Influence of follicular maturation on luteinizing hormone-, cyclic 3',5'-adenosine monophosphate-, forskolin- and cholesterol-stimulated progesterone production in hen granulosa cells

The influence of follicular maturation on progesterone production by collagenase-dispersed hen granulosa cells was measured in short-term incubations. Granulosa cells of the largest follicle (F1) produced up to ten times more progesterone than cells from smaller follicles (F3-F5), not only in response to luteinizing hormone (LH), but also when stimulated by exogenous cyclic AMP or forskolin, both of which raise intracellular cyclic AMP levels by nonreceptor-mediated mechanisms. Moreover, when granulosa cell progesterone synthesis was stimulated by incorporating 25-hydroxy-cholesterol into the incubation medium, an identical pattern was obtained. This could be attributed to a corresponding increase in the specific activity of the mitochondrial cholesterol side-chain cleavage enzyme (20,22 desmolase). An increase in the apparent Vmax was observed without a change in the apparent Km values. Pregnenolone substrate at concentrations which raised progesterone production to levels similar to those observed in response to LH stimulation was utilized equally by granulosa cells of mature and developing follicles. However, at high pregnenolone concentrations, granulosa cells of mature follicles converted significantly more of the precursor to progesterone. Assay of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) showed that the enzyme has two Kms: a low Km present in cells of both mature and developing follicles, and a high Km found only in granulosa cells of more mature follicles. It is concluded that LH-promoted progesterone synthesis in granulosa cells of developing chicken follicles is restricted not so much by the availability of receptors and the competence of the adenylate cyclase/cyclic AMP system, but by the activity of key enzymes, principally the cholesterol-20,22 desmolase.     

Clomiphene and tamoxifen inhibit the cholesterol side-chain cleavage enzyme activity in hen granulosa cells

A radiochemical assay was utilized to study the inhibitory effects of clomiphene and tamoxifen on the cholesterol side-chain cleavage enzyme activity in a mitochondrial preparation of granulosa cells isolated from mature ovarian follicles of laying hens. At saturating substrate concentrations, both clomiphene and tamoxifen were able to suppress enzyme activity in a dose-related manner (IC50 1.8 X 10(-5) M). Double reciprocal plots of kinetic data show that the inhibition is mixed, exhibiting competitive kinetics at low concentrations, whereas at high concentrations, the inhibition is of a non-competitive nature. The competitive inhibition constants as determined from Dixon plots are 2 X 10(-5) M for clomiphene and 2.3 X 10(-5) M for tamoxifen. It is concluded that, in granulosa cells, clomiphene and tamoxifen directly inhibit the mitochondrial cholesterol side-chain cleavage activity. This inhibition may represent an important aspect of the mode of action of clomiphene and tamoxifen.     

FSH induced stimulation of catalase activity in goat granulosa cells in vitro

Reactive oxygen species scavenging enzymes like catalase play diverse role in mammals. The presence of catalase in mammalian ovary is now well established. In the present investigation, changes in catalase activity in granulosa cells isolated from follicles at various stages of differentiation in response to FSH were studied. The follicles were dissected out from goat ovaries and classified as small (<3 mm), medium (3–6 mm) or large (>6 mm). Granulosa cells were isolated from categorized follicles. Results showed that there was a three-fold increase in catalase activity in granulosa cells from large follicles as compared to small and medium follicles. The catalase activity was stimulated significantly when granulosa cells were treated with FSH in vitro. The minimum effective dose that could stimulate catalase activity and estradiol secretion in case of granulosa cells from small and medium sized follicles was 100 ng/ml; for larger follicles, this value was 200 ng/ml. Concomitant to the increase in catalase activity, the estradiol secretion was significantly enhanced when cultured goat granulosa cells were treated with FSH. It was concluded that enzyme catalase may have a functional role in goat ovarian follicular development under endocrine regulation.     

