Development of a long-term serum-free culture system for immature granulosa cells from diethylstilboestrol-treated prepubertal rabbits: influence of androstenedione and fibronectin on FSH-induced cytodifferentiation

Granulosa cells from diethylstilboestrol-treated prepubertal rabbits were cultured for 6 days in M199 with FSH (1-100 ng ml(-1)) in uncoated or fibronectin-coated plates with or without androstenedione to define the time course profile of oestradiol and progesterone secretion, and the possible modulator role of androstenedione and fibronectin during FSH-induced rabbit granulosa cell differentiation. Every 48 h, cultures were photographed and samples of medium were collected and assayed by ELISA for oestradiol and progesterone. FSH increased oestradiol secretion in a dose-dependent manner. Androstenedione augmented FSH-stimulated oestradiol secretion, and led to a decrease in secretion of oestradiol with time in culture. FSH stimulated progesterone secretion in a dose-dependent manner. This was increased by androstenedione with 10 ng FSH ml(-1) (0-96 h) and 1 ng FSH ml(-1) (96-144 h). FSH-stimulated (100 ng ml(-1)) progesterone secretion decreased at 48-96 h. Fibronectin prevented this decrease, without affecting oestradiol or progesterone secretion at other time points. FSH caused cell reaggregation at 48 h. In conclusion, this serum-free culture system is appropriate for the study of mechanisms of rabbit granulosa cell differentiation. FSH induced cytodifferentiation and reaggregation of granulosa cells. Androstenedione appeared to act synergistically with FSH to promote steroidogenesis. Fibronectin sustained progesterone secretion during differentiation.     

In vitro responses of luteinizing rat granulosa cells to human thyroid-stimulating hormone

The effect of human thyroid-stimulating hormone (hTSH) on progesterone (P4) secretion during initial luteinization and subsequent prolactin (Prl)-mediated steroidogenesis by cultured rat granulosa cells was studied. Granulosa cells, obtained from pregnant mare's serum gonadotropin (PMSG)-treated immature female rats, were preincubated for 1, 3, 6, 12, or 24 h in control medium lacking added hormones or in medium containing 1.0 microgram/ml human chorionic gonadotropin (hCG) or hTSH, and maintained subsequently for 6 days in medium containing 1.0 microgram/ml bovine (bPrl). Indices of luteotropic stimulation were provided by: 1) elevated P4 concentrations determined by radioimmunoassay of spent media samples; and 2) cytoplasmic lipid accumulation assessed by osmium tetroxide staining following fixation after 7 days of culture. Progesterone levels in media from cultures exposed to hCG for 24 h were twofold higher than control cultures, whereas those in media from cultures preincubated in hTSH for 24 h were fourfold higher than control levels. Cultures preincubated in 1.0 microgram/ml hCG for as little as 1 h and then maintained for 6 days in Prl secreted significantly more P4 than did control cultures also maintained with Prl for 6 days. Cultures preincubated in hTSH required a 24-h exposure before a significant increase in Prl-mediated P4 secretion was observed. Intensity of cytoplasmic osmiophilia correlated directly with P4 concentration. These results suggest that: 1) hTSH has the ability to promote P4 secretion during initial luteinization and to regulate subsequent Prl-mediated steroidogenesis by cultured rat granulosa cells; and 2) the mechanism by which hTSH stimulates Prl-mediated P4 secretion in this model system may differ from that of hCG.     

Effect of androgens and gonadotropins on progesterone secretion of chicken granulosa cells

A culture system has been used to study the effect of PMSG in vivo pretreatment and androgens on the in vitro secretion of progesterone from avian granulosa cells. PMSG in vivo pretreated cells secreted greater amounts of progesterone than did cells obtained from untreated hens. Testosterone and 5-alpha-dihydrotestosterone significantly increased basal progesterone secretion in PMSG pretreated cells as well as in granulosa cells harvested from non-treated hens. Testosterone or 5 alpha-dihydrotestosterone in combination with FSH or LH were additive and never resulted in a synergistic stimulation of progesterone secretion.     

