Absence Of GnRH Research Articles

ODP335 Mutual Effects of Orexin and BMPs on Gonadotropin Expression by Mouse Gonadotrope LβT2 Cells

Abstract Orexins are neuropeptides that express primarily in the hypothalamus and are produced in two isoforms, orexin A and orexin B. There are two kinds of G protein coupled receptors, orexin type 1 (OX1R) and type 2 (OX2R) receptors. Orexin has been reported to have key roles on sleep-wake regulation and feeding behavior in the central nervous system, whereas its receptors are also expressed in peripheral tissues including the endocrine organs, and orexin affects the regulation of the endocrine system. In our previous experiments, we have revealed the impact of orexins on anterior pituitary functions. For instance, we reported that orexin A plays an inhibitory role in prolactin production through the suppression of endogenous bone morphogenetic protein (BMP) activity in rat pituitary lactotrope GH3 cells. It was also reported that orexin A enhances pro-opiomelanocortin (POMC) transcription by upregulating corticotropin-releasing hormone (CRH) receptor signaling and by downregulating BMP-Smad signaling in mouse corticotope AtT20 cells. However, the effects of orexin on the endocrine function regarding gonadotrope cells remain unclear. We have recently uncovered that core clock genes and BMPs have mutual effects on luteinizing hormone (LH) expression in a phase-dependent manner by mouse gonadotrope LβT2 cells. In the present study, we investigated the effect of orexin on pituitary gonadotropin expression using LβT2 cells, which express OX1R and OX2R, by focusing on the functional involvement of BMP system and clock genes. It was revealed that orexin A stimulation increased LHβ and FSHβ mRNA expression in a concentration-responsive manner in the absence of GnRH, and interestingly, GnRH co-treatment further upregulated LHβ mRNA expression in LβT2 cells. Regarding the interrelationships between the signalings of orexin and BMPs, it was also revealed that orexin A pretreatment enhanced the BMP receptor signaling detected as the Smad1/5/9 phosphorylation, indicating that orexin enables to upregulate the BMP actions in LβT2 cells. In our previous studies, we reported that several BMP ligands such as BMP-6, -7 and 15 expressed in LβT2 cells can promote gonadotropin transcription, in which BMP-6 regulates GnRH-stimulated LH expression by modulating the sensitivity to somatostatin analogs. Based on the present results, it was implied that endogenous orexin can be functionally involved in the underlying mechanisms of gonadotropin expression. Collectively, orexin enhances gonadotropin expression by regulating BMP signaling in gonadotrope cells. Here, we will also discuss functional involvement of clock genes in the regulatory system of gonadotropin secretion induced by orexin and BMPs. Presentation: No date and time listed

Mutual Effects of Orexin and Bone Morphogenetic Proteins on Gonadotropin Expression by Mouse Gonadotrope Cells.

Orexin plays a key role in the regulation of sleep and wakefulness and in feeding behavior in the central nervous system, but its receptors are expressed in various peripheral tissues including endocrine tissues. In the present study, we elucidated the effects of orexin on pituitary gonadotropin regulation by focusing on the functional involvement of bone morphogenetic proteins (BMPs) and clock genes using mouse gonadotrope LβT2 cells that express orexin type 1 (OX1R) and type 2 (OX2R) receptors. Treatments with orexin A enhanced LHβ and FSHβ mRNA expression in a dose-dependent manner in the absence of GnRH, whereas orexin A in turn suppressed GnRH-induced gonadotropin expression in LβT2 cells. Orexin A downregulated GnRH receptor expression, while GnRH enhanced OX1R and OX2R mRNA expression. Treatments with orexin A as well as GnRH increased the mRNA levels of Bmal1 and Clock, which are oscillational regulators for gonadotropin expression. Of note, treatments with BMP-6 and -15 enhanced OX1R and OX2R mRNA expression with upregulation of clock gene expression. On the other hand, orexin A enhanced BMP receptor signaling of Smad1/5/9 phosphorylation through upregulation of ALK-2/BMPRII among the BMP receptors expressed in LβT2 cells. Collectively, the results indicate that orexin regulates gonadotropin expression via clock gene expression by mutually interacting with GnRH action and the pituitary BMP system in gonadotrope cells.

