An in vitro study of the effects of androgens on the cytoskeleton of ovarian granulosa cells with special reference to actin

Ovarian granulosa cells from small antral follicles from immature rats were cultured in a serum-free medium for 1-6 days with or without the presence of 10(-5) M dehydroepiandrosterone (DHEA) or 10(-5) M-androstenedione (delta 4-A). Control cultures reveal that the cells are flattened and contain many filamentous bundles organized as stress fibers, numerous scattered cytoplasmic actin filaments, microtubules and vimentin. Alpha actinin and myosin were shown by immunocytochemistry to have a punctate pattern along the stress fibers. For the most part, cells exposed to androgens did not flatten; however, they assumed a varied shape and contained fewer stress fibers and actin filaments. Many of these cells did not develop stress fibers and those that did develop were fewer in number and displayed--actinin and myosin in a punctate pattern. Microtubules and vimentin filaments remained unaltered when compared to controls. It is believed that the deficiency of actin filaments, coupled with certain other degenerative changes which express themselves in other cellular compartments, leads to an early atresia of the granulosa cell cultured in high concentrations of androgens.     

Non-steroidal inhibition of granulosa cell aromatase activity in vitro

Treatment of cyclic rats with the substituted triazole R151885 (1,1-di (4-fluorophenyl)-2-(1,2,4-triazol-1-yl)-ethanol causes delayed ovulation with suppressed blood oestradiol levels. To determine if R151885 can exert a direct action on ovarian oestrogen biosynthesis, we studied its effect on steroidogenesis in granulosa cell cultures from prepubertal rat ovaries. The cells were incubated for 48 h in medium containing 100 ng human FSH/ml and 10(-7) M testosterone to induce steroidogenic enzymes. When R151885 was also present in the culture medium, there was a marked and concentration-dependent reduction in granulosa cell oestradiol production. Inhibition was half-maximal at approx 3 X 10(-7) M and almost complete at 10(-5) M R151885. Progesterone and 20 alpha-hydroxy-4-pregnen-3-one production were unaffected except by the highest concentration of the substituted triazole (36% inhibition at 10(-5) M). Direct assessment of aromatase activity in the 48-h cultured monolayers (oestradiol formation during a 3-h incubation with 10(-7) M testosterone) was made to determine if the inhibitory effect of R151885 was due to reduced aromatase induction/activation. This was not the case, since cells cultured in the presence of 10(-6) or 10(-5) M R151885 had levels of aromatase up to 60% higher than those cultured in its absence. To determine acute effects of R151885 on testosterone (10(-7) M) aromatization, 3-h incubations were carried out using granulosa cell suspensions with high extant aromatase activity due to stimulation by ovine FSH (100 micrograms sc, twice daily for 2 days) in vivo. The triazole acted as an apparent competitive aromatase inhibitor (apparent Km for testosterone 2.5 X 10(-8) M in the absence of R151885 rising to 4.4 X 10(-8) M in the presence of 10(-7) M R151885). Its potency as an aromatase inhibitor was approximately 10 times greater than that of the naturally occurring steroidal aromatase inhibitor 5 alpha-dihydrotestosterone. Various structurally related substances proved to be even more potent aromatase inhibitors than R151885. The most active were also substituted 4,4'-difluorophenyl derivatives containing an imidazolyl or pyridyl moiety instead of the 1,2,4-triazolyl substituent in R151885. This study has identified a novel series of nonsteroidal substances which have the characteristics of potent and specific inhibitors of testosterone aromatization by rat granulosa cells in vitro.     

Steroid production from 17alpha-hydroxypregnenolone and dehydroepiandrosterone by human granulosa cells in vitro.

Granulosa cells were aspirated 3--4 h before the expected time of ovulation from 10 follicles of 4 patients treated with gonadotrophins: 4 of the follicles were immediately preovulatory. The granulosa cells were cultured for 10 h with 17alpha-hydroxypregnenolone or dehydroepiandrosterone and samples of medium removed at 3 and 10 h were assayed for 6 steroids. Granulosa cells were unable to synthesize androgens from endogenous substrate or undertake conversions via the delta5 pathway, but cells from all follicles were capable of aromatizing exogenous androgens to oestrogens although this capability was reduced in cells from follicles beginning to luteinize. Granulosa cells from preovulatory follicles synthesized more progesterone from endogenous substrate than cells from follicles which had not begun to luteinize. The results provide further support for the two-cell theory of oestrogen biosynthesis whereby granulosa cells aromatize androgens which are synthesized by the thecal cells in vivo.     

