Direct inhibition of rat granulosa cell inhibin production by epidermal growth factor in vitro

The effect of epidermal growth factor (EGF) on inhibin production by rat granulosa cells has been investigated using a recently developed inhibin radioimmunoassay (RIA). Granulosa cells from intact immature diethylstilbestrol (DES)-treated rats were exposed to EGF (1-100 ng/ml) in the presence or absence of FSH for varying periods in vitro. An inhibitory effect of EGF on basal inhibin secretion was evident at day 2 of culture and was sustained over the subsequent 2 days. This action on basal inhibin secretion was dose-dependent, and maximal inhibition to 50% of control was observed at a dose of 100 ng EGF/ml at day 4. EGF also inhibited basal progesterone secretion in a similar manner. EGF caused a dose-dependent inhibition of FSH-stimulated inhibin secretion, with an ID50 (0.5 ng/ml, 0.08 nM) about one-eighth that in the absence of FSH. In addition, EGF also inhibited the stimulation of inhibin production by 8-Br-cAMP and prostaglandin E2. To exclude the possibility that EGF was toxic to the granulosa cells, several biochemical parameters related to cell growth were measured. EGF treatment did not alter cell number but slightly increased [3H]thymidine incorporation into cellular DNA. The effect of EGF on [35S]methionine incorporation into cellular protein was biphasic, being stimulatory at doses less than 10 ng/ml but inhibitory at 100 ng/ml. The present data have demonstrated a direct inhibitory effect of EGF on basal and FSH-stimulated inhibin production by granulosa cells suggesting an important regulatory role of this growth factor in the differentiation of ovarian function.     

The effect of transforming growth factor-beta on follicle-stimulating hormone-induced differentiation of cultured rat granulosa cells

Growth factors have been shown to modulate differentiation of cultured ovarian granulosa cells. Transforming growth factors (TGFs) constitute a family of polypeptide growth factors capable of reversibly inducing anchorage-independent growth in normal cells. Epidermal growth factor (EGF), which has significant structural homology with TGF alpha, has been shown to modulate differentiation of granulosa cells in vitro. Similarly, TGF beta (TGFB) has been found to have significant structural homology with ovarian follicular fluid inhibin. To examine whether TGFB might affect granulosa cell growth or differentiation, rat granulosa cells were cultured in serum-free medium containing insulin for up to 3 days with varying concentrations of TGFB in the presence or absence of FSH. TGFB caused a dose-dependent increase in FSH-stimulated LH/hCG receptor binding, but had no effect on binding in the absence of FSH; TGFB (10.0 ng/ml) further increased FSH-stimulated LH/hCG receptor binding by 48 +/- 8% (P less than 0.02). Similarly, FSH-stimulated progesterone production was increased by TGFB in a dose-dependent manner; TGFB (1.0-10.0 ng/ml) increased FSH-stimulated progesterone production 2- to 3-fold (P less than 0.02). In contrast, EGF (10.0 ng/ml) decreased FSH-stimulated LH/hCG receptor binding by 93 +/- 1% (P less than 0.02). Neither FSH-stimulated intracellular nor extracellular cAMP accumulations were affected by TGFB treatment. However, EGF (10.0 ng/ml) diminished extracellular and intracellular FSH-stimulated cAMP accumulation at 48 and 72 h of culture. Culture protein and DNA content were not significantly affected by TGFB. These results suggest that TGFB may enhance FSH-stimulated LH receptor induction and steroidogenesis by mechanisms that do not further increase net cellular cAMP accumulation; TGFB and EGF can have opposite effects on gonadotropin-dependent differentiation; and products of the TGFB/inhibin gene family may have a capacity for autocrine or paracrine modulation of granulosa cell differentiation.     