The relationship between basal and regulated Gnrhr expression in rodent pituitary gonadotrophs

Hypothalamic GnRH together with gonadal steroids and activins/inhibin regulate its receptor gene (Gnrhr) expression in vivo, which leads to crucial changes in GnRHR numbers on the plasma membrane. This is accompanied by alterations in the gonadotroph sensitivity and responsiveness during physiologically relevant situations. Here we investigated basal and GnRH-regulated Gnrhr expression in rodent pituitary gonadotrophs in vitro. In pituitary cells from adult animals cultured in the absence of GnRH and steroid hormones, the Gnrhr expression was progressively reduced but not completely abolished. The basal Gnrhr expression was also operative in LβT2 immortalized gonadotrophs never exposed to GnRH. In both cell types, basal transcription was sufficient for the expression of functional GnRHRs. Continuous application of GnRH transiently elevated the Gnrhr expression in cultured pituitary cells followed by a sustained fall without affecting basal transcription. Both basal and regulated Gnrhr transcriptions were dependent on the protein kinase C signaling pathway. The GnRH-regulated Gnrhr expression was not operative in embryonal pituitary and LβT2 cells and was established neonatally, the sex-specific response patterns were formed at the juvenile-peripubertal stage and there was a strong correlation between basal and regulated gene expression during development. Thus, the age-dependent basal and regulated Gnrhr transcription could account for the initial blockade and subsequent activation of the reproductive system during development.

β-Arrestin 2 Is a Mediator of GnRH-(1–5) Signaling in Immortalized GnRH Neurons

We have previously demonstrated that the cleavage product of the full-length GnRH, GnRH-(1-5), is biologically active, binds G protein-coupled receptor 173 (GPR173), and inhibits the migration of cells in the immortalized GnRH-secreting GN11 cell. In this study, we attempted to characterize the GnRH-(1-5) intracellular signaling mechanism. To determine whether the signaling pathway mediating GnRH-(1-5) regulation of migration involves a G protein-dependent mechanism, cells were treated with a generic G protein antagonist in the presence and absence of GnRH-(1-5), and a wound-healing assay was conducted to measure migration. G Protein antagonist 2 treatment abolished the GnRH-(1-5) inhibition of migration, indicating that the mechanism of GnRH-(1-5) is G protein coupled. To identify the potential Gα-subunit recruited by GnRH-(1-5) binding GPR173, we measured the second messengers cAMP and inositol triphosphate levels. GnRH-(1-5) treatment did not alter cAMP levels relative to cells treated with vehicle or forskolin, suggesting that GnRH-(1-5) does not couple to the Gαs or Gαi subunits. Similarly, inositol triphosphate levels remained unchanged with GnRH-(1-5) treatment, indicating a mechanism not mediated by the Gαq/11 subunit. Therefore, we also examined whether GnRH-(1-5) activating GPR173 deviated from the canonical G protein-coupled receptor signaling pathway by coupling to β-arrestin 1/2 to regulate migration. Our coimmunoprecipitation studies indicate that GnRH-(1-5) induces the rapid interaction between GPR173 and β-arrestin 2 in GN11 cells. Furthermore, we demonstrate that this association recruits phosphatase and tensin homolog to mediate the downstream action of GnRH-(1-5). These findings suggest that the GnRH-(1-5) mechanism deviates from the canonical G protein-coupled receptor pathway to regulate cell migration in immortalized GnRH neurons.

Hypophysiotropic role of RFamide-related peptide-3 in the inhibition of LH secretion in female rats

Gonadotropin-inhibitory hormone (GnIH), a newly discovered hypothalamic RFamide peptide, inhibits reproductive activity by decreasing gonadotropin synthesis and release in birds. The gene of the mammalian RFamide-related peptides (RFRP) is orthologous to the GnIH gene. This Rfrp gene gives rise to the two biologically active peptides RFRP-1 (NPSF) and RFRP-3 (NPVF), and i.c.v. injections of RFRP-3 suppress LH secretion in several mammalian species. In this study, we show whether RFRP-3 affects LH secretion at the pituitary level and/or via the release of GnRH at the hypothalamus in mammals. To investigate the suppressive effects of RFRP-3 on the mean level of LH secretion and the frequency of pulsatile LH secretion in vivo, ovariectomized (OVX) mature rats were administered RFRP-3 using either i.c.v. or i.v. injections. Furthermore, the effect of RFRP-3 on LH secretion was also investigated using cultured female rat pituitary cells. With i.v. administrations, RFRP-3 significantly reduced plasma LH concentrations when compared with the physiological saline group. However, after i.c.v. RFRP-3 injections, neither the mean level of LH concentrations nor the frequency of the pulsatile LH secretion was affected. When using cultured pituitary cells, in the absence of GnRH, the suppressive effect of RFRP-3 on LH secretion was not clear, but when GnRH was present, RFRP-3 significantly suppressed LH secretion. These results suggest that RFRP-3 does not affect LH secretion via the release of GnRH, and that RFRP-3 directly acts upon the pituitary to suppress GnRH-stimulated LH secretion in female rats.