Luteotropic and luteolytic responsiveness of ovine luteal cells in long-term culture

Ovine luteal cells were collected and plated 36 h (Day 2) after injection of human chorionic gonadotropin (Day 0) to induce ovulation. Cells were maintained (Days 2-12) in Medium 199 containing 5% calf serum, which was replaced daily. Progesterone secretion was not stimulated (p greater than 0.05) by luteinizing hormone (LH, 10 ng/ml or 100 ng/ml) at any time during culture. However, it was enhanced (p less than 0.05) with a 24-h pulse of dibutyryl adenosine 3', 5'-monophosphate (dbcAMP) during early (2.2-fold stimulation over basal; Days 5,6) or mid- (1.7-fold stimulation over basal: Days 8,9) culture if the pulsing medium contained serum, but not if serum had been withdrawn for 24 h. Continuous exposure of cultures to dbcAMP (2 mM, Days 3-12) resulted in continuously stimulated (p less than 0.05) progesterone secretion (range 1.8- to 4.1-fold stimulation). An increased (p less than 0.05) percentage of cells staining positive for 3 beta-hydroxy-delta 5-steroid dehydrogenase-delta 5, delta 4-isomerase (3 beta HSD) activity were recovered on Day 12 in cultures incubated (Days 3-12) with dbcAMP. Incubation of cultures continuously with prostaglandin F2 alpha (PGF2 alpha) produced dose-dependent inhibition (p less than 0.05) of progesterone secretion. Reduced numbers of 3 beta HSD-positive cells were recovered from these incubations. These experiments demonstrate luteotropic (dbcAMP) as well as luteolytic (PGF2 alpha) effects on ovine luteal cells in long-term culture. This study provides evidence that these cultures will be useful for investigating the development of hormonal regulation of luteal function.     

Prepubertal rat ovary: hormonal modulation of beta-adrenergic receptors and of progesterone response to adrenergic stimulation

We have previously shown that, in the rat, ovarian beta-adrenergic receptor content varies during the time of puberty, with values first increasing and then decreasing abruptly on the afternoon of the first proestrus, i.e., at the time of the preovulatory surge of gonadotropins and prolactin (Prl). In the present study, experiments have been conducted to determine: 1) if hormones other than follicle stimulating hormone (FSH) that are known to be involved in regulating prepubertal ovarian function can mimic the facilitatory effect of FSH on progesterone (P) response of granulosa cells to beta-adrenergic stimulation; 2) if beta-adrenergic receptor content of granulosa cells is under hormonal regulation; and 3) whether the facilitatory effect of hormones on the P response to beta-adrenergic stimulation is due to an increased cyclic AMP response to receptor activation. A 48-h in vitro preexposure of granulosa cells from juvenile, 29-day-old ovaries to the pituitary hormones Prl, luteinizing hormone (LH), or FSH showed that only the latter was able to facilitate the subsequent P response to Zinterol, a beta2-adrenergic agonist. Follicle-stimulating hormone also increased basal P release. Of the two nonpituitary hormones examined, the luteinizing hormone-releasing hormone (LHRH) agonist D-(Ala6,Pro9)-LHRH-ethylamide (LHRH-A) failed to affect P responsiveness, whereas corticosterone enhanced both basal P release and P response to Zinterol. This effect was less pronounced than that of FSH. Luteinizing hormone, Prl and corticosterone decreased beta-adrenergic receptor content to different extents, with corticosterone being the most effective and LH the least (50% and 15% decrease, respectively); LHRH-A was ineffective.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of FSH and HCG on progesterone secretion by cultured oocyte-cumulus complexes and granulosa cells from porcine antral follicles: Influence of follicular development

