Regulation of apolipoprotein E synthesis in rat ovarian granulosa cells

Apoprotein E (apo-E) is a surface component of several classes of plasma lipoproteins. It functions as a ligand for receptor-mediated uptake of lipoproteins. Granulosa cells from ovaries of diethylstilbestrol-stimulated hypophysectomized immature rats cultured in serum-free medium with [35S]methionine secretes a 34-kDa protein which reacts with a monospecific anti-rat apo-E antibody and represents 0.2% of total secreted protein. Protease mapping confirms that this protein is apoprotein E. The secreted apoprotein E may be complexed with lipid since it floats in the ultracentrifuge at density less than 1.21 micrograms/ml. Freshly isolated granulosa cells contain receptors for follicle stimulating hormone (FSH) but not for human chorionic gonadotropin (hCG) or prolactin. Apoprotein E secretion is stimulated 2-fold by FSH, but hCG and prolactin have no effect. When granulosa cells develop hCG and prolactin receptors after 48 h of culture with FSH, apoprotein E secretion is not stimulated by addition of FSH, hCG, or prolactin although steroidogenesis is induced. The addition of 10(-7) M androgen plus FSH stimulates a marked increase in progestin synthesis over FSH alone, but androgen has little added effect on apoprotein E secretion. Cholera toxin (1.25 micrograms/ml) and dibutyryl cAMP (5 mg/ml), both of which increase intracellular cAMP, stimulate apo-E secretion 9-fold and 12-fold, respectively. The dibutyryl cAMP effect is dependent on both dose (greater than or equal to 0.5 mg/ml required) and time (onset at 24 h, maximum at 48 h, and back to near baseline at 96 h). Isobutylmethylxanthine, a phosphodiesterase inhibitor, augments FSH-stimulated apoprotein E synthesis 2.5-fold, supporting a role for cAMP in mediating the FSH effect. This is the first demonstration of the hormonal regulation of apoprotein E synthesis in an extrahepatic tissue.     

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.     

The effect of stage of estrous cycle and follicular maturation on ovarian inhibin production in sheep

Twenty-four Scottish Blackface ewes (mean weight 50.0 +/- 0.1 kg with ovulation rate 1.3 +/- 0.1) were randomly divided into 4 groups of 6 animals. Under general anesthesia, following the collection of a timed sample of ovarian venous blood, the ovaries of these animals were collected either on Day 10 of the luteal phase or 12, 24, and 48 h after a luteolytic dose of a prostaglandin (PG) F2 alpha analogue (cloprostenol 100 micrograms i.m.) administered on Day 10. All follicles greater than 3 mm were dissected from the ovaries and incubated in Medium 199 (M199) at 37 degrees C for 2 h, following which the granulosa cells were harvested and incubated in triplicate for 24 h in M199 with or without ovine FSH or ovine LH. Plasma and culture media samples were assayed for inhibin, estradiol (E2), androstenedione (A4), and testosterone (T) by specific RIA. After correcting for hematocrit, ovarian secretion rates were calculated from the product of the plasma concentration and flow rate. The rate of ovarian inhibin secretion during the luteal phase was similar from ovaries categorized on the basis of presence of luteal tissue (1.0 +/- 0.3 and 0.9 +/- 0.5 ng/min for CL present and absent, respectively), confirming that the ovine CL does not secrete appreciable amounts of inhibin. Inhibin secretion was higher (p less than 0.05) at 12 h after PG-induced luteolysis but not at 24 or 48 h compared to values for luteal phase control ewes. Although ovaries containing large estrogenic follicles (greater than or equal to 4 mm in diameter and classified as estrogenic from in vitro criteria) secreted the most inhibin (55%; p less than 0.05), both ovaries containing large nonestrogenic follicles (33%) and small (11%; less than 4 mm in diameter) follicles secreted appreciable amounts of inhibin. This contrasted strongly with E2 where greater than 80% of the steroid was secreted by large estrogenic follicles. The rate of ovarian inhibin secretion was positively correlated (p less than 0.05) with the rate of E2, A4, and T secretion. Overall, there was no significant effect of stage of cycle on follicular inhibin content after 2 h incubation in vitro, release of inhibin by follicles incubated in vitro, or synthesis of inhibin by granulosa cells cultured in vitro. FSH and LH had no effect on the production of either inhibin or estradiol by cultured granulosa cells. Follicular diameter was positively correlated (p less than 0.001) with follicular inhibin and steroid release. Follicular inhibin content after 2 h incubation in vitro was more highly correlated with inhibin release by incubated follicles (r = 0.7; p less than 0.001) than with inhibin synthesis by granulosa cells in vitro (0.4; p less than 0.01).(ABSTRACT TRUNCATED AT 400 WORDS)     

