Relative effect of gonadotropin-releasing hormone (GnRH)-I and GnRH-II on gonadotropin release

Two forms of GnRH (GnRH-I and GnRH-II) are expressed in the hypothalamus of humans and rhesus monkeys, but their relative abilities to stimulate LH and FSH release are unknown. Therefore, young (8-12 yr) and old (21-23 yr) female rhesus monkeys were treated i.v. with bolus injections of either GnRH-I or GnRH-II (dose range, 0.01-10 microg/kg body weight); serial blood samples were remotely collected through a vascular catheter for up to 2 h after injection. Overall, plasma LH concentrations were similarly elevated after treatment with GnRH-I and GnRH-II, and the responses were slightly greater in the younger animals. Although plasma FSH concentrations were unaffected by a single exposure to GnRH-I or GnRH-II, they showed a similar significant increase after repeated exposures (every 2 h for 24 h). In a subsequent experiment, antide, a GnRH-I receptor antagonist, was administered (100 microg/kg body weight) together with a single injection of GnRH-I or GnRH-II (1 microg/kg body weight). As expected, GnRH-I-induced LH release was significantly attenuated by this combined treatment; moreover, GnRH-II-induced LH release was completely blocked. Taken together, these data show that GnRH-II can potently stimulate gonadotropin release in vivo and that this action is likely mediated through the GnRH-I receptor.     

Effect of passive immunization to gonadotropin-releasing hormone (GnRH) using GnRH antiserum on the mitotic activity of gonadotrophs in castrated male rats

In order to reveal the mechanism of elevation in the mitotic activity of gonadotrophs in the pituitary gland of castrated rats, passive immunization to GnRH designed to block the activity of GnRH in castrates was performed, and changes in the mitotic activity of pituitary gonadotrophs and mammotrophs were studied. The increased serum levels of gonadotropins and their subunits in castrates were dramatically suppressed by the administration of rabbit anti-GnRH serum (RAGnRH). However, this treatment had no effect on the serum levels of PRL, suggesting that the passive immunization to GnRH used in this study was only effective in blocking the hormonal activity of GnRH to the gonadotropin secretion. Mitosis of the gonadotrophs in normal rat anterior pituitary was rarely observed, but it was dramatically increased by castration. This elevated mitotic activity of gonadotrophs in the castrated rats was significantly suppressed by the administration of RAGnRH. On the other hand, mitotic activity of mammotrophs was decreased by castration. This diminution in the mitotic activity of mammotrophs was not changed by administration of RAGnRH. These results showed that GnRH is an important factor for stimulation of gonadotroph cell proliferation in castrated rats.     

Annexin 5 messenger ribonucleic acid expression in pituitary gonadotropes is induced by gonadotropin-releasing hormone (GnRH) and modulates GnRH stimulation of gonadotropin release.

Our previous studies on annexin 5, a member of the annexin family of proteins, have shown its expression in the anterior pituitary gland, its preferential distribution in gonadotropes, and its increase after ovariectomy. In the present study, we examined (1) whether annexin 5 is synthesized in gonadotropes, (2) whether its expression is under the control of gonadotropin-releasing hormone (GnRH), and (3) the effect of annexin 5 on gonadotropin release. Large cells, also called castration cells, appeared in anterior pituitary tissue 3 weeks after ovariectomy. These cells have been confirmed to be hyperfunctioning gonadotropes and are easily discriminated from other pituitary cells without immunostaining. Using in situ hybridization with a digoxigenin-labeled ribonucleic acid probe, enhanced expression of annexin 5 messenger ribonucleic acid (mRNA) in these gonadotropes was clearly demonstrated. Northern blot analysis showed an increase in the level of annexin 5 mRNA expression 3 weeks after ovariectomy. It was lessened 3 h after the injection of Cetrorelix (GnRH antagonist, 10 microg i.v.). Administration of a GnRH analog [GnRHa; Des-Gly 10 (Pro9) GnRH ethylamide, 0.2 ml of 2.5 microg/ml saline ten times intraperitoneally at 30-min intervals] significantly increased pituitary annexin 5 mRNA. In primary cultures of anterior pituitary cells, recombinant rat annexin 5 stimulated luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release in a dose-dependent manner. Concomitant administration of annexin 5 (1 microg/ml) and GnRHa augmented the LH and FSH release induced by GnRHa. After a 1-hour incubation, cycloheximide (10 microg/ml) apparently inhibited the LH response to GnRHa, while annexin 5 (2 microg/ml) moderated this inhibition. Further, the antisense oligodeoxynucleotide to annexin 5 mRNA blunted the LH response to GnRHa. It is thus concluded that annexin 5 is synthesized in the gonadotropes under the effect of GnRH, and it is suggested that annexin 5 synthesis mediates at least partly GnRH receptor signaling to stimulate gonadotropin secretion.     

