Inhibitory effect of gossypol on basal and luteinization factor-stimulated progesterone synthesis in porcine granulosa cells.

Gossypol, a polyphenolic aldehyde, inhibits steroidogenesis and the reproductive system in both sexes. The present study was undertaken to investigate whether gossypol may affect progesterone biosynthesis in cultured porcine granulosa cells isolated from small (1-2 mm) follicles (SGC). SGC were cultured with gossypol, NO donor S-nitroso-N-acetylpenicillamine (S-NAP) or the specific NO-synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME), in the presence or absence of follicular fluid isolated from large (5-8 mm) follicles (LFF) or conditioned media (CM) of granulosa cells isolated from large follicles (LGC). Gossypol enhanced the nitrite content in culture media of SGC and inhibited basal progesterone secretion by SGC. S-NAP (10(-3) M) inhibited progesterone secretion and enhanced the formation of cGMP by SGC. L-NAME had no effect on progesterone accumulation by SGC. The stimulatory effect of LFF or CM media on progesterone production by SGC in culture was also inhibited by S-NAP (10(-3)) and gossypol (10(-4) M). Moreover, gossypol inhibited forskolin-stimulated progesterone secretion, as well as substrate-enhanced conversion of 22-OH-cholesterol and pregnenolone to progesterone. These results suggest that the inhibitory effect of gossypol on progesterone secretion in culture of SGC may be mediated via NO generation.     

Glucocorticoid inhibition of fsh-induced estrogen production in cultured rat granulosa cells

The effects of glucocorticoids on the steroidogenesis of ovarian granulosa cells were investigated. Cortisol and dexamethasone inhibited the increase in aromatase activity induced by FSH in cultured rat granulosa cells. In the same cultures progesterone production was stimulated to a maximum of 167% of the control level. This differential effect of glucocorticoids on estrogen and progesterone production by the granulosa cells indicates that glucocorticoids exert specific inhibition of the induction of aromatase by FSH and do not cause a general suppression of granulosa cell activity. In contrast to their inhibition of the FSH induction of aromatase enzymes, glucocorticoids did not interfere with the activity of pre-existing aromatase enzymes. In granulosa cells containing fun aromatase activity, treatment with cortisol and dexamethasone did not inhibit aromatization of androstenedione to estrogens whereas two known aromatase inhibitors (dihydrotestosterone and 4-androstene-3, 6, 17-trione) were effective. These results indicate that the glucocorticoids exert a selective inhibition of the FSH-induction of aromatase activity in rat granulosa cells by a mechanism other than directly interfering with the aromatization reaction.     

Inhibitory effects of interleukin-1 on follicle-stimulating hormone induction of aromatase activity, progesterone secretion, and functional luteinizing hormone receptors in cultures of porcine granulosa cells

Effects of interleukin-1 (IL-1) on FSH-induced differentiation of immature porcine granulosa cells in vitro were examined in short-term (48-h) cultures. IL-1 inhibited FSH induction of aromatase activity and of LH-stimulated cAMP accumulation by granulosa cells. Both these inhibitory actions of IL-1 were concentration-dependent. Significant inhibitory effects were observed with as low as 0.05-0.25 ng/ml of IL-1, with maximal effects at 25 ng/ml. IL-1 also significantly inhibited increases in [125I]iodo-LH binding and progesterone secretion induced by FSH, as well as reducing basal levels of aromatase activity and LH-stimulated cAMP accumulation. Studies on the mechanisms of IL-1 actions on FSH-induced differentiation of immature porcine granulosa cells revealed that IL-1 reduced cAMP accumulation by the cells in response to FSH in a time- and concentration-dependent manner. IL-1 also inhibited induction of aromatase activity and LH-stimulated cAMP accumulation induced by dibutyryl cAMP, suggesting that IL-1 also affects the steps distal to cAMP generation. In contrast, IL-1 had no effect on progesterone secretion induced by dibutyryl cAMP, suggesting that post-cAMP steps of progesterone secretion were unaffected by IL-1.     

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.     