Interactions between follicle-stimulating hormone and growth factors in modulating secretion of steroids and inhibin-related peptides by nonluteinized bovine granulosa cells

The aim was to investigate potential interactions between FSH and intraovarian growth factors in modulating secretion of inhibin A (inh A), activin A (act A), follistatin (FS), estradiol (E2), and progesterone (P4) by bovine granulosa cells cultured under conditions in which a nonluteinized FSH-responsive phenotype is maintained. Cells from 4- to 6-mm follicles were cultured in serum-free medium containing insulin (10 ng/ml) and androstenedione (10(-7) M), and effects of ovine FSH (0.037-3 ng/ml) were tested alone and in combination with insulin-like growth factors (IGF) (LR3 IGF-I analogue; 2-50 ng/ml) and epidermal growth factor (EGF; 0.1-10 ng/ml). Medium was changed every 48 h and cultures ended after 144 h, when cell number was determined. Between 48-96 h and 96-144 h, FSH promoted (P < 0.0001) increases in output of inh A (6-fold), act A (15-fold), FS (6-fold), and E2 (18-fold), with maximal responses (in parentheses) elicited by 0.33 ng/ml FSH during the final period. Higher FSH doses (1 and 3 ng/ml) gave reduced responses for each of the above hormones, whereas P(4) output was maximal (3-fold) at these doses. FSH promoted a slight increase in cell number ( approximately 1.7-fold; P < 0.001). LR3 IGF-I alone markedly increased (P < 0.0001) output of inh A (8-fold), act A (41-fold), FS (12-fold), and E2 (18-fold); this was accompanied by modest increases (P < 0.01) in P4 output ( approximately 2.5-fold) and cell number ( approximately 2-fold). Whereas FSH enhanced inh A, act A, FS, and E2 secretion evoked by lower doses of LR3 IGF-I, it suppressed (P < 0.001) the response to the highest dose. EGF alone promoted a 1.7-fold increase in cell number (P < 0.001) without affecting hormone release; however, it abolished (P < 0.001) FSH-induced secretion of inh A, act A, FS, and E2. Both FSH alone and LR3 IGF-I alone dose-dependently increased the act A:FS ratio ( approximately 3-fold; P < 0.005) and act A:inh A ratio (3-fold to 6-fold; P < 0.001), suggesting that both factors selectively raise activin "tone" and that this could be a key requirement for FSH and IGF-induction of follicular E2 production. This hypothesis was reinforced by the finding that addition of FS, to reduce the act A:FS ratio and sequester secreted activin, markedly suppressed (P < 0.001) FSH (3-fold)-, and LR3 IGF-I (2-fold)-induced E2 output.     

Hormonal requirements for the growth and differentiation of hamster preantral follicles in long-term culture

Preantral follicles from pro-oestrous and oestrous hamsters were isolated enzymically (Stages 1-5) and by microdissection (Stage 6) and cultured for up to 168 h in the absence or presence of 100 ng ovine FSH or LH separately or combined or 1 or 10 micrograms progesterone or estradiol-17 beta in serum-free defined medium and exposed to 1 muCi [3H]thymidine for 24 h before termination. In the presence of insulin and hydrocortisone but not gonadotrophins, the morphology of follicles from pro-oestrous animals at Stages 1-4 (1-4 layers granulosa cells; no theca) were unaffected for up to 48 h whereas for Stages 5 (5-6 layers granulosa cells and developing theca) and 6 (7-8 layers granulosa cells and theca), atresia was prominent by 24 h. FSH significantly reduced the percentage of atretic follicles in Stages 1-5 throughout the culture period; but was effective only up to 96 h for Stage-6 follicles. LH was also effective, albeit to a lesser extent. FSH increased follicular labelling indexes during every 24-h labelling period and, during a pulse-chase period, follicular DNA content and granulosa cell numbers. FSH, but not LH, induced differentiation by 96 h of preantral follicles at Stage 6 into small antral stages (Stages 7-8). FSH and LH together induced almost the same effect as FSH alone. However, neither progesterone nor oestradiol had any significant long-term effects on DNA synthesis and oestradiol induced atresia beyond 24 h. Both FSH and LH induced follicular maturation in vitro as evident from increases in progesterone, androstenedione and oestradiol production. Follicles (Stages 1-4) collected from oestrous hamsters responded to FSH to a lesser extent than did those from pro-oestrous animals, possibly because of in-vivo exposure to periovulatory changes in gonadotrophins; however, an antrum formed in Stage-6 follicles by 72 h.     

