Regulation of the synthetic rate of gonadotropin-releasing hormone receptors in rat pituitary cell cultures by inhibin

Regulation of steady state levels of plasma membrane receptors for GnRH is the arithmetic result of processes that contribute to the appearance of receptors (synthesis, recycling, and unmasking) less those that contribute to the loss of receptors (degradation, internalization, and inactivation). We have adapted the density shift technique to evaluate specifically the rate of synthesis of GnRH receptors in rat pituitary cell cultures. Recently, it has been shown that inhibin can decrease the steady state levels of GnRH receptors in rat pituitary cell cultures and can block homologous up-regulation of GnRH receptors. In the present study we have evaluated the ability of purified inhibin to affect the synthesis rate of GnRH receptors under basal conditions and after exposure of cultured gonadotropes (from female weanling rats) to GnRH. Cells were exposed to inhibin alone (4 or 12 ng/ml) or to GnRH (10(-10) M) plus inhibin (0.4, 4, or 12 ng/ml) in the presence of densely labeled amino acids. GnRH was administered as a 20-min pulse, but inhibin treatment was continued for up to 2 days. After these treatments, GnRH receptors were covalently linked to a radio-labeled photoaffinity probe (125I- Tyr5-[azido-benzoyl-D-Lys6] GnRH) and solubilized with 1% sodium dodecyl sulfate. Newly synthesized GnRH receptors (those that had incorporated the dense amino acids) were separated from previously synthesized receptors (those containing normal amino acids) by velocity sedimentation through sucrose gradients (O-20% sucrose, 1% sodium dodecyl sulfate, and 10 mM Tris-HCl, pH 7.0; centrifuged at 156,000 x g for 24 h). After velocity sedimentation, gradients were fractionated, and the radioactivity in each fraction was quantified. Treatment with inhibin alone had no effect on the synthesis rate of GnRH receptors compared to that of control cultures (t1/2, 23.5 +/- 0.3 vs. 23.3 +/- 0.3 vs. 22.9 +/- 0.9 h for control, 4 ng/ml inhibin, and 12 ng/ml inhibin, respectively). In contrast, inhibin blocked the stimulation of homologous receptor synthesis by GnRH in a dose-dependent manner (t1/2, 12.2 +/- 0.7 vs. 14.0 +/- 0.7 vs. 19.2 +/- 1.5 vs. 20.0 +/- 2.9 h for GnRH alone and GnRH plus 0.4, 4, or 12 ng/ml inhibin, respectively). These data indicate that in rat pituitary cell cultures, inhibin does not decrease basal levels of GnRH receptors by affecting the synthesis rate of receptors, but prevents up-regulation of GnRH receptors by blocking stimulation of GnRH receptor synthesis by homologous hormone.     

Effects of porcine follicular fluid, inhibin-A, and activin-A on goldfish gonadotropin release in vitro.

Inhibin and activin are important reproductive regulators in mammalian species and have been demonstrated to be highly conserved in structure. The present study examines the effects of porcine follicular fluid (pFF; a crude inhibin and activin preparation) and purified porcine inhibin-A and activin-A on goldfish gonadotropin-II (GTH-II) release. In studies using primary cultures of dispersed goldfish pituitary cells in static incubation, treatments with pFF, inhibin-A, and activin-A for 10 h caused dose-dependent increase in GTH-II release. In perifusion studies using goldfish pituitary fragments, basal GTH-II release was significantly elevated after 12-h exposure to 500 micrograms/ml pFF. Furthermore, GnRH-induced GTH-II secretion was potentiated by pretreatment with pFF. When pFF was applied in the form of 5-min pulses, a rapid dose-related stimulation of GTH-II was observed. Similarly, challenges with 2-min pulses of 15, 150, and 1500 pM inhibin-A and activin-A stimulated GTH-II release by goldfish pituitary fragments in a rapid and dose-dependent manner. This acute stimulatory action of inhibin on goldfish GTH-II release was completely abolished after pretreatment with specific inhibin antibodies. The acute actions of inhibin and activin on GTH-II release are probably not due to the release of endogenous GnRH from nerve terminals in the pituitary fragments or binding to the GnRH receptors. First, a specific GnRH antagonist did not block the actions of inhibin and activin. Second, dopamine, a potent inhibitor of GnRH-stimulated GTH-II secretion in goldfish, was only partially effective in decreasing inhibin- and activin-induced GTH-II release. Third, the stimulatory effects of inhibin and GnRH on GTH-II release were additive. These lines of evidence also indicate that the mechanisms mediating inhibin and activin stimulation of goldfish GTH-II release may be somewhat different from those of GnRH. These results demonstrate that in contrast with the usual inhibitory effects of inhibin on GTH release in mammals, both inhibin and activin exert long term and acute stimulatory actions on GTH-II release in the goldfish.     