Hormonal regulation of progesterone secretion by cultured mouse granulosa cells

Secretion of progesterone by granulosa cells from preovulatory follicles of mice was determined during 2 weeks of cell culture in the presence of androgens, estrogen and pituitary gonadotropins. Androstenedione (10(-7) M) and dihydrotestosterone (10(-7) M) stimulated (P less than 0.05) progesterone secretion during the first 11 days of culture. In contrast, 17 beta-estradiol (10(-11)-10(-7) M) did not alter (P greater than 0.10) progesterone secretion throughout the culture period. Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) stimulated (P less than 0.01) the granulosa cells in a dose-dependent manner during the first few days of culture. This luteotropic effect was rapidly lost and at later times when FSH was not effective, LH suppressed (P less than 0.05) progesterone secretion. In the presence of prolactin (Prl) (1 microgram/ml), granulosa cells progressively secreted more progesterone during the first week of culture. After maximal stimulation on Days 7-9, progesterone secretion by Prl-treated cells began to decline, but the amount of steroid produced on Day 13 was still higher (P less than 0.05) than in control cultures. Androstenedione and Prl gave an additive effect on progesterone secretion during Days 3-5 of culture. Thereafter, the androgen, although stimulatory by itself, did not influence the luteotropic action of Prl. Unlike the early effect of androgens, 17 beta-estradiol acted synergistically with Prl to maintain maximal secretion of progesterone during the last 4 days of culture.(ABSTRACT TRUNCATED AT 250 WORDS)     

Diacylglycerol rather than Ca2+ mediates GnRH inhibition of FSH induced steroidogenesis in ovarian granulosa cells.

Treatment of cultured granulosa cells with PLC or GnRH stimulated the rapid generation of DAG and phosphoinositide turnover. The PKC activators PLC (3 mU/ml) and TPA (10(-7)M) or the decapeptide GnRH (10(-6)M) elicited similar inhibitory responses on FSH or cAMP stimulated granulosa cell steroidogenesis. Mobilization of intracellular Ca2+ with A23187 (10(-8)M) was followed by a slight increase in the steroidogenic activity of cultured granulosa cells, whereas elevation of extracellular K+ (50 mM) largely augmented the steroid biosynthetic activity of the granulosa cells. These results suggest that the inhibitory effect of GnRH on granulosa cell steroidogenesis is mediated by generation of DAG, rather than by increases in intracellular Ca2+ concentrations.     

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.     

Granulosa cells inhibit the resumption of meiosis in bovine oocytes in vitro

The oocytes of cattle are not as sensitive as those of laboratory animals to purines, cAMP, or follicular extracts. To study the resumption of meiosis, a method is needed that is capable of inhibiting meiosis completely for a minimum of 24 h. This study was designed to evaluate interrelationships in granulosa-oocyte-cumulus complexes using fresh granulosa cells aspirated from small follicles (1-5 mm) in which the cumulus is normally firmly attached. Selected oocyte-cumulus complexes obtained from a slaughterhouse (n = 2,236) were co-incubated with one of the following: various concentrations of fresh granulosa cells in tissue cultures medium (TCM) 199 or bovine follicular fluid (BFF) either without or after one washing and/or freezing; resuspended granulosa cells previously cultured for 7 days; blood cells; or medium alone. Additionally, oocyte-cumulus complexes were embedded in agar cylinders before incubation with or without cells. The rate of maintenance of intact germinal vesicles (GV) in oocytes after 24 h ranged from 40-77% when 5-100 x 10(6) unwashed cells/ml BFF were used, compared to only 16% in oocytes cultured in BFF alone. The pattern was the same when washed cells were used (30-77%, using 5-100 x 10(6) cells/ml BFF), but they were not as effective as unwashed cells. With TCM-199 and the same five concentrations of cells (5, 10, 25, 50, and 100 x 10(6)/ml), a similar inhibition was obtained with greater than or equal to 25 but not with 5 (3%) or 10 (5%) x 10(6)/ml.(ABSTRACT TRUNCATED AT 250 WORDS)     