Oocyte-cumulus complexes and granulosa cells were harvested from small (1–2 mm), medium (3–5 mm), and large (6–12 mm) porcine antral follicles and cultured for 2 and 3 days. The effects of various doses of purified hCG and human FSH on progesterone secretion and monolayer formation were examined. After a 2-day culture period it was found that FSH was more effective in stimulation of progesterone secretion by cultured oocyte-cumulus complexes than in granulosa cells harvested from small follicles (P < 0.01), whereas hCG was more effective in stimulating progesterone secretion in granulosa cells than in oocytecumulus complexes harvested from large follicles. In contrast, after a 3-day culture period, granulosa cells secreted more progesterone compared to oocytecumulus complexes under control conditions or in the presence of hCG or FSH. After 3 days both FSH and hCG stimulated progesterone secretion by oocytecumulus complexes and granulosa cells; however, the hormone effect was greater upon granulosa cells than oocyte-cumulus complexes. After 3 days of culture in the case of both follicular cell types, there was a greater response to FSH in the case of cells harvested from small compared to large follicles. The reverse was true in the case of hCG responsiveness. Monolayer formation ability of oocyte-cumulus complexes was greater in the case of complexes harvested from small and medium than complexes harvested from large follicles. Addition of hCG to the cultures led to a dose-dependent decrease in monolayer formation by oocyte-cumulus complexes harvested from all sizes of follicles.     

Effect of follicle-stimulating hormone on steroid secretion and messenger ribonucleic acids encoding cytochromes P450 aromatase and cholesterol side-chain cleavage in bovine granulosa cells in vitro

We determined 1) whether the previously observed induction of estradiol secretion in bovine granulosa cells cultured in serum-free conditions is associated with an increase in cytochrome P450 aromatase (P450(arom)) mRNA abundance and 2) whether P450(arom) mRNA levels are responsive to FSH in vitro. Granulosa cells from small (2-4-mm) follicles were cultured in serum-free medium. Estradiol secretion increased with time in culture and was correlated with increased P450(arom) mRNA abundance. Progesterone secretion also increased with time in culture, but P450 cholesterol side-chain cleavage (P450(scc)) mRNA abundance did not. FSH stimulated estradiol secretion and P450(arom) mRNA abundance; the effect was quadratic for both estradiol and P450(arom) mRNA. Estradiol secretion and P450(arom) mRNA levels were correlated. FSH stimulated progesterone secretion and P450(scc) mRNA abundance, although the minimum effective dose of FSH was lower for estradiol (0.1 ng/ml) than for progesterone (10 ng/ml) production. Insulin alone stimulated estradiol secretion and P450(arom) mRNA levels but not progesterone or P450(scc) mRNA abundance. We conclude that this cell culture system maintained both estradiol secretion and P450(arom) mRNA abundance responsiveness to FSH and insulin, whereas P450(scc) mRNA abundance and progesterone secretion were responsive to FSH but not insulin.     

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.     

Episodic secretion of gonadotrophins and ovarian steroids in jugular and utero-ovarian vein plasma during the follicular phase of the oestrous cycle in gilts

