The effect of bovine activin and follicle-stimulating hormone (FSH) suppressing protein/follistatin on FSH-induced differentiation of rat granulosa cells in vitro

The time- and dose-dependent effects of bovine activin A and bovine follicle stimulating hormone (FSH) suppressing protein (FSP) or follistatin on basal and FSH-induced steroidogenesis and inhibin production were studied in granulosa cells from immature, diethylstilbestrol (DES)-treated rats. In the presence of rat FSH (20 ng/ml) which stimulates aromatase activity and the production of progesterone and inhibin, activin (0.3-100 ng/ml) augmented all three parameters, whereas FSP (0.3-100 ng/ml) enhanced progesterone production and attenuated the other two parameters. In the absence of FSH, the basal parameters were unaffected by treatment with either activin or FSP alone, except for a statistically significant increase in basal inhibin in the presence of activin alone (P less than 0.05, at doses of 30 and 100 ng/ml). Neither activin nor FSP influenced the timing of the maxima of FSH-induced activities over 5 days. These findings suggest that activin and FSP, both present in follicular fluid, may play an important role in the local regulation of granulosa cell differentiation.     

Effects of activin and follicle-stimulating hormone (FSH)-suppressing protein/follistatin on FSH receptors and differentiation of cultured rat granulosa cells.

The aim of this study was to investigate the actions of both activin and FSH-suppressing protein (FSP)/follistatin either alone or in combination on FSH receptor number and on the responsiveness of granulosa cells to FSH and LH. Granulosa cells were harvested from diethylstilbestrol-treated immature Sprague-Dawley rats and cultured 48 h in serum-free medium with or without treatment. Activin treatment alone (3-100 ng/ml) resulted in a 4-fold increase in FSH receptor number with no change in binding affinity. This effect of activin was inhibited 31% by FSP (100 ng/ml) treatment which alone had no effect on FSH receptor number. Treatment with activin (100 ng/ml) prevented FSH-induced down-regulation of FSH receptor number, whereas at lower concentrations (3-30 ng/ml) activin enhanced down-regulation of FSH receptor number by 20% (P less than 0.05). In contrast, FSP alone prevented FSH-induced down-regulation by increasing FSH receptor number up to 40-50%. Pretreatment of granulosa cells with activin, but not FSP, for 24 h increased the responsiveness of cells to FSH (20 ng/ml) and LH (40 ng/ml) shown by increases in aromatase activity, progesterone, and immunoreactive inhibin production over and above control in a manner which depended upon activin doses. We conclude that 1) activin enhancement of FSH action on rat granulosa cells may be mediated in part via regulation of FSH receptor number, and 2) the effects of FSP on granulosa cells are likely to be due to its activin binding properties.     

Modulation of differentiation of rat granulosa cells in vitro by interferon-gamma.

The effects of recombinant rat interferon-gamma (rRaIFN-gamma) and rat IFN (RaIFN, a mixture of IFN-gamma and -alpha) on basal and FSH-induced ovarian granulosa cell function were studied. Granulosa cells were harvested from diethylstilboestrol-treated immature rats and cultured (2 x 10(5) viable cells/well per 0.5 ml) in serum-free medium with or without treatment for 48 h. In the presence of FSH (20 ng/ml), rRaIFN-gamma (10-1000 U/ml) significantly inhibited FSH-stimulated aromatase activity (76.4 +/- 2.3% maximum inhibition compared with FSH treatment alone), inhibin (40.4 +/- 3.7%), progesterone (47.7 +/- 8.6%) and 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OHP) (51.8 +/- 1.7%) production in a dose-dependent manner. Furthermore, rRaIFN-gamma inhibited FSH- and forskolin (FSK; 30 mumol/l)-induced extracellular cAMP accumulation (46.0 +/- 6.6% and 29.1 +/- 7.3% respectively). The inhibitory effect of rRaIFN-gamma on FSK-induced cAMP was accompanied by decreased FSK-induced aromatase activity, inhibin, progesterone and 20 alpha-OHP production. rRaIFN-gamma had no detectable effect on aromatase activity, progesterone production and 20 alpha-OHP production in the absence of FSH, but significantly stimulated basal inhibin production by 1.5-fold. rRaIFN-gamma alone also caused a small but significant increase in basal levels of cAMP. The time-course studies showed that FSH-induced aromatase activity and inhibin production were consistently suppressed by rRaIFN-gamma, FSH-induced progesterone and 20 alpha-OHP were inhibited at 1 and 2 days and then stimulated on days 3, 4 and 5 relative to FSH alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development-related effects of recombinant activin on steroid synthesis in rat granulosa cells.

