Progestins inhibit fsh-stimulated steroidogenesis in cultured rat granulosa cells

It has been hypothesized that progesterone (P) exerts a direct inhibitory effect on ovarian follicular development, an effect which could be mediated by P receptors located in granulosa cells. We tested this hypothesis by examining the effect of several progestins on FSH-stimulated estrogen (E), P, and 20α-dihydroprogesterone (DHP) production by cultured rat granulosa cells, and correlated the results with the ability of the progestins to bind to the granulosa cell P receptor. Granulosa cells from immature hypophysectomized DES-treated rats produced 9 ng/ml E, 21 ng/ml P and 29 ng/ml DHP during a 2-day incubation in McCoy's 5a medium containing 10^?7 M androstenedione and 10 ng/ml oFSH. The FSH-induced increase in E production was inhibited by 50 and 95% following concomitant treatment with 3 × 10^?6 and 10^?5 M resp. of R5020, a potent synthetic progestin. Added R5020 at these concentrations also significantly inhibited P and DHP production. R5020 had no effect on granulosa cell viability or plating efficiency, and the inhibitory action of R5020 on E production was reversible. In studies of the specificity of the progestin inhibitory action, the relative abilities of various progestins to inhibit E production were: R5020 > P > DHP > 17α-hydroxyprogesterone (170HP). The relative abilities of these progestins to bind to the ovary P receptor were also: R5020 > P > DHP > 170HP. These results indicate that exogenous progestins directly inhibit the FSH-stimulation of granulosa cell steroidogenesis in vitro and suggest that the progestin effect may be mediated by the P receptor. Such results offer a possible mechanism whereby progesterone could exert a direct but reversible inhibitory action on ovarian follicular development.     

Progestin receptors in human uterine cytosol

Progestin(norethindrone and norethindrone acetate)-binding protein, exhibiting characteristics similar to uterine progesterone receptor, has been identified in human uterine cytosol. The progestin receptor was characterized by sedimentation coefficient 4.2 S; Stokes radius, 39 Å; frictional ratio 1.29; isoelectric pH 4.6; molecular radius 2.7 nm; and molecualr weight in the range 67 000–74 000. The ammonium-sulfate-precipitated progestin-receptor complex was eluted from a DEAE-cellulose column at 0.18 M KC1. The progestin binding was saturable and stereospecific. The sequential variation in receptor concentration (early proliferative, 3800–4300 sites/cell; late proliferative, 9500–11200 sites/cell; early secretory, 4900–6200 sites/cell; late secretory, 1800–2300 sites/cell) was in conformity for progesterone and the progestins, when concurrently measured. Oral administration of norethindrone significantly reduced the cytoplasmic and nuclear receptor concentration for estradiol and progesterone. A significant observation was that the progestins stabilized the progestin receptor by forming a slowly dissociating complex with a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{～ 110?130}\text{min}$ as compared with the progesteronereceptor complex dissociating with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{～41}\text{min}$. Thus, the uterine progestin receptor recognizes progestins in general, although with a varying degree of affinity, and the altered rate constants could be of putative importance in determining the biological potency of the progestins.     

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.     

Effect of 17 beta-estradiol on thyroliberin responsiveness in GH3/B6 rat prolactin cells

GH3/B6 rat prolactin cells were used to analyse at the cellular level the mechanisms by which 17 beta-estradiol (E2) regulates TRH responsiveness of prolactin cells. Before experiments, cells were grown for up to 7 days in 3 different media: normal medium (N) containing 15% horse serum and 2.5% fetal calf serum, CD medium prepared with charcoal-dextran extracted serum and CDE medium supplemented with 4 x 10(-8) M E2. The binding of 3H-TRH (30 min at 37 degrees C) and the TRH-induced percent increase of prolactin release as a function of TRH doses were compared in the 3 conditions. Preculture in E2 enriched medium increased by 50% the number of TRH high-affinity binding sites without modifying their affinity, increased by up to 3 times the percent of the TRH-induced stimulation of prolactin release and improved by one order of magnitude the ED50 of the TRH effect on prolactin release. The presence of HEPES (10 mM) during TRH challenge masked the effect of E2 on the increase in number of binding sites but respected its potentiating effect on prolactin release.     

