Synergistic actions of estradiol and the insulin-like growth factor somatomedin-C on swine ovarian (granulosa) cells

Estradiol amplified synergistically the dose- and time-dependent stimulatory actions of human somatomedin-C on progesterone biosynthesis by cultured swine granulosa cells. This facilitative interaction was not attributable to inhibition of the catabolism of progesterone to 20 alpha-hydroxypregn-4-en-3-one, but, rather, reflected time-dependent stimulation of pregnenolone synthesis measured in the presence of exogenous soluble sterol substrate for cholesterol side-chain cleavage. Moreover, treatment with somatomedin-C was accompanied by increased synthesis of two immunoprecipitable cholesterol side-chain cleavage constituents, viz. cytochrome P-450scc and adrenodoxin. The synergism between estradiol and somatomedin-C was associated with significantly greater specific binding of somatomedin-C in estrogen-treated than control cultures, with no change in apparent receptor affinity. In vitro synergism occurred at somatomedin-C concentrations estimated by sequence-specific immunoassay to be attainable in ovarian follicular fluid in vivo and was specific in that it was not mimicked by the insulin-like peptide relaxin or by epidermal growth factor or fibroblast growth factor. However, high concentrations of insulin-like growth factor II (multiplication-stimulating activity) and insulin were able to interact with estradiol in a facilitative fashion to enhance progesterone production. In addition, the estrogenic component of the synergism was specific, since it was antagonized by the selective antiestrogen LY156758 and was mimicked sparingly by the nonaromatizable androgen 5 alpha-dihydrotestosterone. We conclude that estradiol and somatomedin-C interact synergistically in a time- and dose-dependent manner to enhance the biosynthesis of pregnenolone and progesterone by swine granulosa cells. Since estradiol and somatomedins are present in significant concentrations in the antral fluids of maturing Graafian follicles, we suggest that coordinated trophic effects of estradiol and insulin-like growth factor(s) may effectively prepare granulosa cells for the high rates of progesterone biosynthesis ultimately required after ovulation.     

Estrogen augmentation of gonadotropin-stimulated progestin biosynthesis in cultured rat granulosa cells

The influence of estrogens on gonadotropin-stimulated production of progesterone and 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-P) was examined in primary cultures of rat granulosa cells. Granulosa cells were cultured for 3 days with increasing concentrations of FSH in the presence or absence of either diethylstilbestrol (DES) or estradiol. FSH treatment increased progestin production in a dose-dependent manner, whereas treatment with estrogens alone were ineffective. In contrast, concomitant addition of either DES or estradiol augmented FSH-stimulated production of progesterone and 20 alpha-OH-P. Increasing concentrations of estradiol (10(-10) - 10(-7) M) augmented the stimulatory effect of FSH (30 ng/ml) on progesterone production in a dose-dependent manner with ED50 values of approximately 3 X 10(-9) M. The facilitatory action of estradiol was time-related, becoming significant after 36 h of treatment. Granulosa cells were also cultured for 2 days with FSH to induce functional LH receptors. The FSH-primed cells were treated for an additional 3 days with increasing concentrations of LH (0.3-30 ng/ml) in the absence or presence of DES (10(-7) M). LH stimulated progesterone and 20 alpha-OH-P production in a dose-dependent manner, whereas concomitant addition of DES further enhanced LH-induced progestin biosynthesis. (Bu)2cAMP also increased progesterone and 20 alpha-OH-P production by the granulosa cells; however, concurrent addition of DES did not augment the actions of (Bu)2cAMP. The effect of estrogens on gonadotropin-stimulated cAMP accumulation was also examined. FSH treatment dose-dependently increased cAMP accumulation, whereas concomitant treatment with estradiol further increased the FSH action. Similarly, LH treatment also stimulated cAMP accumulation in FSH-primed cells, whereas concurrent addition of DES further augmented LH action. Thus, the stimulatory effect of estrogens upon gonadotropin-stimulated progestin production may be related to the augmentation of cAMP biosynthesis. The present observations suggest that intraovarian estrogens may act locally to enhance the sensitivity of granulosa cells to FSH and LH, thereby increasing the biosynthesis of progestins and cAMP by the granulosa cells.     