Activin-A modulates gonadotropin-releasing hormone secretion from a gonadotropin-releasing hormone-secreting neuronal cell line.

The recent development of GnRH-secreting neuronal cell lines (GT1-1, GT1-3 and GT1-7 clones) has provided a model system for the study of the neural regulation of GnRH expression and secretion. We report here that activin-A stimulates GnRH secretion by GT1-7 cells in a dose-dependent manner, with an EC50 of approximately 2.5 ng/ml. The maximal response (50% stimulation) was achieved after 2 days of incubation with 20 ng/ml activin-A. Activin-A treatment increased total GnRH (secreted + cellular) in GT1-7 cells, possibly reflecting a stimulation of GnRH biosynthetic rates. The secretory effect of activin-A was also accompanied by a change in the cellular morphology to a more neuronal phenotype. The addition of TGF-beta (10 ng/ml), which is structurally related to activins, did not significantly increase secretion of GnRH by GT1-7 cells illustrating the specificity of the activin effect on this cell line. Although inhibin (20 ng/ml) alone did not directly affect the spontaneous secretion of GnRH, it was able to partially block the stimulatory effect of activin. The present study with the GT1-7 clonal cell line suggests that activin, and perhaps inhibin, might act at hypothalamic sites to regulate reproduction through the control of GnRH production and/or secretion.     

Effect of recombinant inhibin on androgen synthesis in cultured human thecal cells

Effects of inhibin (recombinant human inhibin-A) on ovarian androgen synthesis were tested in vitro using serum-free monolayer cultures of human thecal cells. Treatment for 4 days with inhibin alone at doses between 10 and 100 ng/ml caused modest (approximately 2-fold) increases in production of androgen (androstenedione and dehydroepiandrosterone): similar to the maximal level of stimulation caused by luteinizing hormone (LH) (10 ng/ml) alone but only about one-third of that caused by insulin-like growth factor I (IGF-I) (30 ng/ml) alone. Combined treatment with LH and inhibin elicited additive effects on androgen production whereas LH and IGF-I were synergistic, giving rise to androgen production rates at least 40 times greater than control. Additional presence of inhibin caused up to 10-fold augmentation of the response to LH + IGF-I. Activin (recombinant human activin-A) was previously shown to inhibit LH + IGF-I-induced androgen synthesis in this human thecal cell culture system. In the present study we found that the additional presence of inhibin (greater than 1 ng/ml) completely neutralized this inhibitory action of activin (10 ng/ml). These effects of inhibin were dose-dependent (ED50 1-10 ng/ml) and maximal at approximately 100 ng/ml. Inhibin stimulation of androgen synthesis occurred in the absence of measurable effects on progesterone production, and cell numbers in cultured cell monolayers were unaltered by the protein. It is concluded that inhibin exerts potent and selective stimulation of human thecal cell androgen synthesis in vitro. These results a paracrine role for inhibin(s) in modulating follicular androgen biosynthesis in the human ovary.     

