Regulation of immunoreactive inhibin A and B secretion in cultured human granulosa-luteal cells by gonadotropins, activin A and insulin-like growth factor type-1 receptor

Inhibins are gonadal glycoproteins with endocrine effects on pituitary FSH secretion and para/autocrine effects on ovarian and testicular function. The purpose of this study was to investigate the endocrine and para/autocrine regulation of inhibin A and inhibin B secretion in human ovarian granulosa-luteal cells. The cells were obtained from women undergoing in vitro fertilization, and the primary cultures were treated with FSH, LH, human chorionic gonadotropin (hCG), activin A, 8-bromo cyclic AMP (8-BrcAMP), staurosporine (a protein kinase C inhibitor) and an antagonist of IGF action (type-1 IGF receptor antibody alpha IR3). The secretion of inhibins was measured by ELISA assays capable of reliably distinguishing between inhibin A and B. FSH, LH, hCG and 8-BrcAMP increased inhibin A secretion on average up to 180% (P<0.01), 192% (P<0.05), 210% (P<0.01) and 243% (P<0.01) respectively of the control level, while their stimulatory effect on inhibin B secretion was less pronounced (up to 167%, P<0.01; 139%, P<0.05; 127%, P>0.05; 133%, P>0.05 of the controls respectively). alpha IR3 decreased inhibin A and B secretion down to 70% (P<0.01) and 50% (P<0.01) respectively of the control. Staurosporine decreased inhibin B secretion down to 49% (P<0.01) of the control; its effect on inhibin A secretion was not significant. Activin A increased inhibin B secretion up to fourfold of the control (P<0.05) while its effect on inhibin A secretion was insignificant. We conclude that gonadotropins via the protein kinase A signal transduction pathway are the main positive regulators of inhibin A and B secretion in human granulosa-luteal cells. The protein kinase C signal transduction pathway seems to be important especially for inhibin B secretion. Locally produced IGFs are probably important inducers of the production of both forms of inhibin in human ovaries while activins seem to upregulate inhibin B secretion.     

Effects of activin and follicle-stimulating hormone (FSH)-suppressing protein/follistatin on FSH receptors and differentiation of cultured rat granulosa cells.

The aim of this study was to investigate the actions of both activin and FSH-suppressing protein (FSP)/follistatin either alone or in combination on FSH receptor number and on the responsiveness of granulosa cells to FSH and LH. Granulosa cells were harvested from diethylstilbestrol-treated immature Sprague-Dawley rats and cultured 48 h in serum-free medium with or without treatment. Activin treatment alone (3-100 ng/ml) resulted in a 4-fold increase in FSH receptor number with no change in binding affinity. This effect of activin was inhibited 31% by FSP (100 ng/ml) treatment which alone had no effect on FSH receptor number. Treatment with activin (100 ng/ml) prevented FSH-induced down-regulation of FSH receptor number, whereas at lower concentrations (3-30 ng/ml) activin enhanced down-regulation of FSH receptor number by 20% (P less than 0.05). In contrast, FSP alone prevented FSH-induced down-regulation by increasing FSH receptor number up to 40-50%. Pretreatment of granulosa cells with activin, but not FSP, for 24 h increased the responsiveness of cells to FSH (20 ng/ml) and LH (40 ng/ml) shown by increases in aromatase activity, progesterone, and immunoreactive inhibin production over and above control in a manner which depended upon activin doses. We conclude that 1) activin enhancement of FSH action on rat granulosa cells may be mediated in part via regulation of FSH receptor number, and 2) the effects of FSP on granulosa cells are likely to be due to its activin binding properties.     

Inhibin/activin subunits are costored with FSH and LH in secretory granules of the rat anterior pituitary gland.