Differential Effects of GnRH and Androgens on Cres mRNA and Protein in Male Mouse Anterior Pituitary Gonadotropes

The Cres gene defines a new subgroup in the family 2 cystatins of cysteine protease inhibitors. However, unlike typical cystatins, CRES does not inhibit cysteine proteases but rather inhibits the serine protease prohormone convertase 2, an enzyme with roles in proprotein processing in the neuroendocrine system. Cres is expressed in the gonadotropes and colocalizes with LHbeta, suggesting a role in the regulation of gonadotrope secretion. Our present studies were carried out to examine the regulation of Cres mRNA and protein expression by GnRH and steroid hormones, thus providing clues regarding its role in gonadotropes. Castration profoundly reduced Cres mRNA, while replacement with estradiol (E(2)), testosterone (T), or dihydrotestosterone (DHT) further decreased Cres, suggesting negative regulation by GnRH or steroid hormones. The administration of Antide, a GnRH antagonist, resulted in a 3-fold increase in Cres mRNA, supporting a negative regulation by GnRH. Because all hormonal manipulations in vivo resulted in alterations in steroid hormones, organ culture was used to assess the effects of GnRH independent of steroids. Mouse pituitaries cultured in the absence of GnRH or steroids showed high Cres mRNA levels, while DHT or E(2) resulted in decreases of 25% and 68%, respectively. However, an 85% decrease in Cres mRNA occurred following the administration of GnRH, demonstrating that GnRH, and to a lesser degree E(2), negatively regulate Cres mRNA in gonadotropes. Examination of CRES protein by immunohistochemistry showed that levels were profoundly reduced following castration, while DHT and in part T, but not E(2), restored CRES levels. Castrated mice treated with Antide showed little effect. However, castrated mice treated with Antide + DHT showed a dramatic recovery of CRES, suggesting that androgens act directly at the level of the gonadotrope to regulate CRES protein. Together, our studies suggest that Cres mRNA and protein are low at peak gonadotrope secretory activity, possibly as a means to allow proprotein processing events to occur that are integral to gonadotrope function.

Intra-pituitary regulation of gonadotrophs in male rodents and primates.

Paracrine and autocrine regulation is well established in many organs including the gonads, but the notion of communication among pituitary cells is a relatively new concept. The FSH-beta and GnRH-receptor genes are up-regulated by pituitary activin and down-regulated by pituitary follistatin, and circulating inhibin disrupts this local regulation by functioning as an endogenous competitor of the activin receptor. Activin and follistatin production by folliculostellate cells may play a central role in these responses. alpha-Subunit expression is maintained at high levels in the absence of GnRH through unknown mechanisms. There is evidence that the intra-pituitary regulation of FSH-beta and GnRH-receptor gene expression may activate pubertal maturation in male rats. Finally, there are marked differences in follistatin expression and its regulation by GnRH and androgens in male primates and rats that appear to explain species differences in the differential secretion of FSH and LH, although the physiological significance of these differences is not yet known.

Immunomodulatory actions of gonadal steroids may be mediated by gonadotropin-releasing hormone.