Aminoglutethimide augments follicle-stimulating hormone-induced aromatase activity in cultured porcine granulosa cells

The role of endogenous progestin synthesis in the modulation of FSH-induced aromatase activity was examined. Granulosa cells isolated from nonatretic medium-sized (3-5 mm) follicles of prepubertal pigs were cultured for an initial 48-h period, during which time aromatase activity was induced by FSH in the absence or presence of aminoglutethimide (AG). After induction, the cell monolayers were washed before being cultured for a further 6-h period in the presence of the substrate testosterone (0.5 microM). The aromatase activity was assessed by measuring the accumulation of estradiol during the test period. Basal aromatase activity was negligible and was unaffected by the presence of AG (0.1-100 microM) during the induction period. But when cells were cultured with FSH and AG (0.1-1000 microM) during the induction period, there was a dose-dependent, biphasic increase in the FSH-induced estradiol synthesis during the test period. Maximal enhancement was obtained with 10 microM AG (3.5-fold). Thereafter the aromatase activity declined and, at 1000 microM AG, was significantly (P less than 0.05) inhibited. At the same time, the FSH-stimulated progestin production during the induction period was inhibited in a dose-related fashion by AG. This AG-enhanced aromatase activity was dose and time dependent but was independent of the FSH concentration used. The apparent median effective dose of AG was 2.4 microM and a minimal time of 24 h or less was needed to potentiate the induction of aromatase activity by FSH. If AG was, however, added to the cell cultures during the test period, the FSH-induced aromatase activity was inhibited, showing that AG is an inhibitor of FSH-induced aromatase activity. This action of AG during the test period could be alleviated by the addition of testosterone during the induction period. The viability of the granulosa cells and the total cellular protein were not significantly (P greater than 0.05) altered by AG. These results show that the induction of aromatase activity by FSH could be enhanced by AG, which probably acts by inhibiting progestin production during the induction period, leading us to conclude that endogenous progestins might play an important role in modulating the induction of aromatase activity by FSH.     

The role of cyclic AMP in the induction of estrogen and progestin synthesis in cultured granulosa cells

The role of cyclic AMP in the induction of enzymes involved in estrogen and progestin biosynthesis in undifferentiated granulosa cells was investigated. When granulosa cells from immature hypophysectomized, DES-treated rats were cultured for 2 days in serum-free medium with aromatase substrate (10^?7M androstenedione) together with graded doses of FSH, prostaglandin E₂ (PGE₂), cholera toxin (CT), or dibutyryl cyclic AMP (Bu₂cAMP), there was a dose-related increase in estrogen (E) production. The induction of E production by saturating doses of FSH, PGE₂, CT and Bu₂cAMP required a lag phase of ～24 h, after which the E response increased sharply to maximum levels at day 3 and then declined gradually to day 5. Treatment for 24 h (day 0–1) with FSH, together with 1 μg/ml of either actinomycin D or cycloheximide, completely abolished the stimulatory action of FSH on E production. When the inhibitors were removed, the FSH-induced increases in E returned to near normal levels after a 24-h lag period. Similar effects of the inhibitors upon E production by CT, PGE₂ and Bu₂cAMP were observed. As with E, the production of progesterone and 20 α-dihydroprogesterone was markedly stimulated by FSH, PGE₂, CT and Bu₂cAMP and the results of the time course, dose response and inhibitor experiments were similar to those for E production.These results indicate that FSH induces the de novo synthesis of enzymes required for both estrogen and progestin biosynthesis by undifferentiated granulosa cells and suggest that this action is mediated by cyclic AMP.     

Modulation of FSH-controlled steroidogenesis in rat granulosa cells: direct in-vitro effects of LHRH and ICI-118630

Direct inhibitory effects of LHRH and an LHRH agonist (ICI-118630) on FSH-controlled steroidogenic processes in ovarian granulosa cells were characterized in vitro. Over a 2-day culture period in the presence of testosterone (10(-7) M), FSH (3-3 000 ng/ml) caused dose-dependent increases in the aromatase activity of granulosa cells isolated from oestrogen-pretreated immature rats. Progestogen biosynthesis was stimulated in a similar manner. The presence of LHRH (10(-9) - 10(-7) M) in the culture medium inhibited these responses by right-shifting the dose-response curves. Thus the net effect was one of reduced sensitivity to FSH. ICI-118630 was approximately 10 times more effective than LHRH as an inhibitor of aromatase induction and progestogen biosynthesis in response to FSH. Over a 1-h incubation at concentrations up to 10(-7) M, neither decapeptide had a consistent inhibitory effect on FSH-stimulated granulosa cell cAMP formation either in the presence or absence of 1-methyl-3-isobutyl-xanthine (MIX); but during the 2-day culture, ICI-118630 and occasionally LHRH significantly inhibited aromatase induction by cholera toxin and 2 different cAMP analogues. Over the same range of concentrations, each peptide progressively inhibited the stimulatory effect of MIX on FSH-induced aromatase activity and progestogen biosynthesis. Thus LHRH/ICI-118630 can directly modulate FSH-controlled granulosa cell steroidogenesis in vitro via effects on one or more biochemical loci distal to the FSH-receptor coupled adenylate cyclase system. These experiments have implications for the role of a putative LHRH-like ovarian substance(s) in the local co-ordination of follicular development and function.     