Relationships between luteinizing hormone, follicle-stimulating hormone and prolactin secretion and ovarian follicular development in the weaned sow

Folliculogenesis was studied by assessing development of the largest 10 follicles obtained from 10 sows 48 h after weaning and by analyzing changes in plasma luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin (PRL) for 24 h before weaning until 48 h after weaning. Follicular diameter, follicular fluid volume, and concentrations of estradiol and testosterone and granulosa cell numbers were determined in all follicles, and 125I-hCG binding to theca and granulosa and maximal aromatase activity in vitro was determined in five follicles/sow. Overall, a significant rise in LH, but not in FSH, occurred at weaning, although in individual sows an increase in LH was not necessarily related to subsequent estrogenic activity of follicles. In 9/10 sows, PRL fell precipitously after weaning. In lactation, LH was negatively, and after weaning, positively, correlated with FSH and PRL. Marked variability in follicular development existed within and between sows. Overall, most follicular characteristics were positively correlated to follicular diameter; however, in larger follicles the number of granulosa cells was variable and unrelated to estrogenic activity, which--together with theca and granulosa binding of hCG--increased abruptly at particular stages of follicular development. Differences in maturation of similarly sized follicles from different sows were related to estrogenic activity of the dominant follicles but not to consistent differences in LH, FSH or PRL secretion. Both the dynamics and the control of folliculogenesis in the sow, therefore, appear to be complex.     

Hormonal regulation of cyclic AMP-dependent protein kinase in cultured ovarian granulosa cells. Effects of follicle-stimulating hormone and gonadotropin-releasing hormone

The hormonal regulation of cAMP-dependent protein kinase was examined in granulosa cells from diethylstilbestrol-implanted immature rats. Follicle-stimulating hormone (FSH) increased the number of available cAMP-binding sites in a dose- and time-dependent manner, with a maximum 4-6-fold increase at 50-100 ng/ml between 6 and 48 h of culture after a transient decrease in available sites during the first 6 h. The potent gonadotropin-releasing hormone (GnRH) agonist [D - Ala6]des - Gly10 - GnRH - N - ethylamide (GnRHa) reduced the FSH-induced increase in cAMP-binding sites by approximately 50% at 24 and 48 h of culture. Photoaffinity labeling with 8-azido-[32P] cAMP revealed the existence of one major cAMP-binding protein (Mr = 55,000 +/- 400) which appeared to be the regulatory (R) subunit of type II cAMP-dependent protein kinase. While FSH induced a 5-10-fold increase in the labeling of R II both in vivo and in vitro, GnRHa reduced the amount of R II induced by FSH in granulosa cells cultured for 48 h. The large increase in R II subunit was not accompanied by a corresponding increase in protein kinase activity, which was only enhanced by 50% after 48 h of culture with FSH. Fractionation of granulosa cell cytosol from FSH-treated ovaries on DEAE-cellulose showed a single peak of cAMP-dependent phosphokinase activity with the elution properties of a type II protein kinase. However, the peak of cAMP binding activity (eluted at 0.20 M KCl) was not coincident with the protein kinase activity. FSH transiently stimulated cAMP-dependent protein kinase activity during the first 10-30 min of culture. GnRHa impaired the FSH-induced early increase in protein kinase activity, causing a delay in activation until 60 min. These findings suggest that a large dose- and time-dependent increase in the content of cAMP-binding sites may be a major factor in cAMP-mediated differentiation of granulosa cells. The inhibitory effect of GnRHa on both FSH-induced protein kinase activation during the first minutes of culture and on FSH-induced R II synthesis during the subsequent 48 h of culture could be crucial events in the prevention of granulosa cell maturation by GnRH agonists.     

