Identification, expression, and physiological functions of Siberian hamster gonadotropin-inhibitory hormone

Gonadotropin-inhibitory hormone (GnIH) is a hypothalamic neuropeptide that inhibits gonadotropin secretion in birds and mammals. To further understand its physiological roles in mammalian reproduction, we identified its precursor cDNA and endogenous mature peptides in the Siberian hamster brain. The Siberian hamster GnIH precursor cDNA encoded two RFamide-related peptide (RFRP) sequences. SPAPANKVPHSAANLPLRF-NH(2) (Siberian hamster RFRP-1) and TLSRVPSLPQRF-NH(2) (Siberian hamster RFRP-3) were confirmed as mature endogenous peptides by mass spectrometry from brain samples purified by immunoaffinity chromatography. GnIH mRNA expression was higher in long days (LD) compared with short days (SD). GnIH mRNA was also highly expressed in SD plus pinealectomized animals, whereas expression was suppressed by melatonin, a nocturnal pineal hormone, administration. GnIH-immunoreactive (-ir) neurons were localized to the dorsomedial region of the hypothalamus, and GnIH-ir fibers projected to hypothalamic and limbic structures. The density of GnIH-ir perikarya and fibers were higher in LD and SD plus pinealectomized hamsters than in LD plus melatonin or SD animals. The percentage of GnRH neurons receiving close appositions from GnIH-ir fiber terminals was also higher in LD than SD, and GnIH receptor was expressed in GnRH-ir neurons. Finally, central administration of hamster RFRP-1 or RFRP-3 inhibited LH release 5 and 30 min after administration in LD. In sharp contrast, both peptides stimulated LH release 30 min after administration in SD. These results suggest that GnIH peptides fine tune LH levels via its receptor expressed in GnRH-ir neurons in an opposing fashion across the seasons in Siberian hamsters.     

In vitro effect of ethanol exposure on basal and GnRH-stimulated LH secretion from pituitary cells

The question of whether ethanol's (ETOH's) known suppressive effect on serum luteinizing hormone (LH) could be mediated directly at the anterior pituitary level was addressed by examining the effects of ETOH in vitro on release of LH from cultured male rat pituitary cells. The impact of added ethanol concentrations ranging from 50 to 400 mg% on LH release was examined in the basal state and after stimulation by gonadotropin-releasing hormone (GnRH) at a dose of 5 x 10(-10) M. While ETOH did not significantly suppress basal LH release, secretion stimulated with GnRH was noted to be attenuated with higher doses of ETOH (greater than or equal to 100 mg%) compared to stimulated control cells. It is concluded that ETOH exposure in vitro alters stimulated LH secretion by acting directly on pituitary gonadotropes.     

Does cortisol inhibit pulsatile luteinizing hormone secretion at the hypothalamic or pituitary level?

Elevations in glucocorticoids suppress pulsatile LH secretion in sheep, but the neuroendocrine sites and mechanisms of this disruption remain unclear. Here, we conducted two experiments in ovariectomized ewes to determine whether an acute increase in plasma cortisol inhibits pulsatile LH secretion by suppressing GnRH release into pituitary portal blood or by inhibiting pituitary responsiveness to GnRH. First, we sampled pituitary portal and peripheral blood after administration of cortisol to mimic the elevation stimulated by an immune/inflammatory stress. Within 1 h, cortisol inhibited LH pulse amplitude. LH pulse frequency, however, was unaffected. In contrast, cortisol did not suppress either parameter of GnRH secretion. Next, we assessed the effect of cortisol on pituitary responsiveness to exogenous GnRH pulses of fixed amplitude, duration, and frequency. Hourly pulses of GnRH were delivered to ewes in which endogenous GnRH secretion was blocked by estradiol. Cortisol, again, rapidly and robustly suppressed the amplitude of GnRH-induced LH pulses. We conclude that, in the ovariectomized ewe, cortisol suppresses pulsatile LH secretion by inhibiting pituitary responsiveness to GnRH rather than by suppressing hypothalamic GnRH release.     