Androgen/antiandrogen modulation of cyclic AMP-induced steroidogenesis during granulosa cell differentiation in tissue culture

FSH stimulation of granulosa cell differentiation is believed to be mediated by the intracellular cyclic AMP (cAMP) level. However, steroidogenic enzyme induction in the differentiating granulosa cell is subject to direct modulation by androgenic steroid: in granulosa cell cultures established from ovaries of oestrogen-pretreated, prepubertal rats, potentiating effects of testosterone (T) on FSH induction of oestrogen synthetase (aromatase) and progesterone (P) biosynthesis can be blocked by including a stoichiometric excess of antiandrogen (hydroxyflutamide, SCH 16423) in the culture medium. In this study we used the same experimental model to determine effects of T and SCH 16423 on the induction of steroidogenesis by endogenous cAMP and an exogenous cAMP analogue, 8-bromo-cAMP (8brcAMP). Granulosa cells were cultured in medium containing variable FSH concentration (3–300 ng/ml) with a fixed (100 μm) dose of 3-isobutylmethylxanthine (MIX), or containing a fixed (minimally effective: 10–15 ng/ml) dose of FSH with MIX concentration variable (50–800 μM). By relating steroidogenic endpoints at 48 h to the acute cAMP response (accumulations in the medium) at 1 h, it was deduced that aromatase induction was saturable under conditions where FSH-sensitive cAMP production and the induction of P biosynthesis showed further, proportionate increases.Although T (0.1μM) did not alter acute FSH-responsive cAMP production, its presence throughout the 48 h culture was required for full expression of FSH-induced steroidogenesis in the cell monolayers. When the aromatase response (but not the P response) was ‘supersaturated’ by endogenous cAMP (i.e. culture with FSH plus MIX), SCH16423 was unable to antagonize the potentiating effect of T on aromatase induction while it continued to block T-potentiated P biosynthesis. Steroidogenic induction by cholera toxin (100 ng/ml) was also subject to similar modulation by T and SCH16423. However, the phosphodiesterase-resistant cAMP analogue 8brcAMP (3 mM) not only induced each response (albeit submaximally in the case of aromatase) in the absence of T, but its effects tended to be refractory to androgen/antiandrogen modulation. Accumulations of cAMP in the medium from 48 h cultures which had been incubated with FSH (100 ng/ml) were increased 2–3-fold by the additional presence of T (0.1 μM). This long-term stimulatory effect of T on FSH-dependent cAMP accumulation was blocked by culture in the presence of SCH16423 (10 μM). Thus, androgen potentiation of steroidogenic enzyme induction during FSH-stimulated granulosa cell differentiation may involve a suppression of cAMP catabolism exerted by way of the androgen-receptor system.     

Androgen inhibition of follicle-stimulating hormone-stimulated luteinizing hormone receptor formation in cultured rat granulosa cells

Since LH receptors are decreased in atretic follicles known to contain high androgen levels, we have studied the androgen modulation of LH receptor formation in vitro. Granulosa cells from hypophysectomized, diethylstilbestrol-treated rats were cultured for 3 days with FSH in the presence or absence of nonaromatizable androgens, dihydrotestosterone and 5 alpha-androstane-3 alpha, 17 beta-diol, or a synthetic androgen, R1881 (17 beta-hydroxy-17 alpha-methyl-4,9,11-estratrien-3-one). FSH increased LH receptor content in granulosa cells, while concomitant androgen treatment decreased LH receptor content in a dose- and time-dependent manner, without changing the equilibrium dissociation constant (Kd) for human CG. R1881 (10(-7) M), dihydrotestosterone (10(-6) M), and 5 alpha-androstane-3 alpha, 17 beta-diol (10(-6) M) inhibited LH receptor content by 68%, 65%, and 65%, respectively. Similar to earlier findings, these androgens enhanced FSH-stimulated progesterone biosynthesis and aromatase activity in the same cells. To study their LH responsiveness, androgen-treated cells were washed and reincubated for 2 more days with or without LH. Although basal progesterone production was elevated by R1881 pretreatment, the androgen-pretreated cells were less responsive to LH. Treatment with cyanoketone, an inhibitor of 3 beta-hydroxysteroid dehydrogenase, did not alter the inhibitory effects of R1881 on LH receptors, indicating that the androgen action is not mediated by endogenous progestins. Furthermore, R1881 inhibited the stimulation of LH receptor formation by forskolin, cholera toxin, and 8-bromo-cAMP, suggesting that androgens may inhibit LH receptor induction by affecting post-cAMP events. Estrogen treatment enhanced the FSH induction of LH receptor content, while concomitant addition of R1881 also suppressed the estrogen action. Thus, androgens inhibit FSH-induced functional LH receptors in cultured rat granulosa cells. The androgen effect is exerted, at least partially, at post-cAMP sites and is independent of changes in progestin biosynthesis.     