We recently reported that pituitary gonadotropes, major targets of circulating inhibins and activins, are also capable of synthesizing the inhibin (I) alpha- and inhibin/activin (I/A) beta B-subunits. In the present study, we examined the subcellular distribution of these subunits, with special attention given to determinating the extent to which they might be colocalized with the gonadotropins in secretory granules. Pituitaries from adult male rats were cryofixed, molecular distillation-dried, and resin-embedded. Immunogold staining methods were used to examine concurrently the distributions of an I/A subunit and FSH or LH. I/A subunits were detected only in cells that also labeled positively for a gonadotropin, and, in contrast to the gonadotropins, were sequestered almost exclusively within secretory granules. The I alpha-subunit colocalized with FSH in 31%, and with LH in 36%, of all positively stained granules. The I/A beta B-subunit was found with FSH or LH in about 25% of the granules. Approximately 52-69% of the granules contained FSH or LH alone; 7-18% were positive only for an I/A subunit, and this varied as a function of the particular gonadotropin with which costaining was carried out. Dual staining for the I alpha- and the I/A beta B-subunits indicated that at least 35% of all immunolabeled granules showed positive signals for both subunits. Coupled with methodological considerations to indicate that these estimates of the extent of colocalization are likely to be conservative, these data suggest that inhibin and activin are characteristically copackaged, and presumably coreleased, with the gonadotropins.     

Variation in pituitary expression of mRNAs encoding the putative inhibin co-receptor (betaglycan) and type-I and type-II activin receptors during the chicken ovulatory cycle

Secretion of LH and FSH from the anterior pituitary is regulated primarily by hypothalamic GnRH and ovarian steroid hormones. More recent evidence indicates regulatory roles for certain members of the transforming growth factor beta (TGFbeta) superfamily including inhibin and activin. The aim of this study was to identify expression of mRNAs encoding key receptors and ligands of the inhibin/activin system in the hen pituitary gland and to monitor their expression throughout the 24-25-h ovulatory cycle. Hens maintained on long days (16 h light/8 h dark) were killed 20, 12, 6 and 2 h before predicted ovulation of a midsequence egg (n = 8 per group). Anterior pituitary glands were removed, RNA extracted and cDNA synthesized. Plasma concentrations of LH, FSH, progesterone and inhibin A were measured. Real-time quantitative PCR was used to quantify pituitary expression of mRNAs encoding betaglycan, activin receptor (ActR) subtypes (type I, IIA), GnRH receptor (GnRH-R), LH beta subunit, FSH beta subunit and GAPDH. Levels of mRNA for inhibin/activin betaA and betaB subunits, inhibin alpha subunit, follistatin and ActRIIB mRNA in pituitary were undetectable by quantitative PCR (<2 amol/reaction). Significant changes in expression (P<0.05) of ActRIIA and betaglycan mRNA were found, both peaking 6 h before ovulation just prior to the preovulatory LH surge and reaching a nadir 2 h before ovulation, just after the LH surge. There were no significant changes in expression of ActRI mRNA throughout the cycle although values were correlated with mRNA levels for both ActRIIA (r = 0.77; P<0.001) and beta-glycan (r = 0.45; P<0.01). Expression of GnRH-R mRNA was lowest 20 h before ovulation and highest (P<0.05) 6 h before ovulation; values were weakly correlated with betaglycan (r = 0.33; P = 0.06) and ActRIIA (r = 0.34; P = 0.06) mRNA levels. Expression of mRNAs encoding LH beta and FSH beta subunit were both lowest (P<0.05) after the LH surge, 2 h before ovulation. These results are consistent with an endocrine, but not a local intrapituitary, role of inhibin-related proteins in modulating gonadotroph function during the ovulatory cycle of the hen, potentially through interaction with betaglycan and ActRIIA. In contrast to mammals, intrapituitary expression of inhibin/activin subunits and follistatin appears to be extremely low or absent in the domestic fowl.     

Inverse correlation between baseline inhibin B and FSH after stimulation with GnRH: a study of serum levels of inhibin A and B, pro alpha-C and activin A in women with ovulatory disturbances before and after stimulation with GnRH