Blood samples were collected simultaneously from the jugular and utero-ovarian veins of 13 gilts from Days 11 through 16 of the oestrous cycle. A luteolytic dose (10 mg) of PGF-2 alpha was given on Day 12 to facilitate the natural occurrence of luteolysis and standardize the associated decrease in concentrations of progesterone. The mean interval from PGF to oestrus was 5.5 +/- 0.7 days (mean oestrous cycle length = 17.5 +/- 0.7 days). Mean concentrations, pulse amplitudes and pulse frequencies of oestradiol and progesterone were greater (P less than 0.05) in the utero-ovarian than jugular vein. Secretory profiles of LH and FSH were similar (P greater than 0.05) in plasma collected simultaneously from both veins. Based on these data, temporal relationships among hormonal patterns of FSH and LH in the jugular vein and oestradiol and progesterone in the utero-ovarian vein were examined. Concentrations of progesterone declined (P less than 0.05) between Days 12 and 14, while all secretory variables for oestradiol increased (P less than 0.05) from Day 12 through 16 of the oestrous cycle. The pulsatile secretion of FSH remained relatively constant during the experiment. However, both pulse amplitude and mean concentration tended (P less than 0.2) to be lower on Day 16 compared with Day 12. The episodic secretion of LH shifted from a pattern characterized by high-amplitude, low-frequency pulses to one dominated by numerous pulses of diminishing magnitude between Days 13 and 14. From Days 14 to 16 of the oestrous cycle, 91% of all oestradiol pulses were temporally associated with gonadotrophin pulses composed of both FSH and LH episodes. However, pulses of oestradiol (52%) not associated with an episode of LH and/or FSH were observed on Days 12 and 13. These data demonstrate that during the follicular phase of the pig oestrous cycle substantial oestradiol production occurred coincident with luteolysis and before the shift in the episodic secretion of LH. The pool of follicles which ovulated was probably the source of this early increase in the secretion of oestradiol. Therefore, we propose that factors in addition to FSH and LH are involved in the initial selection of follicles destined to ovulate during the early stages of the follicular phase of the pig oestrous cycle. In contrast, high-frequency, low-amplitude pulses composed of LH and FSH were the predominant endocrine signal associated with oestradiol secretion during the second half of the oestrous cycle.(ABSTRACT TRUNCATED AT 400 WORDS)     

Follicular fluid effects on progesterone secretion are not due to follicle-stimulating hormone or steroids

An antiserum raised against porcine follicle-stimulating hormone (FSH) was unable to eliminate the stimulatory action of fluid from large antral porcine follicles on progesterone secretion by granulosa cells from small antral porcine follicles. The same titers of the antiserum were completely effective at eliminating the effect of 2 micrograms of NIH-FSH-P12, whereas maximal stimulation of progesterone secretion was observed with 0.5 micrograms FSH/ml. The androgen and estrogen concentrations measured in charcoal-treated inhibitory follicular fluid from small porcine antral follicles and from stimulatory follicular fluid from large follicles were added separately and together to culture media supplemented with serum to determine if these low concentrations (5 X 10(-11) to 5 X 10(-10) M) of steroids could mimic the actions of follicular fluid on progesterone secretion. Neither the inhibitory nor the stimulatory actions of the follicular fluids could be mimicked by these low concentrations of steroids. Higher concentration of steroids (10(-8) to 10(-7) M range) did stimulate progesterone secretion as reported by others. Our data indicate that the actions of charcoal-treated follicular fluids on granulosa cell progesterone secretion cannot be explained by difference in FSH or steroid contents between the inhibitory and stimulatory fluids and serum.     

Androgen inhibition of FSH-stimulated progesterone production by granulosa cells of prepubertal pigs

Androgens have been reported to stimulate progesterone production by granulosa cells of several species, and to act synergistically with FSH in stimulation of progesterone accumulation by rat granulosa cells. Studies were undertaken to examine the effect of androgens on FSH-stimulated progesterone production in culture by granulosa cells derived from prepubertal pig ovaries. When included in 24-h culture with FSH, both androstenedione and testosterone caused a reduction in progesterone accumulation, but dihydrotestosterone and androsterone did not. Granulosa cells were cultured for 24 h with FSH and [14C]progesterone with or without testosterone; testosterone did not affect the rate of overall metabolism of [14C]progesterone and it was therefore concluded that testosterone inhibited progesterone synthesis, rather than enhancing its catabolism. 17 beta-Estradiol also inhibited FSH-stimulated progesterone accumulation. To determine whether the action of testosterone was mediated by conversion to estradiol, granulosa cells were cultured with FSH and testosterone with or without an aromatase inhibitor (4-acetoxy-androstenedione). The aromatase inhibitor failed to prevent the testosterone-induced reduction in progesterone accumulation, although it markedly inhibited estradiol accumulation. These results indicate that theca-derived androgens can inhibit FSH-stimulated progesterone production by granulosa cells in the prepubertal pig, independently of estradiol.     