Absence of regular pulsatile LH secretion during pre- and post-implantation periods in sheep

Two experiments were conducted to determine the patterns of LH secretion and to evaluate the LH responses to pulsatile administration of GnRH during early pregnancy in ewes. In experiment 1, pregnant ewes (n=16) were used to determine the concentration of LH in plasma of jugular blood samples collected every 15 min for 6h before (day 10 post-mating) and after (days 20 and 30 post-mating) implantation. In experiment 2, the pituitary LH responses to exogenous pulsatile administration of GnRH were examined on day 10 post-mating in 4 pregnant ewes. A small dose of GnRH (200 ng/ml saline) was injected (i.v.) every 3h and jugular blood samples were collected every 15 min for 12h beginning at the onset of GnRH administration and continuing through the 4th GnRH pulse. During the frequent-sample bleeding at any of the stages of pregnancy examined, LH concentrations oscillated in a pulsatile manner. However, pulsatile LH release occurred irregularly and infrequently. Overall mean LH concentrations, frequency and amplitude of LH pulses were not significantly different between any of the stages of pregnancy examined. Pulsatile administration of GnRH on day 10 post-mating induced regular pulses of LH. In conclusion, these data demonstrate that: (i) pulsatile LH secretion occurs irregularly during early pregnancy, and (ii) the absence of regular pulsatile LH release during early pregnancy is not attributed to a lack of pituitary responsiveness to GnRH.     

Phytoestrogens and gonadotropin-releasing hormone pulse generator activity and pituitary luteinizing hormone release in the rat

Phytoestrogens can produce inhibitory effects on gonadotropin secretion in both animals and humans. The aims of this study were 2-fold: 1) to determine in vivo whether genistein and coumestrol act on the GnRH pulse generator to suppress hypothalamic multiunit electrical activity volleys and associated LH pulses and/or on the pituitary to suppress the LH response to GnRH; and 2) to examine the effect of these phytoestrogens on GnRH-induced pituitary LH release in vitro and to determine whether estrogen receptors are involved. Wistar rats were ovariectomized and chronically implanted with recording electrodes and/or indwelling cardiac catheters, and blood samples were taken every 5 min for 7--11 h. Intravenous infusion of coumestrol (1.6-mg bolus followed by 2.4 mg/h for 8.5 h) resulted in a profound inhibition of pulsatile LH secretion, a 50% reduction in the frequency of hypothalamic multiunit electrical activity volleys, and a complete suppression of the LH response to exogenous GnRH. In contrast, both genistein (1.6-mg bolus followed by 2.4 mg/h for 8.5 h) and vehicle were without effect on pulsatile LH secretion. Coumestrol (10(-5) M; over 2 or 4 h) suppressed GnRH-induced pituitary LH release in vitro, an effect blocked by the antiestrogen ICI 182,780. It is concluded that coumestrol acts centrally to reduce the frequency of the hypothalamic GnRH pulse generator. In addition, the inhibitory effects of coumestrol on LH pulses occur at the level of the pituitary by reducing responsiveness to GnRH via an estrogen receptor-mediated process.     

Disruption of GnRH pulses by anti-GnRH serum and phentolamine obliterates pulsatile LH but not FSH secretion in ovariectomized rabbits

