Regulation of gonadotropin secretion and number of gonadotropin-releasing hormone receptors by inhibin, activin-A, and estradiol.

Primary cultures of ovine pituitary cells were used to characterize the effects of inhibin and activin on the secretion of gonadotropins and on the regulation of number of GnRH receptors in the presence or absence of estradiol. Number of GnRH receptors was determined by the specific binding of a saturating dose of [125I]des-Gly10-D-Trp6-GnRH-ethylamide (GnRH-A). Recombinant human inhibin-A (rh-inhibin-A) or inhibin in porcine and bovine follicular fluid (pFF and bFF, respectively) decreased secretion of FSH in a dose-dependent manner, with maximum inhibition at an inhibin concentration of approximately 0.1 nM. Neither pFF or bFF affected secretion of LH, although rh-inhibin-A caused a modest decrease (p less than 0.05) in secretion of LH. Treatment of cells with rh-inhibin-A, bFF, or pFF approximately doubled the number of GnRH receptors. Scatchard analysis indicated that increases in GnRH-A binding were due to an increase in receptor number rather than a change in affinity. Additionally, rh-inhibin-A, at a dose that doubled numbers of GnRH receptors, increased GnRH-induced LH release above that caused by GnRH alone, indicating that the increase in receptor number leads to increased responsiveness to GnRH. Recombinant human activin-A (rh-activin-A) increased secretion of FSH but did not affect secretion of LH. Number of GnRH receptors was not affected by lower concentrations of rh-activin-A but was decreased (p less than 0.05) by 3.0 nM activin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of antisera directed against follistatin/activin-binding protein peptides.

An attempt was made to develop immunologic probes directed against follistatin/activin-binding protein (ABP), for use in investigating the distribution of ABP in various tissues. Five oligopeptides corresponding to different regions of the predicted ABP amino acid sequence (peptides 1-12, 28-43, 123-134, 270-281 and 300-315) were synthesized chemically, and coupled to Limulus polyphemus hemolymph hemocyanin. The peptide-hemocyanin conjugates were then used to immunize rabbits, and the immunoresponses were monitored by enzyme-linked immunosorbent assay. Reactivity of the antipeptide antisera with ABP was determined by SDS-polyacrylamide gel electrophoresis and immunoblotting analysis. All of the peptides produced immune responses. The antiserum to peptide 123-134 recognized all forms of ABP, whereas the antiserum to peptide 300-315 reacted specifically and sensitively with the long forms of ABP. These two antisera exhibited only a limited cross-reaction with other proteins or none at all. Therefore, they will be useful for studying the distribution of ABP in various tissues.     

Expression of mRNA for activin-binding protein (follistatin) during early embryonic development of Xenopus laevis.

Follistatin is a specific activin-binding protein and is supposed to control activin functions. During Xenopus embryonic development, activin is thought to act as a natural mesoderm-inducing factor. We isolated here the Xenopus follistatin cDNA from Xenopus ovary cDNA library and studied the expression of Xenopus follistatin gene during the course of early embryonic development. The Xenopus follistatin has an 84% homology at the level of deduced amino acid sequence with human and porcine follistatin. Its 3.5 kb mRNA is first expressed at the gastrula stage, when the expression of activin mRNA becomes first detectable, and increased thereafter. Another species of 2 kb mRNA become detectable from early neurula and also increased dramatically in tadpole. These results suggest that the follistatin acts also as a regulator of activin in inductive interactions during amphibian embryonic development.     

Characteristics of pulsatile luteinizing hormone secretion in middle-aged ovariectomized rats

Experiments were performed to characterize the pulsatile patterns of circulating luteinizing hormone (LH) in the middle-aged ovariectomized (OVX) rat. Frequent blood samples were taken from OVX rats at 6, 7-8, and 9-10 mo of age, and LH was measured by radioimmunoassay. Rats had been OVX either 2 wk (STO) or 10-20 wk (LTO) previously. Mean LH levels were significantly lower with increasing age, reflecting effects on both pulse amplitude and pulse frequency. Mean LH levels were higher in LTO than STO groups, reflecting primarily an increase in pulse amplitude, but there was also a small, significant decrease in pulse frequency with increased time following OVX. In a second experiment, a random selection of the rats in the STO groups was tested again 10 wk after OVX. A significantly higher number of 9- to 10-mo-old rats had pulsatile patterns at 10 wk than at 2 wk following OVX. Furthermore, mean plasma LH concentrations were higher in all three groups. We conclude that decreases in several parameters of LH secretion are seen in middle-aged OVX rats, at the time when irregularities are first seen in the estrous cycle in the intact rat.     

