A novel role for somatomedin-C in the cytodifferentiation of the ovarian granulosa cell

The role of somatomedin-C (Sm-C) in the acquisition of granulosa cell progesterone biosynthesis was investigated in vitro in a primary culture of rat granulosa cells cultured for 72 h under serum-free conditions. Basal progesterone accumulation was negligible and remained unaffected by treatment with highly purified Sm-C (50 ng/ml). Whereas treatment with FSH (20 ng/ml) produced a 9-fold increase in progesterone accumulation, the concurrent application of increasing concentrations (0.3-50 ng/ml) of Sm-C brought about dose-dependent increments in the FSH-stimulated accumulation of progesterone with a median effective dose of 4.0 +/- (SE) 0.3 ng/ml and a maximal response 9.6-fold greater than that induced by FSH alone. A monoclonal antibody raised against Sm-C (sm 1.2) produced complete immunoneutralization of the synergistic interaction between FSH and Sm-C, supporting the specificity of the Sm-C effect and arguing against the possible involvement of copurified contaminant(s) in the preparation used. Treatment of granulosa cells with the highest dose of Sm-C tested (50 ng/ml), in the absence or presence of FSH, did not result in significant alterations in cell number, DNA content, plating efficiency or viability. Taken together, our findings indicate that Sm-C is capable of synergizing with FSH in the induction of granulosa cell progesterone biosynthesis. Significantly, this ability of Sm-C to augment differentiated phenotypic expression of the developing granulosa cell is distinct from its well established growth-promoting property and may thus represent a novel biologic effect of this polypeptide.     

Dimeric inhibin A and B production are differentially regulated by hormones and local factors in rat granulosa cells.

In the present study, we have examined the role of hormones and growth factors in regulating dimeric inhibin production in immature rat granulosa cells. Purified granulosa cells from estrogen-primed immature rats were cultured under defined conditions. Inhibins A and B in the culture media were measured using a two-site enzyme-linked immunosorbent assay specific for each dimer. Under basal conditions, granulosa cells produced 14-fold more inhibin A than inhibin B (inhibin A, 2.0; inhibin B, 0.14 ng/ml, measured against human standards; average A/B apparent ratio, 14). Addition of increasing doses of FSH elicited dose-dependent increases in both inhibins, the effects being more pronounced on inhibin A than on inhibin B (9.4- and 4.1-fold increases, respectively; average A/B ratio, 34). Estradiol, when added alone, stimulated inhibin A production 3- to 6-fold, whereas minor changes were observed in inhibin B production. Insulin-like growth factor-I produced a similar stimulation of both inhibins (3-fold stimulation over control). This growth factor, however, induced a marked dissociation in the sensitivity of inhibins A and B to FSH stimulation, with maximal stimulation of inhibin B observed at comparatively lower concentrations of the gonadotropin. Transforming growth factor-beta (TGF-beta, 5 ng/ml) had a more marked stimulatory effect on inhibin B than on inhibin A production (7- to 14-fold vs. 2- to 5-fold for inhibin B and A, respectively). A more pronounced differential stimulation of inhibin B was also exerted by another member of the TGF-beta superfamily, activin A (A/B ratio, 0.66). This preferential stimulation of inhibin B by TGF-beta and activin A was amplified in the presence of FSH. Coculture of rat granulosa cells with freshly isolated bovine oocytes was also associated with a marked stimulation of inhibin B production (100-fold increase) and a comparatively lower stimulation of inhibin A (10-fold increase; A/B ratio, 1). The discrepancy between the proportion of inhibin dimers in serum (A/B ratio, 0.13) and those produced by untreated granulosa cells may suggest that intraovarian factors, such as TGF-beta, activin A, or oocyte-derived factor(s), are responsible for the shift of the ratio toward the predominance of inhibin B.     

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.     

Somatomedin-C as an amplifier of follicle-stimulating hormone action: enhanced accumulation of adenosine 3',5'-monophosphate