Regulation of follicle-stimulating hormone secretion by the interactions of activin-A,dexamethasone and testosterone in anterior pituitary cell cultures of male rats

This study was designed to evaluate the effects of glucocorticoids and gonadal steroids on the expression of inhibin/activin subunits and follistatin of the anterior pituitary and test the hypothesis that resulting changes in the local activin/inhibin/follistatin tone contribute to steroid effects on follicle stimulating hormone (FSH) production from gonadotropes. In primary cell cultures of male rat anterior pituitaries, dexamethasone (DEX) or testosterone (T) stimulated FSH secretion and FSHbeta mRNA and their effects were additive with activin-A. Follistatin (FS288) and inhibin-A antagonized the rise in FSH secretion both in the absence and presence of exogenous activin-A. Despite the similarity in their action on FSH production, DEX and T had opposite effects on follistatin mRNA levels. Follistatin mRNA levels of cultured rat anterior pituitary cells were elevated upon the addition of DEX but attenuated by T. On the other hand, both DEX and T suppressed inhibin/activin betaB mRNA levels while only DEX affected betaA mRNA. In these cells, activin-A stimulated follistatin and inhibin/activin betaB mRNA levels but had no effect on betaA. Together, DEX and activin-A caused a further increase in follistatin mRNA levels while T attenuated the effect of activin-A alone. Both steroids attenuated the effect of activin-A on betaB mRNA accumulation. These results support the possibility that DEX and T, possibly acting on different subsets of anterior pituitary cells, use distinct mechanisms to modify the local activin/inhibin/follistatin circuitry and thereby upregulate FSH production from the anterior pituitary gonadotropes.     

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)     

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.     

Activin-A, inhibin and transforming growth factor-beta modulate growth of two gonadal cell lines

The proliferative and differentiating effects of the gonadal hormones inhibin and activin-A were examined on cell lines derived from the ovary and testis. Activin-A was found to inhibit the growth of CHO-K1 (Chinese hamster ovary) cells in culture, with an IC50 of 3 ng/ml. The maximal response (50% inhibition) required 3 days of incubation in the presence of 40 ng/ml activin-A, and the inhibitory effect was accompanied by morphological changes. Inhibin (10 ng/ml) partially blocked the inhibition of growth by activin. Transforming growth factor-beta (TGF beta), which is structurally related to activin and inhibin, was a very potent inhibitor of the proliferation of CHO-K1 cells, with an IC50 of 0.2 ng/ml and a maximal effect (70% inhibition) at 2 ng/ml. The combination of high concentrations of both TGF beta and activin-A did not result in a greater inhibitory effect than that observed with TGF beta alone, suggesting an overlapping step in the mechanism of action for both factors. In contrast to the results with CHO-K1 cells, differential effects of activin-A and TGF beta were observed in R2C (rat Leydig cell testicular tumor) cells. Activin-A had only a slight effect on proliferation over a 4-day incubation, but inhibited progesterone accumulation ina concentration-dependent fashion within 12 h. TGF beta, on the other hand, was a potent inhibitor of both growth and steroidogenesis in R2C cells. These studies suggest that activin-A and inhibin may regulate proliferation as well as functions of gonadal cells.     

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.     

Regulation of pituitary inhibin/activin subunits and follistatin gene expression by GnRH in female rats

Pituitary inhibin B, activin B, and follistatin are local regulators of FSH. Activin B is a homodimeric molecule (β(B)-β(B)), while inhibin B contains an α and a β(B) subunit. The regulation of gene expression of α, β(B), and follistatin by local and endocrine hormones was examined in pituitaries from female rats and in perifused pituitary cells by RT-PCR. Ovariectomy (OVX) induced an elevation in the mRNA level of α and β(B) subunits and follistatin. Short-term (4 h) treatment of pituitary cells with GnRH decreased both the inhibin α and the inhibin/activin β(B) subunit mRNA levels, while long-term treatment (20 h) with 100 nM GnRH stimulated the expression of both subunits. In contrast, the mRNA level of follistatin was elevated after the short-term GnRH treatment. Long-term exposure of pituitary cells to estradiol and inhibin B suppressed the mRNA expression of β(B) and had no effect on the expression of α subunit and follistatin. Our results demonstrate that the increased expressions of inhibin/activin subunits and follistatin in the post-OVX period can be induced by the lack of gonadal negative feedback, resulting in a high GnRH environment in the pituitary. This study reports for the first time that GnRH administered in high doses and for a long period stimulates the gene expression of inhibin/activin subunits and thereby may contribute to the stimulatory effect of OVX on the expression of these genes.     

