activities of oocytes, whereas GDF9 does both. Although the BMPR-II ECD antagonises these oocyte factors, this receptor-signalling system may not necessarily be the means by which oocytes regulate MGC progesterone synthesis.

Growth differentiation factor-9 stimulates progesterone synthesis in granulosa cells via a prostaglandin E2/EP2 receptor pathway.

Growth differentiation factor-9 (GDF-9), an oocyte-secreted member of the transforming growth factor beta superfamily, progesterone receptor, cyclooxygenase 2 (Cox2; Ptgs2), and the EP2 prostaglandin E(2) (PGE(2)) receptor (EP2; Ptgerep2) are required for fertility in female but not male mice. To define the interrelationship of these factors, we used a preovulatory granulosa cell culture system in which we added recombinant GDF-9, prostaglandins, prostaglandin receptor agonists, or cyclooxygenase inhibitors. GDF-9 stimulated Cox2 mRNA within 2 h, and PGE(2) within 6 h; however, progesterone was not increased until 12 h after addition of GDF-9. This suggested that Cox2 is a direct downstream target of GDF-9 but that progesterone synthesis required an intermediate. To determine whether prostaglandin synthesis was required for progesterone production, we analyzed the effects of PGE(2) and cyclooxygenase inhibitors on this process. PGE(2) can stimulate progesterone synthesis by itself, although less effectively than GDF-9 (3-fold vs. 6-fold increase over 24 h, respectively). Furthermore, indomethacin or NS-398, inhibitors of Cox2, block basal and GDF-9-stimulated progesterone synthesis. However, addition of PGE(2) to cultures containing both GDF-9 and NS-398 overrides the NS-398 block in progesterone synthesis. To further define the PGE(2)-dependent pathway, we show that butaprost, a specific EP2 agonist, stimulates progesterone synthesis and overrides the NS-398 block. In addition, GDF-9 stimulates EP2 mRNA synthesis by a prostaglandin- and progesterone-independent pathway. Thus, GDF-9 induces an EP2 signal transduction pathway which appears to be required for progesterone synthesis in cumulus granulosa cells. These studies further demonstrate the importance of oocyte-somatic cell interactions in female reproduction.

Paracrine actions of growth differentiation factor-9 in the mammalian ovary.

Although the transforming growth factor-beta (TGF-beta) superfamily is the largest family of secreted growth factors, surprisingly few downstream target genes in their signaling pathways have been identified. Likewise, the identities of oocyte-derived secreted factors, which regulate important oocyte-somatic cell interactions, remain largely unknown. For example, oocytes are known to secrete paracrine growth factor(s) which are necessary for cumulus expansion, induction of hyaluronic acid synthesis, and suppression of LH receptor (LHR) mRNA synthesis. Our previous studies demonstrated that absence of the TGF-beta family member, growth differentiation factor-9 (GDF-9), blocks ovarian folliculogenesis at the primary follicle stage leading to infertility. In the present study, we demonstrate that mouse GDF-9 protein is expressed in all oocytes beginning at the type 3a follicle stage including antral follicles. To explore the biological functions of GDF-9 in the later stages of folliculogenesis and cumulus expansion, we produced mature, glycosylated, recombinant mouse GDF-9 using a Chinese hamster ovary cell expression system. A granulosa cell culture system was established to determine the role of GDF-9 in the regulation of several key ovarian gene products using semiquantitative RT-PCR. We find that recombinant GDF-9 induces hyaluronan synthase 2 (HAS2), cyclooxygenase 2 (COX-2), and steroidogenic acute regulator protein (StAR) mRNA synthesis but suppresses urokinase plasminogen activator (uPA) and LHR mRNA synthesis. Consistent with the induction of StAR mRNA by GDF-9, recombinant GDF-9 increases granulosa cell progesterone synthesis in the absence of FSH. Since induction of HAS2 and suppression of the protease uPA in cumulus cells are key events in the production of the hyaluronic acid-rich extracellular matrix which is produced during cumulus expansion, we determined whether GDF-9 could mimic this process. Using oocytectomized cumulus cell-oocyte complexes, we show that recombinant GDF-9 induces cumulus expansion in vitro. These studies demonstrate that GDF-9 can bind to receptors on granulosa cells to regulate the expression of a number of gene products. Thus, in addition to playing a critical function as a growth and differentiation factor during early folliculogenesis, GDF-9 functions as an oocyte-secreted paracrine factor to regulate several key granulosa cell enzymes involved in cumulus expansion and maintenance of an optimal oocyte microenvironment, processes which are essential for normal ovulation, fertilization, and female reproduction.

Barbiturates inhibit progesterone synthesis in cultured Leydig tumor cells and human granulosa cells

Screening drugs used in obstetrical practice for effects on steroid hormone synthesis revealed that phenobarbital inhibited progesterone synthesis in MA-10 Leydig tumor cells. The inhibition was apparently a drug class effect since it could be reproduced by other barbiturates. Barbiturate blockade was reversible and could be bypassed in the MA-10 cells by using 22-hydroxycholesterol. Human granulosa cell progesterone synthesis was also inhibited in a dose dependent fashion by phenobarbital, secobarbital and barbituric acid. Significant inhibition occurred in dose ranges that would be therapeutic for treating epilepsy. From these data we conclude that barbiturates block steroidogenesis by inhibiting cholesterol transport to the cholesterol side chain cleavage enzyme.