Somatomedin-C (Sm-C) has recently been found to amplify the FSH-mediated acquisition of granulosa cell progestin biosynthetic capacity, aromatase activity, and LH receptors, an effect distinct from its established replicative property. To further characterize the cellular mechanism(s) underlying the synergistic interaction of Sm-C with FSH, we have set out to evaluate the intermediary role of cAMP in this regard. Isolated granulosa cells from immature hypophysectomized diethylstilbestrol-treated rats were cultured for up to 3 days under serum-free conditions. The basal extracellular accumulation of cAMP remained unchanged in response to treatment with highly purified Sm-C (50 ng/ml). However, concurrent treatment with increasing concentrations (0.3-50 ng/ml) of Sm-C, produced dose- and time-dependent increments in the FSH-stimulated accumulation of cAMP, with an apparent median effective dose (ED50; mean +/- SE) of 5.1 +/- 0.6 ng/ml, a maximal response 8.8-fold greater than that induced by FSH alone, and a minimal time requirement of 1-2 days. Given increasing concentrations of FSH, treatment with a constant concentration (50 ng/ml) of Sm-C resulted in 1.7-, 5.8-, and 4.3-fold increases in cAMP accumulation for 10, 30, and 100 ng/ml FSH, respectively. The ability of Sm-C to augment FSH-stimulated cAMP accumulation was evident and, in fact, enhanced by ZK62711 (Rolipram; 3 X 10(-6) M)-induced blockade of cAMP-phosphodiesterase activity. Decreasing dilutions (1:64,000 to 1:1,000) of a monoclonal antibody raised against Sm-C (sm 1.2) produced progressive and complete immunoneutralization of the synergistic interaction of Sm-C with FSH, suggesting specificity of action. Taken together, these findings suggest that Sm-C, acting at nanomolar concentrations compatible with its granulosa cell receptor binding affinity (0.6-2.0 nM), is capable of amplifying FSH-stimulated cAMP accumulation in a time- and dose-dependent manner. These observations suggest that the synergistic action of Sm-C is exerted, at least in part, at a site(s) proximal to cAMP generation.     

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.     

Somatomedin-C enhances induction of luteinizing hormone receptors by follicle-stimulating hormone in cultured rat granulosa cells

Recent studies have documented the ability of somatomedin-C (Sm-C) to synergize with FSH in the induction of rat granulosa cell progesterone biosynthesis. This direct cytodifferentiative action of Sm-C proved distinct from its established mitogenic property. To determine whether Sm-C partakes in the differentiation of granulosa cell functions other than progesterone biosynthesis, the role of Sm-C in the acquisition of LH receptors was investigated in vitro. Granulosa cells were obtained from immature, hypophysectomized diethylstilbestrol-treated rats and cultured under serum-free conditions for up to 72 h. Specific granulosa cell LH/hCG binding under basal conditions was relatively low and was not significantly affected by treatment with highly purified Sm-C (50 ng/ml). In contrast, treatment with FSH (oFSH; NIH FSH S14; 20 ng/ml) produced a 3.8-fold increase in LH/hCG binding to 3167 +/- 660 (+/- SE) cpm/10(6) cells. However, concurrent treatment with Sm-C (50 ng/ml) resulted in a 6.1-fold enhancement of the FSH effect. The ability of Sm-C to synergize with FSH in the induction of LH receptors was dose and time dependent, with an apparent median effective dose (ED50) of 6.2 +/- 0.6 (+/-SE) ng/ml, and a minimal time requirement of 24 h or less. Examination of the binding parameters of the LH/hCG receptor revealed that the Sm-C-mediated increase in LH/hCG binding was due to enhanced binding capacity, but not affinity (Kd = 4.4 X 10(-11) M). Decreasing dilutions (1:128,000-1:8,000) of a monoclonal antibody raised against Sm-C (sm 1.2), produced progressive and virtually complete immunoneutralization of the synergistic interaction between Sm-C and FSH, suggesting specificity of action. The ability of Sm-C to enhance LH receptor induction was associated with increased hCG-stimulated progesterone biosynthesis, suggesting that the newly acquired receptors are functional in nature. Our present findings indicate that nanomolar concentrations of Sm-C are capable of enhancing FSH-stimulated LH/hCG-binding capacity, but not affinity, in a time- and dose-dependent fashion. These observations suggest that the direct cytodifferentiative effect of Sm-C is not limited to the induction of progesterone biosynthesis, but other key granulosa cell functions may also be subject to modulation by this regulatory peptide.     

Independent and synergistic actions of somatomedin-C in the stimulation of proteoglycan biosynthesis by cultured rat granulosa cells