Steroid 17 alpha-hydroxylase activity of ovarian granulosa cells from hypophysectomized immature rats treated with pregnant mare's serum gonadotropin (PMS)

Immature hypophysectomized rats were injected with 2mg of diethylstilbestrol to increase granulosa cell numbers and with 20 IU of PMS to stimulate ovarian growth. Steroid 17 alpha-hydroxylase activity of cultured granulosa cell, harvested from mature follicles 48 h after injection of PMS, was demonstrated using a tritium exchange assay with 17 alpha 3H-pregneneolone as substrate. For comparison, aromatase activity of the same cells was examined by a similar assay using 1 beta 3H-testosterone as the substrate. The activities of the two enzymes were similar when expressed in terms of the amount of substrate converted per unit time. While an NADPH generating system in the incubation medium was essential for demonstrating any hydroxylase activity, 10-15% of the total aromatase activity could be found without added cofactor. Attempts to alter hydroxylase activity of granulosa cells by inclusion of LH, FSH or prolactin in the incubation medium were unsuccessful. However, activity could be change by prostaglandins (PG) or agents which can alter PG synthesis. Activity was increased by low concentrations of phospholipase A2 (PLA2), histamine, and arachidonic acid (AA). Large doses of PLA2, or AA, were inhibitory. PGE2, but not PGF2 alpha, increased, while indomethacin decreased, hydroxylase activity. The results clearly indicate that granulosa cells in the rat have a potent 17-hydroxylase system and therefore do not support the widely held contention that lack of this enzyme is one of the bases for the need for two kinds of cells for ovarian estrogen production.     

Biphasic effect of calcium on luteinizing hormone-stimulated cyclic adenosine 3',5'-monophosphate production in granulosa cells of the fowl (Gallus domesticus).

Both cAMP and Ca2+ play important roles in the steroidogenic action of LH in hen granulosa cells. However, the interaction of these intracellular messengers is not fully understood. In the present study we used two calcium ionophores (ionomycin and A23187), as well as trifluoperazine (TFP), an inhibitor of calmodulin, to investigate LH- and forskolin-induced cAMP production in granulosa cells isolated from the largest (F1) preovulatory follicle of White Leghorn laying hens. Between 0.1 and 1.0 microM, both ionophores significantly potentiated cAMP responses to LH in the presence of 0.1 mM extracellular Ca2+. When calcium was omitted from the medium, ionophores had no effect. When either calcium was raised above 1 mM, or the concentration of ionophores was increased above 1 microM, LH-induced cAMP production was drastically inhibited. In the presence of 0.5-2.0 mM calcium, A23187 inhibited forskolin-promoted cAMP synthesis. TFP, while having no effect on basal cAMP, suppressed LH-induced responses and the potentiating effect of ionomycin. It is concluded that for full activation of the adenylate cyclase/cAMP system by LH, Ca-calmodulin is required at a site upstream from the catalytic component of the enzyme. However, high intracellular Ca2+ and/or other effects of ionophores (such as uncoupling of oxidative phosphorylation) inhibit LH-induced cAMP production.     

Plasminogen activator activity and thymidine incorporation in avian granulosa cells during follicular development and the periovulatory period.

The hormonal and second messenger regulation of plasminogen activator (PA) activities in avian granulosa and theca cells has been documented. However, the physiological role(s) of PAs in the avian ovary remains poorly understood. The present studies were designed to evaluate PA activity in hen granulosa cells collected from the most mature (F1) preovulatory follicle at three discrete time points relative to a spontaneous ovulation and from follicles collected at various stages of follicular development. Levels of PA activity in the granulosa layer of the F1 follicle declined by greater than 90% as follicles were collected closer to their anticipated time of ovulation (e.g., from 17-16 h to 0.75-0.15 h; p less than 0.05). Timing of tissue collection was confirmed by evaluation of serum progesterone levels, which peaked as expected at the 6-5-h time point. During follicular development, PA activity was several times greater in rapidly growing follicles (6-12 mm, 1-3 wk prior to ovulation) than in slowly growing (1-5 mm) or preovulatory (F3 and F1) follicles (p less than 0.05). Granulosa cells of these rapidly growing follicles also incorporated significantly higher levels of 3H-thymidine than did granulosa cells of mature follicles (p less than 0.05), suggesting a higher level of DNA synthesis. Similarly, granulosa cells of the mitotically active germinal disc region of the F1 granulosa layer were found to possess at least 3-fold higher (p less than 0.05) levels of PA activity and a 2-fold greater level of 3H-thymidine incorporation than the more mature granulosa cells isolated from the remaining F1 granulosa layer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biphasic effect of gonadotropin releasing hormone on progestin secretion by rat granulosa cells