Differential regulation of gonadotropin subunit gene promoter activity by pulsatile gonadotropin-releasing hormone (GnRH) in perifused L beta T2 cells: role of GnRH receptor concentration

The pulsatile release of GnRH by the hypothalamus is required to stimulate the pituitary-gonadal axis, and variations in GnRH pulse frequency are associated with differential synthesis and release of LH and FSH by pituitary gonadotropes. How gonadotropes differentiate between GnRH pulse frequencies and subsequently differentially regulate the expression of the LH beta and FSH beta genes remains to be determined. In the present study, using a perifusion system that allows us to replicate the GnRH pulsatility occurring in vivo, we have systematically characterized the effects of varying GnRH pulse frequencies on LH beta, FSH beta, alpha, and GnRH receptor (GnRHR) gene promoter stimulation in L beta T2 cells. We demonstrate that LH beta gene promoter activity is stimulated to the greatest extent at higher GnRH pulse frequencies, whereas the FSH beta gene promoter is preferentially stimulated at lower GnRH pulse frequencies, reflecting previous observations in primary rat pituitary cells in vivo and in vitro. By measuring GnRH binding, we demonstrate that cell-surface GnRHR number is increased at higher frequencies of pulsatile GnRH and that this increase precedes the differential regulation of LH beta and FSH beta gene promoter activity. To test the role of GnRHR number in mediating the differential effects of pulsatile GnRH, the rat GnRHR was overexpressed in L beta T2 cells, and the response to pulsatile GnRH was again assessed. Interestingly, although overexpression of GnRHR had no effect on the frequency-dependent regulation of LH beta, the induction of FSH beta gene promoter activity by pulsatile GnRH was reduced, and frequency dependence was abrogated. Our results demonstrate that L beta T2 cells represent a suitable model for the study of the differential regulation of gonadotropin subunit gene expression by pulsatile GnRH. Furthermore, our studies indicate that cell-surface GnRHR density is a critical mediator of this differential regulation.     

Analysis of the radioimmunoassay for gonadotropin-releasing hormone (GnRH): studies on the effect of radioiodinated GnRH.

When GnRH is radioiodinated by the chloramine-T method, two immunoreactive labeled species are formed at pH 6.5 with a chloramine-T: GnRH molar ratio of 11:1, whereas four bands (I, IIa, IIb, and III) are separated by polyacrylamide gel electrophoresis when the hormone is iodinated at pH 7.5 in a system containing a 97:1 molar ratio of chloramine-T:GnRH. Because they were more stable and were more immunoreactive than the other products, band I and band IIa from the latter system were used separately as tracers with Niswender antiserum R-42 in radioimmunoassays for GnRH. The standard curves of each tracer are distinct: when analyzed after log-logit transformation, the band I curve had a mean slope of -3.31 +/- 0.2 (SE) and a 50% B/Bt level of 9 +/- 0.8 pg (n=8) of synthetic GnRH, whereas the band IIa standard curve had a slope of -2.30 +/- 0.6 and a 50% B/Bt value of 20 +/- 0.9 pg (n=11). The sensitivity of both assays is approximately 2.0 pg. Gn RH concentrations in plasma and serum samples assayed with band I were consistently greater than those assayed with band IIa. Normal adult male plasmas assayed with band I measured 21 +/- 0.9 pg/ml, whereas band IIa values were 8 +/- 0.4 pg/ml. No difference between plasma and serum was detected, nor was there any difference among adult men, adult women, prepubertal children, hypogonadal patients, or hypopituitary patients with either assay. Plasma GnRH concentrations were also similar in jugular and vena cava samples from intact and castrated male rats. Because many of the samples were at or below the sensitivity of the band IIa assay, they were concentrated after extraction with either methanol or acid-ethanol. However, endogenous immunoreactive GnRH could not be concentrated by these extraction procedures. As measured in the band IIa assay, hypothalamic extracts from control adult male rats contained 3.1 +/- 0.4 ng while hypothalami from castrated rats contained 1.4 +/- 0.1 ng. Similar but slightly lower values were obtained with band I. In contrast, the GnRH content of pineal glands from intact and castrated male rats was similar (approximately 150 pg) when determined in either assay. These studies emphasize that: 1) the characteristics of the radioiodinated hormone can influence the quantitation of GnRH; and 2) endogenous plasma concentrations of GnRH are much lower than previously reported.     