Differential modulation of gonadotropin secretion by selective estrogen receptor 1 and estrogen receptor 2 agonists in ovariectomized ewes

The objectives of this study were to determine whether activation of estrogen receptor 1 (ESR1; also known as ERalpha), or estrogen receptor 2 (ESR2; also known as ERbeta), or both are required to: 1) acutely inhibit secretion of LH, 2) induce the preovulatory-like surge of LH, and 3) inhibit secretion of FSH in ovariectomized (OVX) ewes. OVX ewes (n = 6) were administered intramuscularly 25 micrograms estradiol (E2), 12 mg propylpyrazoletriol (PPT; a subtype-selective ESR1 agonist), 21 mg diaprylpropionitrile (DPN; a subtype-selective ESR2 agonist), or PPT + DPN. Like E2, administration of PPT, DPN, or combination of the two rapidly decreased (P < 0.05) secretion of LH. Each agonist induced a gradual, prolonged rise in secretion of LH after the initial inhibition, but neither agonist alone nor the combined agonists was able to induce a "normal" preovulatory-like surge of LH similar to that induced by E2. Compared with E2-treated ewes, the beginning of the increase in secretion of LH occurred earlier (P < 0.01) in DPN-treated ewes, later (P < 0.05) in PPT-treated ewes, and at a similar interval in ewes receiving the combined agonist treatment. Like E2, PPT decreased (P < 0.05) secretion of FSH, but the duration of suppression was much longer in PPT-treated ewes. DPN did not alter secretion of FSH in this study. Modulation of the number of GnRH receptors by PPT and DPN was examined in primary cultures of ovine pituitary cells. In our hands, both PPT and DPN increased the number of GnRH receptors, but the dose of DPN required to stimulate synthesis of GnRH receptors was 10 times higher than that of PPT. We conclude that in OVX ewes: 1) ESR1 and ESR2 mediate the negative feedback of E2 on secretion of LH at the level of the pituitary gland, 2) ESR1 and ESR2 do not synergize or antagonize the effects of each other; however, they do interact to synchronize the beginning of the stimulatory effect of E2 on secretion of LH, 3) ESR1 and ESR2 may mediate at least partially the positive feedback of E2 on LH secretion by increasing the number of GnRH receptors, and 4) only ESR1 appears to be involved in the negative feedback of E2 on secretion of FSH.     

Dexamethasone increases inhibin and estradiol secretion mediated by endogenous FSH in equine chorionic gonadotropin (eCG)-primed immature female rats.

In the present study, we have examined whether the effects of dexamethasone on follicle stimulating hormone (FSH) secretion were mediated by hypophysiotropic factors, and whether the increased levels of FSH induced by dexamethasone can stimulate ovarian functions in equine chorionic gonadotropin (eCG)-primed immature female rats. Dexamethasone (500 microg) significantly increased serum concentrations of FSH in hypophysectomized rats implanted with pituitary under the kidney capsule, as well as in intact rats. Serum concentrations of inhibin and estradiol in eCG (2.5, 5 i.u.)-primed rats were significantly increased by simultaneous treatment with dexamethasone (500 microg) and eCG. These simultaneous effects were not confirmed in hypophysectomized rats. The results had shown that hypophysiotropic factors do not mediate the selective increase of FSH secretion caused by dexamethasone. Dexamethasone induces the excess amount of FSH secretion from anterior pituitary and this FSH can stimulate inhibin and estradiol secretion in eCG-primed immature female rat.     

Intracerebroventricular administration of ghrelin rapidly suppresses pulsatile luteinizing hormone secretion in ovariectomized rats.

Ghrelin, an endogenous growth hormone (GH) secretagogue, is shown to increase food intake, which action is similar to that of orexin, also a hypothalamic peptide. Since orexin suppresses pulsatile LH secretion in ovariectomized (OVX) rats, the present study was undertaken to investigate whether ghrelin also suppresses LH secretion. Effects of intracerebroventricularly injected ghrelin (0.1 nmol/0.3 microl) were examined in OVX rats treated with a small dose of 17beta-estradiol (E(2)). After ghrelin injection, pulsatile LH secretions which were ongoing in these E(2)-treated OVX rats were significantly suppressed for about 1 h, whereas GH secretion increased, peaking at 30 min. The main parameter suppressed by ghrelin was the pulse frequency, not the pulse amplitude, suggesting the hypothalamus as the site of ghrelin action. This study provides evidence that ghrelin acts not only in the control of food intake but also in the control of LH secretion.     