The effect of an agonistic gonadotropin releasing hormone (GnRH)-analog (D-Ala6, des-Gly10-NH2-GnRH-ethylamide, GnRHa) on granulosa cell steroidogenesis in the presence or absence of follicle-stimulating hormone (FSH) or luteinizing hormone (LH) was studied. Granulosa cells, isolated from preovulatory follicles of pregnant mare's serum gonadotropin (PMSG)-treated immature rats or from the less mature follicles of untreated immature rats, were cultured for a period of 72 h with daily changes of medium, and progesterone and its metabolite, 20 alpha-dihydro-progesterone (20 alpha-OHP), were assayed in the medium. In granulosa cells from preovulatory follicles, LH and FSH caused a much greater stimulation of steroidogenesis than did GnRHa. There appeared to be no interaction between GnRHa and FSH during the first 10 h, but at 24 h and later the presence of GnRHa clearly inhibited the steroidogenic response to LH and FSH. Steroidogenesis in granulosa cells from immature rats was considerably lower and the effects of GnRHa and FSH alone less pronounced. In these cells, FSH-stimulated progesterone secretion was inhibited by GnRHa only at 72 h. In contrast, 20 alpha-OHP secretion in the same cultures was potentiated by the combined presence of FSH and GnRHa. In conclusion, it seems as though the effects of GnRHa on granulosa cell steroidogenesis varies with exposure time, the initial response being stimulatory and the later inhibitory. Furthermore, the response is also to some extent determined by the maturational stage of the granulosa cells.     

Granulosa cells from pig follicles of different sizes demonstrate maturational differences in their steroidogenic responses to FSH, calcium ionophore A23187, and phorbol diester

Cultures of granulosa cells from small (less than 3 mm), medium (3-6 mm), or large (8-10 mm) pig follicles were treated as follows: (1) basal controls, (2) cyclic adenosine 3',5'-monophosphate (cAMP) pathway agonists (pig FSH: 100 ng/ml; forskolin: 10 microM; dibutyryl cAMP; 1 mM), (3) calcium ionophore A23187 (0.005-1 micrograms), or (4) phorbol 12-myristate 13-acetate (TPA; 0.05-4 ng/ml). The combination of A23187 or TPA together with cAMP agonists was also examined in cultures of granulosa cells from follicles of different sizes. All substances were added at the time of culture, and oestradiol and progesterone were measured in the culture media after 48 h. All cAMP agonists were most potent in their stimulation of steroidogenesis (as a % of control) in cells from small follicles (P less than 0.05) with the exception of forskolin, which increased oestradiol in cells from large follicles to a greater extent than in cells of small follicles (P less than 0.05) (cells from medium follicles demonstrated less stimulation than those from small follicles except in progesterone production, for which FSH was equipotent). With the exception of forskolin, however, granulosa from large follicles showed little (oestradiol) or no stimulation (progesterone) with cAMP agonists. Under basal conditions, A23187 inhibited progesterone in all groups (P less than 0.05), and oestradiol production was reduced in granulosa cells from small follicles (P less than 0.05), unchanged in cells from medium follicles, and significantly stimulated in cells from large follicles. A23187 inhibited the enhanced production of both hormones after administration of cAMP agonists from cells of small and medium follicles (P less than 0.05), with inhibition significantly greater in cells of small follicles compared with medium. In cells from large follicles challenged with cAMP agonists, A23187 inhibited progesterone but stimulated oestradiol production; substitution of TPA (a protein kinase C stimulator) for A23187 gave identical results under basal or FSH-treated cultures of granulosa cells from small-, medium- or large-sized follicles. Our results suggest that TPA, A23187 and cAMP agonists modulate steroidogenesis differently in pig granulosa cells, depending on the stage of maturation of the follicle. Oestradiol production in granulosa cells from large preovulatory follicles may come under the stimulatory control of regulators of protein kinase C as in follicles near ovulation.     