Estrogens are considered to be immunostimulatory, whereas androgens are considered to be immunosuppressive. We hypothesized that the divergent actions of gonadal steroids on the immune system may be mediated indirectly, via their potent divergent feedback effects on the hypothalamic hormone GnRH, which is itself immunostimulatory. We used the GnRH-deficient HPG/Bm mouse in an effort to disentangle the effects of gonadal steroids from the effects of GnRH. We randomized GnRH-deficient mice and their GnRH-sufficient littermates to receive androgens, estrogens, or GnRH. We subsequently measured B and T cell proliferative responses to mitogen and serum IgG levels. We demonstrate that estrogens exert stimulatory effects on B cell proliferation and serum IgG levels in the presence of GnRH but not in the absence of GnRH. Testosterone exerts suppressive effects on B cell function in the presence of GnRH but not in its absence. Androgens and estrogens exerted divergent actions on T cell function irrespective of the presence and absence of GnRH, although responses were markedly attenuated in GnRH-deficient mice. Our data suggest that the immunostimulatory effects of estrogen and the immunosuppressive effects of androgens on B cell function may be mediated indirectly via GnRH.

Fetal development of Leydig cell activity in the mouse is independent of pituitary gonadotroph function.

During fetal development the testes secrete anti-Mullerian hormone and testosterone to induce formation of the male phenotype. Adult Leydig cells secrete testosterone under the control of LH, but the role of the fetal pituitary in regulating fetal Leydig cell function is unclear. To study the early relationship between pituitary and Leydig cell function, we have examined the development of fetal pituitary LH levels and Leydig cell function in normal mice and in hypogonadal (hpg) mice that lack GnRH and, thus, circulating gonadotropins. In normal and hpg mice, pituitary LH content was barely detectable until embryonic day 17 (E17), when levels began to increase significantly in both groups. Pituitary levels of LH in hpg mice were, however, only about 10% of normal at all ages. Full-length LH receptor transcripts were first detectable in fetal testes on E16 in both normal and hpg mice. In normal mice, levels of testicular messenger RNA (mRNA) encoding cytochrome P450 side-chain cleavage and 17alpha-hydroxylase increased from E13 to reach a peak around birth. In hpg mice, levels of mRNA encoding these enzymes were normal until around birth, at which time there was a significant decline. Levels of testicular mRNA encoding 3beta-hydroxysteroid dehydrogenase type I were similar in normal and hpg mice and showed little change during development. Intratesticular testosterone reached a peak on E18 in normal animals before declining again after birth. In hpg mice, intratesticular testosterone levels were normal throughout fetal development and on the day of birth, but were barely detectable by postnatal day 5. Results show 1) that fetal Leydig cell function in the mouse is normal in the absence of endogenous circulating gonadotropins; 2) that Leydig cells become dependent on gonadotropins shortly after birth; and 3) that pituitary LH synthesis can start in the absence of GnRH but is dependent on LH for a normal level of synthesis and secretion.

L-hydroxytryptophan amplifies pulsatile secretion of LH in the follicular phase of normal women.

The hypothalamus possesses serotoninergic fibres which arise from neuronal cell bodies located in the raphe nuclei and have synapse on GnRH-secreting neurones. Hitherto, no firm evidence has been produced to support a role for serotoninergic control of LH in humans. Our first objective was to investigate whether pulsatile administration of L-5-hydroxytryptophan--the immediate precursor of serotonin--affects pulsatile LH secretion in the medium-late follicular phase of normal women. Since the results of the first experiment suggest that L-5-hydroxytryptophan amplifies LH release, our second objective was to investigate whether, in the absence of GnRH, L-5-hydroxytryptophan can release LH. This was done by studying LH response in patients with hypogonadotrophic hypogonadism of hypothalamic origin. Twenty-two normal women (18-25 years old) and 8 patients with hypogonadotrophic hypogonadism (2 men with Kallmann's syndrome and 6 women with anorexia nervosa). Serum LH levels in the 22 subjects (reference population) were monitored at 10-minute intervals over an 8-hour period (1000-1800h) during medium-late follicular phase. In 7 of these subjects, serum LH levels were monitored in their next medium-late follicular phase while L-5-hydroxytryptophan was administered at hourly intervals from 1000 to 1800 h; the peripheral conversion of L-5-hydroxytryptophan to serotonin was inhibited by 150 mg of benserazide at 0930 and 1430h. To investigate whether, in the absence of GnRH, L-5-hydroxytryptophan can release LH, two patients with Kallmann's syndrome were monitored over a 7-hour period and 6 patients with anorexia nervosa over a 9-hour period. After a 2-5 hour control period the subjects received 150 mg of benserazide, and pulsatile L-5-hydroxytryptophan (every 30 minutes in the Kallmann's patients and every 45 minutes in the subjects with anorexia nervosa). LH pulses were identified and analysed according to number, amplitude, interpulse interval and pulse duration. In the normal women, L-5-hydroxytryptophan increased pulse amplitude (mean +/- SD; 3.02 +/- 1.42 IU/l vs. 1.75 +/- 0.98 IU/l before L-5-hydroxytryptophan and 1.90 +/- 1.04 IU/l in the reference population; P < 0.01 in both cases), but had no significant effects on pulse duration, interpulse interval or number of pulses. L-5-hydroxytryptophan had no effect on LH in patients with Kallmann's syndrome. In the anorexia nervosa group, the mean serum LH level increased significantly after L-5-hydroxytryptophan (3.90 +/- 2.46 IU/l vs. 3.06 +/- 1.23 IU/l, P < 0.001) but, as in Kallmann's patients, the three women in this group without residual LH pulsatility did not respond to L-5-hydroxytryptophan. Pulsatile administration of L-5-hydroxytryptophan increases LH pulse amplitude in the follicular phase of normal women. In the absence of GnRH, L-5-hydroxytryptophan does not stimulate pituitary LH secretion.