Activin is structurally related to polypeptide growth factors such as transforming-growth factor-beta, which may have paracrine and/or autocrine functions in the ovaries. We have investigated the action of activin on granulosa cell steroidogenesis in vitro in relation to preovulatory follicular development in vivo. Estrogen-primed immature female rats received no other treatment (nondifferentiated granulosa cells), treatment with ovine (o) FSH (differentiated granulosa cells), or treatment with oFSH followed by human (h) CG (preovulatory granulosa cells) to stimulate preovulatory follicular development. Granulosa cells were isolated and cultured in the presence and absence of recombinant human activin-A using serum-free medium supplemented with 1.0 microM testosterone as an aromatase substrate and hFSH, hLH, forskolin, or 8-bromo-cAMP to stimulate steroid synthesis in vitro. After 48 h, medium was collected for measurement of estradiol (aromatase activity), progesterone, and cAMP. Basal steroid synthesis in nondifferentiated granulosa cells was unaffected by activin, but both aromatase activity and progesterone production induced by treatment with FSH in vitro were dosedependently enhanced up to 10-fold by the presence of activin. FSH-stimulated cAMP production was not measurably altered by activin; however, steroidogenesis induced by forskolin or 8-bromo-cAMP was significantly enhanced by the factor. Thus the effect of activin on steroidogenesis includes action at a subcellular level(s) distal to the production of cAMP. After gonadotropin treatment in vivo, granulosa cell aromatase activity and progesterone production showed divergent responses to activin in vitro. Basal-, FSH-, and LH-stimulated aromatase activity were all enhanced by activin in cultures of differentiated and preovulatory granulosa cells. However, whereas basal progesterone production was stimulated by activin in cultures of differentiated granulosa cells, in preovulatory granulosa cells it was inhibited. Moreover, in vitro stimulation of progesterone production by treatment of both differentiated and preovulatory granulosa cells with FSH or LH was suppressed by the presence of activin. Thus rat granulosa cells display development-related steroidogenic responses to activin, aromatase production becoming enhanced and progesterone production suppressed as follicular maturation progresses. These results further implicate activin as a local modulator of granulosa cell steroid synthesis in the ovaries, although its functional significance has yet to be established.     

Relative effects of activin and inhibin on steroid hormone synthesis in primate granulosa cells.

Ovarian granulosa cells produce inhibin and activin, structurally related proteins with potentials to directly modulate follicular steroidogenesis. The aim of the present study was to compare development-related effects of inhibin-A and activin-A on steroidogenesis in marmoset monkey (Callithrix jacchus) granulosa cells. Granulosa cells from "immature" (< 1.0 mm diameter) and "mature" (> 2 mm diameter) follicles were incubated in serum-free culture medium for 96 h with and without peptide (1-100 ng/mL), in the presence and absence of gonadotropins [human (h) FSH or hLH] (10 ng/mL). Spent medium was collected and stored frozen for progesterone assay. Aromatase activity was determined by incubating cells for a further 6 h in the presence of 1 mumol testosterone and assaying accumulation of oestradiol. Granulosa cells from immature follicles showed characteristically low basal rates of steroid synthesis that were unaffected by treatment alone with either inhibin or activin. Treatment with hFSH stimulated both progesterone production and aromatase activity. Cotreatment with activin and hFSH further enhanced aromatase activity by up to 4-fold. The progesterone response to activin plus hFSH was related to the effect of hFSH in the absence of activin: high-level responsiveness to hFSH was suppressed by activin while low-level responsiveness was enhanced. Inhibin had no significant effect on FSH-responsive progesterone production, but at high concentrations (> 10 ng/mL) it caused slight (up to 30%) reduction in FSH-induced aromatase activity. Granulosa cells from mature follicles showed relatively high basal rates of steroidogenesis, and treatment with inhibin did not influence either basal or gonadotropin responsive steroidogenesis. Treatment with activin had divergent effects on aromatase activity and progesterone synthesis in that it increased both basal and hLH-responsive aromatase activity (up to 11-fold), had no effect on basal progesterone production, and markedly suppressed (by more than 50%) the progesterone response to hLH. These data reveal development-dependent effects of inhibin and activin on granulosa cell steroidogenesis that are likely to have physiological relevance to ovarian function in vivo.     

