Steroidogenesis by equine preovulatory follicles: relative roles of theca interna and granulosa cells

Estrous cycles in mares have several unique characteristics, including the presence of a long period of estrus and the absence of a typical LH surge. Like follicles of other species, equine preovulatory follicles are characterized by their ability to secrete large amounts of 17 beta-estradiol, but it is not clear which follicular cell type is responsible for estradiol synthesis in mares. To better understand the relative roles of theca interna and granulosa cells in follicular steroidogenesis, presumptive ovulatory follicles were obtained from mares during early estrus (first or second day of estrus; n = 4) and during late estrus (fourth or fifth day of estrus; n = 4). Preparations of theca interna and granulosa cells were cultured for 3 days in medium with or without equine LH, FSH, LH plus FSH, or CG (100 ng/ml) in the presence or absence of 0.5 microM testosterone, and culture media were assayed for progesterone, androstenedione, and 17 beta-estradiol. Progesterone was the predominant steroid secreted by granulosa cells in the absence of exogenous testosterone. Its accumulation was significantly higher in cultures of granulosa cells from late vs. early estrus (P less than 0.05), and all gonadotropins stimulated progesterone secretion at both stages of follicular development (P less than 0.05). In contrast, granulosa cells secreted very low amounts of androstenedione in vitro, and only very small amounts of 17 beta-estradiol were produced when cells were cultured in medium without testosterone. However, the addition of testosterone caused a 170-fold increase over control values in estradiol accumulation over 3 days of culture (P less than 0.0001), clearly indicating the presence of a very active aromatase enzyme system in equine granulosa cells. Steroid secretion by theca interna differed in several respects from secretion by granulosa cells. Theca interna from early and late estrous follicles secreted negligible amounts of progesterone in vitro, and equine gonadotropins had no effect on its secretion. Also, theca interna secreted only small amounts of estradiol in vitro, and its accumulation was not increased by the addition of exogenous testosterone. Also, in contrast to granulosa cell cultures, androstenedione was the predominant steroid secreted by theca interna from early and late estrous follicles. In conclusion, this study does not support the current model of equine follicular steroidogenesis, which holds that 17 beta-estradiol biosynthesis derives primarily from the theca interna layer.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of gonadotrophins on progesterone secretion by cultured granulosa cells obtained from human preovulatory follicles

Granulosa cells were obtained from human preovulatory follicles in 31 women undergoing in vitro fertilization and embryo transfer due to tubal infertility. Follicular maturation was stimulated and synchronized by treatment with Clomiphene or human menopausal gonadotrophin (hMG), or both, plus human chorionic gonadotrophin (hCG). Follicles were aspirated by ultrasound guided puncture approximately 34-36 h after the hCG injection. The granulosa cells were washed and suspended in modified medium 199 containing 10% foetal bovine serum and cultured as monolayers for 6-8 days in the absence and presence of hormones and reactants. Progesterone formation was analyzed by RIA. In general, the cells underwent morphological luteinization and secreted high amount of progesterone. Under basal conditions the secretion of progesterone was highest during the first 2 days in culture and then gradually declined. Progesterone secretion was stimulated by human LH, hCG and the adenylate cyclase stimulator forskolin, with a maximal effect between days 2-6. The beta-adrenergic agonist isoproterenol in preliminary experiments potentiated the stimulatory effect of hCG but had no own stimulatory effect. No clear differences in progesterone secretion or responsiveness to in vitro stimulation relating to the various in vivo stimulation protocols were found.     

Progesterone production by cultured preantral rat granulosa cells? Stimulation by androgens.

Progesterone (P) production by isolated rat granulosa cells from preantral follicles was enhanced by addition of androgens to the tissue culture medium. Testosterone (T) at 10(-7), 10(-6), and 10(-4)M as well as 10(-6)M dihydrotestosterone (DHT) increased P production 400 to 700% over paired control cultures. Human chorionic gonadotropin (100 mIU/ml) and 17beta-estradiol (7.8 X 10(-10M) had no effect on P production. P was identified by both a specific radioimmunoassay and sephadex LH-20 column chromatography. The stimulatory influence of T and DHT on these preantral follicular cells is consistent with a direct role for androgens in granulosa cell differentiation.     