Effects of transforming growth factor-beta on the production of immunoreactive insulin-like growth factor I and progesterone and on [3H]thymidine incorporation in porcine granulosa cell cultures

Transforming growth factor-beta (TGF beta) has been reported to enhance many FSH-stimulated functions in rat granulosa cell cultures. We therefore, investigated the actions of TGF beta on cultured porcine granulosa cells. We evaluated the production of immunoreactive insulin-like growth factor I (iIGF-I) and progesterone in short term (3-day) and in longer term (7-day) cultures using porcine TGF beta 1 (pTGF beta 1). TGF beta had a biphasic effect on epidermal growth factor (EGF)-stimulated iIGF-I production in short term cultures. A modest stimulatory effect was apparent at 10 pg/ml; however, this end point was completely inhibited by 1-10 ng/ml. TGF beta also had a slight stimulatory effect on basal iIGF-I production at 1 pg/ml, but not at higher levels. In longer term cultures TGF beta did not have a significant effect on either basal or FSH-stimulated iIGF-I production. In both short and longer term cultures TGF beta markedly inhibited basal and FSH-stimulated progesterone production. We also evaluated the effects of TGF beta on the incorporation of [3H]thymidine into DNA and found that basal and growth factor-stimulated [3H]thymidine incorporation were inhibited. No stimulatory effects of TGF beta on progesterone production or [3H]thymidine incorporation could be detected over the dose range tested (1 pg/ml to 10 ng/ml). The effects of human TGF beta 1 and pTGF beta 2 were compared with those of pTGF beta 1 on basal and EGF-stimulated [3H]thymidine incorporation. Effects of the peptides were qualitatively similar, but pTGF beta 2 was somewhat less inhibitory to EGF-stimulated [3H]thymidine incorporation than pTGF beta 1. The present studies show that in contrast to the well documented stimulatory actions of TGF beta in cultured rat granulosa cells, this growth factor is a predominantly negative regulator of porcine granulosa cells. With the exception of a modest stimulation of iIGF-I production at very low doses, the effects of TGF beta were to potently inhibit both growth and differentiated function. The inhibitory nature of TGF beta should not be overlooked when considering the possible role of this peptide in ovarian development and differentiation.     

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)     

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.     

Interactions between activin and follicle-stimulating hormone-suppressing protein and their mechanisms of action on cultured rat granulosa cells

Direct roles of follicle-stimulating hormone (FSH)-suppressing protein (FSP) and activin in regulation of ovarian granulosa cell differentiation have been reported recently. The present study further investigated the effects of these peptides on steroidogenesis and inhibin production as well as cAMP generation in cultured granulosa cells from immature, diethylstilbestrol (DES)-treated rats. In the presence of FSH (20 ng/ml) and activin (30 ng/ml), which enhanced FSH-induced aromatase activity, progesterone production and inhibin production, FSP (1-100 ng/ml) reversed the stimulating activities of activin in a dose-dependent manner. In addition, activin reversed the inhibitory effects of FSP on FSH-induced aromatase activity and inhibin production. In the presence of FSH, activin enhanced FSH-stimulated extracellular cAMP accumulation, and FSP caused a reduction in extracellular cAMP. Activin but not FSP also stimulated basal cAMP level. In the presence of forskolin, a potent stimulant of adenyl cyclase activity which stimulated extracellular cAMP, aromatase activity, progesterone production and inhibin production, activin augmented the effect of forskolin on all four parameters, whereas FSP significantly enhanced progesterone production without changing the other three parameters. Our findings suggest that activin action on rat granulosa cells may be mediated via regulation of cAMP generation. The action of FSP and FSH and/or activin-dependent, consistent with either an action as an activin binding protein or by a direct action of FSP on the granulosa cells.     

Transforming growth factor beta 1 inhibits gonadotropin action in cultured porcine Sertoli cells.

In the present study, we have tested the effects of transforming growth factor beta 1 (TGF beta 1) on FSH action toward aromatase activity and lactate production in cultured Sertoli cells isolated from immature porcine testes. Whereas treatment of Sertoli cells with FSH resulted in a dose-dependent increase (about 7-fold) in aromatase activity (conversion of testosterone into estradiol) (ED50 = 80 ng/ml FSH), the addition of TGF beta 1 reduced this gonadotropin action. The inhibitory effect of TGF beta 1 on FSH aromatase activity was dose dependent (ED50 = 0.1 ng/ml, 4 pM TGF beta 1) with a maximal decrease (about 40%) observed after a long term (48-h) treatment. TGF beta 1 exerted its inhibitory effect on FSH action at the level(s) of cAMP accumulation, exerting no apparent effect on the gonadotropin receptor or at a site(s) related to cAMP action. TGF beta 1 (2 ng/ml) significantly (P less than 0.002) reduced (52% decrease) FSH-stimulated cAMP levels in cultured porcine Sertoli cells. However, such an inhibitory effect of the growth factor was no longer observed when stimulation of cAMP accumulation with FSH occurred in the presence of methyl isobutyl xanthine (0.5 mM), an inhibitor of cAMP-phosphodiesterase activity. This observation suggests that TGF beta 1 decreased cAMP levels by increasing catabolism of the cyclic nucleotide through an enhancement of cAMP-phosphodiesterase activity. The inhibitory effect of TGF beta 1 was not limited to the action of FSH on aromatase activity but also extended to the gonadotropin action (mediated by cAMP) on lactate production. As for the inhibitory effect of TGF beta 1 on FSH-induced aromatase activity, the inhibitory effect of the growth factor on FSH-stimulated lactate production was dose and time dependent with a maximal decrease (about 30%) observed in the picomolar range (1 ng/ml, 40 pM) after 48 h treatment with TGF beta 1. In conclusion, the present study demonstrates that TGF beta 1 attenuates FSH action on Sertoli cell activity and that such inhibitory action is potentially exerted through a decrease in cAMP levels. Because of the local production of TGF beta 1, it is suggested that the effects of the growth factor reported here might be exerted in the context of the testicular paracrine mechanisms.     