The role of estradiol as a biological amplifier of the actions of follicle-stimulating hormone: in vitro studies in swine granulosa cells

We describe an in vitro system of swine granulosa cells which remain responsive to estradiol (E2) and FSH. In this system, we examined mechanisms by which E2 amplifies the stimulatory actions of FSH. E2 stimulated progesterone production in a dose-dependent manner and enhanced the tropic effects of FSH. The facilitative interaction between E2 and FSH could not be accounted for by mitogenic effects, by a leftward shift in the dose-response curves for FSH or E2, or by catabolism of progesterone to 20 alpha-hydroxypregn-4-en-3-one. E3 also enhanced stimulatory actions of 8-bromo-cAMP, choleratoxin, and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. Lipoproteins were required for maximal interactive effects between E2 and FSH. However, estrogen significantly increased the effects of FSH even in serum- and lipoprotein-free medium. In addition, when de novo cholesterol biosynthesis by granulosa cells was suppressed by Compactin, the actions of E2 and/or FSH were not impeded. In contrast, E2 alone and FSH alone significantly augmented progesterone production in response to the oxygenated sterol 5-cholesten-3 beta, 25-diol, which is an effective substrate for cholesterol side-chain cleavage. In the presence of 5-cholesten-3 beta, 25-diol, the magnitude of the synergism between E2 and FSH was also increased markedly. We conclude that E2 augments the stimulatory actions of FSH or cAMP on steroidogenesis. This facilitative interaction is amplified by exogenous lipoproteins and does not seem to depend upon endogenous cholesterol biosynthesis. Studies with 5-cholesten-3 beta, 25-diol suggest that estrogen and FSH exert significant stimulatory effects at the level of cholesterol side-chain cleavage. These observations reveal important interactions of E2 and fSH in preparing granulosa cells for the high rates of steroidogenesis they ultimately express in the luteinized state.     

Mechanisms subserving insulin's differentiating actions on progestin biosynthesis by ovarian cells: studies with cultured swine granulosa cells

To characterize the nature of insulin action on ovarian cells, an in vitro system of swine granulosa cells was developed in which 3- to 50-fold stimulation of progesterone production was observed in response to insulin under serum-free or sparsely (1%) serum-supplemented conditions. These studies demonstrate that optimal cell density is critical for the full expression of insulin action, and that the dose-dependent responses of granulosa cells to insulin are also significantly influenced by the maturational status of the parent follicle. The striking (greater than 30-fold) responses of medium-sized and healthy preovulatory follicles to insulin were not attributable to the presence or absence of atresia or to a selective inhibition of progesterone's catabolism to 20 alpha-dihydroprogesterone, since 20 alpha-dihydroprogesterone production was also markedly increased in response to insulin. The mechanisms subserving insulin action were explored further by testing the capacity of insulin to 1) increase progesterone accumulation in response to exogenously provided pregnenolone, and 2) stimulate pregnenolone biosynthesis in the presence of trilostane, an inhibitor of pregnenolone metabolism. The effects of insulin were to increase the biosynthesis of progesterone from available pregnenolone and increase the production of pregnenolone from endogenous sterol substrate. The physiological relevance of these differentiative actions of insulin is suggested by insulin's ability to significantly enhance the stimulatory effects of FSH, LH, epinephrine, prostaglandin E2, and cAMP effectors, cholera toxin and 8-bromo-cAMP. In summary, the present studies delineate culture conditions in which swine granulosa cells exhibit a high degree of responsiveness to the stimulatory actions of insulin on progestin biosynthesis. The effects of insulin are critically influenced by cell density, stage of follicle maturation, and the presence or absence of classical ovarian effector hormones with which insulin can interact synergistically. Moreover, our studies indicate that insulin exerts significant actions at several levels of progestin biosynthesis, including the production of pregnenolone, progesterone, and 20 alpha-dihydroprogesterone by granulosa cells. In view of the high concentrations of insulin-like growth factors and somatomedins attained in porcine Graafian follicles, we suggest that trophic actions of insulin or insulin-like peptides and the synergism of insulin with classical ovarian effector hormones are likely to be of physiological importance to the differentiation of granulosa cells in the developing ovarian follicle.     