In vivo action of activin-A on pituitary-gonadal system.

Activin, a dimer of the beta-subunits of inhibin, has been found to stimulate FSH secretion from the cultured pituitary cells. However, in vivo action of activin is poorly elucidated. Daily sc injections of 40 micrograms activin-A over a period of 1-3 days to intact immature female rats caused a significant increase in serum FSH, inhibin, estradiol, uterine weight, and ovarian FSH receptors. Daily sc injections of 5 micrograms or 20 micrograms activin-A for 6 days caused a marked increase in ovarian weight and the development of large ovarian follicles. However, daily sc injections of 20 micrograms activin-A to hypophysectomized immature female rats for 3 days induced no significant changes in ovarian and uterine weight, serum inhibin, estradiol, and progesterone levels. Simultaneous injections of both activin-A and 5 IU PMSG induced a significant increase in ovarian and uterine weight, serum inhibin, and estradiol levels, compared to simultaneous injections of both vehicle and PMSG in the hypophysectomized immature female rats. These results demonstrate that activin-A induces not only an increase of FSH secretion from the pituitary but also a direct autocrine or paracrine ovarian stimulation resulting in an increase of the number of ovarian FSH receptors and ovarian and uterine weight, as well as an increase in the level of inhibin and estradiol secretion from the ovary.     

Action kinetics of inhibin in superfused pituitary cells depend on gonadotropin-releasing hormone treatment

We examined the effects of partly purified inhibin from porcine follicular fluid on FSH and LH release in superfused rat pituitary cell cultures exposed to different GnRH stimuli. Pituitary cells from immature male rats were cultured in chemically defined medium. After 4 days of static culture in the absence of inhibin preparation and GnRH, the cell monolayers were superfused for approximately 10 h at a constant speed (0.15 or 0.25 ml/min) with medium with or without inhibin preparation (1 micrograms/ml). During the superfusion, some cultures were stimulated with GnRH (10 nM) continuously or intermittently (1 min/0.5 h or 6 min/1 h). In the basal condition (no GnRH), inhibin suppressed FSH release after 5 h of exposure (P less than 0.01), whereas LH secretion was not affected. In cultures treated with GnRH pulses (of either frequency), the inhibitory effects on the GnRH-stimulated FSH and LH release were statistically significant (P less than 0.01) after 2 h of exposure, became more pronounced in the next several hours, then remained stable until the end of the experiment. In cultures exposed to GnRH continuously, the suppressing effects of inhibin preparation became significant (P less than 0.01) after 3 h of exposure and were maximal at 4 h (52% and 61% of control values for FSH and LH, respectively). Later, the suppressing effect became less pronounce due to the decreasing rate of gonadotropin secretion in control (no inhibin) cultures exposed continuously to GnRH. The magnitude of FSH and LH suppression after 9 h of exposure to the inhibin preparation was statistically different (P less than 0.05) for different GnRH treatments and was more pronounced with GnRH pulses (24-27% and 54-57% of control values for FSH and LH, respectively) than with cultures exposed to GnRH continuously (77% and 89% of control values for FSH and LH, respectively) or in the absence of GnRH (50% and 92% of control values for FSH and LH, respectively). We conclude that both the kinetics and magnitude of action of the inhibin preparation on FSH and LH release can differ significantly depending on the presence or absence of GnRH as well as on the mode of GnRH stimulation. Of particular importance is the observation that suppressive effects of inhibin preparation decline in cultures that have been desensitized to GnRH after prolonged continuous GnRH exposures. These differences stress the role of GnRH-inhibin interactions in the regulation of gonadotropin secretion and emphasize the importance of the mode of GnRH stimulation in studies concerning inhibin action on pituitary cells in vitro.     