It has been hypothesized that the secretion of gonadotropins, i.e. luteinizing hormone (LH) and follicle-stimulating hormone (FSH), is driven by a synchronized neural network ('pulse generator'). This network, regulated in part by alpha-adrenergic activity, ultimately generates bursts of hypothalamic gonadotropin-releasing hormone (GnRH) release. In this study, we used the push-pull (PP) perfusion technique in ovariectomized rabbits to investigate three aspects of the ('GnRH/gonadotropin pulse generator') hypothesis. The objectives were to determine: (1) if plasma LH and FSH pulses occur concomitantly with mediobasal hypothalamic (MBH-) GnRH pulses, (2) changes in the patterns of pulsatile LH and FSH secretion when pulsatile MBH GnRH signals are interrupted by either local immunoneutralization of GnRH or intravenous infusion of the alpha-adrenergic antagonist phentolamine (PHEN, 4 mg/kg BW), and (3) whether third cerebroventricular (3VT-) GnRH patterns reflect neuronal GnRH release from the MBH. We found that while both plasma LH and FSH patterns were pulsatile, MBH GnRH pulses were significantly coupled only with LH pulses (94% coincidence). Both the local immunoneutralization of MBH GnRH pulses and the PHEN-induced suppression of MBH GnRH pulses obliterated the pulsatile secretion of LH, but not FSH. Neither MBH GnRH nor plasma LH or plasma FSH pulses were concurrent with 3VT GnRH pulses. However, the PP perfusion of the 3VT appeared to alter the pulsatile release of MBH GnRH and pituitary LH. The results support the hypothesis that in the absence of ovarian signals, the 'pulse generator' is maintained by tonic alpha-adrenergic input and that a 'cellular unity' of MBH GnRH release (GnRH pulses) drives the gonadotrophs to secrete LH in pulses. In contrast, the pulsatile release of FSH appears to involve additional nonovarian regulatory events to those controlling LH secretion.     

LH AND FSH SECRETION AND RESPONSES TO GnRH AND TRH IN PATIENTS WITH CLINICALLY FUNCTIONLESS PITUITARY ADENOMAS

Serum concentrations of LH and FSH and their response to the separate administration of GnRH (100 micrograms i.v.) and TRH (200 micrograms i.v.) have been studied preoperatively in 12 patients with a clinically functionless pituitary adenoma, of whom nine (3F: 6M) were found to secrete gonadotrophins in vitro. In three patients with a gonadotrophin-secreting adenoma (GSA) the pulsatile release of LH and FSH was also assessed preoperatively. An elevated serum FSH was recorded in six of nine patients with a GSA, and was subnormal in one, whilst an elevated LH was recorded in only two and was subnormal in six. A doubling of LH occurred in only four of the nine patients after GnRH and in three of six after TRH. None of the three patients with a non-GSA was shown to have an aberrant response to GnRH or TRH. In patients with a GSA, pulsatile release of LH and FSH was usually asynchronous and neither hormone demonstrated any regular harmonic pattern. These data show that in patients with a GSA the serum FSH level is usually elevated but this is not invariable, and the LH may well be low. Pathological responses of LH are frequently found following the administration of either GnRH or TRH and these stimulation tests should be performed separately in patients presenting with a clinically 'non-functioning' pituitary tumour to assist in the preoperative diagnosis. The absence of normal LH and FSH pulsing also appears to be a feature of GS adenomas, and suggests that tumorous gonadotrophin secretion is not under physiological control by hypothalamic GnRH.     

The suppression of pulsatile luteinizing hormone secretion during lactation in the rat