Direct actions of 17 beta-estradiol on progesterone production by highly differentiated porcine granulosa cells in vitro. II. Regulatory interactions of estradiol with luteinizing hormone and cyclic nucleotides

We investigated direct actions of 17 beta-estradiol and LH in the coordinate control of progesterone production by highly differentiated porcine granulosa cells maintained in monolayer culture. The administration of estradiol acutely suppressed both basal and LH-stimulated progesterone synthesis in vitro, i.e within the first 24-36 h of estrogen treatment. In contrast, continuation of estradiol administration alone beyond 48 h significantly augmented progesterone production per 10(5) granulosa cells. Among 12 independent experiments, the absolute stimulatory effects of estradiol were highly correlated (r = 0.991) with basal progesterone production by granulosa cells at the outset of culture, i.e. when steroid synthesis presumably reflected the degree of prior cytodifferentiation attained in vivo. Notably, estrogens also facilitated the dose-dependent actions of LH in a synergistic fashion. Synergism occurred during periods of both maximal and spontaneously declining steroidogenesis in vitro, and could be impeded by specific inhibitors of steroid biosynthesis (10 microM cyanoketone and 50 microM trilostane). In experiments designed to assess granulosa cell responsivity to delayed hormone rechallenge, there was a critical bihormonal requirement for both estradiol and LH in order to sustain maximal long term progesterone secretion. Further investigation of the biochemical mechanisms subserving synergistic effects demonstrated that estradiol was capable of augmenting the stimulatory actions of either exogenously supplied or endogenously generated cAMP. In particular, estradiol markedly enhanced the effects of potent phosphodiesterase resistant analogs of cAMP, 8-bromo-cAMP (0.1 mM), dibutyryl cAMP (2 mM) or 8-thio-cAMP (1 mM). Estradiol also significantly facilitated the stimulatory effects of agents that putatively increase or sustain intracellular pools of cAMP by various well defined mechanisms, i.e. choleratoxin (10 microgram/ml), guanyl-5'-imido-diphosphate (1.0 mM) or 3-isobutyl-1-methylxanthine (0.25 mM). Thus, the current in vitro studies delineate directly major interactions between estradiol and LH in the control of progesterone synthesis by highly differentiated granulosa cells. The present data further indicate that the synergistic stimulation of progesterone production by LH and estradiol is mediated in part by intracellular mechanisms operating distal to LH-stimulated cAMP production. These in vitro observations using physiological concentrations of hormones suggest a critically bihormonal role for estradiol and LH in the facilitation of progesterone secretion in vivo during late follicular phase differentiation of granulosa cells.     

Divergent effects of prolactin on estrogen and progesterone production by granulosa cells of rat Graafian follicles

The effect of PRL on ovarian steroidogenesis was studied in cultured granulosa cells isolated from follicles of mature cycling rats on the morning of proestrus. Ovine PRL 10-1000 ng/ml) inhibited estradiol production but stimulated progesterone biosynthesis in a dose-dependent manner. The effect of PRL was most prominent after 4 days of culture: 1000 ng/ml PRL suppressed estradiol production by 80% but increased progesterone synthesis by 290%, whereas the lower dose of 10 ng/ml inhibited estrogen secretion by 20% without altering progesterone synthesis. The divergent effect of PRL was not shown to be species specific, since ovine, rat and human PRL had similar effects. Using increasing concentrations of androstenedione (the aromatase substrate), estrogen secretion remained suppressed and progesterone production was stimulated by PRL. FSH stimulated both estrogen and progesterone production. The FSH-induced increased in estrogen production was inhibited by concomitant treatment with PRL. In contrast, PRL and FSH had an additive action in stimulating progesterone production. Although LH alone had no effect on steroidogenesis, concomitant treatment with LH and PRL resulted in a stimulation of progesterone production that was additive. This study demonstrates that PRL acts directly on granulosa cells of Graafian follicles of adult cycling rats to stimulate the secretion of progesterone and to suppress estradiol production.     