3 Alpha-hydroxysteroid dehydrogenase and 3 beta-hydroxysteroid dehydrogenase in the ovary of young Mongolian gerbils

The ovaries of sexually mature, pregnant mare serum gonadotropin (PMSG) stimulated, 12 week old Mongolian gerbils were investigated morphologically and enzyme histochemically for the appearance of the 3 alpha-hydroxy-steroid and the 3 beta-hydroxysteroid dehydrogenase during the estrous cycle. Up to ovulation, on day 3 of the estrous cycle, the number of vesicular follicles increases continuously. Primarily atretic follicles can be seen on day 4. On day 5 corpora lutea appear, but they degenerate already by day 6. During the entire estrous cycle, 3 alpha-hydroxysteroid dehydrogenase and 3 beta-hydroxysteroid dehydrogenase activity can be found in the theca of tertiary follicles and in the interstitial cells, whereas the theca of secondary follicles and the granulosa of healthy follicles do not exhibit any enzyme activity. The activity decreases from day 1 till day 6. The granulosa of atretic follicles and the cells of corpora lutea show only weak activity. It may be significant that the intensity of enzyme activity in the ovary and the estrogen level in the plasma are differently correlated to the estrous cycle.     

Effects of conditioned media from murine granulosa cell lines on the growth of isolated bovine preantral follicles

Isolated morphologically normal bovine preantral follicles (40 to 70 microm) were cultured for 8 d in collagen gel in control medium or in 1 of 3 conditioned media from the murine granulosa cell lines GRMO1L, GRMO2 and GE2. The percentages of follicles at Day 1 that remained nomal at Day 8 were similar for follicles cultured in the conditioned and control media (84 to 90%). A significantly higher percentage of follicles cultured in each of 3 conditioned media started to grow (89%; P < 0.05) and their increase in diameter was greater than that of follicles cultured in control medium (72%; P < 0.05). The mitotic activity of the granulosa cells from follicles cultured in conditioned media was increased (P < 0.05) indicated by a higher percentage of nuclei that incorporated BrdU compared with that of follicles cultured in control medium. Follicular viability was measured by the presence of nonspecific esterase activity, active mitochondria and dead cells in cultured follicles using the fluorescent probes calcein-AM, rhodamine 123 and ethidium homodimer-1 in combination with confocal laser scanning microscopy. The percentages of follicles with esterase activity and active mitochondria present in their granulosa were similar for follicles in all groups. Culturing in GRMO2 or GE2 tended to lower the number of granulosa with dead cells. The percentage of follicles with oocytes without esterase activity and active mitochondria was lower (P < 0.05) in follicles cultured in GRMO2 or GE2 compared with those cultured in control medium. Moreover, the percentages of dead oocytes tended to be higher in follicles cultured in GRMO1L and GE2 compared with oocytes of follicles incubated in control medium. Taken together, the conditioned media stimulated follicular growth and granulosa viability as well as enhance mitotic activity of the granulosa cells. However, they negatively affected oocyte viability.     