Characteristics of GnRH Binding in the Gonads and Effects of Lamprey GnRH-I and -III on Reproduction in the Adult Sea Lamprey

In the present study, both lamprey GnRH-I and -III stimulated steroidogenesis and induced ovulation in adult female sea lampreys during their final reproductive stage. One injection of lamprey GnRH-III at 0.1 or 0.2 μg/g lamprey stimulated plasma estradiol levels in lampreys held at each of three water temperatures, 13°, 17°, and 19°, corresponding to increasing stages of maturation. Four successive injections, 3 to 4 days apart, of lamprey GnRH-III at 0.1 or 0.2 μg/g body weight induced ovulation in 100 or 88% of lampreys, respectively, compared to 21% in controls by Day 31. Lamprey GnRH-III also had a direct stimulatory effect on estradiol production in the sea lamprey gonadsin vitro.Lamprey GnRH-III at 100 or 1000 ng/ml stimulated estradiol levels in media incubated with either lamprey ovaries or testes. In contrast to a previous finding in which lamprey GnRH-III was more potent than lamprey GnRH-I in inducing spermiation in adult male sea lampreys (Deragon and Sower, 1994), the results from the present study indicate that lamprey GnRH-I and -III are equally potent in inducing ovulation and stimulating steroidogenesis in female sea lampreys. In addition, GnRH binding sites have been demonstrated for the first time in both the testis and the ovary of the adult sea lamprey using an analog of mammalian GnRH ([D-Lys6] mammalian GnRH) as a labeled ligand. Scatchard analysis suggested the presence of a high affinity binding site in both the testis and the ovary. In summary, lamprey GnRH-III is biologically active in stimulating the pituitary–gonadal axis in adult female sea lampreys. This is the first report demonstrating the presence of a GnRH binding site in the gonads of an Agnathan. The evidence for a direct stimulatory effect of lamprey GnRH in the gonads, the presence of GnRH binding site, and the absence of GnRH in the plasma suggest that, like other vertebrates including rat, rabbit, teleost fish, and human, there may be a GnRH-like factor produced in the gonads of the lamprey and it may act as a paracrine/autocrine modulator of gonadal function. This study further strengthens the paracrine regulatory role of GnRH peptides in the gonads of vertebrates, which appear to be evolutionarily conserved.

Effect of oestradiol on mRNA encoding GnRH receptor in pituitary tissue of orchidectomized sheep passively immunized against GnRH.