The inhibition of LH secretion during lactation may be the consequence of a pituitary insensitivity to GnRH stimulation and/or an inhibition of GnRH release from the hypothalamus. To assess the contribution that these mechanisms may make to the suppression of LH secretion during lactation, we described the pattern of LH secretion in lactating rats and the magnitude of LH secretion in response to a GnRH stimulus. We assessed the effect of the strength of the suckling stimulus (two and eight pups), the length of lactation (5 and 10 days), and the presence of the ovaries on the pattern of LH secretion. We also examined the pattern of LH secretion after removal of a large suckling stimulus. In the intact rat, the pattern of LH secretion during lactation was uniformly nonpulsatile, despite significant differences between animals suckling two and eight pups in pituitary responsiveness to GnRH. In intact rats suckling two pups during day 10 of lactation, significant LH secretion was stimulated by 0.4-ng pulses of GnRH every 50 min, while animals with eight pups secreted little LH in response to the same stimulus. It was concluded that a two-pup suckling stimulus was sufficient to completely suppress pulsatile GnRH release without affecting pituitary function, whereas an eight-pup suckling stimulus also depressed pituitary sensitivity to GnRH. In ovariectomized (ovx) rats suckling two pups, seven of nine animals showed no postcastration rise in LH secretion or evidence of pulsatile LH secretion during day 5 of lactation. In the remaining two animals, a castrate pattern of pulsatile LH secretion was observed, with a LH interpulse interval of 31 +/- 6 min. By day 10 of lactation, all animals suckling two pups had castration patterns of LH secretion, with a LH interpulse interval of 35 +/- 2 min, which was significantly different from the LH interpulse interval of 26 +/- 1 min observed in ovx animals without pups. Therefore, a two-pup suckling stimulus is capable of retarding the increase in LH pulse frequency characteristically seen in the rat after castration. In ovx rats suckling eight pups, the postcastration rise in LH secretion was completely inhibited in all animals examined on days 5 and 10 of lactation, and the pattern of LH secretion was uniformly nonpulsatile. A consistent pattern of pulsatile LH secretion was not reinitiated until 72 h after removal of the suckling stimulus (LH interpulse interval, 31 +/- 2 min).(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of progesterone on pulsatile luteinizing hormone (LH) secretion and LH subunit messenger ribonucleic acid during lactation in the rat

In ovarian-intact lactating rats, removal of the suckling stimulus leads to restoration of pituitary LH beta mRNA levels and pulsatile LH secretion after 72 h, which correlates with a sharp decrease in plasma progesterone concentrations to basal levels. In contrast, in ovariectomized lactating rats, the increase in pituitary LH function is observed by 24 h after pup removal. To determine if progesterone secretion from the ovary participates in the delayed recovery of LH secretion, we treated lactating rats with the progesterone antagonist RU 486 and determined the effects on the time course of recovery of pulsatile LH secretion and LH subunit mRNA after pup removal and on pituitary responsiveness to GnRH. In ovarian-intact lactating rats treated with RU 486, pulsatile LH secretion was observed in about 40% of the rats within 24 h after pup removal (LH interpulse interval, 43.7 +/- 8.3 min) and in about 90% of the rats within 48 h after pup removal (LH interpulse interval, 46.1 +/- 3.6 min). The mean plasma LH level in the RU 486-treated rats was 10.1 +/- 2.2 ng/ml 24 h after removal of pups (control, less than 5 ng/ml) and had increased to 35.1 +/- 6.4 ng/ml 48 h after pup removal (control, 9.1 +/- 2.5 ng/ml). However, RU 486 treatment had no significant effect on LH mRNA subunit levels. To determine whether progesterone acts at the pituitary to block GnRH stimulation of LH secretion, we tested the effects of RU 486 on LH secretion in response to 2- and 5-ng pulses of GnRH. Pituitary responsiveness was tested 24 h after pup removal. We found that both doses of GnRH were effective in stimulating pulsatile LH secretion, and treatment with RU 486 had no significant effect on this response. We conclude from these studies that progesterone secretion from the ovary contributes to the inhibition of LH secretion that occurs after pup removal, since antagonizing progesterone's action resulted in an earlier restoration of pulsatile LH secretion. The increase in LH secretion occurred in the absence of any significant changes in responsiveness of the pituitary to GnRH stimulation or in LH subunit mRNA levels. Therefore, the primary site of action of progesterone would appear to be at the hypothalamus to suppress pulsatile GnRH secretion.     