Potency and selectivity of the aromatase inhibitor R 76,713. A study in human ovarian, adipose stromal, testicular and adrenal cells

The effects of R 76,713 on steroidogenesis were studied in primary cultures of four different human cell types, i.e. ovarian granulosa cells, adipose stromal cells, testicular cells and adrenal cells. In human granulosa cells aromatization of [1 beta, 2 beta-3H]androstenedione (as measured by the release of tritiated water) showed a Km (Michaelis constant) of 78 nM. R 76,713 competitively inhibited aromatization with a Ki (dissociation constant of the enzyme-inhibitor complex) of 1.6 nM. In human adipose stromal cells aromatization was measured by following the conversion of androstenedione to estrone and 17 beta-estradiol. In this system a Km for aromatization of androstenedione of 10.8 nM was found. R 76,713 again showed competitive kinetics with a Ki-value of 0.14 nM. In human testicular cells the synthesis of the androgens testosterone, androstenedione and dehydroepiandrosterone was only inhibited by drug concentrations exceeding 10(-6) M. At 10(-5) M of R 76,713, steroid concentrations were lowered to 56, 64 and 81% of the control for testosterone, androstenedione and dehydroepiandrosterone respectively. Concomitantly, a slight increase in the levels of pregnenolone (138% of the control) and progesterone (133% of the control) was seen. In human adrenal cells the synthesis of cortisol and aldosterone was slightly affected by R 76,713 also at concentrations exceeding 10(-6) M. At 10(-5) M of R 76,713 the concentrations of cortisol and aldosterone were lowered to respectively 59 and 51% of the control. At the same drug concentration the precursors 11-deoxycortisol and 11-deoxycorticosterone rose to 189 and 147% of the control. These results show that in primary cultures of human cells, R 76,713 is a very potent aromatase inhibitor with a selectivity of at least 1000-fold compared to other steps in steroidogenesis.     

Follicle-stimulating hormone and estradiol regulate antrum-like reorganization of granulosa cells in rat preantral follicle cultures

Formation of a fluid-filled antrum results from the actions of FSH and estrogen on preantral ovarian follicles in most mammalian species. To investigate the novel proposal that hormone-regulated cell-cell interactions mediate antrum formation, we isolated preantral follicles from infant (10- or 11-day-old) Wistar rats and cultured them in a substratum-adherent manner in Minimum Essential Medium supplemented with 2 mM hypoxanthine, 3 mg/ml bovine serum albumin, 5 micrograms/ml insulin, 5 micrograms/ml transferrin, and 5 ng/ml selenium. Similar cultures were previously shown to support oocyte growth and acquisition of meiotic competence. In the absence of FSH, follicles attached to the plastic surface and granulosa cells spread-out uniformly around granulosa cell-enclosed oocytes. FSH treatment caused certain follicles to show an increase between culture days 3 and 7 in appearance of conspicuous antrum-like reorganization of the granulosa cells, but without forming a completely enclosed fluid-filled cavity. This response was biphasic over 10-500 ng/ml FSH, with an optimal concentration of 50 ng/ml resulting in a mean of 37.8 +/- 4.7% of follicles showing antrum-like reorganization for 3 similar experiments. Estradiol-17 beta alone at 10(-10)-10(-8) M was without effect on this response, but at 10(-10) and 10(-9) M, it significantly augmented the action of an optimal concentration of FSH by about 2-fold in 4 experiments. In these experiments, the effect of 10(-8) M estradiol was not significantly different from FSH alone, indicating that the response to estradiol was also biphasic.(ABSTRACT TRUNCATED AT 250 WORDS)     