Regulation of cytochrome P450 aromatase messenger ribonucleic acid and activity by steroids and gonadotropins in rat granulosa cells.

Estradiol (E) biosynthesis by the cytochrome P450 aromatase (P450arom) enzyme system increases as preovulatory follicles develop and is subsequently reduced by the ovulatory LH surge. To determine the specific effects of gonadotropins and steroids on expression of P450arom in rat granulosa cells, steady state levels of messenger (m) RNA were examined in vivo and in vitro, with the latter also being related to aromatase enzyme activity and cAMP production. P450arom mRNA and activity were induced in granulosa cells by FSH alone in a dose-, time-, and stage-dependent manner. E enhanced the effects of FSH in vivo and in vitro. The synergistic effect of E with FSH (50 ng/ml) was observed in the absence/presence of serum and was mimicked by a similar concentration (20 nM) of testosterone, dihydrotestosterone, or dexamethasone. In contrast, ovulatory doses of LH (500 ng/ml) or forskolin (10 microM) but not concentrations of progesterone reached in preovulatory follicles (100-1000 nM) acted on differentiated (FSH + E) granulosa cells to cause a rapid loss of P450arom mRNA. Whereas cycloheximide prevented the LH/cAMP-mediated decrease in P450arom mRNA in the differentiated cells, enzyme activity remained unaltered during the same 6-h period. Thus, expression of aromatase mRNA in rat granulosa cells is induced primarily by low FSH/cAMP, enhanced by physiological doses of several steroids (except progesterone), and, once induced, can be rapidly inhibited by elevated gonadotropin/cAMP via a pathway requiring protein synthesis.     

An inhibitory effect of transferrin on differentiation of rat granulosa cells in vitro

The effects of human transferrin (TRF) on granulosa cell function were examined using serum-free cultures of rat granulosa cells obtained from immature, diethylstilbestrol-treated rats. The results show that TRF had dose- and time-dependent inhibitory effects on FSH-induced inhibin and progesterone production with the half-maximal inhibitory dose of 6.1-6.3 micrograms/ml. The inhibitory effect of TRF on FSH-induced inhibin and progesterone production was not reversed by removing TRF and changing medium after 48 h of treatment. TRF also inhibited insulin- and insulin-like growth factor-I (IGF-I)-induced inhibin production in a dose-dependent manner. TRF did not inhibit forskolin- and 8-bromo-cAMP-induced progesterone production but did inhibit inhibin production induced by these agents. TRF had no effect on basal production of inhibin and progesterone. On the other hand, high concentrations of insulin and cortisol completely counteracted the inhibitory effect of TRF on FSH-induced progesterone production but only partially counteracted the inhibitory effect of TRF on FSH-induced inhibin production. Our data suggest that: 1) TRF may be an important negative modulator of the stimulatory actions of FSH or IGF-I and other factors acting on granulosa cells; 2) the inhibitory effects of TRF require the presence of FSH or other factors such as IGF-I or insulin, which facilitate granulosa cell differentiation; and 3) different mechanisms are involved in the modulating effects of TRF on inhibin and progesterone production.     