Endotoxin inhibits pituitary responsiveness to gonadotropin-releasing hormone

Immune/inflammatory challenges powerfully suppress reproductive neuroendocrine activity. This inhibition is generally considered to be centrally mediated via mechanisms that regulate GnRH secretion. The present study provides two lines of evidence that bacterial endotoxin, a commonly used model of immune/inflammatory challenge, also acts to inhibit pituitary responsiveness to GNRH: In the first experiment, pulsatile secretion of GnRH into pituitary portal blood and LH into peripheral blood were monitored in ovariectomized ewes treated with a low dose of endotoxin. Although this treatment only marginally suppressed GnRH pulsatile secretion, it markedly disrupted LH pulsatility. In extreme cases, the low dose of endotoxin blocked LH pulses without inhibiting endogenous GnRH pulses, thereby uncoupling GnRH and LH pulsatile suppression. In the second experiment, we tested the hypothesis that endotoxin inhibits pituitary responsiveness to exogenous GnRH pulses. Hourly pulses of GnRH were delivered to ovariectomized ewes in which endogenous GnRH secretion was blocked. Endotoxin suppressed the amplitude of GnRH-induced LH pulses. Together, these observations support the conclusion that endotoxin inhibits pituitary responsiveness to GNRH:     

Gonadotropin-releasing hormone differentially regulates expression of the genes for luteinizing hormone alpha and beta subunits in male rats.

Gonadotropin-releasing hormone (GnRH) and gonadal steroids regulate synthesis and release of luteinizing hormone (LH). GnRH is secreted intermittently by the hypothalamus, producing pulsatile LH release, and a pulsatile GnRH stimulus is required to maintain LH secretion. We report the regulatory effects of GnRH pulse injections on pituitary concentrations of LH alpha and beta subunit mRNAs in a castrated/testosterone-replaced male rat model. Replacement with physiologic amounts of testosterone decreased concentrations of both LH subunit mRNAs. GnRH pulse injections (10-250 ng per pulse given every 30 min for 48 hr) increased both mRNA concentrations, but the dose response patterns were markedly different. alpha subunit mRNA was increased by all GnRH doses but not the levels seen after castration alone. In contrast, LH beta subunit mRNA concentrations showed a marked dependence on GnRH dose. Maximal responses, to values similar to those in castrates, occurred after 25-ng GnRH pulses, and larger doses produced a smaller increase in LH beta subunit mRNA. Both the acute LH secretory response to GnRH and the number of GnRH receptors followed a pattern similar to the LH beta subunit mRNA concentration and were maximal after the 25-ng GnRH dose. These results show that GnRH can differentially regulate LH subunit mRNAs and suggest that concentrations of LH beta subunit mRNA may be a limiting factor in GnRH-stimulated LH release.     

Rapid augmentation by progesterone of agonist-stimulated luteinizing hormone secretion by cultured pituitary cells

Progesterone acts bimodally at the hypothalamus and at the pituitary gland, the sequelae in vivo being either stimulation or inhibition of gonadotropin secretion depending on a host of preconditions. Pituitary cells in culture were studied to characterize the acute action of progesterone on LH secretion. Preliminary studies established that anterior pituitary cells from adult female rats cultured for three days in 10% charcoal treated fetal bovine serum (c/t FBS) resulted in LH secretory responses to GnRH pulses which were half that for cells cultured in untreated FBS or c/t FBS + 0.2 nM 17 beta-estradiol (E2). Under standardized culture conditions (c/t FBS + E2), GnRH self-potentiation was evident. With this system, 90 min exposure to 200 nM progesterone resulted in a 3-fold augmentation of GnRH-stimulated LH secretion without affecting baseline LH. This action was manifested by 45 but not 15 min of progesterone exposure and was inhibited by simultaneous addition of cycloheximide. The augmentation of agonist-stimulated LH release could be elicited up to 4-5 h after progesterone addition. The estimated half-maximal effect was 10(-9) M, and this concentration of progesterone required E2-pretreatment of the cultured cells. In summary, addition of progesterone to cultured anterior pituitary cells pretreated with E2 leads to a concentration-, time-, and protein synthesis-dependent augmentation of pulsatile GnRH-stimulated LH secretion within 45 min of progesterone exposure. This rapid and unambiguous progesterone action in pituitary cells could function in vivo to define the final magnitude of the preovulatory LH surge.     