The effect of oestradiol on tissue concentrations of GnRH receptor and mRNA encoding GnRH receptor was assessed in orchidectomized sheep (wethers) made deficient in GnRH by passive immunization. Wethers were assigned to one of four groups (n = 6 animals per group). Animals in groups 2 and 4 received ovine anti-GnRH sera (200 ml; i.v.) at passive immunization, while antisera against the carrier protein was administered to wethers in groups 1 and 3. Oestradiol was delivered as a continuous infusion (2 micrograms h-1) to wethers in groups 1 and 2. Animals in groups 3 and 4 were infused with vehicle alone. Anterior pituitary tissue was collected at the end of the 48 h infusion. Anti-GnRH sera induced a rapid reduction in the serum concentration of LH. Continuous delivery of oestradiol resulted in a twofold increase (P < 0.05) in tissue concentration of GnRH receptor. This oestradiol-induced response was manifest even in wethers in which endogenous GnRH had been neutralized by passive immunization. Conversely, infusion of oestradiol increased (P < 0.05), and intravenous administration of anti-GnRH sera decreased (P < 0.05), concentrations of mRNA encoding GnRH receptor in pituitary tissue. When delivered in combination, anti-GnRH sera reduced (P < 0.05), but did not eliminate, the oestradiol-induced augmentation of steady-state concentrations of mRNA encoding GnRH receptor. These data demonstrate that the basal concentration of mRNA encoding GnRH receptor is dependent on continued GnRH stimulation. In contrast, the oestradiol-induced increase in steady-state concentration of mRNA encoding GnRH receptor is manifest even in the absence of GnRH.

Messenger ribonucleic acid for gonadotropin-releasing hormone receptor and numbers of gonadotropin-releasing hormone receptors in ovariectomized ewes after hypothalamic-pituitary disconnection and treatment with estradiol.

To study the regulation of ovine GnRH receptors in the absence of GnRH, hypothalamic input was removed by hypothalamic-pituitary disconnection (HPD). Steady-state concentrations of GnRH receptor mRNA and numbers of GnRH receptors were measured after HPD and subsequent treatment with estradiol. Anterior pituitary glands were collected 24 (n = four), 36 (n = two), 48 (n = four), and 72 h (n = four) after HPD. An additional group of ewes received subcutaneous implants of estradiol 24 h after HPD, and pituitary glands were collected 0 (n = four), 12 (n = four), 24 (n = three), and 48 h (n = four) after exposure to estradiol. Pituitary glands were also obtained from four ovariectomized ewes that did not undergo HPD (OVX controls). At 24 h after HPD, mean number of GnRH receptors had decreased (P < .05) by 73%; however, mean concentration of GnRH receptor mRNA was not different from OVX controls. Relative to HPD ewes, treatment with estradiol increased mean concentrations of GnRH receptor mRNA and mean numbers of GnRH receptors (P < .01 and P < .001, respectively). From these data we conclude that 1) acute removal of GnRH decreases the numbers of GnRH receptors but does not affect steady-state concentrations of GnRH receptor mRNA and 2) estradiol increases the numbers of GnRH receptors and steady-state concentrations of GnRH receptor mRNA via direct effects at the level of the pituitary gland.

Effects of corticosterone and testosterone on pituitary gonadotropin content, secretion, bioactivity and messenger RNA levels in the presence or absence of GnRH in male rats

The effects of corticosterone (B) and testosterone (T) on pituitary and serum bioactive and immunoreactive gonadotropins and on gonadotropin hormone subunit messenger RNA levels were compared in the absence of GnRH. Male rats were implanted with pellets of either cholesterol, B or T. At implantation, 2 and 4 days later half of each group received GnRH antagonist and animals were killed 5 days after implantation. As expected, GnRH antagonist lowered bioactive and immunoreactive serum FSH and LH, pituitary FSH, LHβ and FSHβ mRNA. B treatment alone lowered bioactive and immunoreactive serum FSH and immunoreactive serum LH. B reversed the antagonist effect on bioactive and immunoreactive pituitary FSH and FSHβ mRNA. T alone lowered bioactive and immunoreactive serum FSH and LH levels. T reversed the antagonist effect on bioactive and immunoreactive pituitary FSH. T lowered bioactive and immunoreactive pituitary LH and LHβ mRNA and partially reversed the antagonist effect on FSHβ mRNA. The data suggest that either B or T enhance FSH synthesis by acting directly at the gonadotrope, but that B does not affect LH variables to the same extent as T. The results suggest that in stressed animals, when T levels are reduced, B can substitute for T in sustaining FSH synthesis.

Gonadotropin-releasing hormone stimulates glycoprotein hormone alpha-subunit messenger ribonucleic acid (mRNA) levels in alpha T3 cells by increasing transcription and mRNA stability.