OBJECTIVE: Interest has focused recently on the influences of the polypeptide factors inhibin and activin on the selective regulation of the pituitary secretion of gonadotropins. DESIGN: Measurement of the concentrations of inhibin-related proteins in relation to the changes in pituitary gonadotropin (FSH, LH) parameters, after GnRH stimulation with a bolus injection of 100 microg gonadorelin, in 19 women with ovulatory disturbances. METHODS: Serum levels of inhibin A and B, activin A, and pro alpha-C were measured using sensitive ELISA kits. RESULTS: Within 60 min after GnRH stimulation, FSH values doubled from 5 to 10 mU/ml (P < 0.001). LH increased 12-fold from 2 to 24 mU/ml (P < 0.001). Activin A showed a significant decrease from 0.47 to 0.36 ng/ml (P < 0.001), whereas pro alpha-C increased from 127 to 156 pg/ml (P = 0.039). The median inhibin A concentration did not show a significant change between baseline and the 60 min value, whereas inhibin B was characterized by a minor, but not significant, increase in the median from 168 to 179 pg/ml (P = 0.408). A significant inverse correlation (P = 0.014) with a mean coefficient of correlation of 0.5516 was found, demonstrating a strong relationship between high inhibin B baseline levels and a small increase of FSH after 60 min. CONCLUSION: Our results show an interesting correlation between the baseline inhibin B and the change in FSH before and after GnRH stimulation. A high baseline inhibin B implies only a minor increase of FSH after 60 min.     

Relative effects of activin and inhibin on steroid hormone synthesis in primate granulosa cells.

Ovarian granulosa cells produce inhibin and activin, structurally related proteins with potentials to directly modulate follicular steroidogenesis. The aim of the present study was to compare development-related effects of inhibin-A and activin-A on steroidogenesis in marmoset monkey (Callithrix jacchus) granulosa cells. Granulosa cells from "immature" (< 1.0 mm diameter) and "mature" (> 2 mm diameter) follicles were incubated in serum-free culture medium for 96 h with and without peptide (1-100 ng/mL), in the presence and absence of gonadotropins [human (h) FSH or hLH] (10 ng/mL). Spent medium was collected and stored frozen for progesterone assay. Aromatase activity was determined by incubating cells for a further 6 h in the presence of 1 mumol testosterone and assaying accumulation of oestradiol. Granulosa cells from immature follicles showed characteristically low basal rates of steroid synthesis that were unaffected by treatment alone with either inhibin or activin. Treatment with hFSH stimulated both progesterone production and aromatase activity. Cotreatment with activin and hFSH further enhanced aromatase activity by up to 4-fold. The progesterone response to activin plus hFSH was related to the effect of hFSH in the absence of activin: high-level responsiveness to hFSH was suppressed by activin while low-level responsiveness was enhanced. Inhibin had no significant effect on FSH-responsive progesterone production, but at high concentrations (> 10 ng/mL) it caused slight (up to 30%) reduction in FSH-induced aromatase activity. Granulosa cells from mature follicles showed relatively high basal rates of steroidogenesis, and treatment with inhibin did not influence either basal or gonadotropin responsive steroidogenesis. Treatment with activin had divergent effects on aromatase activity and progesterone synthesis in that it increased both basal and hLH-responsive aromatase activity (up to 11-fold), had no effect on basal progesterone production, and markedly suppressed (by more than 50%) the progesterone response to hLH. These data reveal development-dependent effects of inhibin and activin on granulosa cell steroidogenesis that are likely to have physiological relevance to ovarian function in vivo.     

Regulation of inhibin/activin expression in rat early antral follicles

The aim of the present study was to determine the endocrine activity of cultured early antral follicles (EAF) isolated from prepubertal diethylstilbestrol-treated rats. The effect of steroidogenic substrates and FSH on steroid, inhibin A and B, Pro-αC and activin A production was evaluated. Androsterone was the predominant steroid produced by EAF. The addition of androstenedione, androstenedione + FSH and progesterone stimulated oestradiol production, whereas 25-hydroxycholesterol (25-OH-Chol) increased progesterone production. Inhibin A, B, Pro-αC, and activin A were produced under basal conditions. The predominance of inhibin B over inhibin A was not affected by the addition of androstenedione or progesterone. Inhibin A and activin A production was stimulated by FSH. 25-OH-Chol increased Inha, Inhba and Inhbb mRNA expression and the production of the three molecular forms of inhibins but decreased activin A production. These results show that FSH and the steroid follicular microenvironment differentially modulate the gene expression of inhibin/activin subunits, their assembly and secretion.     