The role of somatomedin-C (Sm-C) in the regulation of granulosa cell proteoglycan biosynthesis was investigated in vitro in a primary culture of rat granulosa cells labeled with [35S]sulfate. Basal [35S]sulfate incorporation into extracellular proteoglycans was increased by 93 percent in response to treatment with highly purified Sm-C (50 ng/ml) by itself. Whereas treatment with a minimally effective dose of FSH (20 ng/ml) alone produced a 43 percent increase over basal levels in extracellular [35S]sulfate-labeled proteoglycans, concurrent treatment with Sm-C yielded a 2.7-fold amplification of the FSH effect. Qualitatively similar results were obtained when [35S]sulfate incorporation into cellular proteoglycans was determined, the latter accounting for approximately one half of the total radioactivity incorporated. Significantly, fractionation of the major extracellular proteoglycan species revealed FSH to favor the exclusive production of dermatan sulfate (1.6-fold increase), whereas Sm-C supported the simultaneous biosynthesis of both heparan and dermatan sulfate (2.5- and 1.8-fold increments, respectively). Moreover, Sm-C proved capable of diverting FSH-driven proteoglycan biosynthesis from the exclusive stimulation of dermatan sulfate towards the enhanced production of heparan sulfate over dermatan sulfate. These findings suggest that while Sm-C may synergize with FSH in stimulating granulosa cell proteoglycan biosynthesis, it is also able to act in tis own right to effect marked quantitative as well as qualitative alterations in proteoglycan economy. Given the possible role of proteoglycans in follicular antrum formation and follicular atresia, our findings raise the possibility that Sm-C of granulosa cell origin may partake in the growth as well as the demise of the developing ovarian follicle.     

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)     

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.     

Somatomedin-C-mediated potentiation of follicle-stimulating hormone-induced aromatase activity of cultured rat granulosa cells

We have recently observed that nanomolar concentrations of exogenously added somatomedin-C (Sm-C) are capable of synergizing with FSH in the induction of cultured rat granulosa cell progesterone biosynthesis and LH receptors without altering granulosa cell survival or replication. To further characterize the cytodifferentiative properties of Sm-C, we have undertaken to investigate whether the acquisition of granulosa cell aromatase activity is also subject to modulation by this intraovarian peptide. Granulosa cells from immature hypophysectomized diethylstilbestrol-treated rats were initially cultured for up to 3 days in an androstenedione-free medium, during which time aromatase activity was induced by FSH in the absence or presence of Sm-C (treatment interval). At the conclusion of this period, the cells were washed and reincubated for an additional 8-h test interval, during which time aromatase activity was estimated. Basal aromatase activity, as assessed by the conversion of unlabeled androstenedione (10(-7) M) to radioimmunoassayable estrogen, was negligible, remaining unaffected by treatment with highly purified Sm-C (50 ng/ml) alone. However, concurrent treatment with Sm-C (50 ng/ml) produced a 7.0-fold increase in the FSH (100 ng/ml; NIH FSH S14)-stimulated accumulation of estrogen. Similarly, Sm-C produced a 6.1-fold increase in FSH-induced aromatase activity, as assessed by the stereospecific generation of tritiated water from [1 beta-3H]androstenedione substrate. Sm-C-potentiated aromatase activity was dose and time dependent, with an apparent median effective dose of 5.0 +/- 1.9 (+/- SE) ng/ml and a minimal time requirement of 24 h or less, but was independent of the FSH dose employed. Although bovine insulin and multiplication-stimulating activity, like Sm-C, proved capable of augmenting aromatase activity (albeit at a substantially reduced potency), little or no effect was observed for either porcine or rat relaxin, a distantly related member of the insulin-like growth factor family. Examination of the apparent kinetic parameters of the aromatase enzyme revealed that the Sm-C-mediated potentiation of aromatase activity was due to enhancement of the apparent maximal reaction velocity, but not substrate affinity (Km = 2.8 X 10(-8) M). Our findings indicate that nanomolar concentrations of exogenously added Sm-C synergize with FSH in the enhancement of the maximal reaction velocity, but not Km, of granulosa cell aromatase in a dose- and time-dependent fashion.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of oxytocin on steroidogenesis by bovine theca and granulosa cells