Orexins, orexigenic hypothalamic neuropeptides, suppress the pulsatile secretion of luteinizing hormone in ovariectomized female rats.

It is well known that feeding disorders are deeply related to reproductive dysfunction, and some feeding regulatory factors may modulate the reproductive function. We examined the effect of orexins, the newly discovered orexigenic hypothalamic neuropeptides, on the pulsatile secretion of LH to clarify their influence on the reproductive function. We administered orexins or saline into the third ventricle of bilaterally ovariectomized (OVX) rats, and measured the serum LH concentration by RIA in blood samples drawn every 6 min for 2 hours to analyze the pulsatile secretion. In the orexin-treated groups, the mean LH concentration and the pulse frequency were significantly reduced (p < 0.01), but the pulse amplitude did not differ significantly. These data indicate that orexins suppress the pulsatile secretion of LH by influencing GnRH neurons at the hypothalamic level.     

Depletion of tuberoinfundibular dopamine does not restore gonadotropin secretion in ovariectomized hyperprolactinemic rats

The effect of depleting tuberoinfundibular dopamine (TIDA) on gonadotropin secretion was studied in rats made hyperprolactinemic by implantation of the prolactin and growth hormone secreting Furth MtTW15 tumor. Implants of the pituitary tumor prevented gonadotropin release in response to castration. Daily injection of 100 mg dl-alpha-methyl-para-tyrosine/kg bw which reduced TIDA levels in tumor bearing rats more than 90% did not restore gonadotropin release. It seems likely that the increased activity of the TIDA system does not mediate the suppression of gonadotropins during chronic hyperprolactinemia.     

Regulation of gonadotropin secretion and puberty onset by neuromedin U

Neuromedin U (NMU), an anorexigenic peptide, was originally isolated from porcine spinal cord in 1985. As NMU is abundant in the anterior pituitary gland, we investigated the effects of NMU on gonadotropin secretion. Both NMU and its receptors, NMUR1 and NMUR2, were expressed in the pituitary gland. NMU suppressed LH and FSH releases from rat anterior pituitary cells. Moreover, NMU-deficient mice exhibit an early onset of vaginal opening. The LHbeta/FSHbeta ratio, which is an index of puberty onset, is high in young NMU-deficient mice. These results indicate that NMU suppresses gonadotropin secretion and regulates the onset of puberty.     

Effects of neuromedin U on the pulsatile LH secretion in ovariectomized rats in association with feeding conditions

We examined the effects of intracerebroventricular injection of neuromedin U (NMU), at a dose that is reported to induce satiety in rats, on the pulsatile luteinizing hormone (LH) secretion in adult ovariectomized (OVX) rats under a normal feeding or a 48-h fasted condition. In OVX rats under the normal feeding condition, injection of NMU (1 nmol/3 microl) significantly decreased the mean LH concentration without affecting the frequency or amplitude of LH pulses, but under the 48-h fasted condition, it significantly decreased the mean LH concentration and the frequency of LH pulses without affecting the amplitude. The interpulse interval was significantly lengthened by NMU injection under the normal and the 48-h fasted condition, but the effect under the 48-h fasted condition was greater than under the normal feeding condition. We also confirmed that the 48-h fasted condition per se did not affect the pulsatile LH secretion in OVX rats. We suggest that NMU and fasting synergistically inhibit the pulsatile LH secretion, even though NMU has been said to act as a satiety factor.     

Regulation of progesterone secretion in human syncytiotrophoblast in culture by human chorionic gonadotropin.

In the present investigation we sought to define the specific sites in the pathway of placental progesterone biosynthesis that underlie the action of human chorionic gonadotropin (hCG). When the cells were challenged with dibutryl cAMP (dbcAMP), forskolin or isobutylmethylxanthine, they produced significantly higher amounts of progesterone which in the presence of the hCG antibody was reduced to the level of the control set of cells. Trophoblast cells cultured in serum free medium with 25-hydroxycholesterol (25-OHC) produced increased amounts of progesterone. In the presence of hCG antibody at a concentration which neutralized the secreted hCG, the steroid production was completely blocked, even when the 25-OHC was added to the medium. Also, direct quantitation of the cytochrome P450 SCC enzyme in the absence of hCG indicated a significant decrease. The exogenous addition of low density lipoproteins (LDL) increased the progesterone secretion by the trophoblast cells in culture. Neutralization of hCG by the antibodies, however, drastically reduced the LDL induced progesterone secretion, which was restored by the addition of dbcAMP to the medium. Based on these findings, we suggest a stimulatory effect of hCG on normal trophoblast cells at the level of LDL utilization and cytochrome P450 SCC enzyme. Since dbcAMP could mimic these actions of hCG, the data suggest a possible autocrine/paracrine role of hCG on the trophoblast cells. An additive effect of hCG and cAMP on progesterone secretion observed in our studies, indicate that apart from hCG, adenylate cyclase activity may also be regulated by other factors.     