17 beta-Estradiol biosynthesis in cultured granulosa and thecal cells of human ovarian follicles: stimulation by follicle-stimulating hormone.

Cellular sites and gonadotropic control of human follicular estrogen secretion have been assessed by culturing the theca and granulosa components separately under different hormonal conditions. Granulosa cells from human follicles were grown in chemically defined media containing gonadotropins and/or testosterone (T) for 24 h. The production of 17 beta-estradiol (E2) by cells cultivated in T-free media with or without FSH was very low during the culture period. There was a highly significant increase (P less than 0.001) in E2 production when T alone was added and a more marked increase was consistently noted in the presence of FSH and T. In all cases, hCG failed to exert any significant effect on E2 production by granulosa cells in the presence or absence of T. No treatments examined altered the E2 production of thecal cells during a 24-h culture period and the amounts of E2 released into media were negligible when compared with levels produced by granulosa cells from the same follicles. It is concluded that granulosa cells but not thecal cells are the prime site of follicular estrogen production and that FSH regulates estrogen secretion by nonluteinized granulosa cells of the human follicle.     

19-Hydroxylation of androgens by rat granulosa cells

The transformation of androgens by rat granulosa cells was examined employing [19-C3H3]-, [1 beta-3H]-, and [1,2,6,7-3H]androgens as substrates. Rat granulosa cell homogenates incubated with [19-C3H3]androstenedione generated [3H] water and [3H]formic acid in a ratio of 8-9, indicating considerable 19-hydroxylation which was not followed by aromatization. This ratio remained relatively constant regardless of the time in the estrous cycle when the ovaries were removed, although there were large differences in the extent of the reactions. Parallel incubations with [1 beta-3H]]androstenedione showed that the aromatization of [19-C3H3]androstenedione in this tissue proceeds with a negative isotope effect of approximately 3, similar to that in human placenta. Incubation of the same substrates with granulosa cell cultures produced [3H]water and [3H]formic acid in ratios of 4-5 and showed a smaller negative isotope effect in the aromatization of [19-C3H3]androstenedione. FSH stimulation of the cell cultures had no influence on the ratio of 19-hydroxylation to aromatization with respect of either the duration of stimulation or the concentration of the pituitary hormone. Incubation of the cell cultures with [1,2,6,7-3H]androstenedione yielded tritium-labeled 19-hydroxy- and 19-oxoandrostendiones and estrogens in relative quantities corresponding to those expected from the [3H]water and [3H]formic acid formation. Virtually all of the products were found in the medium, with only trace quantities located intracellularly. Similarly, incubation of granulosa cell homogenates with [14C]androstenedione yielded [14C]19-oxygenated androgens in excess of [14C] estrogens. These results indicate that rat granulosa cells effect C-19-hydroxylation of androgens greater than that linked to aromatization and that the rat ovaries produce 19-oxygenated androgens in quantities exceeding those of estrogens. The excess 19-hydroxylation is synchronous with aromatization, but it is not known whether it is catalyzed by the same or a different enzyme. The formation of 19-oxygenated androgens in cell cultures indicates that they are distinct metabolites of androgens in the rat ovary and are not merely trapped transient aromatization intermediates.     

Oxytocin and progesterone secretion by bovine granulosa cells of individual preovulatory follicles cultured in serum-free medium

To examine the secretion of oxytocin (OT) and progesterone (P) from a homogeneous population of cells during luteinization, we developed a serum-free culture technique for granulosa cells, obtained from individual preovulatory bovine follicles. Granulosa cells from earlier stages of the follicle development did not have the capability to secrete OT under the in vitro conditions used. For optimal stimulation of the cells the medium (a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12) was supplemented with bovine serum albumin (BSA) and insulin. OT was detectable from day 1 of culture reaching a maximum level between days 2 and 4 and then declined towards day 5. In the absence of insulin OT declined from day 1 onwards and was undetectable from day 4. When cells were cocultured with theca tissue or theca-conditioned medium (TCM), there was an enhancement in OT secretion, but not in P secretion. Other tissues including liver, kidney, aorta, muscle and adrenal incubated with the cells induced a similar increase in OT production. In the presence of insulin progesterone secretion was increased and was correlated with OT production, but did not show a consistent pattern among follicles. We conclude that (a) culture of granulosa cells from an individual follicle in a serum-free medium can be used to study the secretion of OT and P from bovine granulosa cells, (b) insulin is essential for the optimal production of OT and P by these cells, and (c) the addition of theca or other tissues enhanced OT secretion by a mechanism not understood.     