Potent action of RFamide-related peptide-3 on pituitary gonadotropes indicative of a hypophysiotropic role in the negative regulation of gonadotropin secretion

We identified a gene in the ovine hypothalamus encoding for RFamide-related peptide-3 (RFRP-3), and tested the hypothesis that this system produces a hypophysiotropic hormone that inhibits the function of pituitary gonadotropes. The RFRP-3 gene encodes for a peptide that appears identical to human RFRP-3 homolog. Using an antiserum raised against RFRP-3, cells were localized to the dorsomedial hypothalamic nucleus/paraventricular nucleus of the ovine brain and shown to project to the neurosecretory zone of the ovine median eminence, predicating a role for this peptide in the regulation of anterior pituitary gland function. Ovine RFRP-3 peptide was tested for biological activity in vitro and in vivo, and was shown to reduce LH and FSH secretion in a specific manner. RFRP-3 potently inhibited GnRH-stimulated mobilization of intracellular calcium in gonadotropes. These data indicate that RFRP-3 is a specific and potent mammalian gonadotropin-inhibiting hormone, and that it acts upon pituitary gonadotropes to reduce GnRH-stimulated gonadotropin secretion.     

Role of endogenous opioid peptides in mediating progesterone-induced disruption of the activation and transmission stages of the GnRH surge induction process.

How progesterone blocks the E2-induced GnRH surge in females is not known. In this study we assessed whether the endogenous opioid peptides (EOPs) that mediate progesterone negative feedback on pulsatile GnRH secretion also mediate the blockade of the GnRH surge. We treated ovariectomized ewes with physiological levels of E2 and progesterone to stimulate and block the GnRH surge, respectively, using LH secretion as an index of GnRH release. A pilot study confirmed that blocking opioidergic neurotransmission with the opioid receptor antagonist, naloxone (NAL; 1 mg/kg.h, i.v.), could prevent the suppression of pulsatile LH secretion by progesterone in our model. By contrast, antagonizing EOP receptors with NAL did not restore LH surges in ewes in which the E2-induced GnRH surge was blocked by progesterone treatment during the E2-dependent activation stage (Exp 1) of the GnRH surge induction process. However, in ewes treated with progesterone during the E2-independent transmission stage (Exp 2), NAL partially restored blocked LH surges, as indicated by increased fluctuations in LH that, in some cases, resembled LH surges. We conclude, therefore, that the EOPs that mediate progesterone negative feedback on pulsatile GnRH secretion are not involved in blockade of activation of the E2-induced GnRH surge by progesterone, but do appear to be part of the mechanism by which progesterone disrupts the transmission stage.     

Gonadotropin-inhibitory hormone in Gambel's white-crowned sparrow (Zonotrichia leucophrys gambelii): cDNA identification, transcript localization and functional effects in laboratory and field experiments