Ovarian actions of tumor necrosis factor-alpha (TNF alpha): pleiotropic effects of TNF alpha on differentiated functions of untransformed swine granulosa cells.

We have examined interactions between tumor necrosis factor-alpha (TNF alpha), a product of the immune system, and ovarian cells using serum-free monolayer cultures of untransformed swine granulosa cells. Recombinant human TNF alpha, a potent cytoactive product of activated macrophages, bound specifically and with high affinity to intact granulosa cells. Binding sites had an apparent Kd of 0.17 nM (95% confidence interval, 0.065-0.31), and a binding capacity of 80 nmol/micrograms DNA (95% confidence interval, 52-110). The binding capacity of granulosa cells for TNF alpha (but not the binding affinity) was increased approximately 2-fold by treatment with FSH and insulin. The biological effects of TNF alpha on pig granulosa cells were expressed after 48 and 96 h in culture. At the latter time, TNF alpha significantly suppressed insulin- and insulin- plus FSH-stimulated progesterone accumulation, with respective ID50 values of 0.08 +/- 0.008 and 0.06 +/- 0.014 nM, but did not affect basal progesterone accumulation or DNA content. TNF alpha also significantly attenuated the stimulatory effect of combined treatment with FSH and insulin on cAMP generation during 48-96 h of culture. TNF alpha inhibited the stimulatory effects of forskolin, cholera toxin, and the cAMP analog 8-bromo-cAMP on progesterone accumulation, indicating multiple sites of action of this immune modulator. Inhibition of progestin biosynthesis was observed even in the presence of 25-hydroxycholesterol, a soluble oxygenated sterol substrate for the cholesterol side-chain cleavage reaction, and was accompanied by decreased concentrations of specific cellular mRNA encoding cholesterol side-chain cleavage enzyme. There were no changes in the amounts of a constitutively expressed enzyme, phosphoglyceraldehyde dehydrogenase. Inhibitory actions of TNF alpha were specific to de novo steroid hormone biosynthesis, since nanomolar concentrations of this cytokine stimulated accumulation of prostaglandin E2 and prostaglandin F2 alpha basally and during treatment with FSH, cholera toxin, or 8-bromo-cAMP. In contrast, prostaglandin accumulation was not enhanced by interferon-gamma or interleukin-2. In summary, untransformed porcine granulosa cells exhibit specific, high affinity, low capacity saturable binding sites for TNF alpha, and the number of such binding sites can be regulated by combined treatment with insulin and FSH. Granulosa cells are susceptible to the inhibitory actions of TNF alpha on FSH- and insulin-supported progesterone biosynthesis and cAMP accumulation. One important locus of TNF alpha action is blockade of hormonally stimulated increases in specific mRNA encoding the cholesterol side-chain cleavage cytochrome P450 enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

The role of cholesterol esterification in ovarian steroidogenesis: studies in cultured swine granulosa cells using a novel inhibitor of acyl coenzyme A: cholesterol acyltransferase

We have used a novel competitive inhibitor of acyl coenzyme A:cholesterol acyltransferase (ACAT), Sandoz compound 58-035 [3-(decyldimethyl-silyl)N-[2-(4-methyl-phenyl)1-phenylethyl propanamide], to assess the importance of the cholesterol esterification reaction in ovarian steroidogenesis. Compound 58-035 markedly (greater than or equal to 96%) inhibited ACAT activity of swine ovarian microsomes in a dose-dependent (0.1-3.5 micrograms/ml) fashion. In addition, treatment of cultured granulosa cells with this fatty acylamide effectively (greater than or equal to 98%) suppressed hormonally stimulated cholesterol esterification, as assessed by the incorporation of [3H]oleic acid into cholesteryl ester. Accordingly, we used this inhibitor to test the role of cholesterol esterification in ovarian cells. In cultures with limited or no serum supplementation, long term (2- to 6-day) treatment of granulosa cells with compound 58-035 significantly increased basal progesterone production and amplified by 2- to 10-fold the stimulatory actions of trophic hormones, such as estradiol, FSH, estradiol combined with FSH, or insulin. The amplifying effect of ACAT inhibition on hormone-stimulated progesterone production could be mimicked by providing exogenous cholesterol substrate in the form of low density lipoprotein (LDL). Cotreatment with compound 58-035 and LDL resulted in no further augmentation of steroidogenesis. In contrast to the facilitative effects of compound 58-035 in longer term cultures, this ACAT inhibitor did not alter progesterone biosynthesis acutely (2-20 h) in swine or hamster ovarian cells. These observations suggest that there is an obligatory partitioning of some sterol into the ester pool in granulosa cells. In times of diminished availability of cholesterol, inhibition of the esterification pathway can make additional cholesterol available for use in steroid hormone biosynthesis. Thus, in the intact Graafian follicle, where LDL cholesterol delivery to granulosa cells and intracellular cholesteryl ester stores are limited, regulation of the ACAT reaction may significantly modulate rates of progesterone biosynthesis. The present results indicate that the use of a selective inhibitor of cholesterol esterification can permit one to probe the functional significance of the esterification reaction in steroidogenic cells.     

