Regulation Of GnRH Receptors Research Articles

Characterization of the Mechanism of Inhibin α-Subunit Gene in Mouse Anterior Pituitary Cells by RNA Interference

Inhibin, a member of the transforming growth factor-β [TGF-β] superfamily, is a suppressor of follicle-stimulating hormone [FSH] release through pituitary–gonadal negative feedback loop to regulate follicular development. In this study, Inhibin α-subunit [Inha] gene was knocked down successfully in mice primary anterior pituitary cells at both transcriptional and translational levels by RNAi-Ready pSIREN-RetroQ-ZsGreen Vector mediated recombinant pshRNA vectors. The results indicated that inhibin silencing significantly promoted apoptosis by up-regulating Caspase-3, Bax and Bcl-2 genes without affecting p53 both at transcriptional and translational levels. Furthermore, it markedly impaired the progression of G1 phase of cell cycle and decreased the amount of cells in S phase [as detected by flow cytometry]. Inhibin silencing resulted in significant up-regulation of mRNA and protein expressions of Gondotropin releasing hormone receptors [GnRHR] and down-regulated mRNA levels of β-glycans with parellel change in the amount of its protein expression. Silencing of inhibin-a significantly increased [P<0.05] activin-β concentration without affecting FSH and LH levels in anterior pituitary cells. These findings revealed that up regulation of GnRH receptors by silencing inhibin a-subunit gene might increase the concentration of activin-β in the culture medium. Inhibin a silencing resulted in increased mRNA and protein expressions of inhibinβ which may demonstrate that both inhibin subunits co-participate in the regulation of reproductive events in anterior pituitary cells. This study concludes that inhibin is a broad regulatory marker in anterior pituitary cells by regulating apoptosis, cellular progression and simultaneously by vital fluctuations in the hormonal signaling.

The pituitary potential for opportunistic breeding in the Cape dune mole-rat, Bathyergus suillus

In this paper we investigated the effect of sex and season on baseline and post-GnRH challenge luteinising hormone (LH) levels in a solitary, seasonally breeding mole-rat. Circulating basal concentrations of luteinising hormone (LH) were found to differ significantly with season in both sexes. However, no significant difference was found in circulating basal LH concentration between the sexes either within or out of the breeding season. The magnitude of the LH response to an exogenous pharmacological overdose of GnRH both in and out of the breeding season in males and females respectively was not significant. This finding suggests that there is no down regulation of GnRH receptors on the pituitary during the non-breeding season. Cape dune mole-rats thus have the potential for opportunistic breeding outside of the typical breeding period. We argue that this represents an adaptation to limited and brief opportunities for mating in this xenophobic and aggressive species.

Regulation of gonadotropin-releasing hormone receptors by protein kinase C: inside out signalling and evidence for multiple active conformations.

Desensitization and internalization of G protein-coupled receptors can be mediated by phosphorylation within the C-terminal tail, facilitating beta-arrestin binding and targeting the receptor for internalization. Type II GnRH receptors (GnRH-Rs) show such regulation, but type I GnRH-Rs lack C-tails and are not rapidly desensitized or internalized. Here we show contrasting susceptibility of type I (human and sheep) and II (Xenopus) GnRH-Rs to regulation by protein kinase C (PKC). When human (h) or Xenopus (X) GnRH-Rs were expressed using recombinant adenovirus, PKC activation increased radioligand binding to XGnRH-Rs but not to hGnRH-Rs. A dominant-negative dynamin mutant (K44A) inhibited internalization of XGnRH-Rs (but not hGnRH-Rs) without influencing PKC regulation of XGnRH-R binding. PKC activation increased the affinity of XGnRH-Rs for the type II GnRH ligand and increased effects of low concentrations of GnRH-II on the [Ca(2+)](i) but had no effect on type I ligand binding to hGnRH-Rs, sGnRH-Rs or XGnRH-Rs, or to chimeric receptors with the XGnRH-R C-tail added to a type I receptor. Binding of type II ligand to human or sheep receptors was also unaffected but was increased in the chimeras. Mutation of both PKC-phosphorylation consensus sites in the XGnRH-R tail did not prevent the PKC-mediated increases in binding or alter agonist-induced translocation of beta-arrestin2/green fluorescent protein or inhibition of inositol phosphate accumulation by beta-arrestin2/green fluorescent protein. Thus, it appears that there are two distinct active conformations of XGnRH-Rs (differing in affinity for type I and II ligands) and that these cells exhibit a novel form of inside-out signaling in which PKC feeds back to influence receptor affinity.

Negative regulation of gonadotropin-releasing hormone and gonadotropin-releasing hormone receptor gene expression by a gonadotrophin-releasing hormone agonist in the rat hypothalamus.