Secretion of follicle-regulatory protein by porcine granulosa cells

Follicle-regulatory protein (FRP) affects ovarian steroidogenesis and thus follicular maturation. However, secretion of FRP by cells from different-sized follicles as well as the modulation of FRP production by gonadotropins and locally produced steroids are unknown. To evaluate which cell type secretes FRP, theca and granulosa cells were obtained from porcine follicles. In addition, the effects of follicle-stimulating hormone (FSH) and steroids on FRP secretion from granulosa cells of small (less than 3 mm), medium (3-6 mm), and large (greater than 8 mm) porcine follicles and theca cells of large follicles were determined. Granulosa cells were obtained from follicular aspirates, whereas theca cells were recovered after digestion of the stereomicroscopically removed thecal layer. Both were cultured in monolayer in serum-free medium. Granulosa cells were treated as follows: 1) control; 2) FSH (250 ng/ml); 3) progesterone (500 ng/ml, 3 micrograms/ml), or estradiol-17 beta (500 ng/ml, 4 micrograms/ml), or dihydrotestosterone (500 ng/ml, 1 microgram/ml); 4) FSH + progesterone, or estradiol-17 beta, or dihydrotestosterone. Theca cells received the same treatment except that human chorionic gonadotropin (hCG) (5m IU/ml) was used in place of FSH. At 48 or 96 h, media were removed and FRP was quantitated by an Enzyme-Linked Immunosorbent Assay (ELISA). FRP was identified in granulosal medium from follicles of all sizes, but was not present in thecal cultures. At 48 h, granulosa cells from small and medium-sized follicles produced more FRP (20.04 +/- 4.4, 35.42 +/- 4.1 immunoreactive units [IRU]) than cells from large (3.53 +/- 0.97 IRU) follicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cholinergic inhibition of follicle-stimulating hormone-induced progestin production by cultured rat granulosa cells

The influence of cholinomimetics on follicle-stimulating hormone (FSH)-induced progestin production was studied in a primary culture of rat granulosa cells. Cells were cultured for 2 days with FSH and delta 4-androstenedione in the presence or absence of increasing concentrations of cholinergic agonists. Although ineffective as stimulators of steroidogenesis by themselves, the three nicotinic receptor-selective agonists lobeline, dimethylphenylpiperazinium iodide (DMPP), and phenyltrimethylammonium iodide (PTMA) inhibited FSH-induced progesterone and 20 alpha-hydroxypregn-4-en-3-one production in dose-dependent fashions. The rank order of inhibitory potencies was lobeline greater than DMPP greater than PTMA with IC50 values of 2 X 10(-6) M, 3 X 10(-5) M, and 3 X 10(-4) M, respectively. In contrast, the muscarinic receptor-selective agonists muscarine and bethanechol failed to inhibit steroid production. The inhibitory effect of lobeline on the time course of FSH-induced induced steroid production indicated an immediate inhibitory action; however, this inhibition was readily reversed upon removal of the drug. Further studies demonstrated that the FSH-stimulated increase in intracellular cAMP levels, as well as progesterone production induced by cholera toxin and forskolin (agents that stimulate cAMP production) and by dibutyryl cAMP (a cAMP analog), were also suppressed by lobeline. The present observations indicate that nicotinic, but not muscarinic, cholinergic agonists inhibit progesterone biosynthesis in cultured granulosa cells and suggest that endogenous acetylcholine may play a modulatory role in ovarian steroidogenesis.     

The effects of dehydroepiandrosterone (DHEA) and its metabolites on the polycystic ovarian condition (PCO): cystogenic changes of rat granulosa cells in vitro

During mammalian folliculogenesis, granulosa cells (GCs) are initially steroidogenically quiescent, later proliferate, and subsequently commence to hormonally differentiate, first producing estrogen and later, in the preovulatory stage, secreting both estrogen and progesterone. In this study and elsewhere, we have used follicle-stimulating hormone with a combination of growth factors in vitro to simulate the above in vivo conditions. In a previous study, we used dehydroepiandrosterone (DHEA) to accomplish the polycystic ovary condition (PCO) in rats. In the latter model, there were high circulating levels of DHEA and its metabolite, androstenedione. In the present study, we investigated the effects of high levels of DHEA (10⁻⁵M) and its metabolites, androstenedione, androstenediol and dehydroepiandrosterone sulfate on the quiescent, proliferative, and steroidogenically differentiating stages of GCs cultured in a serum-free medium for up to 10 days. In addition to possessing the regularly occurring organelles, when cultured with the aforementioned androgens, the GCs acquired endoplasmic reticulum of the smooth variety which is associated with steroidogenesis. The radioimmunoassay data showed that GCs cultured in the quiescent and proliferative stages in the presence of the androgens, no longer remain in these stages but proceed to differentiate in a preovulatory direction by producing both estrogen and progesterone. This study supports our hypothesis that high circulating levels of DHEA and/or its metabolites have most effect during the quiescent and proliferative stages of granulosa cells, with regard to their structure and their steroidogenic activities.     