Estrogen regulates low density lipoprotein metabolism by cultured swine granulosa cells

To test estrogen's possible regulation of lipoprotein metabolism by granulosa cells, swine granulosa cells were cultured under serum-free conditions in the presence or absence of estradiol. Treatment with estradiol significantly enhanced high affinity, saturable, [125I]iodo-low density lipoprotein (LDL) binding with a median 2.85-fold (range 2.3- to 5.6-fold, n = six experiments) increase in the calculated number of LDL receptors and no change in the apparent dissociation constant (Kd) for LDL binding (Kd = 3.4 +/- 0.92 micrograms/ml in control and 4.0 +/- 0.87 micrograms/ml human LDL in estradiol-treated cultures). Estradiol also significantly increased [125I]iodo-LDL internalization by granulosa cells and augmented the maximal rate of LDL degradation by 2.0 to 2.5-fold without altering the apparent Michaelis-Menten constant (Km) for this process. Estrogen's dose-dependent enhancement of [125I]iodo-LDL binding, internalization, and degradation could be observed at minimum estradiol concentrations of approximately 100 ng/ml and was accompanied by increased progesterone secretion by granulosa cells. Further studies indicated that estrogen's stimulation of LDL internalization and degradation was not simply attributable to increased rates of nonspecific bulk fluid-phase pinocytosis (assessed with [125I]iodo-polyvinylpyrrolidone) or increased steroidogenesis per se (tested by blocking cholesterol side-chain cleavage with aminoglutethimide). We conclude that estradiol amplifies LDL binding by swine granulosa cells by increasing the number of high affinity, saturable LDL receptors with no alteration in their apparent affinity. Moreover, estrogen action is accompanied by enhanced rates of progesterone production in the presence of LDL, and increased rates of LDL internalization and degradation, which could not be accounted for simply by accelerated nonspecific bulk fluid-phase pinocytosis. We suggest that the significant facilitative actions of estradiol on lipoprotein binding and metabolism are likely to assist in preparing granulosa cells for the increased rates of progesterone biosynthesis ultimately required in functional corpora lutea.     

Follicle-stimulating hormone regulates low density lipoprotein metabolism by swine granulosa cells

FSH amplified the stimulatory effect of low density lipoprotein (LDL) on progesterone biosynthesis by primary cultures of swine granulosa cells in a synergistic manner. The mechanisms subserving this synergism included the following. 1) FSH increased by 2.1-fold the number of specific high affinity low density lipoprotein (LDL) receptors on granulosa cells, with no change in apparent binding affinity. 2) FSH augmented by 1.8- and 3.6-fold the maximal rates of [125I]iodo-LDL internalization and degradation, respectively, without altering half-maximally effective concentrations of LDL supporting these processes. 3) FSH increased significantly the mass (micrograms) of free and esterified cholesterol (measured by fluorometry) contained in granulosa cells. 4) FSH stimulated the intracellular accumulation of free [3H]cholesterol and [3H]cholesteryl ester from exogenous [3H]cholesteryl linoleate-labeled LDL and amplified [3H]progesterone secretion by granulosa cells exposed to this source of lipoprotein-borne sterol. 5) The stimulatory effects of FSH on LDL degradation and cholesterol side-chain cleavage activity were mimicked by other activators of granulosa cell cAMP production, e.g. forskolin and cholera toxin. We conclude that FSH and LDL synergistically enhance progesterone biosynthesis by swine granulosa cells via cellular mechanisms that result in significantly augmented binding, internalization, and degradation of LDL in concert with increased effectual utilization of sterol substrate in the cholesterol side-chain cleavage reaction. These observations are consistent with a key trophic role for FSH in regulating the sterol-metabolizing ability and steroidogenic differentiation of granulosa cells and indicate that such actions of FSH can be accounted for by activation of the cAMP-effector pathway.     