There exists evidence for the presence of ultrashort loop feedback circuits of gonadotropin-releasing hormone (GnRH) secretion in the hypothalamus. It is, however, uncertain whether a similar mechanism is involved in the regulation of GnRH gene expression in vivo. Furthermore, little is known about the regulation of GnRH receptor (GnRHR) expression in the brain. In the present study, we examined the regulation of GnRH and its receptor gene expression by GnRH in vivo. A GnRH agonist, [D-Ala6, des-Gly10]GnRH-ethylamide (des-Gly GnRH), was administered by intracerebroventricular (i.c.v.) injection via the lateral ventricle of ovariectomized and estradiol (OVX + E)-treated rats. The amounts of GnRH and GnRHR mRNA were measured in the preoptic area (POA) and posterior mediobasal hypothalamus (pMBH) micropunch samples from individual rat brain slices by respective competitive reverse transcription-polymerase chain reactions. The i.c.v. administration of des-Gly GnRH significantly decreased GnRH and GnRHR mRNA expression in a dose-and time-related manner: des-Gly GnRH (6 ng) suppressed GnRH and GnRHR mRNA expression within 2 h, and the suppression was maintained without significant variation until 8 h after treatment. Treatment with Antide, [N-Ac-D-Nal(2)1, pCl-D-Phe2, D-Pal(3)3, Lys(Nic)5, D-Lys(Nic)6, Lys(iPR)8, D-Ala10]GnRH (10 ng), a potent GnRH antagonist, did not alter GnRH mRNA expression, but prevented des-Gly GnRH-induced suppression of GnRH mRNA expression. Antide alone decreased GnRHR mRNA expression, but failed to alter agonist-induced suppression of GnRHR mRNA expression. These results demonstrate the existence of an ultrashort loop feedback mechanism for GnRH gene expression in the POA, along with homologous down-regulation of GnRHR mRNA expression in the pMBH.

Hormonal Regulation of Gonadotropin-Releasing Hormone Receptors and Messenger RNA Activity in Ovine Pituitary Culture

Previous studies demonstrate that gonadotroph responsiveness to GnRH, GnRH binding, and the apparent number of GnRH receptors are all increased by 17 beta-estradiol (E) or inhibin (IN) in ovine pituitary cultures. Progesterone (P) attenuates these effects. To explore differences between the effects of IN and E on GnRH binding, a detailed time-course study was performed. The results indicate that after 48 h, IN had a greater effect on binding of a GnRH agonist (5-fold increase) than E (3-fold increase), but was slower to act initially. A combined treatment of IN and E gave a partially additive effect at 48 h (6.5-fold increase). The mechanism of receptor regulation in this system is not known, but could involve synthesis, recycling, or modification of GnRH receptors. To investigate the contribution of altered receptor biosynthesis to the regulation of receptor levels, a functional Xenopus oocyte-based assay for GnRH receptor mRNA activity was employed. After 48 h of treatment, IN or E each led to a 7- to 8-fold increase in GnRH receptor mRNA activity. Treatment with both hormones led to a 19-fold increase. The increase in mRNA activity induced by either hormone was greatly attenuated by P. Modulation of GnRH receptor mRNA levels suggests that IN, E, and P regulate responsiveness to GnRH in the ovine pituitary at least in part by altering de novo synthesis of GnRH receptors. The differing time courses of action, as assayed by GnRH binding, and the additivity of effects at the mRNA level suggest that IN and E alter mRNA levels via different mechanisms.

Copper induces luteinizing hormone release and desensitization of pituitary gonadotropes

Copper stimulated LH release from cultured rat pituitary cells in a dose- and time-dependent manner. After 4 h of incubation with 10 μ M Cu 2+, LH release was stimulated by 3-fold. The release of LH stimulated by Cu 2+ was Ca 2+ dependent, thus excluding the possibility that the releasing activity of this divalent cation was due to a toxic effect on pituitary cells. The stimulatory action of Cu 2+ is substantially mediated via the GnRH-receptors since Cu 2+ inhibited 125I-Buserelin binding and since a GnRH-antagonist blocked most of the Cu 2+-stimulated LH release (80%). Both GnRH (1 μM) and Cu 2+ (10 μM) induced desensitization of pituitary cells to a subsequent stimulation of either GnRH (0.5 nM) or Cu 2+ (10 μM). However, in contrast to GnRH, Cu 2+ did not induce down regulation of GnRH receptors. These findings suggest that the Cu 2+ effects are mainly mediated through the GnRH receptors.

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.

Properties and regulation of GnRH receptors in the anterior pituitary and the testis of the rat: different response of Leydig cell LH and GnRH receptors to hormonal treatments.