The neuropeptide control of gonadotropin secretion is primarily through the stimulatory action of the hypothalamic decapeptide, GnRH. We recently identified a novel hypothalamic dodecapeptide with a C-terminal LeuPro-Leu-Arg-Phe-NH2 sequence in the domestic bird, Japanese quail (Coturnix japonica). This novel peptide inhibited gonadotropin release in vitro from the quail anterior pituitary; thus it was named gonadotropin-inhibitory hormone (GnIH). GnIH may be an important factor regulating reproductive activity not only in domesticated birds but also in wild, seasonally breeding birds. Thus, we tested synthetic quail GnIH in seasonally breeding wild bird species. In an in vivo experiment, chicken gonadotropin-releasing hormone-I (cGnRH-I) alone or a cGnRH-I/quail GnIH cocktail was injected i.v. into non-breeding song sparrows (Melospiza melodia). Quail GnIH rapidly (within 2 min) attenuated the GnRH-induced rise in plasma LH. Furthermore, we tested the effects of quail GnIH in castrated, photostimulated Gambel's white-crowned sparrows (Zonotrichia leucophrys gambelii), using quail GnIH or saline for injection. Again, quail GnIH rapidly reduced plasma LH (within 3 min) compared with controls. To characterize fully the action of GnIH in wild birds, the identification of their endogenous GnIH is essential. Therefore, in the present study a cDNA encoding GnIH in the brain of Gambel's white-crowned sparrow was cloned by a combination of 3' and 5' rapid amplification of cDNA ends and compared with the quail GnIH cDNA previously identified. The deduced sparrow GnIH precursor consisted of 173 amino acid residues, encoding one sparrow GnIH and two sparrow GnIH-related peptides (sparrow GnIH-RP-1 and GnIH-RP-2) that included Leu-Pro-Xaa-Arg-Phe-NH2 (Xaa=Leu or Gln) at their C-termini. All these peptide sequences were flanked by a glycine C-terminal amidation signal and a single basic amino acid on each end as an endoproteolytic site. Although the homology of sparrow and quail GnIH precursors was approximately 66%, the C-terminal structures of GnIH, GnIH-RP-1 and GnIH-RP-2 were all identical in two species. In situ hybridization revealed the cellular localization of sparrow GnIH mRNA in the paraventricular nucleus (PVN) of the hypothalamus. Immunohistochemical analysis also showed that sparrow GnIH-like immunoreactive cell bodies and terminals were localized in the PVN and median eminence respectively. Thus, only the sparrow PVN expresses GnIH, which appears to be a hypothalamic inhibitory factor for LH release, as evident from our field injections of GnIH into free-living breeding white-crowned sparrows. Sparrow GnIH rapidly (within 2 min) reduced plasma LH when injected into free-living Gambel's white-crowned sparrows on their breeding grounds in northern Alaska. Taken together, our results indicate that, despite amino acid sequence differences, quail GnIH and sparrow GnIH have similar inhibitory effects on the reproductive axis in wild sparrow species. Thus, GnIH appears to be a modulator of gonadotropin release.     

Interaction between cortisol and arachidonic acid on the secretion of LH from ovine pituitary tissue.

In several species, glucocorticoids act directly on the pituitary gonadotroph to suppress the gonadotrophin-releasing hormone (GnRH)-induced secretion of the gonadotrophins, especially LH. A mechanism for this action of these adrenal steroids has not been established, but it appears that the glucocorticoids influence LH release by acting on one or more post-receptor sites. This study investigated whether glucocorticoids disrupt GnRH-induced LH release by altering the liberation of arachidonic acid from plasma membrane phospholipids, a component of GnRH-induced LH release. Using perifused ovine pituitary tissue, it was established that exposure of gonadotrophs to 1-1000 nmol cortisol/l for 4 h or longer significantly reduced GnRH-stimulated LH release with the maximal inhibitory effect being observed after 6 h of exposure to cortisol. This suppressive effect of cortisol could be reversed by administration of arachidonic acid, which in its own right could stimulate LH release from ovine pituitary tissue. Furthermore, the inhibitory effect of cortisol on GnRH-stimulated LH release could be directly correlated with decreased pituitary responsiveness to GnRH-stimulated arachidonic acid liberation, consistent with our hypothesis that glucocorticoids can suppress GnRH-induced secretion of LH by reducing the amount of arachidonic acid available for the exocytotic response of GnRH.     

Gonadotrophin-releasing hormone modulation of gonadotrophins in the ewe: evidence for differential effects on gene expression and hormone secretion.

While the regulation of gonadotrophin secretion by gonadotrophin-releasing hormone (GnRH) has been well documented in both rats and sheep, its role in the synthesis of gonadotrophin subunits remains unclear. We have investigated the effects of the specific inhibition of GnRH by a GnRH agonist on the expression of gonadotrophin subunit genes and the subsequent storage and release of both intact hormones and free alpha subunit. Treatment with GnRH agonist for 6 weeks abolished pulsatile LH secretion, reduced plasma concentrations of FSH and prevented GnRH-induced release of LH and FSH. This was associated with a reduction of pituitary LH-beta mRNA and FSH-beta mRNA levels (to 5 and 30% of luteal control values respectively), but not alpha mRNA which was significantly increased (75% above controls). While there was a small decrease in the pituitary content of FSH (30% of controls), there was a drastic reduction in LH pituitary content (3% of controls). In contrast to the observed rise in alpha mRNA, there was a decrease in free alpha subunit in both the pituitary and plasma (to 30 and 80% of control levels). These results suggest that, while GnRH positively regulates the expression of both gonadotrophin beta-subunit genes, it can, under certain circumstances, negatively regulate alpha-subunit gene expression. Despite the complete absence of LH and FSH in response to GnRH, there remained a basal level of beta-subunit gene expression and only a modest reduction (50%) in the plasma levels of both FSH and LH, suggesting that there is a basal secretory pathway.(ABSTRACT TRUNCATED AT 250 WORDS)     