Receptor-mediated actions of growth hormone releasing factor on granulosa cell differentiation

GRF promotes follicular maturation and ovulation when administered with FSH in the treatment of infertility. Such actions could be mediated by stimulation of GH secretion and insulin-like growth factor I production, but the known actions of the structurally related hormone, vasoactive intestinal peptide (VIP), on granulosa cell function suggested that GRF may also act directly on the ovary to stimulate follicular development. Radioligand binding and activation studies, performed in granulosa cells from immature estrogen-treated rats, revealed a common receptor for VIP and rat (r) GRF in the ovary. Specific binding of [125I]VIP to granulosa cells was saturable and dependent on time and temperature. The relative potencies of VIP-related peptides for inhibition of radioligand binding were: VIP greater than rGRF greater than peptide histidine isoleucinamide greater than [His1,Nle27] human GRF(1-32)NH2 greater than secretin. In binding studies with the potent GRF agonist, [125I] [His1,Nle27]GRF(1-32)NH2, relative potencies were: rGRF(1-43)OH greater than [His1,Nle27]human GRF(1-32)NH2 greater than VIP greater than peptide histidine isoleucinamide greater than secretin. Glucagon and gastric inhibitory peptide, other peptides of the glucagon superfamily, and unrelated peptides including CRF and beta-endorphin, did not inhibit binding of either radioligand to ovarian receptors. In cultured granulosa cells, rGRF and VIP stimulated cAMP formation, consistent with coupling of their receptors to the adenylate cyclase system, and potentiated FSH-induced cAMP production. Both peptides also amplified FSH-induced progesterone biosynthesis, aromatase activity, and LH receptor formation. These observations demonstrate that rGRF is a potent cAMP-mediated agonist in the rat ovary and acts on a common VIP/GRF receptor in maturing granulosa cells. It is likely that the potentiating effect of administered GRF on gonadotropin-stimulated follicular development in vivo is in part mediated by direct actions of the peptide on the VIP/GRF receptor. Also, since GRF is present in the gonads, it is possible that the locally-produced peptide promotes follicular maturation by paracrine modulation of the stimulatory action of FSH on granulosa cell function.     

Cortisol metabolism in lymphocytes from cancer-bearing patients

A study was conducted to determine the pattern of cortisol metabolism by lymphocytes obtained from four groups of subjects: 27 male and female patients suffering from various types of malignancy other than malignancy of lymphatic tissues; and 26 healthy male and female controls. Known concentrations of cells were incubated with 1,2-³H-cortisol and the products were isolated by thin-layer and paper chromatography. Three metabolites were found to be produced by lymphocytes from both normal and cancer-bearing patients: 20α-hydroxycortisol, 20β-hydroxycortisol, and tetrahydrocortisol. Cells from the female control group were found to be more active than those from the male controls, while cells from cancer-bearing patients were markedly more active than the normal cells, regardless of sex. It is suggested that this finding of increased metabolism of cortisol by lymphocytes from patients with different types of malignancy other than lymphoma may provide the basis for a new diagnostic aid.     