Effects of follicular size and FSH on granulosa cell apoptosis and atresia in porcine antral follicles

The purpose of this study was to establish a culture model for isolated intact porcine antral follicles and investigate the relationship between granulosa cell apoptosis and follicular atresia. Small (<3 mm), medium (3–5 mm) and large (>5 mm) healthy porcine follicles were isolated and cultured in serum‐free TCM199 with or without follicular stimulating hormone (FSH). Microscopic identification of healthy follicles was confirmed by histology. A spontaneous onset of apoptotic cell death in granulosa cells was observed from cultured antral follicles. The apoptotic rate of granulosa cells from small follicles cultured for 24 hr was higher than those of large and medium follicles, accompanied with high FasL mRNA abundance in granulosa cells. Supplementation with 3 or 5 IU/ml FSH significantly inhibited the percentage of granulosa cells that became apoptotic. FSH did not significantly alter estradiol secretion from cultured follicles. Progesterone secretion significantly decreased after culture for 48 hr, coinciding with the morphological changes observed. FasL and Fas mRNA were expressed in the healthy, early atretic, and progressed atretic porcine follicles regardless of follicular size. However, FasL but not Fas mRNA levels increased during follicular atresia. Addition of FSH significantly decreased FasL rather than Fas mRNA levels in granulosa cells and could attenuate apoptosis. Small follicles seemed to be more susceptible to atresia as compared to medium and large follicles. Mol. Reprod. Dev. 77: 670–678, 2010. © 2010 Wiley‐Liss, Inc.     

The effect of tumor necrosis factor/cachectin on follicle-stimulating hormone-induced aromatase activity in cultured rat granulosa cells

We investigated the effects of tumor necrosis factor (TNF)/cachectin on follicle-stimulating hormone (FSH)-induced aromatase activity in cultured rat granulosa cells using the stereospecific transfer of 3H from [1 beta-3H] androstenedione into 3H2O. TNF (10 pg/ml-10 ng/ml) inhibited FSH (250 ng/ml)-induced aromatase activity in a concentration-dependent manner, and 10 ng/ml of TNF completely abolished the FSH-induced aromatase activity. A time course analysis of the effects of TNF showed that TNF had no effect on induced aromatase activity, but inhibited the further induction of the enzyme by FSH. TNF (10 ng/ml) also inhibited the ability of TGF beta (1 ng/ml) to enhance aromatase activity and increase progesterone synthesis. Thus, TNF is a component of the complex array of proteins that modulate ovarian function and, as such, may play a physiological role in the regulation of the granulosa cell. In view of its association with cachexia, it may also play a pathophysiological role in the suppression of reproductive function during chronic illness.     

Luteinization factor-stimulated steroidogenesis in porcine granulosa cells

Luteinization stimulator (LS), an intrafollicular compound of preovulatory (5-8 mm) follicles, increased both the basal and gonadotropins-stimulated production of progesterone by immature (1-3 mm) granulosa cells. The mechanism by which LS enhance steroidogenesis was investigated by studying the modulation of progesterone biosynthesis from exogenous cholesterol and pregnenolone in cultured porcine granulosa cells in serum-free medium. Progesterone production by cultured granulosa cells was stimulated by FSH, while treatment with 22-OH-cholesterol further enhanced the gonadotropin action. The activity of LS was found in cell conditioned media obtained after 3-day cultivation of preovulatory granulosa cells. Conversion of 22-OH-cholesterol into progesterone by granulosa cells isolated from small follicles was significantly stimulated in the presence LS in culture media. Also, progesterone production by granulosa cells in the presence of pregnenolone was increased considerably. Concomitant treatment with LS led to a further augmentation in progesterone synthesis. Endogenous formation of pregnenolone was inhibited by aminoglutethimide. Thus, LS enhancement of progesterone production in cultured porcine granulosa cells is associated with an increase in the activity of cytochrome P450 cholesterol side-chain cleavage and 3beta hydroxysteroid dehydrogenase enzymes.     

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.     