Epidermal growth factor increases inhibin synthesis by isolated segments of rat seminiferous tubules

The effect of epidermal growth factor (EGF) on the production of immunoreactive inhibin by adult rat isolated seminiferous tubules in vitro has been investigated. EGF (0.1-1000 ng/ml) added to cultures of seminiferous tubules from adult rats caused a dose-dependent increase in inhibin content in the tubules without changing the amount secreted into the media. However, after continuous stimulation with EGF for periods in excess of 5 days, an increase in inhibin secretion was observed. In the presence of 10 and 100 ng FSH/ml, EGF (10 ng/ml) produced a further increment in the inhibin content of the tubules, but this effect was not found with FSH concentrations of 500 or 1000 ng/ml. EGF also increased the tubule content of inhibin after the addition of 100 micrograms dibutyryl cyclic AMP/ml but no effect of EGF was observed on the FSH- or dibutyryl cyclic AMP-induced secretion of inhibin into the medium. The effect of EGF on inhibin content in the tubules was partially suppressed by the addition of 4 beta-phorbol-12 beta-myristate-13 alpha-acetate (20 ng/ml). Insulin (1-100 ng/ml) decreased basal inhibin secretion without changing the inhibin content of tubules and this effect was antagonized by EGF (10 ng/ml) with insulin doses of 1-50 ng/ml whereas, at 100 ng/ml, the effect of EGF on tubule inhibin content was reversed. The addition of EDTA (2 mmol/l) resulted in an inhibition of basal and EGF-induced inhibin production. These data demonstrate a stimulatory effect of EGF on inhibin production by isolated seminiferous tubules which is inhibited by insulin and phorbol esters, both stimulators of protein kinase C activity.     

Antagonistic interactions of transforming growth factors in the regulation of granulosa cell differentiation

The role of transforming growth factors (TGFs) in the acquisition of granulosa cell aromatase activity was investigated in vitro in a primary culture of granulosa cells harvested from immature, diethylstilbestrol-treated rats. Basal aromatase activity, as assessed by the generation of radioimmunoassayable estrogen, was negligible, remaining unaffected by treatment with either TGF alpha or TGF beta applied by themselves at the 10 ng/ml dose level. Whereas treatment with FSH produced a substantial increase in the extent of aromatization, concurrent treatment with TGF beta (0.01-10 ng/ml) resulted in dose-dependent augmentation of the FSH effect with an apparent median effective dose of 224 +/- (SE) 32 pg/ml (ca. 9 pM), and a maximal effect 3.6-fold greater than that induced by FSH alone. In contrast, concomitant treatment with TGF alpha (0.01-10 ng/ml) resulted in dose-dependent attenuation of FSH action with an apparent median inhibitory dose of 330 +/- (SE) 40 pg/ml (ca. 60 pM), and a maximal inhibitory effect of 91 +/- (SE) 2%. However, combined treatment with identical (10 ng/ml) maximally effective doses of both TGFs had little or no effect on the FSH-stimulated accumulation of estrogen, suggesting mutual neutralization by the opposing actions of these peptides. Further evaluation of the antagonistic interaction of the TGFs revealed it to be dose-dependent in that maximally effective doses of TGF alpha (10 ng/ml) partially overcame the stimulation of aromatase activity brought about by relatively low (less than 0.3 ng/ml) but not higher (greater than 1 ng/ml) concentrations of TGF beta, thereby shifting the TGF beta dose-response curve to the right. Treatment with either TGF had no significant effect on granulosa cell DNA content or synthesis, plating efficiency or viability. Taken together, these findings suggest that picomolar concentrations of exogenously provided TGF alpha TGF beta exert potent but diametrically opposed effects on the acquisition of granulosa cell aromatase activity and that the interaction between these two peptides is antagonistic in nature. Our findings further suggest that these direct cytodifferentiative effects of the TGFs may represent intrinsic novel properties of these peptides distinct from their well-established role in the regulation of cellular growth.     