Occurrence of immunoreactive Activin/Inhibin beta(B) in thyrotropes and gonadotropes in the bullfrog pituitary: possible Paracrine/Autocrine effects of activin B on gonadotropin secretion

Occurrence of immunoreactive activin/inhibin beta(B) in the bullfrog (Rana catesbeiana) pituitary was investigated immunocytochemically by use of antibody against Xenopus activin/inhibin beta(B) subunit. Thyrotropes were demonstrated to contain activin/inhibin beta(B)-immunoreactive substances. Moreover, immunoelectron microscopy revealed that in the secretory granules of thyrotropes and, to a lesser extent, in those of gonadotropes, activin/inhibin beta(B)-immunoreactive substances were present. Based on this observation, we investigated the effect of activin B on the release of gonadotropins from dispersed anterior pituitary cells of the bullfrog. Activin B stimulated the release of not only follicle-stimulating hormone (FSH) but also luteinizing hormone (LH) dose dependently. Under the culture conditions used in this experiment, inhibin B, as well as follistatin, did not affect the basal levels of LH and FSH, but they suppressed the activin-induced release of these hormones. This is the first study on the effect of activin on pituitary hormone secretion in lower tetrapods.     

Direct inhibitory effect of gonadotropin-releasing hormone upon luteal luteinizing hormone receptor and steroidogenesis in hypophysectomized rats

The effects of gonadotropin-releasing hormone (GnRH) and its potent agonist [des-Gly10, D-Leu6-N alpha Me) Leu7, Pro9,NHEt-GnRH (GnRH-A)] on ovarian luteal functions maintained by PRL were studied in vivo and in vitro. Hypophysectomized, diethylstilbestrol-treated female rats were primed with FSH for 2 days, followed by an ovulating dose of LH or hCG. Two days later, ovarian luteal functions were maintained by daily injections of 250 microgram PRL for 3 days. PRL treatment increased the serum progesterone level from 13.0 +/- 0.5 to 298 +/- 24 ng/ml and increased the ovarian hCG-binding capacity from 5.8 +/- 1.3 to 584 +/- 86 ng bound hCG/ovary. In contrast, concomitant treatment with GnRH or GnRH-A resulted in dose-dependent decreases in the PRL-induced increase of serum progesterone and ovarian LH/hCG receptor content. GnRH at 100 microgram/day caused a 60% decrease in serum progesterone and an 80% decrease in ovarian LH receptor content, whereas GnRH-A was effective at a 1-microgram dose level. Neither GnRH nor GnRH-A affected the binding affinity (Kd) of ovarian LH receptor. The direct inhibitory effects of GnRH and GnRH-A upon granulosa-luteal cell function were also tested in vitro. FSH treatment for 2 days induced functional LH and PRL receptors in cultured PRL, increased (by approximately 3-fold) progesterone production by these granulosa-luteal cells, whereas concomitant treatment with GnRH-A inhibited progesterone production in a dose-dependent manner. Thus, these studes demonstrated that GnRH and GnRH-A exert direct inhibition on ovarian luteal functions by decreasing LH receptor and progesterone production in vivo as well as inhibiting progesterone production by cultured granulosa-luteal cells in vitro.     

Activin A serum levels and aging of the pituitary-gonadal axis: a cross-sectional study in middle-aged and elderly healthy subjects

Aim of the study was to investigate activin A serum concentration in healthy adult males and post-menopausal females over a wide age-range and its correlation to gonadotropins, inhibin B and testosterone concentrations. The study included 73 males (aged 30-101 years) and 42 postmenopausal females (aged 50-104 years). Blood samples were collected after an overnight fast to measure serum activin A, inhibin B, LH, FSH, and gonadal steroid levels. A significant increase in serum activin A levels over age in both genders, especially in the oldest age-groups, was observed. Serum inhibin B and testosterone concentrations showed a sharp decrease in male subjects, reflecting the age-related decrease of testicular function and by consequence serum FSH and LH significantly increased. In female subjects LH and FSH levels were very high in subjects in their 50s and showed a continuous decline due to pituitary aging. Simple and multivariable regression analyses demonstrated the lack of correlation between activin A and FSH in both males and females. In conclusion, a steep increase in activin A levels is present during aging in both genders, especially in the last decades of life. The physiologic role and site of production of activin A in old subjects remain to be clarified.     