Progesterone secretion by highly differentiated human granulosa cells isolated from preovulatory Graafian follicles induced by exogenous gonadotropins and human chorionic gonadotropin

Human granulosa-luteal cells were harvested from preovulatory Graafian follicles at the time of oocyte retrieval for in vitro fertilization after induction of follicle maturation by sequential injections of menopausal gonadotropins and hCG. Such highly differentiated granulosa cells produced large quantities of progesterone basally (6.8 pg/cell X 2 days) in monolayer culture. Human LH significantly increased progesterone biosynthesis after 6, 12, 48, 96, or 144 h in culture, with a maximal increase of 8- to 20-fold occurring at 96 h. The stimulatory effect of LH could be observed under serum-free conditions and was maximal in the presence of 4% serum. Human granulosa-luteal cells also exhibited significant stimulatory responses to hCG, prostaglandin E2, or the cAMP effectors 8-bromo cAMP, choleratoxin, or forskolin in serum-free incubations. Concentrations of 17 beta-estradiol that are attained physiologically in ovarian follicles in vivo markedly suppressed basal and LH (or cAMP)-stimulated progesterone production in vitro (maximal suppression, greater than 90%). The nonaromatizable androgen 5 alpha-dihydrotestosterone also inhibited progesterone production, but by no more than 45-50% even at supraphysiological concentrations. Estradiol's blockade of progesterone synthesis was associated with a corresponding increase in pregnenolone accumulation. The present studies indicate that human granulosa-luteal cells isolated from preovulatory follicles induced with exogenous gonadotropins and hCG secrete large quantities of progesterone in vitro. Such cells retain stimulatory responses to human LH, hCG, prostaglandin E2, and classical cAMP effectors in serum-free incubations. Moreover, physiological concentrations of 17 beta-estradiol suppress progesterone production, probably by inhibiting cellular conversion of pregnenolone to progesterone. Thus, the present in vitro system permits an investigation of hormone action in well differentiated, human granulosa-luteal cells isolated from preovulatory Graafian follicles that have a defined endocrine history of prior gonadotropin exposure in vivo.     

Estrogens, progestogens, and androgens enhance the follicle-stimulating hormone-stimulated plasminogen activator production by cultured rat granulosa cells

We have previously shown that FSH caused a dose-dependent increase in plasminogen activator (PA) production by cultured rat granulosa cells. PA production was assayed by culturing granulosa cells, obtained from immature diethylstilbestrol-treated rats, on [125I]fibrin plates and determining the extent of fibrinolysis after the addition of the specific substrate plasminogen. To study the effect of ovarian steroids on FSH-stimulated PA production, concomitant treatment with FSH and estradiol [(E2) 10(-7) M], progesterone [(P) 10(-6) M], testosterone [(T) 10(-7) M] or 5 alpha-dihydrotestosterone [(DHT) 10(-7) M] were tested in these cultures. Treatment with E2 significantly enhanced the FSH-stimulated PA production by 35-210% over the whole range of the FSH dose response curve. The augmentation of FSH-stimulated PA production by E2 was not affected by the concomitant treatment with the antiprogestin RU 486 (10(-6) M). Similarly, P significantly enhanced the FSH-stimulated PA production by 34 to 90%. The stimulating action of P on FSH-dependent PA production was blocked by the simultaneous treatment with RU 486. Concomitant treatment with T significantly increased the FSH-induced PA production by 22-60%. That the effect of T was not due to the aromatization to E2 was evidenced by the augmentation by DHT of FSH-stimulated PA production. DHT caused a significant 20-39% enhancement in the responsiveness of the granulosa cells to FSH by further increasing PA production. The enhancement of PA production by androgen is decreased partially by concomitant treatment with the antiandrogen flutamide (10(-5) M) and the antiprogestin RU 486. These in vitro data demonstrated the importance of estrogens, progestogens, and androgens in modulating the stimulating effect of FSH on PA production by immature rat granulosa cells.     