Site of action of androgens on follicle-stimulating hormone-induced aromatase activity in cultured rat granulosa cells

This paper describes experiments on cultured granulosa cells isolated from ovaries of immature rats designed to locate the site of action of androgens on FSH-induced aromatase activity. Treatment of cells during a 36-h induction period with (Bu)2cAMP, 8- BrcAMP , FSH, prostaglandin E2, or cholera toxin resulted in induction of aromatase activity measured as 17 beta-estradiol accumulation during a 6-h test period with testosterone (5 X 10(-7) M) added to medium as substrate. Presence of testosterone (5 X 10(-7) M) during the induction period enhanced the effects of FSH, cholera toxin, and prostaglandin E2 on aromatase activity, but not those of the cAMP analogs. The effects of culturing and steroids on responsiveness of granulosa cells to FSH (measured as FSH-stimulated cAMP production during a 1-h test period) were examined. The data showed that culturing in medium alone for 36 h resulted in a decrease in the ability of FSH to stimulate cAMP production when compared to that of freshly isolated cells. After culture with testosterone (5 X 10(-7) M), dihydrotestosterone (DHT) (5 X 10(-7) M), or 17 beta-estradiol (5 X 10(-7) M), responsiveness was at least partially restored. After treatment with progesterone (5 X 10(-7) M), FSH stimulation of cAMP production was not significantly different from that of cells cultured in medium alone. Hydroxyflutamide (5 X 10(-5) M), an antiandrogen known to block androgen-receptor interaction, abolished the effect of DHT and depressed the effect of testosterone on responsiveness of granulosa cells to FSH. Cells treated for 36 h with testosterone (5 X 10(-7) M) bound significantly more [125I]iodo-FSH than cells cultured in medium alone. Although DHT (5 X 10(-7) M) slightly increased FSH binding, the effect was not statistically significant. These results suggested that androgens regulate granulosa cell aromatase activity not only as substrates, but also by acting at a site before cAMP production (possibly at the level of the FSH receptor) in the control of FSH-induced enzyme activity.     

Studies on the synergistic effect of androgen on the stimulation of progestin secretion by FSH in cultured rat granulosa cells: a search for the mechanism of action.

Cultures of granulosa cells from immature hypophysectomized DES-treated rats were unable to maintain progestin production of more than 48 h in medium without hormone supplementation or in the presence of FSH only. Production of progestin (20alpha-dihydroprogesterone, as measured by radioimmunoassay) remained unimpaired in the presence of androstenedione (Ad) and was markedly increased in the presence of both Ad and FSH. The combined treatment with FSH and Ad during the first 48 h of culture brought about persistent changes in the cultured cells, since progestin accumulation did not decline upon subsequent removal of these hormones during days 3 and 4 of culture. Dibutyryl cyclic AMP (DBC) was able to mimic the changes in steroidogenic capability induced by the combined action of FSH and Ad. The extent of [125I]-FSH binding, FSH-stimulable cAMP accumulation and cyclic 3',5'-nucleotide phosphodiesterase activity were not affected by addition of Ad to the culture medium. Ad synergized with DBC in the stimulation of progestin accumulation in granulosa cell cultures. It is suggested that androgen acts at a step in the regulation of progestin biosynthesis distal to cAMP production.     

Tumor necrosis factor alpha inhibition of follicle-stimulating hormone-induced granulosa cell estradiol secretion in the human does not involve reduction of cAMP secretion but inhibition at post-cAMP site(s)

Tumor necrosis factor α (TNF) inhibits follicle-stimulating hormone-(FSH) induced estradiol secretion by granulosa cells in several species, including humans. One major inhibitory effect of TNF in rat granulosa cells is at the level of stimulatable adenylyl cyclase, resulting in reduced cAMP concentrations. The purpose of the present study was to investigate whether a reduction in cAMP secretion could account for the inhibitory effects of TNF on FSH-induced estradiol in human granulosa cells. Granulosa cells were taken from ovaries of premenopausal women undergoing oophorectomy for reasons unrelated to ovarian pathology. Women in this study were in various stages of the menstrual cycle or exhibited irregular cycles. Granulosa cells from follicles ranging from 5 to 10 mm diameter were subjected to culture for 48 and 96 h. Granulosa cells were cultured with human FSH (2 ng/mL) and testosterone (1 μM) in the presence and absence of human TNF (20 ng/mL). Media were collected at 48 h, fresh media and hormones added, and cultures continued for an additional 48 h. Accumulation of cAMP, progesterone, and estradiol in media were determined by radioimmunoassay (RIA). FSH induced significant increases in cAMP, progesterone, and estradiol by 96 h of culture. TNF inhibited the secretion of estradiol at 96 h without reducing the accumulation of cAMP and progesterone in media. Similar results were observed in the presence of 0.1 mM isobutylmethylxanthine (D3MX), a phosphodiesterase inhibitor that would prevent metabolism of cAMP to AMP. To determine whether TNF would inhibit the ability of cAMP to induce estradiol and progesterone secretion, granulosa cells were incubated with 0.1 mM cAMP in the presence and absence of TNF. TNF consistently inhibited the ability of cAMP to increase estradiol secretion. These results indicate that a pathway for TNF inhibition of FSH- or cAMP-induced estradiol secretion in human granulosa cells is at post-cAMP sites rather than inhibition of FSH-stimulatable adenylyl cyclase.     