Oxytocin (OT) is secreted during the final stages of bovine follicular development. To test OT's potential role as a regulator of follicular steroidogenesis, theca and granulosa cells were isolated from bovine preovulatory follicles 48 h after initiation of luteolysis with prostaglandin F2 alpha, and cultured with graded doses of OT (0, 0.5, 5, 50, and 500 mIU/ml). Granulosa cells were cultured with testosterone (0.5 microM) in either defined medium or medium containing 10% fetal bovine serum in the presence or absence of FSH (300 ng/ml); medium was collected and replaced daily for 5 days. In defined medium, oxytocin alone significantly increased progesterone production by granulosa cells (P less than 0.001) in a dose-dependent manner; over 5 days, doses of 0.5, 5, 50, and 500 mIU/ml OT caused 1.7-, 2.0-, 2.2-, and 2.6-fold increases. FSH enhanced progesterone 5-fold, but no dose of OT increased progesterone in the presence of FSH. OT also elevated progesterone in serum-containing medium (P less than 0.005), but the magnitude of its effects was lower (1.07-, 1.1-, 1.2-, and 1.4-fold increases with 0.5, 5, 50, and 500 mIU/ml OT). OT had little effect on estradiol secretion by granulosa cells cultured with or without FSH. To test the specificity of OT's effects on progesterone production by granulosa cells, granulosa cells were treated with graded doses of an OT antagonist (0, 1, 10, 100, and 1000 ng/ml) in the presence or absence of OT (5 and 50 mIU/ml). Progesterone production by granulosa cells in the presence of the antagonist alone was similar to production in control cultures. The stimulatory effects of 5 and 50 mIU OT were completely abolished in the presence of 100 or 1000 ng antagonist, respectively (P less than 0.01). Preparations of theca interna were cultured in defined medium with graded doses of OT (0, 0.5, 5, 50, and 500 mIU/ml) in the presence or absence of LH (300 ng/ml), with collection and replacement of medium at 3, 6, 12, 24, 48, and 72 h. LH alone increased both progesterone (12-fold) and androstenedione (4-fold) production over controls. However, no dose of OT significantly affected either progesterone or androstenedione production. These results show that OT stimulates progesterone production by granulosa cells, and thus, suggest that OT regulates steroidogenesis in bovine granulosa cells in vivo.     

Somatomedin-C synergizes with follicle-stimulating hormone in the acquisition of progestin biosynthetic capacity by cultured rat granulosa cells

We have recently shown that nanomolar concentrations of somatomedin-C (Sm-C), are capable of enhancing the FSH-mediated (but not basal) accumulation of progesterone (Po) by cultured rat granulosa cells. To further characterize this direct cytodifferentiative effect of Sm-C, granulosa cells from immature, hypophysectomized, diethylstilbestrol-treated rats were cultured under serum-free conditions for up to 96 h. Concurrent treatment with highly purified Sm-C (50 ng/ml) produced 10.2- and 3.6-fold increments in the FSH (20 ng/ml)-stimulated accumulation of Po and 20 alpha-hydroxy-4-pregnen-3-one, respectively. Sm-C-augmented Po biosynthesis was dose- and time dependent, but was independent of the FSH dose employed. Significantly, this effect of Sm-C could not be accounted for by enhancement of cellular viability or plating efficiency, nor by an increase in the number of cells, or their DNA synthesis. Furthermore, specific inhibition of DNA synthesis with cytosine-1-beta-D-arabinofuranoside was without significant effect on the ability of SM-C to enhance FSH-supported Po biosynthesis. Insulin, like Sm-C, also synergized with FSH in the induction of Po biosynthesis. However, insulin [ED50 = 19.2 +/- 1.6 (SE) micrograms/ml] was approximately 4800-fold less potent than Sm-C [ED50 = 4.0 +/- 0.3 (SE) ng/ml] in this regard, and exerted little or no effect at concentrations presumed to saturate the putative high affinity granulosa cell insulin receptor. Although maximal stimulatory doses of Sm-C (75 ng/ml) or insulin (100 micrograms/ml) produced comparable increments in FSH-supported Po biosynthesis, combined treatment with maximal doses of both peptides did not prove additive. Pertinently, the direct cytodifferentiative effect of Sm-C is exerted at (nanomolar) concentrations compatible with its receptor-binding affinity as observed in all other cell types studied. Thus, Sm-C is not likely to be acting through the putative high affinity insulin receptor but rather through its own high affinity recognition sites. Similarly, the cytodifferentiative action of high dose insulin may reflect the consequences of its cross-interaction with the putative Sm-C, rather than the insulin receptor. These findings are in keeping with the suggestion that the granulosa cell may be the site of Sm-C reception and action and that Sm-C of intraovarian or circulatory origin may participate in the differentiation, as well as replication, of the developing granulosa cell.     

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.     