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)     

A novel mechanism for the induction of aromatase in ovarian cells in vitro: role of transforming growth factor alpha-induced protein tyrosine kinase

In the developing follicle, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are the primary stimulators of steroidogenesis in granulosa and theca cells. The steroidogenic actions of both these gonadotropins are largely if not exclusively mediated through cAMP. Previous studies have shown that EGF and TGF alpha do not affect basal estrogen production but attenuate FSH-stimulated estrogen production in vitro in rat granulosa cells. Here we present evidence that TGF alpha stimulates basal estrogen production in prepubertal porcine ovarian granulosa and theca cells in culture under defined conditions. In granulosa cells, TGF alpha is more potent than FSH in stimulating estrogen production. LH does not stimulate estrogen production in prepubertal porcine theca cells but rather attenuates that stimulated by TGF alpha. Treatment of follicular cells with genistein (inhibitor of protein tyrosine kinase) attenuates TGF alpha-induced estrogen biosynthesis suggesting that the action of TGF alpha is mediated through protein tyrosine kinase. These studies provide evidence for an alternative cAMP-independent and TGF alpha-induced tyrosine kinase-dependent mechanism for the induction of ovarian aromatase.     

Follicle-stimulating hormone and somatomedin-C stimulate inhibin production by rat granulosa cells in vitro

The direct effect of somatomedin-C (Sm-C) and FSH on inhibin production by rat granulosa cells in vitro has been examined. FSH stimulated accumulation of inhibin in culture media in a dose-dependent manner with maximal stimulation (6-fold) being observed at a dose of 300 ng FSH/ml. Addition of Sm-C (30 ng/ml) either alone or in the presence of FSH (3-300 ng/ml) increased inhibin production (up to 5-fold). Sm-C alone was effective over the physiological dose range of 3-100 ng/ml. Concomitant addition of FSH (100 ng/ml) and Sm-C (3-100 ng/ml) resulted in a significant increase in inhibin production at all doses of Sm-C. The dose-dependent effects of FSH and Sm-C were also time dependent with a synergistic effect apparent after 48 h of culture. The Sm-C induced FSH inhibitory activity of granulosa cell culture media was confirmed as authentic inhibin by the demonstration of a dose-dependent neutralization of this activity by a monoclonal antibody raised against purified bovine inhibin. The data indicate a direct role for both FSH and Sm-C in ovarian inhibin production and provide additional evidence for an autocrine-paracrine role for Sm-C in granulosa cell differentiation.     

Ovarian transforming growth factor-beta (TGF beta): cellular site(s), and mechanism(s) of action

It is the objective of the experiments reported herein to examine the possible relevance of transforming growth factor-beta (TGF beta) to theca-interstitial cell function, and to further characterize the established interaction of TGF beta with the granulosa cell. In examining the interaction of TGF beta (10 ng/ml) with murine theca-interstitial cells, no significant effect was observed on either basal or human chorionic gonadotropin (hCG)-stimulated androsterone accumulation. In contrast, given murine granulosa cells, TGF beta (10 ng/ml) produced dose- and time-dependent augmentation of follicle-stimulating hormone (FSH)-supported aromatase activity with a minimal and median effective doses of 20 +/- 3 and 123 +/- 24 pg/ml, respectively and a minimal time requirement of less than or equal to 48 h. The ability of TGF beta to augment FSH hormonal action could not be accounted for by alteration(s) of specific FSH binding (13965 +/- 298 and 12614 +/- 694 cpm/4 X 10(5) cells for FSH and FSH + TGF beta). However, TGF beta proved capable of exerting a direct upregulatory effect on stimulatable adenylate cyclase activity, further enhancement occurring at site(s) distal to cAMP generation (dibutyryl cyclic AMP (Bt2cAMP) = 1.4 +/- 0.2 ng/culture; Bt2cAMP + TGF beta = 4.1 +/- 0.6 ng/culture). Taken together, our findings are in keeping with the notion that TGF beta, possibly of intraovarian origin, comprises the central signal of autocrine or paracrine loop(s) capable of amplifying gonadotropin action at the level of the granulosa, but not theca-interstitial cell.     