Interferon gamma inhibits luteinized human granulosa cell steroid production in vitro

OBJECTIVE: The purpose of this study was to determine whether interferon gamma affects luteinized human granulosa cell progesterone, estrone, and estradiol production in the presence and absence of associated white blood cells by either cytotoxic or antiproliferative mechanisms. STUDY DESIGN: Luteinized granulosa cells were isolated by Percoll centrifugation from women during in vitro fertilization cycles. Some cell suspensions were further treated with anti-CD45 magnetic immunobeads to remove associated white blood cells. Granulosa cells with and without white blood cells were cultured in the presence of interferon gamma (0.5 to 50 ng/ml) for 48 hours. Medium was changed at 24-hour intervals, and spent medium was assayed for progesterone, estrone, and estradiol. In separate experiments granulosa cell viability was assessed with the tetrazolium salt reduction assay. RESULTS: Interferon gamma significantly inhibited granulosa cell progesterone production in both basal and human chorionic gonadotropin stimulated cells cocultured with white blood cells in a concentration-dependent manner, whereas cells cultured free of white blood cells demonstrated less inhibition. In the absence of interferon gamma a more profound increase in granulosa cell progesterone synthesis was found in human chorionic gonadotropin-stimulated cultures without associated white blood cells. Interferon gamma inhibited granulosacell estrone and estradiol production in basal cultures containing white blood cells in both a time- and concentration-dependent manner. Estrone production was not affected by interferon gamma in human chorionic gonadotropin-stimulated granulosa cell cultures containing white blood cells, whereas estradiol secretion was decreased at 48 hours with 50 ng/ml interferon gamma. Both estrone and estradiol synthesis were inhibited by 50 ng/ml interferon gamma in granulosa cel cultures free of white blood cells. In cultures free of interferon gamma, granulosa cell estrone and estradiol secretion was not affected by human chorionic gonadotropin stimulation compared with basal controls regardless of the presence or absence of white blood cells. All concentrations of interferon gamma used had no effect on granulosa cell viability at any time point tested. CONCLUSION: Our data suggest that interferon gamma affects granulosa cell steroid production both independently and in synergy with associated white blood cells and further supports the hypothesis that interferon gamma may be an important intraovarian regulator of ovarian steroid production during the luteal phase.

The involvement of inositol phosphoglycan mediators in the modulation of steroidogenesis by insulin and insulin-like growth factor-I

We have investigated the mechanisms by which insulin and insulin-like growth factor-I (IGF-I) regulate the synthesis of progesterone by swine ovary granulosa cells. Analysis of the cell density dependence of the effects of insulin and IGF-I showed that the induction of progesterone synthesis by these growth factors is consistent with an autocrine or paracrine model of action, which involves the insulin- and IGF-I-stimulated release of a soluble factor(s) into the culture medium. We have tested the hypothesis that this soluble factor(s) may be structurally related to inositol phosphoglycans, a class of putative second messengers of the action of insulin. Consistent with this hypothesis, we isolated an activator of pyruvate dehydrogenase (PDH) phosphatase from culture medium obtained from cells treated with insulin or IGF-I. Further analysis showed that specific antibodies raised against the inositol phosphoglycan anchor of the variant surface glycoprotein of Trypanosoma brucei blocked the activation of PDH phosphatase by the material isolated from the culture medium, suggesting a close structural relationship between this putative PDH phosphatase activator and inositol phosphaglycans. Pertussis toxin treatment, shown to inhibit the generation of inositol phosphoglycans in other systems, was found to inhibit the effects of insulin on progesterone synthesis in granulosa cells. Finally, the stimulatory effects of insulin and IGF-I on progesterone synthesis by intact granulosa cells were markedly inhibited by the addition of antiinositol phosphoglycan antibodies to the culture medium. Based on these observations, we propose that the release of inositol phosphoglycans into the extracellular medium plays an important role in the signaling mechanisms by which insulin and IGF-I regulate the synthesis of progesterone in swine ovary granulosa cells.

The involvement of inositol phosphoglycan mediators in the modulation of steroidogenesis by insulin and insulin-like growth factor-I.