Effects of increasing the frequency of low doses of gonadotropin-releasing hormone (GnRH) on gonadotropin secretion in GnRH-deficient men

The effects of increasing the frequency of pulsatile GnRH administration on LH and FSH responsiveness were studied in five GnRH-deficient men who had achieved normal sex steroid levels during prior long term GnRH replacement. Intravenous doses of GnRH were employed that had previously been demonstrated to produce LH and FSH levels in each subject similar to those in normal men. Both acute and chronic changes in pituitary responses were studied after progressive increases in GnRH frequency (from every 120 to 60 min, from 60 to 30 min, and from 30 to 15 min) during three 12-h admissions, each separated by 7 days. During the two intervals between the studies GnRH frequency was 60 and 30 min, respectively. Pituitary responses were characterized by determining the mean serum LH and FSH levels, LH pulse amplitudes, and mean LH and FSH levels which were normalized for the frequency of GnRH administration (nLH and nFSH). As the frequency of GnRH stimulation was increased acutely, mean serum LH levels rose progressively, in contrast to both LH pulse amplitude and nLH levels which decreased, while serum testosterone (T) concentrations remained constant. No further evidence of gonadotroph desensitization occurred after chronic GnRH administration at either 60- or 30-min intervals. At higher frequencies of GnRH stimulation, discrete pulses of LH were not always apparent after injections of GnRH, and in two men, marked destabilization of the gonadotroph responses occurred. Even without detectable LH pulses, serum T levels did not decline during administration of GnRH at intervals as rapid as 15 min. In contrast, there was no change in mean FSH concentrations, although nFSH values decreased progressively as the GnRH frequency was increased. nFSH levels fell to a greater degree than nLH after each increase in GnRH frequency. Thus, pituitary gonadotroph responsiveness to a fixed dose of GnRH decreased as the frequency of GnRH stimulation increased. FSH responsiveness decreased to a greater degree than did LH. Gonadotropin secretory responses are destabilized at higher frequencies of GnRH administration. Pulsatile LH stimulation of the testes does not appear necessary to maintain T secretion.     

Dexamethasone suppresses gonadotropin-releasing hormone (GnRH) secretion and has direct pituitary effects in male rats: differential regulation of GnRH receptor and gonadotropin responses to GnRH

Endogenous or exogenous glucocorticoid excess leads to the development of hypogonadotropic hypogonadism, but the site(s) and mechanisms of glucocorticoid action are uncertain. We studied the effects of various doses of dexamethasone (Dex) on the hypothalamic-pituitary-gonadal axis in intact and castrate testosterone-replaced (cast + T) male rats and attempted to determine possible sites of Dex effects. A dose-dependent suppression of basal gonadotropin secretion was induced by 5 days of Dex treatment (20, 100, 500, or 2,500 micrograms/kg.day), and the highest dose completely abolished the postcastration rise in pituitary GnRH receptor number (GnRH-R) and serum gonadotropin levels. Administration of exogenous GnRH (0.02-200 micrograms/day over 2 days) resulted in a dose-dependent induction in GnRH-R in both intact and cast + T rats, but the effect was significantly (P less than 0.01) augmented in Dex-treated animals. In contrast, acute LH and FSH responses to GnRH (10, 25, 50, 100, or 250 ng, iv) were significantly blunted in Dex-treated rats. The data suggest that 1) Dex suppresses hypothalamic GnRH secretion, thereby preventing the postcastration rises in GnRH-R and gonadotropins; 2) at the pituitary level, Dex dissociates GnRH-R and gonadotropin responses to GnRH, augmenting GnRH-R induction by GnRH and suppressing gonadotropin responses to GnRH at a postreceptor site; and 3) the model of Dex-treated rats may be useful to study differential GnRH regulation of GnRH-R and gonadotropin secretion.     