Transforming growth factor beta and follicle-stimulating hormone promote rat granulosa cell proliferation

Rat granulosa cells isolated from the ovaries of diethylstilbestrol-primed immature rats were treated with estrogen, FSH, and growth factors to determine those factors that were required to promote DNA synthesis. Estrogen and FSH, previously shown to stimulate the incorporation of [3H]thymidine into rat granulosa cell DNA in vivo, were ineffective in vitro. Epidermal growth factor, insulin-like growth factor 1 (IGF1), and fibroblast growth factor did not influence DNA synthesis whereas transforming growth factor beta (TGF beta) alone had a significant effect. Neither estradiol-17 beta (5 X 10(-8)-5 X 10(-6) M) nor IGF1 augmented the actions of TGF beta and FSH. FSH did not influence the actions of epidermal growth factor or IGF1 but dramatically augmented the effect of TGF beta on DNA synthesis. FSH and TGF beta also stimulated [3H]thymidine incorporation into the DNA of granulosa cells isolated from immature rats not treated with diethylstilbestrol. The increase in [3H]thymidine incorporation into DNA stimulated by TGF beta and FSH resulted subsequently in an increase in cell number. The response of the cells to TGF beta in the presence of a constant level of FSH (10 ng/ml) was dose dependent, 2.5 ng/ml being the minimal effective concentration. In the presence of antibody specific for TGF beta the bioactivity of the TGF beta was neutralized indicating that the growth promoting activity was due to TGF beta and not due to contaminants. In this paper, we have shown that the combined actions of FSH and TGF beta influence DNA synthesis and the proliferation of rat granulosa cells. Interactions between FSH and TGF beta may be important in regulating aspects of rat granulosa cell growth in addition to exerting pronounced effects on cytodifferentiation.     

Epidermal growth factor influences growth and differentiation of rat granulosa cells

The interactions of epidermal growth factor (EGF) with transforming growth factor beta (TGF beta), insulin-like growth factor-I (IGF-I), and FSH in the modulation of DNA synthesis and differentiated functions were examined in cultures of granulosa cells isolated from the ovaries of immature rats primed with diethylstilbestrol. EGF alone or in the presence of FSH had no effect on [3H]thymidine incorporation into the DNA of granulosa cells; however, EGF inhibited FSH plus TGF beta-induced DNA synthesis. In contrast, when FSH was omitted from the culture medium, EGF acted in concert with TGF beta, and TGF beta plus IGF-I, to promote DNA synthesis. EGF therefore has opposing actions on DNA synthesis; it inhibits or stimulates depending upon the presence or absence of FSH and consequently upon the endocrine environment in the follicle. As shown previously EGF alone had no effect on basal aromatase activity. EGF however inhibited FSH-induced and FSH plus IGF-I-induced aromatase activity. In this paper we show that EGF also inhibited the FSH-induced aromatase activity in the presence of TGF beta, which augmented FSH action on this system. The action of EGF on 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) was different from the effect of EGF on aromatase. In the absence of FSH, EGF induced 3 beta-HSD activity in the presence or absence of TGF beta. EGF augmented the action of FSH on 3 beta-HSD, and this interaction was further enhanced by TGF beta. These observations emphasize the multifunctional nature of EGF in influencing the growth and differentiation of immature rat granulosa cells. EGF can inhibit or stimulate growth and differentiated functions (aromatase and 3 beta-HSD), the response depending on the context of the signals that the cell receives from its endocrine and microenvironment.     

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.     

Follicle-stimulating hormone increases concentrations of messenger ribonucleic acid encoding cytochrome P450 cholesterol side-chain cleavage enzyme in primary cultures of porcine granulosa cells.