Follicle-stimulating hormone regulation of cytochrome P-450 side-chain cleavage messenger ribonucleic acid accumulation by porcine granulosa cells isolated from small and medium follicles

The experiments described here were conducted to examine regulation of cytochrome P-450 side-chain cleavage (SCC) mRNA accumulation in porcine granulosa cells isolated from small (1-4-mm) and medium (5-6-mm) follicles. Granulosa cells were cultured under the following conditions: 1) for 48 h or 96 h with 0, 50, or 200 ng/ml porcine FSH; 2) for 96 h with 200 ng/ml FSH and aminoglutethimide (100 microM); and 3) for 96 h with forskolin (100 microM). Total RNA was extracted and examined by Northern and dot-blot hybridization analysis, and culture media were assayed for progesterone concentration. Northern blot analysis revealed a single band approximately 2.1 kb in size. Accumulation of SCC mRNA by granulosa cells was both FSH dose- and culture time-dependent (p less than 0.05) with maximal increases approximately 4.5 times control levels. Aminoglutethimide reduced progesterone production by about 80% while having no effect on granulosa cell accumulation of SCC mRNA compared to cells stimulated with 200 ng/ml of FSH. Forskolin-treated cells produced significantly more progesterone than did cells treated with FSH, but accumulation of SCC mRNA was similar. In response to FSH, concentration of SCC mRNA did not vary with follicle size, but granulosa cells from small follicles produced significantly more progesterone than did those from medium follicles. These results demonstrate that concentration of SCC mRNA in cultured porcine granulosa cells is FSH dose-dependent, does not vary significantly in cells from small- and medium-sized follicles, and is correlated with progesterone production, but may not parallel progesterone secretion. This last observation indicates that control at sites other than SCC mRNA can affect progesterone production.     

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.     

Androstenedione, dehydroepiandrosterone and testosterone in ovarian vein plasma and androstenedione in peripheral arterial plasma during the bovine oestrous cycle

Catheters were placed in the carotid artery via a facial artery (n = 12) and in the ovarian vein (n = 12), and, in conjunction, electromagnetic flow meters were placed around the ovarian artery (n = 6) in cyclic beef cows. Androstenedione was quantitatively the highest and dehydroepiandrosterone the lowest of the ovarian androgens measured. Ovarian androgens were correlated positively with each other (P less than 0.05) but not with ovarian blood flow or day of the cycle. There was a trend for spikes of androgen release (ovarian vein concentration x ovarian blood flow) from the ovary to be greatest during the period of decreasing progesterone and CL regression. However, only with testosterone were spikes of release different (Days--13 to--9 less than Days -8 to -4; P less than 0.05; Day 0 = oestrus). The dynamic changes in ovarian androgens noted in this study were compatible with the concept of continuous follicular development and atresia throughout the oestrous cycle.     

Biosynthesis and release of oxytocin by granulosa cells derived from preovulatory bovine follicles: effects of forskolin and insulin-like growth factor-I.

Granulosa cells derived from preovulatory bovine follicles were cultured in the presence of insulin-like growth factor-I (IGF-I, 10-100 ng/ml), forskolin (10 microM), or a combination of the two agents. Forskolin alone was the most potent stimulator of both oxytocin (OT) and progesterone (P4) secretion. The two hormones had different patterns of secretion during the course of incubation. OT production peaked on Day 5 of culture and declined thereafter, whereas P4 rose gradually to a peak between Days 7 and 9. The addition of IGF-I to forskolin did not augment OT release beyond that achieved with forskolin alone, but it did maintain higher levels of OT secretion beyond the Day-5 peak. Two antisera, (antiserum I and antiserum II) directed against OT and its C-terminally extended forms, respectively, were used to identify the OT forms in culture media and granulosa cell and corpus luteum extracts. Fully processed OT was detected only in small amounts (0.43 ng/mg protein) in granulosa cell extracts, whereas the corpus luteum extracts contained 6 ng/mg protein. However, granulosa cells that had been incubated with forskolin contained stores of the OT precursor oxytocin-neurophysin, which is found in young corpora lutea. These data indicate that forskolin (whose action probably mimics gonadotropin action) is an effective stimulator of OT biosynthesis and release in cultured bovine granulosa cells.     