Transforming growth factor beta enhances basal and FSH-stimulated inhibin production by rat granulosa cells in vitro

Transforming growth factor beta (TGF beta) caused a dose-dependent increase in both basal and follicle-stimulating hormone (FSH)-stimulated inhibin production by rat granulosa cells in culture. The TGF beta dose-response curve in the absence of FSH was approximately parallel to that in the presence of either a minimally effective dose (1 ng/ml) or a maximally effective dose (30 ng/ml) of FSH, suggesting an additive effect of these two agents on inhibin production. There was also a suggestion of an increased sensitivity of granulosa cell inhibin production to FSH when the cells were coincubated with TGF beta. The time course study showed that similar to FSH, the stimulatory effect of TGF beta on basal and FSH-stimulated inhibin production was evident on day 1 and was maximal by day 4. In addition, epidermal growth factor (EGF) reduced FSH-stimulated inhibin production with an ID50 value of 1.3 ng/ml. Coincubation of cells with EGF and 1 ng TGF beta/ml enhanced greatly the inhibitory action of EGF on FSH-induced inhibin production (ID50 less than 0.1 ng/ml). It is concluded that: (1) TGF beta directly stimulates inhibin production by rat granulosa cells and the combined effect with FSH was largely additive, (2) the inhibitory effect of EGF on FSH-induced inhibin production was enhanced by TGF beta, (3) individual members of the TGF beta/inhibin gene family regulate ovarian function, not only by direct action on follicle cells but also indirectly by influencing the production rate of other members of that family.     

Regulation of Leydig cell function in primary culture by inhibin and activin

Inhibin and activin are gonadal glycoproteins that selectively inhibit and stimulate FSH release, respectively. Previously we have reported that transforming growth factor-beta inhibited hCG-stimulated testosterone formation in mature Leydig cells. In the present study we evaluated the effects of other members of the transforming growth factor-beta family, inhibin and activin, on Leydig cell function. We found that activin (0.1-10 ng/ml) had no effect on basal testosterone formation, but inhibited hCG-stimulated testosterone formation in a dose-dependent manner. Activin (10 ng/ml) inhibited hCG-stimulated testosterone formation by 42%. Activin also inhibited hCG-stimulated cAMP formation. In the presence of activin (5 ng/ml), forskolin (10 microM)- and 8-bromo-cAMP (0.1 mM)-induced testosterone formation were reduced about one third. Conversions of pregnenolone and progesterone to testosterone were also blocked by activin. Interestingly, [125I]hCG binding to Leydig cells and forskolin-induced cAMP formation were not affected by the addition of activin. In contrast to activin, inhibin (0.1-10 ng/ml) had no effect on hCG-induced testosterone formation at any concentration used. However, the inhibitory effects of activin on Leydig cell function were reversed by the concomitant addition of inhibin. Our results suggest that activin inhibits testosterone formation by the Leydig cells derived from normal mature rats. Multiple steps of the steroidogenic pathway are affected by testosterone. Inhibin alone has no effect, but reverses the inhibitory action of activin.     

Bifunctional role of transforming growth factor-beta during granulosa cell development

Regulatory actions of transforming growth factor-beta (TGF beta) on granulosa cell function were analyzed in cells cultured from the ovaries of diethylstilbestrol-implanted rats. In the presence of a suboptimal concentration of FSH (5 ng/ml) that increased LH receptors by 100-fold during a 72-h culture, TGF beta augmented this response in a dose-dependent manner with a maximal effect at 16 pM. In contrast, the growth factor inhibited the LH receptor response to an optimal dose of FSH (50 ng) by up to 50% and was inactive in the absence of gonadotropin. TGF beta also enhanced the formation of cAMP by 5 ng FSH and partially inhibited the effects of higher FSH concentrations. However, the actions of TGF beta were more prominent on LH receptor induction than on cAMP production with either low or high amounts of FSH. In addition, TGF beta had little effect on cAMP production stimulated by cholera toxin or forskolin, but amplified the actions of these ligands as well as that of 8-bromo-cAMP on LH receptor expression. TGF beta also modulated the steroidogenic activity of the granulosa cells, with increased production of progesterone in response to 5-100 ng FSH. The bifunctional actions of TGF beta on FSH-induced LH receptor formation were observed throughout a 96-h culture period. However, the presence of the growth factor was not required for the first 24 h of culture, indicating that TGF beta alters the later events involved in LH receptor formation. TGF beta augmented the stimulatory actions of 5 ng FSH on LH receptors in the absence or presence of insulin, but its inhibitory effect on these receptors was only observed in cells treated with insulin. These results indicate that TGF beta modifies FSH action during granulosa cell development in a biphasic manner. TGF beta can exert stimulatory or inhibitory effects depending upon the concentration of FSH and the presence of insulin, and these are due to alterations in cAMP action as well as cAMP production. Autocrine and/or endocrine actions of TGF beta during granulosa cell differentiation may be involved in the processes of follicle selection and development.     