FSH is the primary hormonal inducer of ovarian follicle maturation and a critically important regulator of steroidogenesis in granulosa cells. We examined possible molecular mechanisms subserving FSH action by assessing concentrations of cytochrome P450 cholesterol side-chain cleavage (P450scc) mRNA in porcine granulosa cells maintained in serum-free culture. Cellular concentrations of specific P450scc mRNA were measured by Northern blot hybridization using a 32P-labeled 1-kilobase porcine cDNA clone. Specificity was tested by estimating the granulosa cell mRNA content of the constitutively expressed enzyme, glyceraldehyde-3-phosphate dehydrogenase. Steroidogenesis was evaluated by measuring concomitant progesterone accumulation in the culture medium. Treatment with ovine FSH (100 ng/ml) increased P450scc mRNA concentrations in a time-dependent fashion, with significant effects on both P450scc mRNA concentrations and progesterone accumulation by 4 h and a maximal increase (8- to 10-fold) at 48 h. FSH dose-response studies at 48 h revealed a significant stimulatory effect of 30 ng/ml FSH on P450scc mRNA accumulation and progesterone production, with a maximal effect at 100 ng/ml FSH. To examine the role of cAMP in mediating granulosa cell P450scc mRNA accumulation, granulosa cells were treated with forskolin, cholera toxin, 8-bromo-cAMP, 8-bromo-cGMP, 5'AMP, or cAMP analogs that differentially stimulate the two isoenzymes of protein kinase-A. Increased specific P450scc mRNA accumulation and progesterone production occurred in response to each agent except 5'AMP and 8-bromo-cGMP. No effects of these agents were observed on glyceraldehyde-3-phosphate dehydrogenase mRNA. To assess possible feedback effects of steroid or sterol on FSH-stimulated P450scc mRNA concentrations, granulosa cells were treated with aminoglutethimide to block or with low density lipoprotein to stimulate steroid production. Inhibition of sterol utilization by the cholesterol side-chain cleavage enzyme had no effect on basal or FSH-stimulated concentrations of P450scc mRNA, but markedly suppressed progesterone production. Low density lipoprotein, which increases intracellular sterol, also did not alter basal or FSH-stimulated P450scc mRNA accumulation, suggesting that neither the utilization nor the availability of sterol regulates specific P450scc mRNA levels. Estradiol alone did not increase P450scc mRNA accumulation, but did augment progesterone production. Treatment of granulosa cells with estradiol and FSH produced a synergistic increase in progesterone concentrations, but did not affect FSH-stimulated P450scc mRNA accumulation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibin A is a follicle stimulating hormone-responsive marker of granulosa cell differentiation, which has both autocrine and paracrine actions in sheep

The aim of these studies was to examine the origin, control and local actions of inhibin A in monovular species, using the sheep as a model. Experiment 1 examined the pattern of mRNA expression for the inhibin subunits in relation to follicular size and pattern of expression to other differentiative markers in granulosa (P450 aromatase) and thecal cells (P450 17alpha-hydroxylase). Experiment 2 examined the pattern of inhibin A production, in relation to oestradiol, by granulosa cells induced to differentiate in vitro with follicle-stimulating hormone (FSH). Experiment 3 examined possible paracrine and autocrine actions of inhibin A by determining the effect of addition of human recombinant inhibin A and/or antiserum to inhibin on gonadotrophin-stimulated cellular differentiation. The results of Experiment 1 showed that expression of mRNA encoding inhibin subnuits alpha, beta(A) and beta(B) is greater (P<0.05) in large oestrogenic follicles than in small follicles but that only expression of the inhibin beta(A) subunit differs (P<0.05) between large oestrogenic and non-oestrogenic follicles. Expression of 17alpha-hydroxylase, but not of the luteinising hormone (LH) receptor, in thecal cells was related to both the size and the oestrogenicity of antral follicles, in a manner similar to that of the inhibin subunits. Experiment 2 demonstrated that the production of inhibin A by sheep granulosa cells is FSH-responsive after prolonged exposure (P<0.001) and precedes the production of oestradiol by around 48 h in the differentiative cascade induced in granulosa cells by FSH. The results of Experiment 3 showed that inhibin A can augment gonadotrophin-stimulated steroid production by both granulosa and theca cells (P<0.01), and that the addition of antiserum to inhibin can inhibit FSH-stimulated oestradiol production by granulosa cells from both small and large follicles (P<0.001). We conclude that inhibin A is an FSH-responsive marker of granulosa cell differentiation which has both autocrine and paracrine actions in sheep.     

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.