Concomitant inhibition of pulsatile luteinizing hormone (LH) and stimulation of prolactin release by prostacyclin (PGI2) in ovariectomized (OVX) conscious rats

Prostacyclin (PGI2) or its stable metabolite, 6-keto-PGF1 alpha (1-5 micrograms) in 2.5 microliter 0.05 M phosphate buffer (pH 7.4), was injected into the third ventricle (3 V) of ovariectomized (OVX), freely moving rats. Control animals received 2.5 microliter of buffer. In the initial experiments a control blood sample was taken and then the PGI2 was injected and frequent samples taken thereafter. With this protocol injection of 2 micrograms of PGI2 produced a significant decrease in mean plasma LH only at 60 min after its injection (p less than .05), while the higher dose (5 micrograms) decreased plasma LH concentrations at 30 and 60 min (p less than .01 and p less than .001, respectively). In subsequent experiments, blood was removed from indwelling external jugular vein cannulae every 5-6 min during 2 hours and plasma LH and PRL levels were determined by radioimmunoassay. LH pulses were monitored and several parameters of LH pulsation were calculated during the hour before and after injection of phosphate buffer, PGI2 or 6-keto-PGF1 alpha. Intraventricular injection of phosphate buffer failed to modify the characteristic pulsatile release of LH and did not alter plasma PRL levels. The amplitude of LH pulses was significantly reduced by PGI2 and the inhibitory effect was dose-related. Even a dose of 1 microgram produced a significant reduction in pulse height and the response was graded with maximal reduction occurring with the 5 microgram dose which essentially abolished the LH pulses. Following the microinjection of 6-keto-PGF1 alpha, no significant changes were observed in plasma LH values and the pulses of the hormone. Five micrograms PGI2 considerably elevated plasma PRL values during the 20-25 min following its 3V injection, whereas the same dose of 6-keto-PGF1 alpha produced only a very slight stimulatory effect. Since PGI2 had no effect to alter LH release by cultured pituitary cells in vitro, it is concluded that PGI2 can act on structures near the 3V to inhibit pulsatile release of LHRH.     

Regulation of gonadotropin secretion in the anterior pituitary

Studies on the regulation of gonadotropin secretion in dissociated pituitary cell cultures are described. Initial studies employing a ferritin-labelled analogue of gonadotropin hormone releasing hormone (GnRH) to localize its receptor sites on the gonadotropin cell surface that while these receptor sites initially have a random monodisperse distribution, binding of the ligand causes coarse aggregation and internalization of the GnRH receptor. These events are not due to the multivalency of the ligand and probably reflect redistributive events in vivo. By using an octapeptide analogue GnRH that binds to the GnRH receptor but lacks gonadotropin releasing activity in conjunction with sequence-specific antisera it is shown that antibodies that bind the octapeptide can induce the octapeptide to release gonadotropin. These data suggest that receptor aggregation is important in GnRH stimulation. Finally immunocytochemical studies are described in which golg-protein-A-antibody complexes are used to identify gonadotropins on ultrathin frozen sections of porcine pituitary cells. These studies indicate that in porcine gonadotropin cells the majority of the secretory granules contain both luteinizing hormone and follicle-stimulating hormone.     

Influence of estrogen and progesterone on the induction of mammary carcinomas by 7,12-dimethylbenz(a)anthracene in ovariectomized rats.