Herbimycin, a tyrosine kinase inhibitor with Src selectivity, reduces progesterone and estradiol secretion by human granulosa cells

The purpose of the present study was to investigate whether the tyrosine kinase inhibitor herbimycin with some selectivity to block Src would alter the stimulatory effects of follicle-stimulating hormone (FSH) and cyclic adenosine monophosphate (cAMP) on estradiol secretion by human granulosa cells. Granulosa cells were taken from ovaries of premenopausal women undergoing oophorectomy for reasons unrelated to ovarian pathology. Granulosa cells from follicles ranging from 5–20 mm in diameter were subjected to culture. Granulosa cells were cultured with human FSH (2 ng/mL) or cAMP (0–1 mM) and testosterone (1 μM) in the presence and absence of herbimycin (0–2 μM). Media were collected at 24, 48, and 72 h. Accumulation of cAMP, progesterone, and estradiol in the media was determined by radioimmunoassay. Herbimycin dose dependently inhibited the ability of FSH to induce increases in progesterone and estradiol secretion. Although herbimycin increased (p < 0.0001) the accumulation of cAMP in response to FSH, this was evident only at the high concentrations of herbimycin (2 μM). To determine whether herbimycin would inhibit the ability of exogenous cAMP to induce estradiol and progesterone secretion, granulosa cells were incubated with 0–1 mM cAMP in the presence and absence of various doses of herbimycin. Herbimycin inhibited cAMP-induced estradiol and progesterone secretion in granulosa cells. The results from seven experiments indicate that herbimycin inhibits FSH stimulation of estradiol and progesterone secretion and that this inhibition may be, in part, at post-cAMP site(s).     

Insulin-like growth factor type I (somatomedin-C) stimulates high density lipoprotein (HDL) metabolism and HDL-supported progesterone biosynthesis by swine granulosa cells in vitro

Type I insulin-like growth factor (IGF-I) stimulated high density lipoprotein (HDL)-promoted progesterone production by swine granulosa cells cultured under serum-free conditions in vitro. In the presence of pure human IGF-I (50 ng/ml), the half-maximally effective concentration of swine HDL was 16 micrograms/ml (67% confidence limits; 15-17 micrograms/ml) after 2 days of exposure to this growth factor, 5.4 (2.6-9.8) micrograms/ml after 4 days, and 3.8 (1.2-4.8) micrograms/ml after 6 days. Maximal progesterone production increased approximately 10-fold in the presence of IGF-I and HDL on day 2, 125-fold on day 4, and 330-fold on day 6. The facilitative action of IGF-I on HDL-supported progesterone biosynthesis was accompanied by time-dependent stimulatory effects of IGF-I on trypsin-releasable HDL, trypsin-resistant cell-associated HDL, and degraded HDL (P less than 0.01). Moreover, incubation of swine granulosa cells with [3H]cholesteryl oleate-labeled HDL demonstrated that IGF-I exerted a time-dependent stimulatory effect on [3H]free cholesterol and [3H]cholesteryl ester accumulation in granulosa cells, and significantly augmented the secretion of [3H]progesterone (separated by two-dimensional TLC). In addition to the ability of IGF-I to amplify the cellular acquisition of radiolabeled sterol, this growth factor also increased the total mass of cellular cholesteryl ester and total cellular cholesterol as measured by microfluorometric assay (P less than 0.01). We conclude that IGF-I facilitates the effective delivery of HDL-derived sterol substrate into the steroidogenic pool of ovarian cells. Such observations offer an additional role for the differentiative actions of this somatomedin in the expression of full steroidogenic potential by granulosa-luteal cells.     