Androgens and FSH affect androgen receptor and aromatase distribution in the porcine ovary

In the present study the authors investigated whether androgens could interact with FSH to induce aromatase and androgen receptor expression in porcine granulosa cells. Dissected whole porcine follicles (small, medium, and large) were incubated for 8 hours in M199 medium supplemented with testosterone (10(-7) M), FSH (100 ng/ml) or both those hormones. After incubation, the follicles were fixed and immunostained to visualise androgen receptor and aromatase. In cultures of granulosa cells isolated from small and large follicles, oestrogen secretion was measured by appropriate RIA. Incubation of follicles with testosterone and FSH increased aromatase immunoreactivity in preantral and early antral (i.e. small) follicles. The immunostaining for androgen receptor was slightly higher in medium follicles, while such hormonal stimulation had no effect on small and large follicles. Moreover, granulosa cells isolated from small follicles cultured with both testosterone and FSH produced more estradiol than control cultures (40 pg vs. 100 pg/10(5) cells). The level was relatively close to that obtained in the culture of control granulosa cells isolated from large preovulatory follicles (105 pg/10(5) cells). These results indicate that testosterone acts synergistically with FSH to increase aromatase expression in the small porcine follicles.     

Effect of rate of change of luteinizing hormone concentration on in-vitro progesterone secretion within rat corpora lutea during differentiation.

Immature rats were injected with pregnant mares' serum gonadotrophin followed by human chorionic gonadotrophin (hCG). Ovaries were removed 0, 2, 5 or 8 days after hCG and either prepared for morphometric analysis or perifused with 0, 5 or 30 ng luteinizing hormone (LH)/min. In a second study, ovaries were removed on Day 2 or 8 and perifused with 0.1 mg 8-br-cyclic adenosine 5'-phosphate/ml (8-br-cAMP). On Day 0, the granulosa cells of the preovulatory follicles were small (53 +/- 0.5 microns2) with a cytoplasmic to nuclear (Cy:Nu) ratio less than or equal to 1.5. By Day 2, corpora lutea (CL) were present and composed of 95% small luteal cells (diameter less than 125 microns2, Cy:Nu greater than or equal to 3.0) and 5% large luteal cells (diameter greater than 125 microns2, Cy:Nu ratio greater than or equal to 3.0). The percentage of large luteal cells increased to 36 +/- 7% by Day 5, suggesting that they are derived from a select population of small luteal cells. Basal progesterone secretion increased from 38 +/- 5 on Day 0 to 1010 +/- 48 pg/mg/ml on Day 8. The rate of 5 ng LH/min stimulated progesterone secretion on Days 0, 2 and 8; 30 ng LH/min stimulated progesterone secretion on Days 0, 2 and 8, but not on Day 5; 8-br-cAMP stimulated progesterone secretion on both Days 2 and 8. These data demonstrate that once granulosa cells are induced to luteinize they lose their capacity to secrete progesterone in response to 5 ng LH/min and do not regain their responsiveness to LH rate until they completely differentiate. The loss of this LH responsiveness appears to be due to an inability to stimulate sufficient intracellular cAMP concentrations, since cAMP stimulates progesterone secretion on both Days 2 and 8.     