Regulation of inhibin subunit gene expression by FSH and estradiol in cultured rat granulosa cells

Roles of follicle-stimulating hormone (FSH) and sex steroids in regulating the expression of mRNA species encoding the alpha-, beta A- and beta B-subunits of inhibin were studied in cultured granulosa cells from immature rat ovaries. Inhibin subunit mRNAs were detected by Northern blot analysis of total RNA extracted from granulosa cell monolayers which had been incubated for 48 h in serum-free medium containing FSH (100 ng/ml) and/or a steroid (10(-6) M): estradiol (E), testosterone (T) or 5 alpha-dihydrotestosterone (DHT). Levels of mRNA encoding each inhibin subunit in untreated (control) cultures were low. In cultures treated with FSH alone, levels of inhibin alpha-, beta A- and beta B-subunit mRNA were approximately 60-fold, 70-fold and 66-fold greater than control, respectively. In cultures treated with E alone, levels of inhibin alpha- and beta B-subunit mRNA were elevated approximately 4-fold and 2-fold, respectively, but the level of inhibin beta A-subunit mRNA was not measurably affected. Treatment with T or DHT alone had no consistent effect on the levels of any inhibin subunit mRNA. The stimulatory effects of FSH were not consistently altered by the presence of either androgen or estrogen. These results confirm the role of FSH in regulating inhibin alpha-subunit gene expression and provide direct evidence that both inhibin beta-subunit genes are inducible by FSH in granulosa cells. All three inhibin subunit mRNAs followed the same pattern, suggesting that their expression is coordinately regulated by FSH during granulosa cell differentiation.     

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.     

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)     

GnRH receptors in cultured rat granulosa cells: mediation of the inhibitory and stimulatory actions of GnRH

The regulatory actions of FSH and the GnRH agonist [D-Ala6]des-Gly10-GnRH N-ethylamide (GnRHa) upon ovarian GnRH receptors were studied in granulosa cells obtained from ovaries of hypophysectomized diethylstilbestrol-treated rats. When granulosa cells were cultured for 48 h in the presence of FSH (5-250 ng/ml) the binding of 125I-GnRHa to granulosa cell receptors was increased in a dose-dependent manner, to a maximum of 3-4 fold above the control value. Addition of FSH (100 ng) also caused a dose-dependent increase of more than 100-fold in the accumulation of cAMP and progesterone in the culture medium. In freshly prepared cells, Scatchard analysis of GnRHa binding data revealed an equilibrium constant (Ka) of 1.1 x 10(10) M-1 and GnRH receptor concentration fo 401 fmoles/mg protein. Granulosa-cell GnRH receptors decreased during culture without FSH, but were maintained in the presence of 100 ng FSH at 580 femoles/mg protein, with Ka of 0.8 x 10(10) M-1. This action of FSH on GnRH receptors was significantly reduced by 10(-8) M GnRHa. Also, GnRHa concentrations of 10(-10) and 10(-8) M caused inhibition of FSH-induced cAMP and progesterone accumulation. In cells cultured with GnRHa alone, there was a slight enhancement of GnRH receptors by GnRHa concentrations up to 10(-8) M, and a decrease below control levels with higher amounts. Also, GnRHa concentrations from 10(-8) to 10(-5) M caused a 3-4-fold increase in cAMP accumulation in the absence of FSH. These results demonstrate that FSH maintains GnRH receptors in cultured granulosa cells, and that GnRHa attenuates this effect, as well as the other actions of FSH on granulosa cell maturation. It is also evident that GnRHa itself can slightly stimulate cAMP production and partially maintain GnRH receptors, but at high concentrations causes loss of the homologous receptor sites. These findings also emphasize the ability of GnRH agonists to exert both positive and negative direct effects on rat granulosa cell function.     

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.     

Growth factors regulate immunoreactive insulin-like growth factor-I production by cultured porcine granulosa cells

The effects of various growth factors on the production of immunoreactive insulin-like growth factor I (iIGF-I) in short term (3-day) cultures of porcine granulosa cells was investigated. Epidermal growth factor (EGF) was shown to be a potent dose-dependent stimulator of iIGF-I production, achieving a 3.6-fold stimulation at a dose of 10 ng/ml. Transforming growth factor-alpha (10 ng EGF equivalents/ml) was also stimulatory. Platelet-derived growth factor (10 ng/ml) had no effect of its own, but enhanced EGF-stimulated iIGF-I production. The acidic and basic fibroblast growth factors (100 ng/ml) had no effect alone or in combination with EGF. Transforming growth factor-beta (10 ng/ml) had no effect of its own, but inhibited EGF-stimulated iIGF-I production. The interactive effects of EGF and FSH (200 ng/ml) on iIGF-I production were investigated in short term and longer term (7-day) cultures. In short term cultures under conditions optimized for EGF-dependent iIGF-I production, FSH had no effect of its own and inhibited EGF action. Conversely, in longer term cultures optimized for FSH-dependent iIGF-I production, EGF had no effect of its own and inhibited FSH action. Thus IGF production by cultured porcine granulosa cells is regulated in a complex manner and is highly dependent on the culture conditions. Our results suggest that IGF production in the ovary may also be regulated in a complex manner which is dependent on the developmental state of the follicle.     

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.