Anastomosis of the ovarian vein to the hepatic portal vein in sheep induces ovarian hyperstimulation associated with increased LH pulsatility, but only in the absence of the contralateral ovary

In this study, two experiments were performed, the first of which examined the ovarian response in ewes that were subject to unilateral ovariectomy (ULO) at different intervals (0-14 days) after surgical anastomosis (AN) of the ovarian vein to the mesenteric vein (n=7 ewes), or sham operation (SO; n=4 ewes). Hypertrophy and development of multiple follicular and luteal structures on AN ovaries were observed after ULO, while SO ovaries remained of normal size and appearance after ULO. The second experiment involving 11 ewes (five AN; six SO) aimed to clarify the mechanism by which AN following ULO-induced ovarian hypertrophy and increased follicle development. The results confirmed that there were more large (>5 mm) follicles on AN compared with SO ovaries; however, their rate of atresia was similar. Oestradiol and progesterone concentrations in follicular fluid of class 1 follicles (5-9 mm) were higher in AN ovaries than those in control follicles of the same size collected in the late follicular phase of an induced oestrous cycle. In AN ewes, intrafollicular progesterone concentrations increased while follicular aromatase activity and intrafollicular oestradiol, inhibin A, follistatin and activin A concentrations all decreased as follicle size increased. Oestradiol and progesterone concentrations were substantially higher in ovarian venous blood than in hepatic venous blood, both in AN and SO ewes, whereas inhibin A levels were not significantly modified by passage through the liver in either group. Mean plasma LH concentration, and LH pulse frequency and amplitude increased markedly after AN but were not affected by SO. Plasma FSH showed only a small transient increase after AN, presumably due to the maintenance of inhibin feedback. Injection of prostaglandin F(2)(alpha) 4 days later did not further modify LH or FSH secretion in either group. Full ovariectomy (FO) 9-14 days after AN or SO increased LH secretion markedly in SO ewes but to a lesser degree in AN ewes; FO induced a large and rapid increase in FSH levels in both groups. In conclusion, AN of the ovary to the liver via the mesenteric vein provides a useful model for studying the feedback between the ovary and the hypothalamo-pituitary system and the mechanisms controlling follicle development. The present results indicate that the pattern of LH secretion is an important factor controlling the terminal phase of follicle development in the ewe.     

Interactions between activin and follicle-stimulating hormone-suppressing protein and their mechanisms of action on cultured rat granulosa cells

Direct roles of follicle-stimulating hormone (FSH)-suppressing protein (FSP) and activin in regulation of ovarian granulosa cell differentiation have been reported recently. The present study further investigated the effects of these peptides on steroidogenesis and inhibin production as well as cAMP generation in cultured granulosa cells from immature, diethylstilbestrol (DES)-treated rats. In the presence of FSH (20 ng/ml) and activin (30 ng/ml), which enhanced FSH-induced aromatase activity, progesterone production and inhibin production, FSP (1-100 ng/ml) reversed the stimulating activities of activin in a dose-dependent manner. In addition, activin reversed the inhibitory effects of FSP on FSH-induced aromatase activity and inhibin production. In the presence of FSH, activin enhanced FSH-stimulated extracellular cAMP accumulation, and FSP caused a reduction in extracellular cAMP. Activin but not FSP also stimulated basal cAMP level. In the presence of forskolin, a potent stimulant of adenyl cyclase activity which stimulated extracellular cAMP, aromatase activity, progesterone production and inhibin production, activin augmented the effect of forskolin on all four parameters, whereas FSP significantly enhanced progesterone production without changing the other three parameters. Our findings suggest that activin action on rat granulosa cells may be mediated via regulation of cAMP generation. The action of FSP and FSH and/or activin-dependent, consistent with either an action as an activin binding protein or by a direct action of FSP on the granulosa cells.     

The effects of activin on follicle-stimulating hormone secretion and biosynthesis in human glycoprotein hormone-producing pituitary adenomas