Low molecular weight substance from rat ovary induces steroidogenesis in cultured granulosa cells

Ovaries from immature intact rats contain an apparently low molecular weight substance which mimics the action of follitropin (FSH) on ovarian granulosa cells in culture. Similar to FSH action, the ovarian substance (OS) induced cell-shape changes followed by intensive progestin production. Like FSH action, OS-induced steroidogenesis reversibly ceased upon washing the factor from the cultured cells, and could be blocked in the presence of cycloheximide or alpha-amanitin. Although OS stimulated aromatase activity in granulosa cells, it failed to elicit LH responsiveness in the cultured cells. Androstenedione synergistically augmented OS-induced progestin production and aromatase activity. OS itself synergistically augmented FSH-induced progestin but did not have any effect on FSH-induced aromatase activity. In contrast to FSH action which is mediated via cAMP formation, OS doses which evoked extensive synthesis of progestin products failed to stimulate significant increases in intracellular cAMP accumulation. These results suggest the existence of a putative intraovarian hormone-like substance which can mimic some effects of the gonadotropins on the follicular granulosa cell differentiation and may facilitate FSH action at yet unknown stages of the follicular development.     

Regulation of inhibin production by rat granulosa cells

Inhibin production by cultured granulosa cells from immature diethylstilbestrol (DES)-primed rats was studied in relation to estradiol and progesterone production. The inhibin content in culture media was assayed with a specific radioimmunoassay (RIA) using an antibody to porcine 32 kDa inhibin that recognizes rat inhibin as well. Inhibin production was about 10 ng/ml/2 X 10(4) cells/72 h at the basal levels and was maximally stimulated with 25 ng/ml of follicle stimulating hormone (FSH) to 45 ng/ml which was 4.5 times the basal levels, with an ED50 value of 2.0 ng/ml. A cyclic AMP analog (dibutyryl cyclic AMP) or reagents that promote cAMP production were also effective in inhibin production, indicating that FSH stimulates inhibin production through a cAMP-dependent pathway. Luteinizing hormone (LH) was not effective in producing inhibin from freshly prepared granulosa cells, whereas granulosa cells pre-incubated with FSH for 48 h because responsive to LH regarding inhibin production. Testosterone sensitized the granulosa cells to the FSH stimulation, whereas hydrocortisone (4 ng/ml) decreased the sensitivity of granulosa cells by increasing the ED50 value for inhibin production by FSH about 10 times. A similar effect was observed regarding estradiol production, while progesterone production due to stimulation by FSH was enhanced by the hydrocortisone treatment. Insulin and platelet extract both stimulated inhibin production and enhanced the maximal response of inhibin production due to stimulation by FSH without altering, or even increasing the ED50 values. Epidermal growth factor (EGF), (D-Leu6)Des-Gly10-LHRH N-ethylamide (GnRH agonist) and 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent protein kinase C activator, inhibited both inhibin production and estradiol or progesterone production. Consequently, the regulation of inhibin production was similar to that of estradiol production, but markedly different from that of progesterone. However, inhibin and estradiol production were modulated differently by various growth factors and hormones. These phenomena might account for possible discrete changes in the plasma levels of inhibin and estradiol in vivo.     