We have investigated the mechanisms by which insulin and insulin-like growth factor-I (IGF-I) regulate the synthesis of progesterone by swine ovary granulosa cells. Analysis of the cell density dependence of the effects of insulin and IGF-I showed that the induction of progesterone synthesis by these growth factors is consistent with an autocrine or paracrine model of action, which involves the insulin- and IGF-I-stimulated release of a soluble factor(s) into the culture medium. We have tested the hypothesis that this soluble factor(s) may be structurally related to inositol phosphoglycans, a class of putative second messengers of the action of insulin. Consistent with this hypothesis, we isolated an activator of pyruvate dehydrogenase (PDH) phosphatase from culture medium obtained from cells treated with insulin or IGF-I. Further analysis showed that specific antibodies raised against the inositol phosphoglycan anchor of the variant surface glycoprotein of Trypanosoma brucei blocked the activation of PDH phosphatase by the material isolated from the culture medium, suggesting a close structural relationship between this putative PDH phosphatase activator and inositol phosphaglycans. Pertussis toxin treatment, shown to inhibit the generation of inositol phosphoglycans in other systems, was found to inhibit the effects of insulin on progesterone synthesis in granulosa cells. Finally, the stimulatory effects of insulin and IGF-I on progesterone synthesis by intact granulosa cells were markedly inhibited by the addition of antiinositol phosphoglycan antibodies to the culture medium. Based on these observations, we propose that the release of inositol phosphoglycans into the extracellular medium plays an important role in the signaling mechanisms by which insulin and IGF-I regulate the synthesis of progesterone in swine ovary granulosa cells.

In vitro effects of dihydrotestosterone on granulosa cell production of estrogen and progesterone

To investigate the direct effects of androgens on follicle development, intact, immature female rats were given 8 IU PMSG (0 hour) and four injections of either vehicle or dihydrotestosterone (DHT), I mg/kg body weight, at 0, 12, 24, and 36 hours after PMSG. Granulosa cells from small (<200 μm), medium (200 to 400 μm), and large (>400 μm) follicles were isolated and cultured in the presence or absence of 0.5 μm DHT in vitro for 48 hours, and the medium was assayed for progesterone and estrogen. Results show that DHT caused an increase in progesterone accumulation in all granulosa cells, regardless of follicle size. However, DHT inhibited estrogen accumulation in granulosa cells from different-size follicles and the inhibition varied depending on the duration of androgen exposure in vivo. The inhibition of estrogen accumulation was seen in granulosa cells from small follicles without prior exposure to DHT in vivo, while an inhibition of estrogen accumulation was seen in granulosa cells from medium and large follicles exposed to DHT treatment in vivo. Taken together, the results of experiments with in vivo and/or in vitro DHT treatment show that the androgen increases granulosa cell progesterone synthesis regardless of follicle size. However, the estrogen accumulation by granulosa cell is dependent on follicle size and duration of DHT exposure.

Influence of follicular maturation on luteinizing hormone-, cyclic 3',5'-adenosine monophosphate-, forskolin- and cholesterol-stimulated progesterone production in hen granulosa cells.

The influence of follicular maturation on progesterone production by collagenase-dispersed hen granulosa cells was measured in short-term incubations. Granulosa cells of the largest follicle (F1) produced up to ten times more progesterone than cells from smaller follicles (F3-F5), not only in response to luteinizing hormone (LH), but also when stimulated by exogenous cyclic AMP or forskolin, both of which raise intracellular cyclic AMP levels by nonreceptor-mediated mechanisms. Moreover, when granulosa cell progesterone synthesis was stimulated by incorporating 25-hydroxy-cholesterol into the incubation medium, an identical pattern was obtained. This could be attributed to a corresponding increase in the specific activity of the mitochondrial cholesterol side-chain cleavage enzyme (20,22 desmolase). An increase in the apparent Vmax was observed without a change in the apparent Km values. Pregnenolone substrate at concentrations which raised progesterone production to levels similar to those observed in response to LH stimulation was utilized equally by granulosa cells of mature and developing follicles. However, at high pregnenolone concentrations, granulosa cells of mature follicles converted significantly more of the precursor to progesterone. Assay of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) showed that the enzyme has two Kms: a low Km present in cells of both mature and developing follicles, and a high Km found only in granulosa cells of more mature follicles. It is concluded that LH-promoted progesterone synthesis in granulosa cells of developing chicken follicles is restricted not so much by the availability of receptors and the competence of the adenylate cyclase/cyclic AMP system, but by the activity of key enzymes, principally the cholesterol-20,22 desmolase.

Steroidogenesis by Granulosa and Sertoli Cells

Granulosa and Sertoli cells have been isolated from the gonads of immature rats, and have been maintained in monolayer cultures in a chemically‐defined medium. The hormonal requirements of these cell types for the synthesis of steroids have been studied: Granulosa cells contain cholesterol side‐chain cleavage activity and synthesise progesterone when cultured in the presence of FSH and testosterone (or DHT). Sertoli cells on the other hand cannot synthesise steroids de novo. Granulosa and Sertoli cells are similar in that they are unable to convert significant amounts of progesterone to androgens. In the presence of exogenous testosterone as substrate, granulosa cells and immature Sertoli cells can synthesise estradiol and estrone when stimulated with FSH. In summary, FSH stimulates estrogen synthesis from testosterone in both cell types, and acts synergistically with testosterone (or DHT) to stimulate progesterone synthesis in granulosa cells. FSH is therefore involved in controlling the steroid environment within the gonads.