Changes in circulating and testicular levels of inhibin A and B and activin A during postnatal development in the rat

This study describes the testicular levels of inhibin/activin subunits by Northern analysis and in situ hybridization and serum and testicular levels of inhibins A and B and activin A by enzyme linked immunosorbent assays (ELISA) during postnatal development in the rat. We show that serum inhibin A levels are less than 4 pg/ml throughout postnatal life. Serum inhibin B levels peak at 572 +/- 119 pg/ml (mean +/- se) at d 40 post partum (pp) before falling to 182 +/- 35 pg/ml in mature males. Serum activin A decreases from 294 +/- 29 pg/ml at d 6 to 132 +/- 27 pg/ml at maturity. Within the testis, inhibin A levels fall from 0.330 +/- 0.108 ng/g at d 15 to less than 0.004 ng/g at maturity. Inhibin B levels peak at 43.9 +/- 4.2 ng/g at d 6 before falling to 1.6 +/- 0.13 ng/g at maturity. Testicular activin A levels fall from 18.6 +/- 2.2 ng/g at d 6 to 0.094 +/- 0.013 ng/g at maturity. Northern profiles of testicular inhibin/activin subunits correlate with immunoreactive levels demonstrated by ELISA. In situ hybridization suggests that beta(A) and beta(B) subunit expression is largely restricted to the seminiferous tubule, particularly Sertoli cells, spermatogonia, and primary spermatocytes. These data support the view that inhibin B is the major inhibin in the male rat and that levels relate to Sertoli cell number and activity. Furthermore, the demonstration of high local concentrations of activin A during the period of Sertoli cell proliferation and the onset of spermatogenesis support its proposed role because a modulator of testicular development and function.     

Localization and regulation of the activin-A dimer in human placental cells.

Subunits of activin and inhibin and their mRNAs are present in human placental and decidual cells. However, evidence for the presence of intact activin dimers in the human placenta and their regulation has been lacking. Using a monoclonal antibody raised against the human activin-A dimer, we examined the cellular localization of immunoreactive activin-A dimer in human placentas of different gestational ages (8-41 weeks). In addition, we determined the effects of culture and various potential regulators on the cellular accumulation of immunoreactive activin-A dimer in trophoblast cells from human first trimester placentas. Activin-A dimer was found in both cyto- and syncytiotrophoblast cells of all gestational ages studied. Immunoreactive activin-A also was detected in placental Hofbauer cells in first and second trimester placentas as well as in cells of the placental membranes. Exposure of these cells to cAMP, GnRH, activin, inhibin, transforming growth factor-beta, dexamethasone, and interleukin-1 did not significantly change the intensity of immunostaining for activin-A dimer. These results together with previous data suggest that placental cells are a source of activin-A and that activin-A may be a paracrine and/or endocrine regulator of feto-maternal interactions during pregnancy.     

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.     

Follistatin binds to both activin and inhibin through the common subunit

Inhibin, activin, and follistatin are three families of polypeptides originally isolated and characterized from ovarian follicular fluid based on their modulation of FSH release from pituitary cell culture. In addition to their effects on FSH synthesis and secretion, inhibin and activin have other biological functions. By contrast, the physiological significance of follistatin was obscure, until it was discovered that follistatin is a binding protein to activin. Since activin binds to follistatin, it is imperative to determine the nature of the activin/follistatin binding complex. Moreover, because inhibin contains a beta-subunit derived from activin, it is important to determine whether inhibin will also bind follistatin. Using a double-ligand blotting technique, we have determined that activin-A has two binding sites for follistatin, whereas inhibin-A has only one binding site for follistatin. Therefore, these results suggest that follistatin binds to both activin and inhibin through the common beta-subunit.     