Pulsatile GnRH stimulates gonadotropin secretion, whereas continuous exposure to GnRH causes pituitary desensitization and suppressed levels of LH and FSH. At the level of gene expression, continuous GnRH also causes partial or complete suppression of the LH beta and FSH beta genes, but expression of the alpha-subunit gene is stimulated without evidence of desensitization. In this report, we examined the transcriptional and posttranscriptional mechanisms by which GnRH controls alpha-gene expression using the gonadotrope-derived alpha T3 cell line. Continuous GnRH caused a 4- to 5-fold accumulation of alpha mRNA over 72 h, without evidence of a decline. In contrast, measurements of alpha-gene transcription, either by nuclear run-on assays or using a stably integrated alpha LUC reporter gene, revealed that GnRH caused a transient increase in alpha-promoter activity, followed by a decline after 4-6 h. The prolonged accumulation of alpha mRNA at a time when transcriptional activity had abated was accounted for by independent effects of GnRH on alpha mRNA stability. After prior treatment with GnRH, its removal either by washout or using a GnRH receptor antagonist caused an abrupt decline in steady state alpha mRNA levels (t1/2, < 2 h). Readdition of GnRH prevented the decay in alpha mRNA, and experiments using the transcriptional inhibitor actinomycin-D confirmed that this effect of GnRH did not require transcription. Consistent with these results, pulse-chase analyses of mRNA stability demonstrated that GnRH increased the alpha mRNA half-life 6.7-fold, from 1.2 h in the absence of GnRH to 8.0 h in the presence of GnRH. We conclude that GnRH induces a transient burst of alpha-gene transcription that is accompanied by marked induction of mRNA stability.

Effect of Recombinant Inhibin on Follicle-Stimulating Hormone Secretion by the Female Rat: Interaction with a Gonadotropin-Releasing Hormone Antagonist and Estrogen*

There is considerable support for the hypothesis that one component of FSH secretion is independent of GnRH. The ability of follicular fluid preparations containing inhibin to suppress FSH release in the absence of GnRH drive has suggested that this component might be responsive to gonadal proteins. However, follicular fluid contains several proteins that either stimulate or inhibit FSH secretion, thus complicating interpretation of the results. The recent availability of recombinant human (rh) inhibin-A has allowed us to specifically investigate the effect of this protein on FSH secretion by rats whose GnRH release and/or effect was blocked by a specific antagonist, [Ac-D2Nal1,DCpa2, D3Pa13,Arg5, D5-(p-methoxyphenyl) 5-oxo-2-aminopentanoic acid16, DAla10]GnRH, or by estradiol (E2). In a first experiment, the GnRH antagonist (100 micrograms/kg, injected sc 21 h earlier) lowered plasma immunoactive FSH levels by 36%, and rh-inhibin-A (25 micrograms/kg, injected iv 6 h earlier) lowered them by 44%. The combination of both treatments decreased FSH values by 82% (P less than or equal to 0.01 vs. either compound alone). Inhibin and the antagonist also significantly (P less than or equal to 0.01) decreased FSH bioactivity. A second experiment investigated the independent or combined effects of inhibin and E2. E2 (40 micrograms/kg, injected sc at -36 and -12 h) lowered FSH levels by 53%, and inhibin (25 micrograms/kg, injected iv at -6 h) decreased FSH concentrations by 37%. Administration of both compounds produced a 63% inhibition (P less than or equal to 0.05 vs. inhibin alone; P less than or 0.01 vs. E2 alone). Finally, we studied the interaction among the GnRH antagonist, inhibin, and E2. In this protocol plasma FSH levels in rats injected with inhibin and the antagonist did not show an additional decrease after treatment with E2. These results indicate that plasma FSH levels in rats whose GnRH receptors or secretion are blocked can be further lowered by inhibin. Thus, the ability of rh-inhibin-A to interfere with FSH secretion in the ovariectomized rat appears to involve a mechanism independent of the GnRH drive.