FSH influences follicle viability, oestradiol biosynthesis and ovulation rate in Romney ewes

Injection of steroid-free bovine follicular fluid (bFF; 2 X 5 ml s.c. 12 h apart) into anoestrous ewes lowered plasma FSH concentrations by 70% and after 24 h had significantly (P less than 0.01) reduced the number of non-atretic follicles (greater than or equal to 1 mm diam.) without influencing the total number of follicles (greater than 1 mm diam.) compared to untreated controls. Hourly injections of FSH (10 micrograms i.v. NIH-FSH-S12) for 24 h did not influence the number of non-atretic follicles but did negate the inhibitory effects of bFF on follicular viability. Hourly injections of FSH (50 micrograms i.v., NIH-FSH-S12) + bFF treatment for 24 h significantly increased the total number of non-atretic follicles, and particularly the number of medium to large non-atretic follicles (greater than 3 mm diam.) compared to the untreated controls (both P less than 0.01). The 10 micrograms FSH regimen (without bFF) significantly increased aromatase activity in granulosa cells from large (greater than or equal to 5 mm diam.; P less than 0.01) but not medium (3-4.5 mm diam.) or small (1-2.5 mm diam.) follicles compared to controls. The 10 micrograms FSH + bFF regimen had no effect on granulosa-cell aromatase activity compared to the controls. However, the 50 micrograms FSH plus bFF regimen increased the aromatase activity of granulosa cells from large, medium and small non-atretic follicles 2.6-, 8.3- and greater than or equal to 11-fold respectively compared to that in the control cells. Ewes (N = 11) that ovulated 2 follicles had significantly higher plasma FSH concentrations from 48 to 24 h and 24 to 0 h before the onset of a cloprostenol-induced follicular phase (both P less than 0.01) than in the ewes (N = 12) that subsequently ovulated one follicle. Hourly FSH treatment (1.6 micrograms i.v., NIAMDD-FSH-S15) for 24 h but not for any 6 h intervals between 48 and 24 h or 24 and 0 h before a cloprostenol-induced luteolysis also resulted in significant increases (P less than 0.05) in the number of ewes with 2 ovulations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Secretion of IGF-1 by ovine granulosa cells: effects of growth hormone and follicle stimulating hormone

Insulin-like growth factor-1 (IGF-1) is implicated in follicle development and is considered to mediate the actions of growth hormone (GH) and gonadotrophins at the ovarian level. However, the expression and secretion of IGF-1 by the ovary are controversial, partly because of species and cell-type specificity. The present study investigated whether IGF-1 is produced by ovine granulosa cells and whether its production is regulated by GH and follicle stimulating hormone (FSH). Follicles (>/=4.0 mm) were obtained from ewes during seasonal anoestrus. Granulosa cells were cultured for a total period of 96 h in Dulbecco's modified Eagle's medium (DMEM)/Ham's F-12 medium supplemented with BSA (0.1%, w:v), transferrin (0.5 microg/ml) and testosterone (100 ng/ml). In the first set of experiments, cells were incubated in the presence of bovine calf serum (BCS) (2.5%) for the initial 48 h of culture. The cells were then cultured for the next 48 h in medium without BCS, but containing either GH (0, 2, 20, and 200 ng/ml) or FSH (0, 20, 200, and 2000 ng/ml). The medium was assayed for oestradiol (E), progesterone (P) and IGF-1. There were six wells per treatment and the experiment was carried out four times. Control granulosa cells maintained both IGF-1 and E secretion, with only low levels of progesterone output. In all experiments, both GH and FSH produced significant (P<0.001) dose-related increases in E, IGF-1 and P secretion into the medium. The maximum responses to GH (20 or 200 ng/ml) were 402% for E and 528% for IGF-1 compared with controls. The maximum responses to FSH (200 or 2000 ng/ml) were 460% for E and 514% for IGF-1. The objective of the second set of experiments was to determine the effect of the progestogenic status of cells on IGF-1 production. Granulosa cells were cultured both in the presence and absence of BCS (2.5% in the medium) during the initial 48 h of culture. For the next 48 h, cells were cultured in serum-free medium. Addition of BCS to the medium during the initial 48 h of culture stimulated progesterone production. However, it did not affect either IGF-1 or oestradiol secretion between 49 and 96 h of culture, or the cell numbers at the end of culture. In conclusion, (1) IGF-1 is secreted by granulosa cells irrespective of their progestogenic status and (2) concomitant increases in E and IGF-1 production by granulosa cells as a result of GH and/or FSH treatment suggest a role for GH and FSH in the regulation of ovarian function.     