Glucocorticoid-induced cell-size changes and nuclear fragility in rat thymocytes

The events preceding glucocorticoid-induced lymphocytolysis have been studied in isolated rat thymocytes. Incubation of thymocytes at 37°C in the presence of 1 μM dexamethasone resulted in the progressive appearance of pyknotic cells of modal diameter 4.6 μm, distinct from normal cells of diameter 5.2 μm. The rate of appearance of the pyknotic cells was determined by selective electronic cell counting, and was shown to be accompanied by increased nuclear fragility. The production of pyknotic cells was glucocorticoid-specific, dose-dependent, blocked by cycloheximide, and preceded the loss of cell viability as determined by dye exclusion. The pyknotic cells were separated from the non-pyknotic cells by density-gradient centrifugation and shown to be solely responsible for the observed nuclear fragility.     

Protein kinase B is obligatory for follicle-stimulating hormone-induced granulosa cell differentiation

Although FSH receptors are linked to the cAMP second messenger system, additional intracellular signaling pathways appear to be required for the induction of aromatase and the LH receptor during granulosa cell differentiation. We employed adenovirus vectors to modulate specific intracellular signaling systems in undifferentiated granulosa cells to identify the signaling pathway(s) that may be involved in the FSH-mediated induction of aromatase and the LH receptor. Expression of either the constitutively activated human LH receptor D578H or the constitutively active human G(s)alpha Q227L resulted in increased cAMP production without increasing aromatase activity or mRNA levels for the LH receptor. To explore the contributions of other pathways, we expressed the constitutively activated forms MAPK kinase (MEK) and protein kinase B (PKB). Neither MEK nor PKB alone increased estrogen or progesterone production by undifferentiated granulosa cells. Stimulation of granulosa cells by FSH in the presence of the constitutively active PKB, but not MEK, led to an amplification of FSH-induced aromatase and LH receptor mRNA levels, whereas a dominant negative PKB vector completely abolished the actions of FSH. The expression of the constitutively active PKB in combination with the constitutively active LH receptor D578H, the constitutively active G(s)alpha Q227L, or 8-bromo-cAMP led to an induction of aromatase as well as LH receptor mRNA comparable to that seen in cells stimulated with FSH alone. These results demonstrate that PKB is an essential component of the FSH-mediated granulosa cell differentiation and that both PKB and G(s)alpha signaling pathways are required.     

Cyclosporine differentially affects estrogen and progestin synthesis by rat granulosa cells in vitro

Potential side-effects of the immunosuppressive drug cyclosporine (also cyclosporin A, CsA) on ovarian endocrine function have been investigated using granulosa cells isolated from immature estrogen-primed rats and cultured in a chemically defined medium. The FSH-dependent differentiation of steroidogenic pathways for estrogen and progestin secretion was shown to be differentially affected by CsA in vitro, at drug concentrations that approximate immunosuppressive concentrations in blood of humans or animals. CsA at 0.1-1 microgram/ml synergistically enhanced FSH-stimulated aromatase activity as measured by the conversion of exogenous testosterone to 17 beta-estradiol, while production of the progestins (progesterone + 20 alpha-hydroxypregn-4-en-3-one + pregnenolone) was little affected at up to 0.1 microgram/ml CsA and reduced at higher concentrations. CsA alone did not stimulate basal steroid secretion. The action of CsA to augment FSH-stimulated induction of aromatase activity was seen both in the presence or absence of testosterone. The effects of CsA (1 microgram/ml), either stimulatory on aromatase activity or inhibitory on progestin secretion, were in general increased with greater times of cell exposure throughout the culture period, although the temporal effects on 17 beta-estradiol and the progestins were not identical following delayed addition or removal of CsA from the culture medium. Higher concentrations of CsA (3-10 micrograms/ml) were generally toxic to granulosa cells as indicated by marked decreases in 17 beta-estradiol and progestin secretion and in incorporation of [3H]leucine. These results suggest that therapeutic concentrations of CsA might directly influence ovarian function by differentially modulating the FSH-dependent steroidogenic pathways of granulosa 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.