Induction of granulosa cell differentiation by forskolin: stimulation of adenosine 3',5'-monophosphate production, progesterone synthesis, and luteinizing hormone receptor expression

The effects of forskolin on the acquisition of differentiated functions in cultured ovarian granulosa cells were compared with the actions of FSH and prostaglandin E2 (PGE2). In 48-h granulosa cell cultures from immature diethylstilbestrol-treated rats, 100 microM forskolin caused a 45-fold increase in cAMP accumulation and stimulated progesterone production from undetectable levels (less than 0.2 ng/ml) to 80 ng/ml. The forskolin-induced increase in cAMP was similar to the maximum response to FSH, and progesterone production was about 50% of that elicited by FSH. PGE2 also enhanced cAMP and progesterone production in a concentration-dependent manner, with a maximum 8-fold increase in cAMP accumulation and an increase in progesterone to 5.6 ng/ml when the PGE2 concentration was 10 micrograms/ml. The time course of forskolin-stimulated cAMP production was notable for its rapid rise to the maximum level during the first 24 h of culture, followed by a plateau for up to 72 h. This contrasted with FSH-stimulated cAMP production, which increased progressively for up to 72 h when measured at 24-h intervals. LH receptor levels were low in untreated cells and after exposure to the various stimuli for 24 h, but increased 9- to 11-fold after culture with FSH or forskolin for 48-72 h. PGE2-induced LH receptor formation was about 20% of that seen after FSH stimulation. Forskolin enhanced cAMP and progesterone production in response to FSH and choleragen, but impaired the effects of these ligands on LH receptor formation. Exposure of the cultured cells to a potent GnRH agonist inhibited forskolin-induced progesterone and LH receptor synthesis, but did not influence forskolin-stimulated cAMP production. These results demonstrate the ability of forskolin to serve as a nonhormonal stimulator of granulosa cell differentiation and indicate the importance of cAMP in this process, as well as the ability of GnRH agonists to exert inhibitory effects on post-cAMP steps in cellular maturation.     

Hormonal regulation of granulosa cell inhibin biosynthesis

The hormonal regulation of inhibin production by cultured granulosa cells from immature hypophysectomized, estrogen-treated rats was examined using a specific RIA which detects the N-terminal portion of the inhibin alpha-chain. The RIA measured bioactive inhibin of Mr about 32,000 in granulosa cell conditioned media fractionated by fast protein liquid chromatography. In the presence of 10(-7) M androstenedione, FSH stimulated inhibin production in a dose-dependent manner during a 2-day culture. Inclusion of a phosphodiesterase inhibitor decreased the EC50 for FSH from 2.6 to 0.8 ng/ml (n = 3). The stimulatory effect of FSH could be mimicked with forskolin (an adenyl cyclase activator) and with a cAMP analog, (Bu)2cAMP, consistent with FSH action mediated through a cAMP dependent pathway. Intracellular levels of inhibin were unmeasureable, suggesting that inhibin is not stored to any great extent by the granulosa cells. This finding was consistent with in vivo studies which showed that whereas FSH treatment for 2 days doubled serum inhibin levels when compared with basal levels, there was no increase in the concentration of extractable inhibin in ovarian tissue. Granulosa cells which had been exposed to 20 ng/ml FSH for 2 days to induce LH receptors produced inhibin in response to both LH and human CG during the subsequent 2-day culture, with the levels of inhibin equalling the amount inducible by FSH. In contrast, neither PRL nor terbutaline, a beta 2-adrenergic agonist, had any effect on inhibin production even though receptors for these hormones are also induced by FSH. GnRH was found to inhibit the FSH-stimulated production of inhibin (IC50, 10(-7) M), consistent with previous observations that GnRH can act at the ovarian level to inhibit granulosa cell differentiation. This inhibition by GnRH could be reversed by inclusion of a specific GnRH antagonist. On the other hand, another regulatory peptide, vasoactive intestinal peptide, slightly stimulated inhibin production. The effect of several growth factors was also tested. Insulin-like growth factor I raised not only FSH-stimulated inhibin levels, but basal levels as well. Insulin was also effective, but only at 100-fold higher concentration. Epidermal growth factor inhibited FSH-stimulated inhibin production (IC50 = 0.1 ng/ml), whereas fibroblast growth factor had no effect. Thus, granulosa cell inhibin secretion is regulated by FSH and LH but not by PRL, presumably via a cAMP-mediated pathway.(ABSTRACT TRUNCATED AT 400 WORDS)     

Follicle-stimulating hormone increases c-fos mRNA levels in rat granulosa cells via a protein kinase C-dependent mechanism