Involvement of transforming growth factor alpha in the regulation of rat ovarian X-linked inhibitor of apoptosis protein expression and follicular growth by follicle-stimulating hormone

The expression of X-linked inhibitor of apoptosis protein (XIAP), a member of a family of intracellular antiapoptotic proteins, is induced by FSH during follicular development in vivo. Whether the XIAP up-regulation by FSH (100 ng/ml) is a direct action of the gonadotropin and is important in the control of granulosa cell proliferation during follicular growth is unclear. The overall objective of the present study was to examine whether the FSH-induced XIAP expression and granulosa cell proliferation during follicular development is mediated by the secretion and action of intraovarian transforming growth factor alpha (TGFalpha). In rat follicles cultured for 2 and 4 days, FSH stimulated estradiol production, TGFalpha secretion, XIAP expression, and follicular growth. The theca cells are the primary follicular source of FSH-induced TGFalpha, as indicated by in situ hybridization. Intrafollicular injection of a neutralizing anti-TGFalpha antibody (50-200 ng/ml; immunoglobulin G as control) or addition of estradiol-antagonist ICI 182780 (0.5-100 nM) to the culture media suppressed FSH-induced XIAP expression and follicular growth. The effect of ICI 182780 could be partially reversed by high concentrations of estrogen (250 and 500 nM). Whereas TGFalpha (10-20 ng/ml) significantly increased granulosa cell XIAP content and proliferation in primary granulosa cell cultures, FSH alone was ineffective in eliciting the mitogenic response. Our results support the hypothesis that FSH stimulates granulosa cell proliferation via theca TGFalpha secretion and action in response to increased granulosa cell estradiol synthesis, and that XIAP up-regulation in response to FSH suppresses granulosa cell apoptosis and facilitates FSH-induced follicular growth.     

Transforming growth factor beta regulates the inhibitory actions of epidermal growth factor during granulosa cell differentiation

The effects of transforming growth factor beta (TGF-beta) on epidermal growth factor (EGF) receptor content and EGF action were studied in cultured granulosa cells from immature diethylstilbestrol-implanted rats. During follicle-stimulating hormone (FSH)-induced differentiation in vitro, EGF receptors increased by 20-fold as measured by the binding of 125I-EGF to the intact cells. Addition of TGF-beta during the 48-h culture period amplified the stimulatory effects of FSH on EGF receptors up to 2-fold, with ED50 and maximal concentrations of 2.5 and 8 pM, respectively. Also TGF-beta alone in amounts from 1.6 to 16 pM increased EGF receptor content 4-fold. The stimulatory effects of TGF-beta were due to increased numbers of EGF receptors/cell, since the growth factor had no effect on the Kd (3-5 X 10(-11) M) of the high-affinity EGF binding site. TGF-beta action was observed within 20 h of granulosa cell culture and was maximal by 48 h of a 96-h culture. The stimulatory actions of TGF-beta in gonadotropin-induced cells were exerted through the cAMP effector system of the granulosa cell, since the growth factor also amplified the induction of EGF receptors by cholera toxin, forskolin, and 8-bromo-cAMP. The augmentation of EGF receptors by TGF-beta resulted in a parallel 2-fold increase in the inhibitory effects of EGF on FSH-induced cAMP production and luteinizing hormone receptor expression during granulosa cell development. TGF-beta did not increase granulosa cell numbers during culture although it elevated [3H]thymidine incorporation into DNA by 2-fold over that of FSH-treated cells. These results indicate that TGF-beta regulates the effects of both FSH and EGF during granulosa cell differentiation and provides evidence that ovarian function may be controlled by the combined actions of gonadotropins and multiple growth factors.     

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.     

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.     