A postulated role for naturally occurring aromatase inhibitors in follicle selection

The studies reviewed here indicate that follicle regulatory protein (FRP) alters aromatase and 3B-hydroxysteroid dehydrogenase activity in porcine, human, and rat granulosa cells. The inhibitory effect of FRP on granulosal aromatase activity depend upon the response of the cell to FSH: large amounts of FSH can partially overcome FRP inhibition while relatively small amounts of FSH sensitize the granulosal aromatase system to FRP. Although androgens potentiate FSH-mediated granulosal functions, they also sensitize granulosa cell steroidogenic enzymes to inhibition by FRP. The demonstration that FRP acts primarily on granulosa cells of less mature antral follicles to inhibit aromatase supports the hypothesis that FRP may facilitate follicle selection and suggests a role for FRP in atresia. Most of the effects of FRP on granulosal activities reflect an interplay between the systemic endocrine and local paracrine systems. That FRP functions, at least in part, by modulating follicular response to FSH is consistent with the hypothesis that paracrine effectors are important mediators of folliculogenesis in the presence of gonadotropins.     

Effects of ovarian theca cells on granulosa cell differentiation during gonadotropin-independent follicular growth in cattle

We investigated the effects of theca cells or FSH on granulosa cell differentiation and steroid production during bovine early follicular growth, using a co-culture system in which granulosa and theca cells were cultured on opposite sides of a collagen membrane. Follicular cells were isolated from early antral follicles (2-4 mm) that were assumed to be in gonadotropin-independent phase and just before recruitment into a follicular wave. Granulosa cells were cultured under serum-free conditions with and without theca cells or recombinant human FSH to test their effects on granulosa cell differentiation. Messenger RNA levels for P450 aromatase (aromatase), P450 cholesterol side chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), LH receptor (LHr), and steroidogenic acute regulatory protein (StAR) in granulosa cells were measured by real-time quantitative RT-PCR analysis. FSH enhanced aromatase mRNA expression in granulosa cells, but did not alter estradiol production. FSH also enhanced mRNA expression for P450scc, LHr, and StAR in granulosa cells, resulting in an increase in progesterone production. In contrast, theca cells enhanced aromatase mRNA expression in granulosa cells resulting in an increase in estradiol production. Theca cells did not alter progesterone production and mRNA expression in granulosa cells for P450scc, 3beta-HSD, LHr, and StAR. The results of the present study indicate that theca cells are involved in both rate-limiting steps in estrogen production, i.e., androgen substrate production and aromatase regulation, and that theca cell-derived factors regulate estradiol and progesterone production in a way that reflects steroidogenesis during the follicular phase of the estrous cycle.     

Regulation of steroid and steroid sulfate production and aromatase activity in cultured human granulosa-luteal cells

The regulation of the production of steroids and steroid sulfates and the activity of aromatase in human luteinized granulosa cells were investigated. The cells were cultured for 48 h in the presence or absence of hCG and FSH. Basal production of pregnenolone (Pre, 0.3 +/- 0.03 ng/micrograms protein) and progesterone (P, 19.3 +/- 1.7 ng/micrograms protein) were high compared with that of other steroids beyond P in the steroidogenic pathway. The concentration of 17 alpha-hydroxyprogesterone (17-OHP) was lower 0.17 +/- 0.06 ng/micrograms and that of other steroids in the 4-ene and 5-ene pathways and steroid sulfates less than 0.05 ng/micrograms. Both hCG and FSH (100 ng/ml) stimulated the production of Pre and P 3- to 5-fold, but only minimal stimulation of other steroids and steroid sulfates was observed. Aromatase activity of granulosa-luteal cells was measured from the rate of formation of 3H2O from 1 beta-[3H]androstenedione (1 beta[3H]A) after exposing the cells to hCG, FSH or estradiol (E2) for 48 h. Basal aromatase activity was relatively low, but hCG and FSH stimulated aromatase 8- and 4-fold, respectively. The incubation of granulosa-luteal cells with E2 did not affect basal aromatase activity, but E2 augmented FSH-stimulated aromatase 1.4-fold (P less than 0.025). The results suggest that there is low 17 alpha-hydroxylase and steroid sulfokinase activity in human granulosa-luteal cells. Aromatase activity in these cells is regulated by both hCG and FSH, and intra-ovarian estrogens may regulate granulosa cell aromatase activity.     