The androgen receptor does not mediate progestin regulation of progesterone biosynthesis in cultured rat granulosa cells

In the present investigation the influence of androgens and progestins on the FSH modulation of progesterone biosynthesis was studied in cultured rat granulosa cells. Cells obtained from the ovaries of immature estrogen treated rats were cultured for three days in serum free medium or in medium supplemented with FSH or CPA, with or without reduced androgen DHT or the synthetic progestin R5020 alone or in combination with the anti-androgen CPA. Treatment with FSH increased pregnenolone, progesterone and 20 alpha-OHP accumulation in the culture medium 20-, 14- and 7-fold, respectively. Furthermore FSH increased the activity of the enzyme 3 beta-HSD. Concurrent treatment with DHT or R5020 augmented the FSH stimulated steroidogenesis of cultured cells. The androgen enhancement of FSH stimulated steroidogenesis of cultured granulosa cells was blocked by concomitant treatment with CPA, whereas treatment of cultures with anti-androgen did not affect the stimulatory effect of the synthetic progestin R5020.     

In vitro steroidogenesis by primary to antral follicles in the hamster during the periovulatory period: effects of follicle-stimulating hormone, luteinizing hormone, and prolactin

Hamster ovarian follicles at Stages 1 to 10 (Stages 1-4: follicles with 1-4 layers of granulosa cells (GC); Stages 5-7: 5-10 layers GC plus theca; Stages 8-10: antral follicles) were isolated on the morning of proestrus or estrus and incubated for 2 h in the absence or presence of follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin (Prl), progesterone (P4), 17 alpha-hydroxyprogesterone (17OHP), or androstenedione (A). Steroid accumulations in the media were measured by radioimmunoassay (RIA). On proestrus, without any hormonal stimulus, consistent accumulation of P4 through estradiol-17 beta (E2) occurred in low amounts only from Stage 6 and on; both FSH (5-25 ng) and LH (1-25 ng) significantly stimulated steroidogenesis by Stage 6-10 follicles, and the effects of FSH, except for Stage 10, were largely attributable to LH contamination. However, 25 ng FSH significantly stimulated A production by Stages 1-4, whereas 1-25 ng LH was ineffective. On estrus, follicles at all stages, especially 1-6, showed significant and dose-dependent increases in P4 production in response to FSH; both FSH and LH significantly stimulated P4 and 17OHP accumulation from Stage 5 onwards; however, there was no increase in A and E2 compared to controls. Even the smallest estrous follicles showed a shift to predominance of P4 accumulation. On proestrus, Prl had a negative influence on LH-induced accumulation of P4 and 17OHP by Stages 7-9 and 6-8, respectively, without affecting A or E2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Culture of bovine granulosa cells in a chemically defined serum-free medium: the effect of insulin and fibronectin on the response to FSH

Granulosa cells from fully differentiated bovine follicles were cultured in serum-free medium for 4 days. At the end of culture, the number of viable cells was low (10-15% of cells plated on day one) and only progesterone secretion responded to FSH. Insulin increased the number of viable cells at the end of culture (ED50 # 70 ng/ml) and stimulated progesterone secretion (ED50 # 50 ng/ml); the secretion of oestradiol-17 beta over basal value was evident only for concentrations of 1000 and 10,000 ng/ml. FSH acted synergistically with insulin to modify steroid secretion. In the presence of 50 ng/ml of insulin, dose-response studies indicated that secretion of progesterone was maximal at 10 ng/ml of FSH and plateaud thereafter, while oestradiol output peaked at 2 ng/ml of FSH, decreasing at higher concentrations. When cells were seeded in wells precoated with fibronectin, a comparison with cells cultured on plastic showed an increase (30-40%) in the number of viable cells at the end of culture and in oestradiol secretion but a decrease in progesterone output. These results indicate that granulosa cells from large bovine follicles, cultured in a serum-free medium containing insulin, maintain their steroidogenic potency for at least 4 days. Moreover, they show that oestradiol and progesterone synthesis are differentially sensitive to FSH concentrations and that fibronectin increases oestradiol secretion in response to FSH.