The effects of activin on pituitary FSH biosynthesis have been previously characterized using primary rat pituitary cultures; however, little is known of the effects of activin on FSH biosynthesis and secretion in human pituitary tissue. Production of intact glycoprotein hormones and free subunits is increasingly recognized in pituitary tumors; however, the regulation of gonadotropins in such tumors has not been addressed. We have investigated the effects of human recombinant activin on glycoprotein hormone biosynthesis and secretion in primary cultures of 12 human glycoprotein hormone-producing pituitary adenomas and compared this with the effects of activin in normal rat anterior pituitary cells. In 33% of the human pituitary tumors studied, significant (P less than 0.05) increases in FSH beta secretion occurred in response to incubation with 20 ng/mL activin for 24 h (19-287% stimulation), without changes in the production of intact FSH. A Northern analysis performed on cells derived from one tumor indicated that FSH beta mRNA levels increased 350% after activin treatments; however, FSH secretion did not parallel the mRNA changes. None of the human glycoprotein hormone-producing tumors significantly increased FSH secretion in response to activin. To validate the biological activity of recombinant human activin-A and to confirm time and dose conditions for the human tumor cultures, we also examined its ability to stimulate FSH production in rat pituitary cultures. Activin (20 ng/mL) added to the culture medium significantly increased FSH secretion and steady state levels of FSH beta mRNA after 24 h. These data indicate that some glycoprotein hormone-producing pituitary tumors treated with purified activin have discordant responses of intact gonadotropins and free subunit responses. In contrast to responses in normal rat gonadotrophs, FSH beta biosynthetic pathways may be uncoupled from intact FSH secretion in a subset of glycoprotein hormone-producing pituitary adenomas.     

Regulation of activin A and inhibin B secretion by inflammatory mediators in adult rat Sertoli cell cultures

The regulation of Sertoli cell activin A and inhibin B secretion during inflammation was investigated in vitro. Adult rat Sertoli cells were incubated with the inflammatory mediators, lipopolysaccharide (LPS), interleukin-1beta (IL-1beta), IL-6 and the IL-1 receptor antagonist (IL-1ra) over 48 h in culture. Activin A, inhibin B and IL-1alpha were measured in the culture medium by specific two-site ELISAs. Both IL-1beta- and LPS-stimulated activin A and inhibited inhibin B secretion. LPS also stimulated the production of IL-1alpha in the cultures. In contrast to IL-1beta, IL-6 had no effect on activin A, although it did have a significant inhibitory effect on inhibin B secretion. Ovine follicle-stimulating hormone (FSH) and the cAMP analogue dibutyryl cAMP opposed the actions of IL-1 and LPS by suppressing activin A and IL-1alpha secretion and by stimulating inhibin B. Blocking IL-1 activity in the cultures by addition of an excess of IL-1ra completely prevented the response of activin A to exogenous IL-1beta, and reduced the response to LPS by 50%. In the presence of IL-1ra, basal secretion of inhibin B was increased, but IL-1ra was unable to reverse the suppression of inhibin B by LPS. These data indicate the importance of both IL-1 isoforms in regulating secretion of activin A and inhibin B by mature Sertoli cells during inflammation. The data also establish that inflammation exerts its effects on activin A and inhibin B secretion via other pathways in addition to those mediated by IL-1, and that hormonal stimulation by FSH and cAMP moderates the Sertoli cell response to inflammation. Interference with the complex interactions between these cytokines and hormones may contribute to the disruption of reproductive function that can accompany infection and illness in men.     

Effect of activin A and inhibin A on expression of the inhibin/activin beta-B-subunit and gonadotropin receptors in granulosa cells of the hen

Activin A has been shown to be abundant in the theca layer of the large pre-ovulatory follicles of the hen whereas inhibin A is produced in the granulosa layer. The purpose of this study was to investigate the effects of activin A and inhibin A on granulosa cell expression of inhibin beta-B-subunit, FSH receptor (FSHR), and LH receptor (LHR). Granulosa cells were isolated from the F1, F3+F4, and small yellow follicles (SYF; 6-12 mm diameter) of laying hens and pooled according to size. The cells were dispersed and plated in the presence of 0, 10, or 50 ng/ml recombinant human activin A (n=5 replicate cultures). RNA was subsequently extracted from the cells and Northern blots performed. Cell proliferation was determined for all treatments. An identical set of experiments was performed in which the granulosa cells were treated with recombinant human inhibin A (n=4 replicate cultures). Treatment with activin A at 50 ng/ml significantly (p<0.05) increased expression of beta-B-subunit for granulosa cells from all follicles. This dose also significantly increased expression of FSHR in granulosa cells from all follicles (p<0.05) and increased expression of LHR in cells from F1 and F3+F4 follicles (p<0.01) with no significant effect on cells from the SYF. Overall, activin A treatment significantly (p<0.05) decreased cell proliferation at the 50 ng/ml dose. Inhibin A had no significant effect on expression of beta-B-subunit, FSHR or LHR at any dose. There was a moderate stimulatory effect of inhibin A on granulosa cell proliferation. These results suggest that activin A may have an important role in regulating granulosa cell responsiveness to gonadotropins while also modulating follicle development by attenuating cell proliferation.     