Pulsatile GnRH stimulation increases steady-state mRNA levels for FSHβ, LHβ, and α subunits in superfused pituitary cell cultures

The pituitary secretion of FSH and LH is controlled by pulsatile GnRH stimulation of specific magnitude and frequency, and changes in the mode of stimulation can dramatically alter the pattern of gonadotropin secretion. We explored the effects of different GnRH treatments on the secretion of gonadotropins and levels of mRNA for the gonadotropin subunits in superfused cultures of immature rat pituitary cells. After 4 days of stationary culture in the absence of GnRH, the cells were exposed during superfusion to different GnRH treatments for up to 20 h. Each GnRH pulse (6 min/h, 10 n M) elicited pulsatile release of both gonadotropins, causing a 2- to 3-fold increase of mean hourly secretion of FSH and 5- to 6-fold increase of LH. The magnitude of gonadotropin responses after 4, 10, and 20 h of stimulation with GnRH pulses was not significantly different. In contrast, mRNA levels for different subunits varied depending on the duration of GnRH exposure. Only FSHβ mRNA was increased (2.5 ±0.4-fold, P < 0.05) after 4 h of stimulation, and only after 10 h was the a subunit mRNA also significantly increased (2.1 ± 0.3-fold, P < 0.05). After 20 h of GnRH exposure, both mRNA levels became further elevated (4-to 7-fold vs no GnRH) and the LHβ mRNA increased significantly (5.7 ± 0.9-fold, P < 0.05). The effects of continuous (20 h) exposure to GnRH on mRNA levels were different from those of pulsatile treatment. A stimulatory effect comparable to that of pulsatile stimulation was observed only for the a subunit, whereas FSHβ mRNA was significantly reduced ( P < 0.05 vs no GnRH) and LHβ mRNA level was not affected. These studies demonstrated, for the first time, that mRNA steady-state levels for all three gonadotropin subunits can be up-regulated by GnRH in vitro, but following different time intervals, and that the mode and duration of GnRH treatment can profoundly affect the results. Our results also indicate that caution must be exercised when interpreting data from experiments conducted in vitro with continuous or short-term GnRH treatments.

Interrelationship between the Actions of Testosterone and Primate Sertoli Cell Inhibin in the Control of Gonadotropin Secretion by Cultured Pituitary Cells*

There is accumulating evidence that the differential regulation of LH and FSH secretion in the male is partly accomplished by the direct actions of testosterone (T) and inhibin on the pituitary. The present study was designed to examine the interaction between T and inhibin, in the presence and absence of GnRH, using dispersed pituitary cells in monolayer culture and cells perifused with pulses of GnRH from intact, 2-week castrated, and castrated T-replaced young adult male rats. The effect of partially purified inhibin from primate Sertoli cell culture medium (pSCI) to suppress basal FSH secretion was similar with pituitary cells from intact and castrated rats. T increased basal FSH secretion in the presence or absence of pSCI but did not alter the dose-dependent suppression of FSH by pSCI with cells from either intact or castrate rats. Castration increased basal FSH and LH secretion, whereas only basal FSH release was increased with cells from T-replaced castrates. T pretreatment increased the action of pSCI to suppress GnRH-stimulated FSH and LH release from perifused pituitary cells. These data indicate that T and inhibin exert opposite but independent effects on basal FSH release. The action of inhibin to suppress basal FSH secretion is not impaired by the absence of T and inhibin subsequent to castration. By contrast, the actions of T and inhibin to suppress GnRH-stimulated gonadotropin secretion are coordinated and interrelated.

Effects of a novel gonadotropin-releasing hormone antagonist (ORG 30850) on gonadotropin and prolactin secretion by rat pituitary cells in culture

The effect of a new GnRH antagonist (ORG 30850 ANT) on FSH, LH, and PRL secretion was studied using male rat pituitary cells in monolayer cell culture. In the absence of GnRH, ORG 30850 ANT did not alter spontaneous FSH and LH secretion into culture medium or the cell content of these hormones. In the presence of GnRH (10(-8) mol/l), ORG 30850 ANT significantly and dose-dependently inhibited FSH and LH secretion into culture medium while increasing their cell content. Conversely, in the presence of a single dose of ORG 30850 ANT, FSH and LH secretion rose significantly when subjected to increasing amounts of GnRH, whereas the hormonal cell content diminished. Furthermore, inhibition of GnRH-induced FSH and LH release by ORG 30850 ANT was not changed by pre-incubation with the GnRH antagonist regardless of the pre-incubation time. The inhibitory effect of the GnRH antagonist was observed early, with its peak occurring within 6 h of culture. These short-term studies indicate that ORG 30850 ANT specifically inhibits GnRH-induced gonadotropin release into culture medium, exerts no effect on the rate of gonadotropin production in the presence or absence of GnRH, competitively and reversibly inhibits the binding of natural GnRH to its receptors, and does not lead to any modifications in PRL secretion.

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.