Recent evidence has been presented that follicle-stimulating hormone (FSH) stimulates the induction of granulosa cell c-fos protooncogene mRNA in vivo (Pennybacker and Herman (1989) J. Cell Biol. 109, 151A; Delidow et al. (1990) Endocrinology 126, 2302–2306), yet the mechanisms by which FSH induces c-fos mRNA expression have not been delineated. To elucidate the mechanisms of FSH-dependent c-fos mRNA expression, we measured the time and dose dependence of c-fos mRNA levels using Northern blot analysis in intact ovaries and cultured granulosa cells in response to FSH. In intact ovaries, FSH-induced c-fos mRNA expression was time dependent with maximal expression at 90 min post FSH injection, while in cultures of granulosa cells obtained from estrogen-primed immature female rats, c-fos mRNA levels were highest after 30 min exposure to FSH and at a concentration of 100 ng/ml. Neither 8-bromo adenosine 3′,5′-cyclic monophosphate (8-br-cAMP), at doses ranging from 0.1 to 10 mM, nor 100 μM forskolin (in the presence or absence of 200 μM isobutyl-methylxanthine) or luteinizing hormone (LH, 100 ng/ml) were able to mimic FSH-induced c-fos mRNA expression in granulosa cell cultures. However, tetradecanoyl-13-phorbol acetate (TPA, 200 nM) was able to induce c-fos mRNA expression. The protein kinase C (PKC) inhibitors H-7 (0.3–30 μM) and staurosporine (0.75 μg/ml) blocked FSH-induced c-fos mRNA expression in cultured granulosa cells while HA 1004, an inhibitor of cGMP- and cAMP-dependent protein kinases at 30 μM had no effect on TPA-induced c-fos expression, and only minimally inhibited FSH-induced c-fos expression. Both FSH (100 ng/ml) and forskolin (3 μM) increased progesterone production in cultured granulosa cells. These data support the hypothesis that FSH specifically induces c-fos mRNA expression by a PKC-dependent mechanism and that the cAMP arm of the FSH response pathway is operant in these cells.     

Growth hormone enhances follicle-stimulating hormone-induced differentiation of cultured rat granulosa cells

Suppression of serum GH levels in immature rats is associated with delayed onset of puberty and decreased ovarian steroidogenic responsiveness to FSH. To investigate possible direct effects of GH on the differentiation of ovarian cells, granulosa cells from hypophysectomized estrogen-treated rats were cultured with FSH in the presence or absence of GH for 3 days. FSH stimulated granulosa cell LH receptor formation and steroid production in a dose-dependent manner. Concomitant treatment with GH increased LH receptor content by enhancing the action of low doses of FSH without substantial increases in the maximal response. This increase was due to an elevation in the receptor number rather than changes in their affinity for hCG. At 3 ng/ml FSH, concomitant treatment with ovine or bovine GH increased LH/hCG binding in a dose-dependent manner, with 300 ng/ml GH increasing the FSH action by about 3-fold. LH receptors in the GH-treated cells were functional, as indicated by the enhanced cAMP production of these cells in response to LH treatment. The cellular protein content in the FSH-treated cultures was slightly increased by GH (18%), but cell number and viability were unaffected. The change in cell protein content could not account for the increases in the amount of LH receptors. In addition to its effects on LH/hCG receptor content, GH also augmented FSH-stimulated progesterone and 20 alpha-hydroxy-4-pregnen-3-one production in a dose-dependent manner, with 100 ng/ml GH causing significant increases in FSH-induced progesterone production. In contrast, GH treatment did not significantly affect FSH-stimulated estrogen production. The augmentating effects of GH on LH receptor formation and progestin biosynthesis were associated with an enhancement of FSH-stimulated cAMP production. In addition, GH increased forskolin- and 8-bromo-cAMP-induced LH receptor formation and progestin production. Thus, GH-augmented LH receptor induction and progestin biosynthesis may be due to both increased cAMP production and enhanced action of cAMP. The present data have demonstrated that GH augments gonadotropin-stimulated differentiation of ovarian granulosa cells, suggesting an important regulatory role of GH in follicular growth and pubertal development.