Inverse effects of activin and inhibin on the synthesis and secretion of FSH and LH by ovine pituitary cells in vitro

Little information is available on the effects of activin and inhibin on the synthesis and secretion of pituitary gonadotrophins in species other than the rat. In this in-vitro study, ovine pituitary cell cultures derived from immature sheep were used to investigate the effects of recombinant human activin-A and native Mr 32,000 bovine inhibin on basal and gonadotrophin-releasing hormone (GnRH)-induced release of FSH and LH. Residual cellular contents of FSH and LH were also determined, allowing total content/well to be calculated. Activin-A promoted a dose-dependent increase in basal (+72%; P less than 0.001) and GnRH-induced (+25%; P less than 0.001) release of FSH as well as in the residual cell content (+114%; P less than 0.001) and total FSH content/well (+67%; P less than 0.001). Conversely, inhibin significantly (P less than 0.001) suppressed each aspect of FSH production examined, confirming that in sheep, as in rats, activin and inhibin exert opposing effects on pituitary FSH production. In contrast to the rat, however, in which activin is reported to have no effect on LH secretion, exposure of sheep pituitary cells to activin-A promoted a dose-dependent suppression (-42%; P less than 0.001) of GnRH-induced LH release. This was associated with a corresponding increase (P less than 0.001) in residual cellular content of LH. Consistent with a previous report from this laboratory, inhibin had the opposite effect and significantly enhanced (+47%; P less than 0.001) GnRH-induced LH release. This was associated with a corresponding fall (P less than 0.01) in residual cellular content of LH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pituitary follistatin and activin gene expression, and the testicular regulation of FSH in the adult Rhesus monkey (Macaca mulatta).

In rats, FSHbeta gene expression and FSH secretion are increased and decreased, respectively, by pituitary activin and follistatin. Because little information is available on the paracrine control of FSH secretion in the primate, follistatin and activin/inhibin beta(B) messenger RNA (mRNA) levels were measured in pituitaries of adult male rhesus monkeys 6 weeks after castration or sham surgery (n = 5/group). Follistatin mRNA was determined by quantitative RT-PCR assay using oligonucleotide primers designed to span exons 3-5 of the human follistatin gene. Activin/inhibin beta(B) mRNA levels were measured by ribonuclease protection. Orchidectomy resulted in a 100-fold increase in plasma FSH concentrations and a 60-fold rise in those of LH. In castrated monkeys, levels of mRNA encoding FSHbeta, LHbeta, alpha- subunit, and GnRH receptor (GnRH-R) were increased 21-, 2.1-, 1.7-, and 1.7-fold, respectively (P < 0.01). Levels of pituitary follistatin and activin/inhibin beta(B) mRNAs, however, were similar in castrated and intact animals. These data suggest that the paracrine control of FSH secretion in the male differs substantially in primates and rodents. Specifically, the relatively greater postcastration rise in FSHbeta gene expression and FSH secretion in the adult male monkey may result because in this species pituitary follistatin gene expression does not increase after orchidectomy, as it does in the rat.     

Ovine follicular fluid suppresses the ovarian secretion of androgens, oestradiol and inhibin