Inhibitory effects of the porcine follicular fluid on estradiol and progesterone secretion by cultured rat granulosa cells

The effect of porcine follicular fluid on estradiol and progesterone secretion was examined using a rat granulosa cell culture with FSH and testosterone in the medium. Follicular fluids from small (less than 5 mm) and large (greater than 6 mm) follicles (SFFI, LFF1) were treated with charcoal, and then fractionated by filtration through an Amicon XM-50 and an PM-10 membrane. The addition of 25% SFF1 and LFF1 into the culture system significantly inhibited estradiol and progesterone secretion (P less than 0.005). These inhibitory activities were observed in PM-10 retentates (10,000-50,000 MW) and filtrates (less than 10,000 MW) of SFF1 and LFF1. The addition of XM-50 filtrates (less than 50,000 MW) of SFF1 and LFF1 caused a dose-dependent inhibition of estradiol and progesterone secretion. The dose-response relationship between the filtrates and estradiol secretion was linear with a significant correlation coefficient. The addition of the filtrates exerted no inhibitory effect on the growth of the cells cultured. XM-50 filtrate of LFF1 from a batch with a low ratio of small/large follicles showed a lower inhibitory activity on estradiol secretion than that of LFF1, while the inhibitory activities in both filtrates on progesterone secretion were almost equivalent. These results suggest that the follicular fluid of small porcine follicle contains nonsteroidal regulators capable of inhibiting estradiol and progesterone secretion by cultured rat granulosa cells, and that the estradiol secretion inhibitor activity decreases in the fluid of large follicle while the progesterone secretion inhibitor activity does not decrease in it.     

Presence of an aromatase inhibitor, possibly heat shock protein 90, in dominant follicles of cattle.

In cattle, it has been suggested that follicular fluid has direct modulatory effects on follicular growth and maturation. In the first part of this study, an in vitro test using aromatase activity of follicular wall fragments as an end point was validated for cattle follicles and was used to test whether follicular fluid (from dominant or non-dominant follicles) modulates aromatase activity. Fluid from dominant follicles at a concentration of 24 or 12% (obtained during the luteal and follicular phases, respectively) significantly inhibited aromatase activity. Inhibitory activity was low or absent in fluid from non-dominant follicles. FSH-stimulated aromatase activity was also reduced by fluid from dominant follicles, but not to a greater extent than in basal conditions. Finally, charcoal-treated fluid from dominant follicles retained its inhibitory activity. In contrast, ovarian venous serum draining a dominant follicle had no activity at the three concentrations tested (6, 12 and 24%). In the second part of the study, identification of the compounds involved in this modulatory activity was attempted using SDS-PAGE. Comparison of the fluorographs from de novo synthesized proteins stored in follicular fluid (inhibitory medium) with those secreted in incubation medium (inactive medium) demonstrated that one protein (90 kDa, pI 5.8) was significantly (P < 0.05) more abundant in fluid from dominant follicles (2.0 +/- 0.09%) than in the culture medium (1.3 +/- 0.1% of the total proteins). This protein had characteristics similar to those of heat shock protein 90 (hsp 90). Therefore, in the final part of the study, the presence of hsp 90 in ovarian cells and follicular fluid was investigated using immunohistochemistry and western blot analysis. After immunohistochemistry, a positive signal was detected mainly in the granulosa cells of larger follicles and to a smaller extent in thecal cells and oocytes. Western blot analysis also demonstrated the presence of hsp 90 in follicular wall fragments and fluid. When blotting was achieved on a sample of follicular fluid resolved by two-dimensional PAGE, the spot detected had a similar location to that at 90 kDa and pI 5.8. Addition of purified hsp 90 to bovine follicles in vitro depressed aromatase activity by altering the K(m) value (and possibly the Vmax value) of the enzyme. It is proposed that hsp 90 is a functional regulator of follicular maturation through its action on aromatase.     