R5020 enhances PGE2 stimulated steroidogenesis in cultured rat granulosa cells.

Progesterone biosynthesis and metabolization to 20 alpha-hydroxyprogesterone was stimulated in granulosa cells cultured in the presence of 20 ng/ml of follicle stimulating hormone (FSH) or increasing concentrations of PGE2 (10(-9)-10(-7)M). Concurrent treatment with the synthetic progestin R5020 (10(-6) M) enhanced the FSH or PGE2 stimulated progesterone and 20 alpha-hydroxyprogesterone accumulation in culture media, as well as delta 5-3 beta-hydroxysteroid dehydrogenase activity in granulosa cell homogenates. These findings may represent another example of an autocrine control mechanism in which the steroidogenic product of the granulosa cell exerts an ultra-short loop regulation of its own production.     

Response of porcine theca and granulosa cells to GH during short-term in vitro culture

In Experiment 1, the influence of exogenous GH on steroid secretion by granulosa and theca interna cells recovered from small (1-3 mm), medium (4-6 mm) and large (8-12 mm) follicles was tested. In the second experiment, theca cells (Tc) and granulosa cells (Gc) obtained from large follicles were cultured separately or in two types, Tc/Gc co-culture, where both types of cells were mixed in one well or Gc and Tc were separated by cell culture membrane inserts. In the third experiment, the influence of GH on the morphology of Gc and Tc cells and activity of Delta(5),3beta-hydroxysteroid dehydrogenase (3beta-HSD) was studied. Cells were grown in the control medium (M199+5% of calf serum) or supplemented with 100 ng/ml GH. Testosterone (10(-7) M) was added as the aromatase substrate to granulosa cells cultures. The media were assayed after 48 h of culture for progesterone and oestradiol by RIA. GH added to the culture media had no effect on oestradiol and progesterone secretion by granulosa cells isolated from small and medium follicles while it stimulated both oestradiol and progesterone secretion by Gc isolated from large preovulatory follicles. A stimulatory effect on oestradiol secretion by Tc isolated from all size follicles was observed. GH did not stimulate progesterone secretion by Tc isolated from small follicles but stimulated progesterone secretion by Tc isolated from medium and large preovulatory follicles. Both co-culture systems exhibited synergistic effect on oestradiol secretion. The stimulatory effect on progesterone secretion under the influence of GH was observed in Gc cultured alone and Tc cultured alone. In contrast, the secretion of progesterone was attenuated in both co-culture systems and the addition of GH further augmented this attenuation. A statistically significant increase in oestradiol secretion was observed in all culture conditions. The addition of GH to the culture medium stimulated the activity of 3beta-HSD compared with the control culture from both types of cells. In conclusion, the present studies indicate that there are direct and follicular development stage dependent actions of GH on steroidogenesis of porcine follicular cells.     

Steroidogenic and morphological characteristics of granulosa and thecal compartments of the differentiating rabbit corpus luteum in culture

On the day after ovulation, the thecal tissue and associated mural granulosa lutein cells of the rabbit corpus luteum were separated from the granulosa lutein 'core' by dissection and these tissues were cultured separately or together (whole corpus luteum) in defined medium for 10 days on stainless-steel grids. The medium was changed completely every 24 h. Replicate tissues were cultured with testosterone (10 ng/ml), but no other hormones were added to the medium. Progesterone production increased during the first 2 days of culture for whole corpus luteum, granulosa lutein cells and the thecal compartment which also included granulosa lutein cells. After 3 days, the production of progesterone declined gradually, but was still detectable on Day 10. The production of the metabolite, 20 alpha-dihydroprogesterone, by whole corpus luteum was equal to or greater than that of progesterone. Without the addition of testosterone, the granulosa lutein cells produced little (10 pg/culture) oestradiol during 1 day of culture, but the thecal compartment and whole corpus luteum each produced about 100 pg/culture on Day 1 and declining quantities over the next 2 days. In the presence of testosterone added to the medium, the formation of oestradiol was greatly increased for all tissues for 5-6 days of culture, after which time oestradiol was no longer detectable with or without testosterone in medium. Transmission electron microscopy of cells after 10-12 days of culture revealed fine structure that is characteristic of luteal cells, including abundant smooth endoplasmic reticulum, lipid droplets, and junctions between the luteal cells. The corpus luteum in culture resembles the corpus luteum in situ in that steroidogenesis and differentiation can proceed for a period after ovulation without extrinsic hormonal stimulation.     