Activin stimulation of inhibin secretion and messenger RNA levels in cultured granulosa cells

Recent reports suggest that activin (the dimer of inhibin beta subunits with FSH-releasing activity) has specific receptors on ovarian granulosa cells. The present study examined the effects of purified porcine activin on inhibin secretion and mRNA levels in granulosa cells obtained from immature, estrogen-treated rats. Cells were cultured for 48 h in culture media, or media containing FSH (10 ng/ml) and/or activin (30 ng/ml). Western blot analyses performed with affinity-purified antisera to inhibin alpha- and beta A-subunits revealed that treatment with either FSH or activin increased the secretion of inhibin alpha beta dimer (Mr 30,000), with a further increase after cotreatment. These results were confirmed by an inhibin alpha-subunit RIA, which revealed 7-, 14-, and 71-fold increases in the secretion of immunoreactive inhibin-alpha by activin, FSH, and activin plus FSH, respectively. TGF beta, a structural homolog of activin, also stimulated inhibin release, whereas follistatin was ineffective. Total RNA from cultured cells was hybridized with 32P-labeled inhibin alpha-subunit cRNA or beta-actin cDNA probes, and inhibin-alpha message levels were normalized with beta-actin mRNA levels. Northern blot analysis revealed that treatment with FSH and activin increased hybridization of a 1.5 kilobase (kb) message, corresponding to the inhibin alpha-subunit mRNA. Slot blot analyses indicated a 6- and 8-fold stimulation of inhibin alpha-subunit mRNA levels by FSH and activin, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Follicle-stimulating hormone regulation of inhibin alpha- and beta(B)-subunit and follistatin messenger ribonucleic acid in cultured avian granulosa cells

FSH regulation of inhibin alpha-, beta(B)-subunit and follistatin mRNA was investigated in cultured chicken granulosa cells, which were isolated and pooled according to size from the F(4) + F(5) follicles, small yellow follicles (SYF), and large white follicles (LWF). In experiment 1 (four replicate experiments), granulosa cells were cultured, and the effect of FSH (50 ng/ml) on the growth of cells from the different follicles was examined at 24 and 48 h of culture. Cell viability was >95% for all of the granulosa cell cultures at 24 and 48 h. At 24 h, the number of granulosa cells in both the FSH-treated and the untreated cultures for all follicle types was numerically greater than the number of cells originally plated. At 48 h, FSH-treated cultures for all follicle types had twice (P: < 0. 05) the number of cells as the untreated cultures. In experiment 2 (three replicate experiments), FSH increased expression of the mRNA for inhibin alpha-subunit in LWF granulosa cells at 4 and 24 h to detectable levels and increased inhibin alpha-subunit protein accumulation to detectable levels by 24 h in granulosa cells from the LWF. FSH also increased (P: < 0.05) mRNA levels for the inhibin alpha-subunit at 4 and 24 h in SYF granulosa cells and at 24 h in F(4) + F(5) granulosa cells. The effects of FSH on follistatin and ss(B)-subunit were variable with respect to follicle development and culture duration. These results suggest that FSH plays an important role in stimulating the production of mRNA and protein for the inhibin alpha-subunit in small prehierarchical follicles.     

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.     

Preovulatory biosynthesis and granulosa cell secretion of immunoreactive oxytocin by goat ovaries

Oestrous cycles of goats were synchronized hormonally. Immunoreactive oxytocin was undetectable (less than 0.1 ng/mg protein) in media from granulosa cells isolated before the LH surge for small (1-2 mm), medium (3-5 mm) and large (greater than 5 mm diameter) follicles when cultured for 24 h without or with added hormones. Granulosa cells from large and medium, but not small, follicles isolated 6-12 h after spontaneous preovulatory LH surges secreted high concentrations of oxytocin (4-12 ng/mg protein). Addition of PGE-2 (1 microgram/ml) caused a further significant (P less than 0.05) increase in oxytocin secretion by cultured granulosa cells, whereas PGF-2 alpha, FSH and LH were ineffective when added to culture media. Ovarian venous blood and granulosa cells were collected at 0, 6, 12 or 18 h after GnRH injection in hormonally synchronized goats. Peripheral serum LH values were increased significantly in all but 2 of 22 goats within 2 h of GnRH injection. At the earliest sampling time after GnRH (6 h), ovarian venous levels of oxytocin were increased significantly from basal levels of 0.4 pg/ml to 2.4 pg/ml. Oxytocin concentrations in follicular fluid increased from a basal value of 67 pg/ml to 155 pg/ml by 6 h and to 372 pg/ml by 18 h after GnRH injection. Oxytocin secretion by cultured granulosa cells was not increased significantly by 6 h (0.1 ng/mg protein) but rose to 1.4 and 3.5 ng/mg protein at 12 and 18 h, respectively. Approximately parallel increases occurred in progesterone in ovarian venous blood and granulosa cell culture media over the same time period. (ABSTRACT TRUNCATED AT 250 WORDS)     