Stimulatory action of follicular fluid components on maturation of granulosa cells from small porcine follicles

Follicular fluid obtained from large (6-12 mm) porcine follicles (LFF) was investigated to determine its stimulatory activity on progesterone secretion and on follicle stimulating hormone (FSH) induction of 125I-human chorionic gonadotropin (hCG)-luteinizing hormone (LH) binding sites in porcine granulosa cells in a 4-day culture. Incubation of granulosa cells harvested from small porcine follicles (1-2 mm) with 50% LFF led to stimulation of LH/hCG binding sites and progesterone secretion. After partial purification of pooled LFF or proteins precipitated with 90% ethanol on Sephadex G-100 the greatest stimulatory activity was found in the second protein peak eluted from the column. Chromatography of part of the active fraction on DEAE Sephacel using a continuous gradient of NH4HCO3 yielded seven protein fractions. The second fraction, which eluted early, contained the majority of the stimulatory activity which was purified about 32-fold compared to native LFF. In contrast, addition of follicular fluid recovered from small porcine follicles inhibited FSH induction of LH/hCG binding sites and progesterone secretion. It can be concluded, that maturation of granulosa cells from small follicles may be enhanced by protein(s) present in LFF, but not in fluid recovered from less mature follicles.     

Distribution of delta-5 -3beta-hydroxysteroid dehydrogenase activity in the Graafian follicle of the sheep.

The localization of delta-5 -3beta-hydroxysteroid dehydrogenase (3 beta-HSD) has been examined in ovarian follicles in vivo and in vitro, and related to oestrogen and progesterone production. In vivo, during the oestrous cycle, enzyme activity was restricted to the theca interna of the one or two most advanced follicles in each animal, but was present only between Day 2 and 5 and between Day 13 and ovulation. High levels of oestrogen were found in the ovarian venous blood only when follicles containing 3 beta-HSD were present. When sheep were injected with PMSG, the theca interna in a number ofsmall follicles acquired 3 beta-HSD activity and began to secrete oestrogen within 12 hr of the injection. The enzyme was not detected in the membrana granulosa of any follicles before ovulation but within a few hours of ovulation, 3 beta-HSD activity was present in the granulosa lutein cells. In vitro, large activated follicles exhibited 3 beta-HSD activity in the theca interna and secreted high levels of oestrogen into the culture medium. When LH was added to the medium oestrogen secretion was inhibited; within 48 hr, the follicles were secreting high levels of progesterone, and 3 beta-HSD activity was present in both the membrana granulosa and the theca interna. Dibutyryl cyclic adenosine monophosphate mimicked the effect of LH in suppressing oestrogen secretiion, but did not induce production of progesterone; the distribution of 3 beta-HSD activity infollicles treated with this nucleotide was the same as in those cultured in control medium.     

Local regulation of primate granulosa cell aromatase activity

Granulosa cells produce inhibin and activin, proteins implicated in the local regulation of preovulatory follicular development. To assess interactions among FSH, LH, inhibin and activin on primate granulosa cell aromatase activity, we studied primary granulosa cell cultures from the ovaries of the common marmoset (Callithrix jacchus), a monkey with an ovarian cycle similar in length to the human cycle. The distinctive action of activin was augmentation of gonadotropin-responsive aromatase activity throughout antral follicular development. FSH-stimulated aromatase activity in granulosa cells from immature follicles was augmented many fold by picomolar amounts of activin. In cell cultures from preovulatory follicles, the presence of activin stimulated basal aromatase activity in the absence of gonadotropin, as well as augmenting the action of LH. Thus, locally produced activin has the potential to modulate aromatase activity in developing ovarian follicles. By contrast, inhibin or inhibin -subunit purified from bovine follicular fluid had minimal effects on aromatase activity. The only significant effect was slight suppression of FSH-inducible aromatase activity in granulosa cells from immature follicles at an inhibin concentration of 100 ng/ml. The finding that inhibin has a negligible effect on aromatase activity in granulosa cells from mature follicles suggests that it is unlikely to exert a physiologically significant influence on aromatase activity in vivo. However, evidence from other studies suggests that inhibin might affect aromatization indirectly through acting locally to modulate thecal androgen (aromatase substrate) production. Therefore, both inhibin and activin have the potential to contribute at different levels to paracrine and autocrine regulation of follicular oestrogen synthesis.     