Inhibition of progesterone secretion from granulosa cells by estradiol and androgens in the domestic hen

We previously reported no difference in progesterone (P4) secretion from the granulosa layer of the largest follicle (F1) of the domestic hen regardless of the maturity of the F1 follicle. However, coincubation of the granulosa and thecal layers resulted in inhibition of P4 secretion from the less mature F1, but not from the more mature F1. The goal of this study was to determine if estradiol (E2) and androgens secreted by the thecal layer suppress P4 production by the granulosa cells. We removed the granulosa layer from less mature F1 follicles and dispersed granulosa cells (1 x 10(5)) were incubated (3 h) in triplicate with one of these treatments: control, E2, testosterone (T), androstenedione (A), and dihydrotestosterone (DHT; at concentrations of 1 x 10(-7), 1 x 10(-6), and 1 x 10(-5) M), LH (100 ng) as well as LH plus E2, T, A, and DHT at the same concentrations. P4 secretion was measured in the medium and cells, and the experiment was replicated seven times. We found a dose-related suppression of basal and LH-stimulated P4 production by all steroids. In a second experiment (n = 3-5), we tested the specificity of the androgens in suppressing P4 production by granulosa cells by using the aromatase inhibitor 7-(4'-amino)phenylthio-4-androstene-3,17-dione. This compound did not reduce the effectiveness of T in suppressing P4 production. Finally in Exp 3 (n = 4-7), E2 and T were tested individually and in combination at concentrations of 1 X 10(-8)-1 X 10(-5) M. We found a possible synergistic effect, in that the combination of E2 plus T suppressed P4 to a greater degree than either steroid alone. Our results indicate that 1) E2 and androgens suppress basal and LH-stimulated P4 production by granulosa cells in a dose-related manner; 2) androgen suppression of P4 production is not mediated by aromatization to estrogen; and 3) the suppressive effects of E2 and androgens may be synergistic. We conclude that E2 and androgens secreted by the thecal layer may regulate P4 production by the granulosa layer.     

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.     

Comparative effects of androgens and catecholestrogens on progesterone production by porcine granulosa cells

The objective of the present studies was to evaluate and compare the effects of 5 alpha-dihydrotestosterone (DHT) to those of 2-hydroxyestradiol (2-OH-E2) and 2-methoxyestradiol (2-MeO-E2) on progesterone production in cultured porcine granulosa cells. Granulosa cells were exposed to various treatments of DHT, 2-OH-E2 and 2-MeO-E2 in the absence or presence of follicle stimulating hormone (FSH) for 4 days and concentrations of progesterone in medium and cell numbers were determined. In the absence of FSH, maximally effective concentrations of DHT (1 micrograms/ml) and 2-OH-E2 (4 micrograms/ml) stimulated progesterone production (ng/10(5) cells/48 h) to 2.2 +/- 0.2- and 10.8 +/- 2.2-fold of controls (n = 4 experiments), respectively. In the presence of 200 ng/ml FSH, progesterone production stimulated by 1 micrograms/ml DHT and 4 micrograms/ml 2-OH-E2 was 5.4 +/- 1.1- and 15.5 +/- 6.0-fold of controls (n = 4 experiments), respectively. Thus, FSH appeared to enhance the response of both DHT and 2-OH-E2. The dose-response of DHT was biphasic in the presence and absence of FSH, such that progesterone production in the presence of 8 micrograms/ml DHT was similar to basal progesterone production. Concurrent treatment with saturating concentrations of 2-OH-E2 and DHT resulted in fully additive increases in progesterone production. Testosterone mimicked the effect of DHT. In comparison, concurrent treatment of saturating concentrations of 2-MeO-E2 and DHT or 2-MeO-E2 and 2-OH-E2 resulted in progesterone production that was only partially additive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thyroid-stimulating antibody bioassay using porcine thyroid cells cultured in follicles