Circulating inhibin alpha concentrations in infant, prepubertal, and adult male rhesus monkeys (Macaca mulatta) and in juvenile males during premature initiation of puberty with pulsatile gonadotropin-releasing hormone treatment

Circulating inhibin alpha concentrations were determined in infant, juvenile, and adult male rhesus monkeys with a RIA employing antisera to a synthetic fragment of the alpha-subunit of porcine inhibin. Binding of tracer, [DSer1,Nle5]human inhibin alpha(1-25)-Gly-125I-Tyr, to antibody was inhibited by standard, [DSer1,Nle5]human inhibin alpha(1-25)-Gly-Tyr. and by plasma from adult male monkeys in a parallel fashion. Castration in adults resulted in a 5-fold decline in the levels of immunoreactivity in plasma. Mean (+/- SE) plasma inhibin alpha concentrations in infants and adults (322.9 +/- 51.9 and 460.1 +/- 43.9 pg/ml, respectively) were significantly higher (P less than 0.05) than those in juveniles (191.3 +/- 28.3 pg/ml). Moreover, initiation of puberty in juvenile males, 13-18 months of age, with a chronic (10- to 12-week) intermittent iv infusion of GnRH (0.1 microgram/min for 3 min every 3 h) resulted in a progressive rise in circulating inhibin alpha that plateaued, after 5 weeks of pituitary stimulation, at concentrations (343.9 +/- 38.2 pg/ml) comparable to those of infants and adults and twice those observed before initiation of the pulsatile infusion of GnRH. Circulating FSH concentrations increased during the first week of GnRH stimulation from 2.7 +/- 0.1 ng/ml before treatment to 6.0 +/- 1.2 ng/ml, where they remained for the duration of the experiment. Testosterone secretion during the initiation of precocious puberty occurred in discrete episodes that were robustly correlated with GnRH-induced LH discharges. In contrast, changes in circulating inhibin alpha concentrations over the 3-h interval between GnRH pulses were unremarkable. Activation of Sertoli and Leydig cells during initiation of puberty in the juvenile males, as reflected by circulating inhibin alpha and testosterone concentrations, respectively, occurred with similar time courses. At the time of orchidectomy, 10-12 weeks after initiation of GnRH treatment, testicular tissue was prepared for histological examination. In spite of a 2-fold gain in testicular weight and in hypertrophy of Sertoli cells in association with GnRH stimulation, maturation of the germinal epithelium did not progress past prophase I spermatocytes, and the number of these latter cells was meager. These findings indicate that the testis of the infant primate, like that of the adult, secretes significant amounts of inhibin, and that the quiescent Sertoli cell of the juvenile males may be readily provoked by appropriate gonadotropin stimulation into producing inhibin. The results also fail to provide evidence for the view that changes in circulating inhibin concentrations are robustly related, in an inve     

The frequency of gonadotropin-releasing hormone stimulation determines the number of pituitary gonadotropin-releasing hormone receptors

Gonadotropin-releasing hormone (GnRH) induces both synthesis and release of pituitary gonadotropins, but rapid or slow frequencies of stimulation result in reduced LH and FSH secretion. We determined the effects of frequency of GnRH stimulation on pituitary GnRH receptors (GnRH-R). Castrate male rats received testosterone implants (cast + T) to inhibit endogenous GnRH secretion. GnRH pulses were injected by a pump into a carotid cannula and animals received GnRH (25 ng/pulse) at various frequencies for 48 h. In control animals (saline pulses) GnRH-R was 307 +/- 21 fmol/mg protein (+/- SE) in cast + T and 598 +/- 28 in castrates. Maximum GnRH-R was produced by 30-min pulses and was similar to that seen in castrate controls. Faster or slower frequencies resulted in a smaller GnRH-R response and GnRH given every 240 min did not increase GnRH-R over saline controls. Equalization of the total GnRH dose/48 h (6.6 ng/pulse every 7.5 min or 200 ng/pulse every 240 min) did not increase receptors to the maximum concentrations seen after 30-min (25 ng) pulses. Serum LH responses after 48 h of injections were only present after 30-min pulses, and peak FSH values were also seen after this frequency. Serum LH was undetectable in most rats after other GnRH frequencies, even though GnRH-R was increased. These data show that GnRH pulse frequency is an important factor in the regulation of GnRH-R. A reduction of GnRH-R is part of the mechanism of down-regulation of LH secretion by fast or slow GnRH frequencies, but altered frequency also exerts effects on secretory mechanisms at a site distal to the GnRH receptor.