Actions of growth factors in the follicle

In this paper we have examined the possibility that soluble factors produced by the thecal and granulosa cells may be essential local modulators of follicular development. The observations that insulin could influence both the growth and the differentiation of granulosa cells were important in establishing the concept that peptides could act as amplifiers of the actions of gonadotrophins. Insulin alone did not influence aromatase activity significantly but acted synergistically with FSH to augment aromatase activity in rat granulosa cells. Unlike aromatase activity, insulin alone was able to significantly stimulate 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity, the maximum level achieved approaching that obtained with high concentrations of FSH. To determine if insulin could influence other parameters of granulosa cell function in addition to steroidogenesis, we measured a component of extracellular matrix, fibronectin, previously shown to be inhibited by FSH. Treatment with insulin independently inhibited the increase in fibronectin secretion observed in control cultures. Also, insulin alone was able to stimulate quiescent bovine granulosa cells to incorporate [3H]thymidine into DNA under serum-conditions. The concentration of insulin required in these experiments was higher than physiological levels suggesting that other insulin-like growth factors may be involved. Our work and that of others has shown that IGF1 can mimic the actions of insulin and is effective at lower concentrations. A possible source of IGF1 production in the follicle was sought initially by collecting rat granulosa cell conditioned medium, and assessing biological activity and immunoreactivity. Conditioned medium augmented the actions of FSH on aromatase activity and alone stimulated 3 beta-HSD, indicating the presence of insulin-like bioactivity. A positive reaction on immunoblots using specific antiserum confirmed the presence of immunoreactive IGF1. Conditioned medium from thecal cells contained a growth-promoting activity (TcGF) that did not augment FSH-induced aromatase activity. The production of growth factors locally within the follicle may represent the self-amplifying mechanism that enables the dominant follicle to complete its developmental program and ovulate.     

Effects of transforming growth factor-β1 and activin-A on in vitro porcine granulosa cell steroidogenesis

The mammalian ovarian cycle is a strictly regulated process that is dependent on the intimate interactions among the 3 cell types in the follicle — theca, granulosa, and oocyte. The cycle has been shown to be controlled by gonadotropins as well as locally produced peptide factors. In this study, an in vitro culture system was used to study the roles of 2 locally produced ovarian peptide factors, transforming growth factor-β₁ (TGF-β₁) and activin-A, on porcine granulosa cell steroidogenesis. Gonadotropin stimulated cultured porcine granulosa cells (from medium-sized follicles) were pretreated with 100 ng/ml follicle-stimulating hormone (FSH) for 48 h and then treated with 1 ng/ml TGF-β₁, 100 ng/ml activin-A, TGF-β₁ plus activin-A, or received no treatment (control) for 48 h, From our previous studies, the concentrations of the 2 growth factors were determined to produce maximal antisteroidogenic effects in porcine granulosa cells. Progesterone (P₄) production, estradiol-17β (E₂) production, and aromatase activity for gonadotropin-stimulated porcine granulosa cells treated with TGF-β₁, activin-A, and TGF-β₁ plus activin-A were significantly (P < 0.05) reduced fromthat of the control. The same procedures were conducted on basal steroidogenesis studies in which no pretreatment with FSH was performed. Both P₄ and E₂ production and aromatase activity for porcine granulosa cells treated with TGF-β₁, activin-A and TGF-β₁ plus activin-A were significantly (P < 0.05) inhibited compared with the control. Our results indicate that both TGF-β₁ and activin-A can inhibit FSH-stimulated and basal steroidogeneses in porcine granulosa cells and, thus, may act as local atretic factors during follicular development. When the 2 growth factors were given in combination at concentrations that would produce maximal steroidogenic inhibition, they were not able to produce a synergistic effect. These results are consistent with the current theory that TGF-β₁ and activin-A may act via the same messenger system, a serine-threonine kinase.     