Isolated porcine thyroid cells were cultured in agarose-coated dishes and allowed to reform follicles with normal polarity. The thyroid cells reaggregated into follicles were compared with cells cultured in monolayer for cAMP responsiveness to TSH and thyroid-stimulating antibody (TSAb). The cells in follicles were sensitive to TSH stimulation and responded to the hormone at concentrations as low as 3.3-10 microU/ml with an increase in cAMP production. In contrast, 10-50 microU/ml TSH were required to elicit a cAMP response in cells cultured in monolayer using identical conditions. cAMP responsiveness to TSAb also was greater in the cells organized into follicles. TSAb was detected in serum from 89.4% of 66 untreated patients with hyperthyroid Graves' disease using thyroid follicles, but TSAb was detected in serum from only 60% of the patients when assayed using cells in monolayer. The assay using thyroid follicles was used to measure TSAb in 27 euthyroid patients who were euthyroid while receiving thionamide therapy and compared with 20-min thyroid 131I uptake after T3 suppression. TSAb was present in 11 of 12 nonsuppressible patients and in 5 of 15 suppressible patients. TSAb was positively correlated with 20-min 131I thyroid uptake. We conclude that thyroid cells cultured in follicles are suitable for measuring TSAb.     

Steroidogenesis of cultured purified pig Leydig cells: effects of lipoproteins and human chorionic gonadotropin

Dispersed Leydig cells were prepared from pig testes and purified in a discontinuous Percoll gradient. About 95% of these cells stained for 3 beta-hydroxysteroid dehydrogenase. The cells were cultured in a chemically defined medium. Testosterone production was low (2 +/- 0.4 ng/10(6) cells/day) under basal conditions, but increased by 8- to 10-fold on the third day of daily human CG (hCG) treatment. Addition to the medium of both human and pig low density lipoprotein (LDL) produced a dramatic increase in both basal (8-fold) and acute hCG-stimulated (5-fold) testosterone production, whereas both human and pig high density lipoprotein were far less effective. Furthermore the effect of lipoproteins was synergistic with that of hCG. The effects of human LDL on both basal and hCG-stimulated testosterone productions were dose-dependent. Maximum effect was achieved at a protein concentration of 10-40 micrograms/ml with an ED50 of about 4 micrograms/ml. Three days of pretreatment with hCG or (Bu)2cAMP alone induced Leydig cell steroidogenic refractoriness to both hCG and (Bu)2cAMP stimulation. Concomitant treatment with LDL restored the steroidogenic capacity, but only partially. Production of pregnenolone and testosterone of desensitized cells was significantly higher than that of control cells under basal conditions, but was 60% and 40% lower, respectively, after acute hCG stimulation. Moreover, the conversion of exogenous pregnenolone to testosterone by desensitized cells was only 60% of that of control cells. These results show that the de novo synthesis of cholesterol is able to account for only 25% of the maximal steroidogenic capacity of pig Leydig cells and that hCG-induced steroidogenic desensitization is only partially due to cholesterol depletion.     

Catecholestrogens stimulate progestin secretion by cultured porcine granulosa cells

The enzymatic metabolism of estradiol (E2) to the catecholestrogens, 2-hydroxyestradiol (2-OH-E2) and 4-hydroxyestradiol (4-OH-E2) in granulosa cells has been reported. Therefore, we evaluated the effects of these compounds and compared them to those of E2 on porcine granulosa cells cultured in serum-free medium. Cultures of granulosa cells were exposed to various treatments of E2, 2-OH-E2, 4-OH-E2 and(or) follicle-stimulating hormone (FSH) for 4 days and concentrations of progesterone in medium and cell numbers were determined. After 4 days of treatment, 2-OH-E2 and 4-OH-E2 stimulated basal progesterone production by granulosa cells, but 4-OH-E2 was less effective than 2-OH-E2. 2-OH-E2 (1 microgram/ml) stimulated progesterone production by 3.3 +/- 0.6-fold (n = 6 experiments), whereas E2 (1 microgram/ml) stimulated progesterone production 9.9 +/- 1.7-fold (n = 6 experiments). 2-OH-E2 at 4 micrograms/ml further stimulated progesterone production to 10.7 +/- 2.2-fold above controls (n = 9 experiments), whereas 4 micrograms/ml of E2 did not cause further stimulation of progesterone production. Thus, the average potency of 2-OH-E2 was less than E2. Concurrent treatment with 2-OH-E2 (4 micrograms/ml) and saturating concentrations of E2 resulted in further significant increases in progesterone production above the effects of either single treatment both in the absence and presence of FSH (200 ng/ml).(ABSTRACT TRUNCATED AT 250 WORDS)     