Fibronectin stimulates growth but not follicle-stimulating hormone-dependent differentiation of rat granulosa cells in vitro

Since fibronectin is a secretory product of immature rat granulosa cells in culture and may contribute to the follicular microenvironment in vivo, we have studied the effects of this adhesion factor on follicle-stimulating hormone (FSH)-dependent differentiation in short-term (2-3-day) cultures and on growth and protein synthesis in long-term (12-day) cultures. In comparison with cells plated on tissue culture plastic, those plated on an optimal fibronectin-coated substratum showed much greater cell spreading. There were no short-term effects of this morphological change on FSH-stimulation of cyclic AMP production, apparent activities of aromatase or cholesterol side-chain cleavage enzymes, or acquisition of luteinizing hormone (LH) responsiveness in cultured cells. However, progesterone metabolism to 20 alpha-hydroxypregnan-4-en-3-one was increased. Only cultures on fibronectin showed increases between days 3 and 9 in protein (2.5-fold) and DNA (1.4-fold) contents. Cells cultured on fibronectin also showed greater uptake and incorporation of [3H]leucine in comparison with cells cultured on plastic. FSH treatment caused cell aggregation and rounding and delayed the increase in protein content of cells cultured on fibronectin. The results presented demonstrate that the principal direct effect of fibronectin-mediated adhesion on rat granulosa cells is to enhance cell maintenance and growth, while having no generalized action on FSH-dependent differentiation.     

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.     

Oocyte-mediated suppression of follicle-stimulating hormone- and insulin-like growth factor-induced secretion of steroids and inhibin-related proteins by bovine granulosa cells in vitro: possible role of transforming growth factor alpha

The objective was to investigate the potential role of the oocyte in modulating proliferation and basal, FSH-induced and insulin-like growth factor (IGF)-induced secretion of inhibin A (inh A), activin A (act A), follistatin (FS), estradiol (E(2)), and progesterone (P(4)) by mural bovine granulosa cells. Cells from 4- to 6-mm follicles were cultured in serum-free medium containing insulin and androstenedione, and the effects of ovine FSH and IGF analogue (LR3-IGF-1) were tested alone and in the presence of denuded bovine oocytes (2, 8, or 20 per well). Medium was changed every 48 h, cultures were terminated after 144 h, and viable cell number was determined. Results are based on combined data from four independent cultures and are presented for the last time period only when responses were maximal. Both FSH and IGF increased (P < 0.001) secretion of inh A, act A, FS, E(2), and P(4) and raised cell number. In the absence of FSH or IGF, coculture with oocytes had no effect on any of the measured hormones, although cell number was increased up to 1.8-fold (P < 0.0001). Addition of oocytes to FSH-stimulated cells dose-dependently suppressed (P < 0.0001) inh A (6-fold maximum suppression), act A (5.5-fold), FS (3.6-fold), E(2) (4.6-fold), and P(4) (2.4-fold), with suppression increasing with FSH dose. Likewise, oocytes suppressed (P < 0.001) IGF-induced secretion of inh A, act A, FS, and E(2) (P < 0.05) but enhanced IGF-induced P(4) secretion (1.7-fold; P < 0.05). Given the similarity of these oocyte-mediated actions to those we observed previously following epidermal growth factor (EGF) treatment, we used immunocytochemistry to determine whether bovine oocytes express EGF or transforming growth factor (TGF) alpha. Intense staining with TGFalpha antibody (but not with EGF antibody) was detected in oocytes both before and after coculture. Experiments involving addition of TGFalpha to granulosa cells confirmed that the peptide mimicked the effects of oocytes on cell proliferation and on FSH- and IGF-induced hormone secretion. These experiments indicate that bovine oocytes secrete a factor(s) capable of modulating granulosa cell proliferation and responsiveness to FSH and IGF in terms of steroidogenesis and production of inhibin-related peptides, bovine oocytes express TGFalpha but not EGF, and TGFalpha is a prime candidate for mediating the actions of oocytes on bovine granulosa cells.