5 Alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase activities in isolated canine prostatic epithelial cells

Kinetic parameters of two enzymes, 5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase, have been calculated for freshly isolated canine prostatic epithelial cells separated into secretory and non-secretory cells on the basis of their density in Percoll gradients. For 5 alpha-reductase, a Km value of 3 X 10(-6) M was obtained in both epithelial cell types, and similar Vmax values of 1.7 X 10(-12) and 1.9 X 10(-12) moles of dihydrotestosterone formed/min/10(6) cells (P greater than 0.50) were calculated in secretory and non-secretory cells, respectively. The Km of 3 alpha-hydroxysteroid dehydrogenase (dihydrotestosterone----3 alpha-androstanediol) varied between 2.2 and 2.8 X 10(-6) M (P greater than 0.50) with respective Vmax's of 9 and 24 X 10(-12) moles of 3 alpha-androstanediol formed/min/10(6) cells (P less than 0.005) in secretory and non-secretory cells. For the reverse reaction, that is the transformation of 3 alpha-androstanediol to dihydrotestosterone, the Km's obtained were 0.4 and 0.5 X 10(-6) M (P greater than 0.50) with Vmax's of 14 and 19 X 10(-12) moles of dihydrotestosterone formed/min/10(6) cells (P less than 0.50) in secretory and non-secretory cells, respectively. Vmax values for 3 alpha-hydroxysteroid dehydrogenase are 10-fold higher than the ones for 5 alpha-reductase. Moreover, Km values for the reaction 3 alpha-androstanediol----DHT are 5-fold lower than those calculated for the reverse reaction and for 5 alpha-reductase. This could explain why the major metabolic pathway in the canine prostate gland is the conversion of 3 alpha-androstanediol to dihydrotestosterone.     

Effect of diverse mammalian gonadotropins on estrogen and progesterone production by cultured rat granulosa cells

The ability of gonadotropins from six mammalian species to stimulate estrogen and progesterone production was investigated in granulosa cells of hypophysectomized estrogen-primed immature female rats. Granulosa cells were cultured for 2 days in the presence of delta 4-androstenedione (10(-7) M) with or without various gonadotropin preparations. Treatment with follitropin (follicle-stimulating hormone, FSH) from human, rat, ovine, porcine, equine, and bovine origins resulted in dose-dependent increases in steroidogenesis from negligible amounts to maximal levels of approximately 4-8 and 12-30 ng/10(5) cells for estrogen and progesterone, respectively. The ED50 values of the FSH preparations for stimulation of steroidogenesis were: human: 1-4 ng/ml; ovine: 2.5-30 ng/ml; rat: 1.6-4.0 ng/ml; porcine: 7.5-20 ng/ml; equine 2.5-6 ng/ml; and bovine greater than 100 ng/ml. Lutropin (luteinizing hormone, LH) from rat, ovine, bovine, and porcine origins, human chorionic gonadotropin (hCG), the alpha-subunit of human FSH and the beta-subunit of human LH were ineffective in stimulating steroidogenesis, indicating the specificity of the assay system for FSH. In a high concentration (600 ng/ml), the beta-subunit of human FSH-stimulated steroidogenesis to a small extent. Furthermore, pregnant mare serum gonadotropin and equine LH also caused a dose-dependent stimulation of estrogen and progesterone production, the half-maximal response values (ED50) being 1.8-4 and 7.5-10 ng/ml, respectively. This is consistent with previous in vivo and in vitro findings, showing the potent FSH activities of these hormones. Thus, the cultured rat granulosa cell system provides a sensitive assay for measuring FSH activities of gonadotropins from various mammalian species.     

Effect of dibutyryl cyclic adenosine monophosphate on granulosa cell proliferation, oocyte growth and meiotic maturation in isolated mouse primary ovarian follicles cultured in collagen gels.