Short-term effects of FSH in vitro on granulosa cells of individual sheep follicles

In Romanov ewes at Day 13 or 14 of the cycle, granulosa cells originating from individual follicles were studied in short-term incubations for aromatase activity and thymidine incorporation. The study was performed on 76 follicles of different sizes (2-7 mm diameter) and degree of atresia, as assessed by histological examination of smears of granulosa cells. As atresia progressed, the labelling index and aromatase activity of granulosa cells decreased. In normal follicles, when follicular diameter increased, the labelling index decreased, while aromatase activity of granulosa cells and oestradiol-17 beta concentration in follicular fluid increased. There was a negative relationship between oestradiol concentration in follicular fluid and the labelling index of granulosa cells in vitro (rs = -0.75; P less than 0.01), suggesting an inverse relationship between growth and differentiation of granulosa cells in normal sheep follicles. In normal small and medium-sized follicles (2-6 mm), incubation with FSH (100 ng/ml) for 2 h increased significantly the labelling index of granulosa cells. In normal medium-sized follicles (4-6 mm), incubation with FSH (50 ng/ml) for 1 h decreased the aromatase activity of granulosa cells. From these results, it is suggested that FSH acts mainly on cells in the G1 phase of the cell cycle, which are steroidogenically active, and makes them move into the S phase where their steroidogenic activity is temporarily inhibited.     

Aromatase activity in granulosa cells: Regulation by growth factors

This study describes the effects of insulin, insulin-like growth factor 1 (IGF₁), and epidermal growth factor (EGF) on the aromatase activity of granulosa cells isolated from immature rat ovaries. None of the growth factors alone influenced the basal level of aromatase activity, but did modulate follicle-stimulating hormone (FSH)-induced aromatase activity. Insulin and IGF₁ augmented the action of a sub-optimal concentration of FSH (5 ng/mL) on aromatase activity in a dose-dependent manner. In contrast, EGF (1–10 ng/mL) was effective in inhibiting aromatase activity maximally stimulated by FSH. Since insulin and IGF₁ had opposing actions to those of EGF on FSH-induced aromatase activity, we examined the interactions between the growth factors. EGF inhibited the actions of both FSH and insulin on aromatase activity. Both IGF₁ and EGF increased the [³H]thymidine incorporation into the DNA of bovine granulosa cells , IGF₁ being a more potent mitogen. Whereas EGF inhibited the actions of IGF₁ on aromatase activity, it did not inhibit the effects of IGF₁ on the growth of granulosa cells. In summary, growth factors influence both the differentiation and growth of granulosa cells, and may be important regulators of follicular development.     

Effect of genistein on steroidogenic response of granulosa cell populations from porcine preovulatory follicles

Genistein affects reproductive processes in animals. However, the mechanism of its action is not fully elucidated and differs among species. The objectives of the current study were: 1/ to establish an in vitro model of granulosa cell culture for studying the intracellular mechanism of phytoestrogen action in porcine ovary; 2/ to determine an in vitro effect of genistein on basal and FSH-stimulated P(4) and E(2) production by porcine granulosa cell populations (antral, mural, total) isolated from large, preovulatory follicles. Granulosa cells were isolated from large (> or =8 mm), preovulatory follicles and separated into antral and mural cell subpopulations. Cells were allowed to attach for 72 h (37 degrees Celsius, 10% serum, 95% air/5% CO2) and than cultured for next 48 hours with or without serum (0, 5 and 10%), FSH (0, 10 or 100 ng/ml) and genistein (0, 0.5, 5 or 50 microM). Basal P(4) and E(2) production did not differ among antral, mural and unseparated granulosa cells isolated form porcine preovulatory follicles. Only mural cells tended to secrete less P(4) and E(2) than other cell populations. FSH stimulated P(4) production in a dose dependent manner in all cell populations and culture systems. Genistein inhibited in a dose dependent manner basal and FSH-stimulated P(4) production by antral, mural and unseparated granulosa cells. However, genistein did not affect E(2) production by granulosa cells. In addition, viability of porcine granulosa cells was not affected by the pyhytoestrogen except the highest dose of genistein. It appears that genistein may be involved in the regulation of follicular function in pigs. Moreover, unseparated porcine granulosa cells may provide a suitable in vitro model for studying the intracellular mechanism of phytoestrogen action in porcine ovary.     

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.     

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.     

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.