RFamide-related peptide-3 receptor gene expression in GnRH and kisspeptin neurons and GnRH-dependent mechanism of action

RFamide-related peptide-3 (RFRP-3) is known to inhibit the activity of GnRH neurons. It is not yet clear whether its G protein-coupled receptors, GPR147 and GPR74, are present on GnRH neurons or on afferent inputs of the GnRH neuronal network or whether RFRP-3 can inhibit gonadotropin secretion independently of GnRH. We tested the following: 1) whether GnRH is essential for the effects of RFRP-3 on LH secretion; 2) whether RFRP-3 neurons project to GnRH and rostral periventricular kisspeptin neurons in mice, and 3) whether Gpr147 and Gpr74 are expressed by these neurons. Intravenous treatment with the GPR147 antagonist RF9 increased plasma LH concentration in castrated male rats but was unable to do so in the presence of the GnRH antagonist cetrorelix. Dual-label immunohistochemistry revealed that approximately 26% of GnRH neurons from male and diestrous female mice were apposed by RFRP-3 fibers, and 19% of kisspeptin neurons from proestrous female mice were apposed by RFRP-3 fibers. Using immunomagnetic purification of GnRH and kisspeptin cells, single-cell nested RT-PCR, and in situ hybridization, we showed that 33% of GnRH neurons and 9-16% of rostral periventricular kisspeptin neurons expressed Gpr147, whereas Gpr74 was not expressed in either population. These data reveal that RFRP-3 can act at two levels of the GnRH neuronal network (i.e. the GnRH neurons and the rostral periventricular kisspeptin neurons) to modulate reproduction but is unable to inhibit gonadotropin secretion independently of GnRH.     

Hypophyseal actions of pulsatile gonadotropin-releasing hormone in the ewe: development and application of a new experimental model

Ovariectomy of ewes during seasonal anestrus and immediate replacement with subcutaneous Silastic progesterone implants which maintained a midluteal-phase level of circulating progesterone obliterated pulsatile luteinizing hormone (LH) secretion for up to 2 weeks without preventing a normal response of the pituitary to exogenous pulses of gonadotropin-releasing hormone (GnRH). Consideration was given to the possibility that such 'progesterone-suppressed ewes' would be useful as an animal model for isolating the pituitary from pulsatile GnRH secretion, and for testing the hypophyseotropic actions of exogenous GnRH. Two experiments were conducted using this progesterone-suppressed ewe as an animal model. In the first, the amplitude of LH pulses elicited by episodic delivery of GnRH was found to depend upon the frequency of exogenous GnRH pulses. Hourly frequency produced larger LH pulses than a 30-min frequency of GnRH. In the second experiment, LH surges were induced in progesterone-suppressed ewes by a combined treatment of estradiol and GnRH in patterns designed to approximate those secreted in the follicular phase of the estrous cycle. Our findings suggest that the progesterone-suppressed ewe is a suitable animal model for studying the hypophyseotropic actions of GnRH. Further, they are consistent with two hypotheses concerning the regulation of the tonic and surge modes of LH secretion. (1) The inverse relationship between LH pulse frequency and amplitude observed in a number of situations can be accounted for, at least in part, by a differential response of the pituitary to GnRH. (2) Progesterone can block the LH surge by an action on the brain and an inhibition of pulsatile GnRH release.