Isolated primary follicles from 10-day-old mice were cultured in a collagen gel matrix for 6 days in Minimum Essential Medium + foetal calf serum, followed by culture in unsupplemented medium (control) or in medium containing hypoxanthine (2 mM) or dibutyryl cyclic adenosine monophosphate (dbcAMP, 0.25 mM) for a further 3 or 6 days. Less than 10% of oocytes resumed meiosis during the culture period in all groups. At recovery, the diameter of oocytes at the germinal vesicle stage was recorded and their ability to resume meiosis was determined. Hypoxanthine had little effect on oocyte growth and meiotic competence, but culture in dbcAMP resulted in oocytes that were larger (60.2 +/- 0.6 microns) than those of controls (55.8 +/- 0.5 microns) and more competent to resume meiosis than were controls (42.9% and 10.8%, respectively). The addition of dbcAMP to the culture medium induced a 4-5-fold increase in the number of granulosa cells oocyte compared with controls (3757 +/- 423 and 838 +/- 93, respectively). These results indicate that increased oocyte growth and meiotic competence is primarily mediated via dbcAMP effects on the granulosa cells.     

Prostaglandin involvement in follicle-stimulating hormone-induced proliferation of granulosa cells from chicken prehierarchical follicles

The aim of the present study was to evaluate the role of prostaglandin (PG) on proliferation of granulosa cells from prehierarchical small yellow follicles (SYF) of buff laying hens. The granulosa layers were separated by mechanic method and dispersed into single cells. After 16 h pre-incubation in 0.5% FCS medium, the medium was replaced with serum-free medium, which was supplemented with 10 microg/ml insulin, 5 microg/ml transferrin and 3 x 10(-8)M selenite. Cells were challenged with PGE1 and FSH for 24 h and then assessed for proliferation. The results showed that PGE(1) (0.1-10 ng/ml) had a similar proliferating effect as FSH on granulosa cells, and these stimulating effects were restrained by the PGE receptor antagonist SC19220 at 10(-7) to 10(-5)M. Prostaglandin synthase antagonist indomethacin (10(-7) to 10(-5)M) suppressed FSH-induced increase in the number of granulosa cells in a dose-dependent manner. Downstream activation of protein kinase A by forskolin-activated adenylate cyclase resulted in elevated proliferation of granulosa cells, an effect unobserved by phorbol-12-myristrate-13-acetate-activated protein kinase C. In addition, PGE1-stimulated proliferation of granulosa cells was hindered by H89 (PKA inhibitor) but not by H7 (PKC inhibitor). Furthermore, the proliferating cell nuclear antigen labeling index (PCNA-LI) of granulosa cells displayed similar changes with the number of cells. These results indicated that PGE1 promoted the proliferation of granulosa cells from SYF and was also involved in mediating FSH-stimulated intracellular PKA signal transduction.     

Regulation of ovarian progestin production by epidermal growth factor in cultured rat granulosa cells

The modulation of ovarian steroidogenesis by epidermal growth factor (EGF) was investigated in cultured rat granulosa cells. Granulosa cells, obtained from ovaries of immature, hypophysectomized, estrogen-treated rats, were incubated for 2 days with EGF, follicle-stimulating hormone (FSH), or EGF plus FSH. Treatment with EGF did not affect estrogen production, but stimulated progestin (i.e. progesterone and 20 alpha-hydroxy-pregn-4-en-3-one) production in a dose-dependent manner. Stimulation of progestin production by EGF appears to be the result of an increase in pregnenolone biosynthesis as well as increases in the activities of 20 alpha-hydroxysteroid dehydrogenase and 3 beta-hydroxysteroid dehydrogenase/isomerase. Treatment with FSH increased both estrogen and progestin production by cultured granulosa cells. When cells were treated concomitantly with EGF, FSH-stimulated estrogen production was inhibited, while progestin production was further enhanced. The EGF enhancement of FSH-stimulated progestin production appears to be the result of synergistic increases in pregnenolone biosynthesis and 20 alpha-hydroxysteroid dehydrogenase activity, resulting in substantial increases in 20 alpha-hydroxypregn-4-en-3-one but not progesterone production. The effects of EGF were shown to be time-dependent. The concept of a direct action of EGF on rat granulosa cells is reinforced by the demonstration of high affinity (Kd approximately 3 X 10(-10) M), low capacity (approximately 5,000 sites/cell) EGF binding sites in these cells. Thus, EGF interacts with specific granulosa cell receptors to stimulate progestin but to inhibit estrogen biosynthesis.