The properties (association and dissociation rates, affinity and specificity) of GnRH receptors in the rat pituitary and the Leydig cells are very similar. In addition, in vivo administration for 5 or 11 days of 10 micrograms of GnRH to adult rats resulted in an increase (p less than 0.01) in both Leydig cell and pituitary GnRH receptors. In contrast, the same treatment caused a significant decrease (p less than 0.01) of Leydig cell LH receptor number. Furthermore, testosterone propionate (500 micrograms/day), which had no effect on Leydig cell LH receptors, caused a significant increase (p less than 0.01) of testicular GnRH receptors but a decrease (p less than 0.01) of pituitary GnRH receptors. Estradiol 17 beta valerate (5 micrograms/day) reduced (p less than 0.01) both testicular (5 days) and pituitary (5 and 11 days) GnRH receptors. Such treatment for 5 days also decreased (p less than 0.05) the Leydig cell LH receptors. No effect of dexamethasone and 17-hydroxyprogesterone was seen after 5 or 11 days of treatment. Thus, although pituitary and Leydig cell receptors appear similar, their regulation by exogenous hormone treatments is different. Furthermore, the regulation of Leydig cell GnRH and LH receptors also appears very different, suggesting different roles of the receptors in the control of the Leydig cell function.

Photoaffinity labeling of gonadotropin releasing hormone receptors in control and desensitized pituitary cells

Gonadotropin releasing hormone (GnRH), preincubated with cultured rat pituitary cells, induced down regulation of GnRH receptors in a time- and dose-dependent manner. The specific binding was inhibited by 50% after 30 min and maximal inhibition (70%) was obtained after 75 min preincubation with 1 μM GnRH. Preincubation of the cells for 2 h with 10 nM GnRH inhibited the specific binding by 20%, reaching a plateau of 70% inhibition with 0.1 μM GnRH. Concomitantly, exposure of the cells to GnRH caused a time- and dose-dependent desensitization of LH release. The responsiveness of the desensitized cells was not parallel to the binding capacity and was inhibited to a greater extent (93%). Photoactivation of GnRH receptors with iodinated [azidobenzoyl-D-Lys 6]GnRH in control and desensitized cells resulted in the identification of a single specific band with the same apparent molecular weight of 60K daltons. These results indicate that structural alterations of GnRH receptors are not associated with GnRH-induced desensitization. Therefore, desensitization may involve conformational changes in the receptor or more likely a post-receptor mechanism.

Role of Estradiol in Inducing an Ovulatory-Like Surge of Luteinizing Hormone in Sheep 1

Two experiments were performed to examine the effect of estradiol on secretion of luteinizing hormone (LH) and on the number of receptors for gonadotropin-releasing hormone (GnRH) after down regulation of GnRH receptors in ovariectomized ewes. In the first experiment, ovariectomized ewes were administered one of four treatments: Group 1) infusion of GnRH i.v. for 40 h; Group 2) injection of 100 micrograms estradiol i.m.; Group 3) infusion of GnRH i.v. for 16 h followed immediately by an injection of 100 micrograms estradiol i.m.; and Group 4) infusion of GnRH i.v. for 40 h plus injection of 100 micrograms estradiol i.m. after the 16th h of infusion. Ewes in Groups 1, 3 and 4 responded to the infusion of GnRH with an immediate increase in serum concentrations of LH, with maximum values occurring between 2 and 4 h after the start of infusion; serum concentrations of LH then began to decline and were approaching the pretreatment baseline within 16 h. Administration of estradiol resulted in a surge of LH regardless of whether the pituitary had been desensitized by infusion of GnRH or not. In all cases the magnitude of the surge was similar to that induced by the initial infusion of GnRH. In Groups 2 and 3 the surge of LH began at 12.3 +/- 0.1 and 11.9 +/- 0.1 h after administration of estradiol. In contrast, the ewes in Group 4 had a surge of LH beginning 3.7 +/- 0.1 h after administration of estradiol.(ABSTRACT TRUNCATED AT 250 WORDS)

Homologous regulation of gonadotropin-releasing hormone receptors in cultured pituitary cells.

Specific receptors for gonadotropin-releasing hormone (GnRH) in cultured rat pituitary cells were increased by subnanomolar concentrations of GnRH agonists and decreased by high concentrations of these peptides. The antagonist [D-Phe2, Pro3, D-Phe6]GnRH did not alter GnRH binding capacity and blocked the increase in sites induced by GnRH. These findings provide direct evidence for the homologous regulation of GnRH receptors by physiological concentrations of the hypothalamic peptide, an action that could mediate the cyclical and postcastration increases in GnRH receptors and responsiveness of the pituitary gonadotrophs.