Localization of some steroidogenic enzymes in the ovary of the rabbit

The distribution of delta 5-3 beta-HSD, peroxidase and cytochrome oxidase in immature, sexually mature and pregnant rabbit ovary has been studied histochemically. Corpora lutea are found only in pregnant rabbits. delta 5-3 beta-HSD is present in the theca interna of mature follicles, corpora lutea and interstitial gland cells but is absent in the granulosa cells of both developing and mature follicles. The granulosa cells of mature and developing follicles, hypertrophied theca interna and the luteal cells all show intense cytochrome oxidase activity. Peroxidase is present in the corpora lutea only. It is suggested that delta 5-3 beta-HSD in the theca interna and interstitial gland cells is the enzyme responsible for steroid synthesis in the ovaries of immature as well as sexually mature rabbits, while peroxidase and delta 5-3 beta-HSD present in the corpora lutea together regulate luteal steroidogenesis during pregnancy. The intense cytochrome oxidase activity together with peroxidase and delta 5-3 beta-HSD confirms the observations that this tissue is a site of intense oxidative activity.     

Effect of the protein kinase C inhibitor Ro 31-8220 on Ca2+-responses, induced by somatotropin, in swine granulosa cells

Somatotropin effect on Ca2+ responses in pig granulosa cells from antral follicles was investigated using fluorescent dye Fluo-3 AM and chlortetracycline. Ro 31-8220 increased the entry of extracellular calcium and the exit of calcium from intracellular stores. In Ca-free medium Ro 31-8220 exerted no influence on the level of calcium in granulosa cells. The effect of somatotropin on pig granulosa cells is associated with PKC activation. These data suggest the involvement of PKC in the changes of calcium in pig granulosa cells activated by somatotropin.     

Follicular fluid modulation of functional LH receptor induction in pig granulosa cells

Follicular fluid was collected from small (1-2 mm), medium (3-5 mm) and large (6-12 mm) follicles of pigs, treated with charcoal to remove steroids, and tested for effects on the induction of functional LH/hCG receptors in cultures of granulosa cells from small antral pig follicles. Granulosa cells were cultured for 2, 4 or 6 days in Medium 199 + 10% pig serum. Granulosa cells cultured in the presence of purified human FSH (0.1 microgram/ml, LER 8/117), insulin (1 mU/ml), cortisol (0.01 microgram/ml) and thyroxine (10(-7) M) accumulated a 4- to 8-fold increase in LH/hCG receptors compared to control cultures. The amounts of cyclic AMP and progesterone secreted after exposure to ovine LH (1 microgram/ml: NIH-S19) were also increased 2-3-fold and 80-100-fold, respectively. Exposure to FSH alone resulted in lower amounts of LH/hCG receptors with a concomitant decrease in optimum LH responses. Addition of 12.5-50% follicular fluid obtained from small (1-2 mm) follicles led to a dose-dependent inhibition of the FSH plus insulin, cortisol and thyroxine induction of LH/hCG receptors after 4 days of culture. Fluid from medium follicles showed reduced ability to inhibit LH/hCG receptor induction, and fluid from large follicles exerted only a slight inhibition or no inhibition of receptor induction. Fluid from medium-sized and large follicles exerted a progressive dose-dependent stimulation of progesterone secretion by the granulosa cell cultures. The inhibitory activity was precipitated primarily with 70% ethanol and to a lesser degree by 36 and 90% ethanol. These studies demonstrate that induction of functional LH/hCG receptors in cultures of pig granulosa cells from immature follicles is enhanced by including insulin, cortisol and thyroxine, in addition to FSH, in the culture medium, and that follicular fluid modulates both receptor induction and progesterone secretion as a function of follicular maturation.