The influence of estrogen on mammary carcinogenesis was studied in female Sprague-Dawley rats ovariectomized at the age of 36 days and given injections of 17 beta-estradiol (group I:0, II:1, III:10, IV:100, V:1000 micrograms/2 days) between the ages of 36 and 250 days and a single oral dose of 20 mg of 7,12-dimethylbenz(a)anthracene (DMBA) at the age of 50 days. No palpable mammary carcinomas were detected up to the age of 135 days. At the age of 135 days, each group was divided into two subgroups (a and b). Rats of the second subgroup (Ib, IIb, IIIb, IVb and Vb) were given additional injections of progesterone (P; 4 mg/2 days) between the ages of 135 and 250 days. At the age of 250 days, the incidence of mammary carcinoma was significantly higher in rats from group IIIb than in groups Ib and IIIa, and that in group IVa was also higher than in group Ia. The incidence in group IVb was significantly lower than in group IVa. The carcinomas in group IIIb were palpable papillo-tubular adenocarcinomas and those in group IVa were secretory micro-adenocarcinomas. These results indicate that the induction of mammary carcinomas by DMBA is totally inhibited by ovariectomy and/or high doses of estrogen, but that mammary carcinomas are initiated by DMBA under hormonal conditions in which suitable levels of estrogen are present. They also suggest that the growth of DMBA-induced mammary carcinomas in the rats from group III were accelerated by additional injections of P and that those in rats from group IV were inhibited by additional P.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of the pulsatile releases of luteinizing and follicle-stimulating hormones in ovariectomized hamsters

We have combined for modifications of common radioimmunoassay (RIA) techniques to increase the sensitivity of the gonadotropin assays by an order of magnitude compared with those generated according to the instructions provided by the National Pituitary Agency. The four modifications are: a) enzymatic radioiodination, b) purification of radiolabeled hormones by Sephadex and concanavalin A chromatography, c) reduced first antibody concentration, and d) a prolonged incubation time. These methods increase the sensitivities of the RIAs and allow for the quantitation of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels in small volumes of plasma. We have used these methods to measure the changes in pulse frequency and amplitude of LH and FSH in ovariectomized hamsters after a variety of neuroendocrine manipulations. Alterations in catecholaminergic neurotransmission affect the frequency and amplitude of LH but not FSH release, and suggest that the hypothalamic mechanisms responsible for LH releasing hormone (LHRH)-mediated LH release are distinct from those that regulate FSH secretion. Further, alterations in LHRH-pituitary interactions (elicited by injections of LHRH antisera or a potent LHRH agonist), suggest the existence of separate control mechanisms responsible for LH and FSH release at the level of the adenohypophysis. Combined, these studies provide further evidence for complex and separate neuroendocrine regulatory control over the secretion of each gonadotropin.     

The effects of porcine follicular fluid inhibin on gonadotropin secretion in the rabbit

Porcine follicular fluid (pff), treated with charcoal to remove steroids, was used to determine whether inhibin is active in the laboratory rabbit. When pff (5 ml/4 kg body weight) was injected (ip) into does that had been castrated 2 weeks earlier, there was a significant decline in blood follicle-stimulating hormone (FSH) levels; the decline lasted for 8-12 h. Blood levels of luteinizing hormone (LH) were suppressed, but only briefly at 3 h after injection. In other experiments, intact does which had been injected with pff 9 h and 10 min before receiving a single, i.v. injection of luteinizing hormone-releasing hormone (LHRH) (10 micrograms/kg body weight) showed a sharp reduction in the concentration of LH in the blood samples collected 15, 30 and 60 min after LHRH administration. Secretion of FSH responded poorly to LHRH stimulation, and pff had little suppressive action on blood levels. Having established that the pff preparation had inhibin activity, its action on the postovulatory surge of FSH secretion was next examined. This release of FSH, which occurs 6 to 36 h after ovulation, has been hypothesized to be required for the establishment of pregnancy by stimulating the growth of the ovarian follicles supplying the luteotropic estradiol. To test this hypothesis, pff was injected into rabbits every 8 h for the first 5 days of pregnancy and found to block the postovulatory FSH surge. The patterns of secretion of LH and progesterone in the same pff-injected animals were, however, not altered from normal pregnancy patterns by pff.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of activin-binding protein by rat granulosa cells in vitro

We have developed an assay method for activin-binding protein, which exploits its high affinity for sulfated polysaccharides. We used this method to investigate the production of activin-binding protein by rat ovarian granulosa cells, in vitro. The production of activin-binding protein by granulosa cells was dependent on the cell density; the maximum was observed at 6 x 10(5) cells/ml. Follicle-stimulating hormone (FSH), but not luteinizing hormone (LH), enhanced production significantly. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and ligand blotting analyses of the activin-binding protein secreted by rat granulosa cells demonstrated it was the same protein molecule as that purified from rat ovaries. It is inferred from these results that the granulosa cell is a source of ovarian activin-binding protein and that its secretion is regulated by FSH.