Factors influencing follicle-stimulating hormone-responsive steroidogenesis in marmoset granulosa cells: effects of androgens and the stage of follicular maturity

Preovulatory changes in the steroidogenic function of primate granulosa cells were studied using the cyclic marmoset (Callithrix jacchus) as a model. Antral follicles (greater than or equal to 0.5 mm diameter) were dissected from mid-late follicular phase ovaries (7 days after prostaglandin-induced luteolysis) and classified by diameter as small (0.5-1.0 mm), medium (1.1-1.9 mm) or large (greater than or equal to 2.0 mm). Granulosa cells from follicles in each size category were isolated and pooled to assess steroid biosynthesis. The aromatase activity of freshly isolated granulosa cells from large follicles was 200 times greater than that of small follicles, confirming their relatively advanced preovulatory status. Granulosa cells were cultured for 48 h in the presence and absence of human (h) FSH (0.1 ng/ml), with and without 0.1 microM androgen (testosterone or 5 alpha-dihydrotestosterone), to assess basal and hormone-responsive steroidogenesis (progesterone accumulation in culture medium and aromatase activity in washed granulosa cell monolayers). Basal granulosa cell steroidogenesis increased with follicular size, and there was a development-related pattern of response to hFSH and androgen. hFSH responsiveness (maximum fold-stimulation induced by hFSH) declined with follicular size, being 2-6 times greater for granulosa cells from small vs. large follicles. On the other hand, hFSH sensitivity increased with follicular size; the dose of hFSH giving 50% of the maximum response (ED50) for cells from large follicles being 10-20 times less than that of cells from small follicles. For granulosa cells from small follicles, treatment with 0.1 microM androgen in the presence of hFSH led to dramatic (up to 16-fold) enhancement of steroidogenic responses to hFSH. In contrast, for granulosa cells from large follicles, the presence of androgen substantially inhibited aromatase activity stimulated by hFSH and had weak inhibitory effects on progesterone accumulation. These results show that granulosa cell steroidogenesis becomes increasingly sensitive to hFSH during preovulatory follicular development in marmosets. The marked ability of androgen to directly augment hFSH-responsive steroidogenesis in vitro is lost during preovulatory development, such that androgen acts in mature granulosa cells to suppress hFSH-stimulated aromatase activity. These observations are evidence of development-dependent changes in granulosa cell responses to FSH and androgens which may contribute to the control of preovulatory follicular development in primates.     

Differentiation of rat ovarian thecal cells: evidence for functional luteinization

To determine if thecal cells of rat preovulatory (PO) follicles become functionally luteinized, theca from small antral (SA) and PO follicles were isolated before and 8 h after iv injection of an ovulatory dose (10 IU) of hCG. Thecal explants were cultured for 30 days in Dulbecco's Modified Eagle's Medium-Ham's F-12 medium containing 1% fetal calf serum (FCS) with or without 5 ng/ml ovine LH or 10 microM forskolin. Whereas theca from SA, hCG-treated SA, and PO follicles were dependent on LH or forskolin to maintain progesterone (greater than 10 ng/ml) and androstenedione (greater than 10 ng/ml) accumulation, luteinizing theca (hCG-treated PO) accumulated more than 10 ng/ml progesterone and more than 2 ng/ml androstenedione with or without LH or forskolin for 30 days. Granulosa cells were isolated from these same follicles and cultured under similar conditions, including 10 ng/ml testosterone and 25 ng/ml ovine FSH. Only granulosa cells isolated from luteinizing follicles (hCG-treated PO) maintained progesterone (greater than 20 ng/ml) and estradiol (10 ng/ml) accumulation with or without FSH or forskolin for 30 days. Basal concentrations of cAMP were 5 to 10-fold higher in thecal and granulosa cells from luteinizing follicles than in these tissues isolated from SA or PO follicles. We conclude that thecal cells as well as granulosa cells of rat PO follicles respond to the LH/hCG surge by becoming functionally luteinized, less dependent on LH, and capable of maintaining an increased accumulation of basal cAMP. Furthermore, the data suggest that one luteinizing thecal explant produces a similar amount of progesterone as one follicle equivalent of luteinizing granulosa cells. Thus, luteinized theca have the potential of contributing significantly to progesterone secretion by the mature rat corpus luteum.     