An experiment was conducted to examine the effect of steroid-free ovine follicular fluid (oFF) on ovarian hormone secretion. Eight Merino x Finnish Landrace ewes in which the left ovary and vascular pedicle had been autotransplanted to a site in the neck were studied during the breeding season. Luteal regression was induced in all animals by injection of cloprostenol (100 micrograms, i.m.) on day 10 of the luteal phase. Four of the eight animals were treated with steroid-free oFF (3 ml, s.c.) in the early follicular phase, 24 and 36 h after injection of cloprostenol. Samples of both ovarian and jugular venous blood were collected at 4-h intervals from 20 h before until 96 h after injection of cloprostenol. Ovarian and jugular venous blood samples were also collected at 10-min intervals from 48 to 52 h after injection of cloprostenol to investigate the pattern of pulsatile secretion of ovarian hormones. Samples were assayed for oestradiol, androstenedione, testosterone and inhibin and the ovarian secretion rates calculated. Both injections of oFF resulted in a fourfold increase in the concentration of inhibin in jugular venous plasma within 4-8 h of administration (P less than 0.01) with concentrations remaining increased (P less than 0.05) until 56 h after cloprostenol (32 h after the first oFF injection). Following oFF injection there was a profound (100%; P less than 0.001) and prolonged decrease in the peripheral concentration of FSH until 60 h after cloprostenol at which time the concentration of FSH increased five- to sixfold (P less than 0.001) to a peak lasting 24 h. In contrast to FSH, the concentration of LH in jugular venous plasma rose immediately following oFF treatment and continued to increase, exhibiting a profile similar to that described for FSH. No preovulatory LH surge was detected in any of the oFF-treated ewes while untreated ewes had an LH surge within 58.0 +/- 1.2 (S.E.M.) h. Within 8 h of the first injection of oFF the ovarian secretion rate of oestradiol, androstenedione and inhibin began to decline to reach a nadir of less than 1 ng/min within 32-36 h (56-60 h after cloprostenol; P less than 0.01). Testosterone secretion, already barely detectable, did not change significantly following injection of oFF but remained low for 36 h following oFF and did not exhibit the increase observed over this period in controls. After injection of oFF the episodic secretion of oestradiol, androstenedione, testosterone and inhibin was markedly suppressed in spite of numerous pulses of LH.(ABSTRACT TRUNCATED AT 400 WORDS)     

Age-related analysis of inhibin A, inhibin B, and activin a relative to the intercycle monotropic follicle-stimulating hormone rise in normal ovulatory women

Previous studies have reported that the monotropic rise in FSH in older women is associated with decreased inhibin B and/or A levels and increased levels of activin A. Whereas most investigators have found decreased follicular-phase inhibin B, the roles of inhibin A and activin A as modulators of the FSH rise are unclear. The objectives of this study were to determine whether deficiencies in circulating levels of inhibin A, inhibin B, and/or activin A exist during the intercycle interval in ovulatory older (age, 40-45 yr; n = 16), compared with younger women (age, 20-25 yr; n = 13). Blood samples were obtained daily throughout one menstrual cycle and the follicular phase of the subsequent cycle and were analyzed for LH, FSH, estradiol, inhibin A and B, and activin A. Despite significant FSH elevation, no deficiencies in inhibin A, activin A, or estradiol were detected in older subjects. In fact, inhibin A was significantly higher in older participants during the intercycle phase (P = 0.01), whereas inhibin B was significantly lower. Thus, the monotropic rise in FSH does not appear to result from changes in inhibin A or activin A, supporting the concept that inhibin B plays a critical role in mediating the FSH rise in older women.     

Gonadotropins and inhibins along the development of a luteinized rat ovarian tumor

Luteinized intrasplenic ovarian tumors develop in response to high circulating gonadotropins. The relationship between tumor development, gonadotropins and inhibins was studied. Tumor-bearing animals were sacrificed weekly along the first 6 weeks of development. Inhibins were measured by enzyme-linked immunosorbent assay (ELISA), serum gonadotropins, GH and IGF-1 by RIA. Inhibin subunit mRNAs were determined by Northern blot. Tumor histology was examined. Ovarian grafts grew significantly along development. LH increased ten-fold on week 1; a further significant increment was observed on week 3. FSH peaked on weeks 1 and 2 and fell significantly thereafter. Serum inhibins markedly increased on weeks 3-5. Tumor inhibin A content and mRNA levels for alpha and beta A subunits also increased on week 3. Inverse correlations between inhibins and FSH and direct correlations between inhibins and LH were observed. Tumor inhibin A and IGF-1 contents correlated significantly. Increasing levels of luteinization were observed along tumor development. These luteinized tumors develop mainly in response to LH, since growth continues under FSH inhibition. The active inhibin secretion and the positive correlation between inhibins and LH suggests that LH may be the main driving force behind this production, while growth factors produced by the gonads may also participate in their regulation.