FSH-induced aromatase activity in porcine granulosa cells: non-competitive inhibition by non-aromatizable androgens

The aim of this study was to examine the inhibitory effect of the non-aromatizable androgens on FSH-stimulated aromatase activity in porcine granulosa cells. The cells were isolated from medium-sized follicles (4-6 mm) of prepubertal pigs, and cultured under chemically defined conditions in the presence of FSH (1 microgram/ml, NIADDK-oFSH-S13) with and without the androgens for an initial 48-h induction period. Subsequently, the spent medium was replaced with fresh medium containing only testosterone as substrate and the cells were reincubated for a further 6 h. The conversion of this steroid to oestradiol-17 beta during this latter 'test' period was taken as a measure of the aromatase activity. The addition of 5 alpha-dihydrotestosterone (DHT) into cultures of FSH-stimulated cells during the induction period resulted in a definite dose-dependent inhibition (30-70%) of the aromatase activity expressed in the test period. This inhibitory action, of the mixed non-competitive type, is characterized by a decrease in the apparent Vmax and an increase in the Km value, suggestive of an androgen inhibition of FSH-stimulated aromatase synthesis. This inhibition was also shown by the other 5 alpha- and 5 beta-reduced androgens: 5 beta-androstanedione was the most effective, while DHT was the least. Other steroids such as pregnenolone and progesterone were inhibitory, but testosterone and diethylstilboestrol were stimulatory. These results suggest an important mechanism for the intrafollicular control of oestrogen synthesis, involving a possible reciprocal relationship between aromatase and 5 alpha-reductase activities.     

Steroidogenesis of porcine granulosa cells from small and medium-sized follicles: effects of follicle-stimulating hormone, forskolin, and adenosine 3,'5'-cyclic monophosphate versus phorbol ester

We studied the effects of porcine FSH, forskolin, and (Bu)2cAMP [agents that stimulate steroidogenesis via the adenylate cyclase-cAMP pathway (cAMP system)] either alone or with concomitant addition of phorbol 12-myristate 13-acetate (TPA; a phorbol ester that activates protein kinase-C) on steroidogenesis in porcine granulosa cells cultured from small (less than 3 mm) and medium-sized (3-6 mm) ovarian follicles. We attempted to determine if granulosa cells from different maturational states had different responses to these agonists and antagonists. Cells were cultured in serum-free medium 199 supplemented with insulin (10 micrograms/ml), transferrin ( 5 micrograms/ml), and androstenedione (2.5 X 10(-7) M) for 48 h. Levels of progesterone (P) and estradiol (E2) were determined in spent medium by RIA. We found that FSH, forskolin, and cAMP all stimulated secretion of E2 and P in a dose-dependent manner in both developmental groups. When TPA was added alone to cultures, P levels were stimulated at low doses of TPA but inhibited at higher doses in granulosa from both sized follicles, whereas cells from both small- and medium-sized follicles demonstrated reductions in E2. TPA was also found to inhibit FSH-, forskolin-, and cAMP-induced steroidogenesis in a dose-dependent manner in cells from the two groups of follicles. The stimulatory effects of any of the secretagogues on E2 secretion were inhibited by TPA to a significantly greater extent in granulosa cells from small follicles. Although inhibition of FSH- and forskolin-induced P secretion by TPA was also greater in granulosa cells from small follicles, cAMP-treated cells did not show this differential inhibition. Thus, it appears that modulators of the protein kinase-C system regulate steroidogenesis differently in granulosa cells from small and medium follicles. These differences may involve alterations in the interplay between the protein kinase-C and cAMP pathways.