Thyroid stimulating hormone inhibits rat granulosa cell steroidogenesis in primary culture

It is not known why hypothyroidism predisposes the ovary to develop cystic follicles in hCG-treated rats. This study examined the effect of TSH on steroidogenesis in cultured granulosa cells. Granulosa cells were isolated from the ovaries of hCG-treated Long-Evans rats. Cells were cultured in medium with either 0 or 100NM: testosterone or 25(OH)cholesterol and treated with 0, 2.5, 10 or 20 ng/ml of TSH. TSH reduced 3β-HSDI activity in these cells to 12% of control. Progesterone secretion decreased by 72% and estradiol secretion was non-detectable during the second day of treatment with 20 ng/ml of TSH. We conclude that TSH is capable of regulating the secretion of progesterone and estradiol by cultured granulosa cells.     

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.     

Effect of insulin-like growth factor I on steroidogenesis in cultured human granulosa cells

The effect of IGF-I on steroidogenesis in human granulosa cells was studied. Granulosa cells were obtained from follicles of both natural and stimulated cycles. The cells were cultured 4 to 6 days and the effect of IGF-I (1 to 100 micrograms/l) on basal, LH- and FSH-stimulated steroidogenesis was studied. It was found that in granulosa cells from follicles of natural cycles, FSH as well as IGF-I significantly stimulated progesterone and estradiol production in a majority of the experiments. A synergistic effect of FSH and IGF-I could be seen when low (1 and 10 micrograms/l) concentrations of the two hormones were used. Also in granulosa luteal cells from stimulated cycles a stimulatory effect of IGF-I on estradiol as well as progesterone production was observed. The present results suggest that IGF-I in combination with gonadotropins has a physiological role in the human follicle in controlling differentiation of the granulosa cells.     

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.     

Developmental changes in luteinizing hormone/human chorionic gonadotropin steroidogenic responsiveness in marmoset granulosa cells: effects of follicle-stimulating hormone and androgens

Factors regulating LH/hCG responsiveness in primate granulosa cells were examined in the marmoset monkey (Callithrix jacchus). Granulosa cells were isolated and pooled from small antral (0.5-1.0 mm) and large preovulatory (greater than or equal to 2 mm) follicles from mid- to late follicular phase ovaries of cyclic marmosets. The cells from small and large follicles were cultured in serum-free medium for 48 h in the absence or presence of increasing concentrations of hCG (0.1-100 ng/ml) with or without 0.1 microM androgen [testosterone or 5 alpha-dihydrotestosterone (DHT]). Granulosa cells from small follicles were also cultured in the absence or presence of a constant concentration of human FSH (30 ng/ml) with or without androgen for 48 h before exposure to hCG for an additional 48 h. Steroidogenic responsiveness was assessed by measuring progesterone accumulation in culture medium and aromatase activity in washed monolayers. Granulosa cells from large follicles showed dose-dependent increases in both progesterone accumulation and aromatase activity in response to treatment with hCG. In contrast, granulosa cells from small follicles were unresponsive to hCG. However, pretreatment of granulosa cells from small follicles for 48 h with FSH stimulated hCG responsiveness. The effects of both testosterone and DHT on hCG-stimulated aromatase activity and progesterone accumulation by granulosa cells from large preovulatory follicles were inhibitory. Testosterone and DHT also suppressed basal (no hCG) progesterone accumulation in these cells, but had no effect on basal aromatase activity. The effects of androgens on FSH-induced hCG responsiveness in immature granulosa cells were variable. The results show a development-related increase in marmoset granulosa cell responsiveness to LH/hCG and provide evidence that FSH and androgens interact to regulate the onset and expression of this critical event during preovulatory follicular development in the primate ovary.