Topical absorption of gonadotropin-releasing hormone (GnRH) in goldfish

The studies reported here show that exogenous GnRH can be absorbed by goldfish from the intraperitoneal (ip) cavity, gill surface, or surrounding water. Mammalian rather than teleost GnRH was applied in order to ensure that GnRH measurement in plasma did not reflect the native form. A radioimmunoassay (RIA) specific to mammalian GnRH was used to measure the concentration of absorbed GnRH; validation for this approach was provided by HPLC and cross-reactivity studies in which mammalian GnRH was shown not to be present in control goldfish brain or pituitary extracts. Plasma concentration of GnRH was highest at the first sampling time, 4 min after administration, for all three routes. For intraperitoneal injection, plasma concentration was halved in 12 min, a period comparable with the half-life in rats. The pituitary content of GnRH also increased rapidly during the first 4 min after ip injection and remained high for 60 min. Absorption of GnRH from the gill was equally effective with water or dimethyl sulfoxide (DMSO) as vehicle.     

Dose-dependent switch in response of gonadotropin-releasing hormone (GnRH) neurons to GnRH mediated through the type I GnRH receptor

Pulsatile release of GnRH provides central control of reproduction. GnRH neuron activity is likely synchronized to produce hormone pulses, but the mechanisms are largely unknown. One candidate for communication among these neurons is GnRH itself. Cultured embryonic and immortalized GnRH neurons express GnRH receptor type I (GnRHR-1), but expression has not been shown in adult GnRH neurons. Using mice that express green fluorescent protein (GFP) in GnRH neurons, we tested whether adult GnRH neurons express GnRHR-1. GFP-positive (n = 42) and -negative neurons (n = 22) were harvested from brain slices, and single-cell RT-PCR was performed with cell contents. Fifty-two percent of the GnRH neurons tested expressed GnRHR-1, but only 9% of non-GnRH hypothalamic neurons expressed GnRHR-1; no false harvest controls (n = 13) were positive. GnRHR-1 expression within GnRH neurons suggested a physiological ultrashort loop feedback role for GnRH. Thus, we examined the effect of GnRH on the firing rate of GnRH neurons. Low-dose GnRH (20 nm) significantly decreased firing rate in 12 of 22 neurons (by 42 +/- 4%, P < 0.05), whereas higher doses increased firing rate (200 nm, five of 10 neurons, 72 +/- 26%; 2000 nm, nine of 13 neurons, 53 +/- 8%). Interestingly, the fraction of GnRH neurons responding was similar to the fraction in which GnRHR-1 was detected. Together, these data demonstrate that a subpopulation of GnRH neurons express GnRHR-1 and respond to GnRH with altered firing. The dose dependence suggests that this autocrine control of GnRH neurons may be not only a mechanism for generating and modulating pulsatile release, but it may also be involved in the switch between pulse and surge modes of release.     

Luteinizing hormone release and gonadotropin-releasing hormone (GnRH) receptor internalization: independent actions of GnRH

Three different approaches are described which provide independent and new evidence that gonadotropin-releasing hormone (GnRH) internalization and GnRH-stimulated LH release are distinct actions of the releasing hormone. 1) Removal of GnRH from medium bathing the pituitary cell cultures resulted in the prompt return of LH release to basal levels. This finding indicated that a continuous supply of externally applied GnRH is required for the stimulation of LH release. 2) Covalent immobilization of D-Lys6-des-Gly10-Pro9-ethylamide GnRH (a GnRH agonist) on agarose beads resulted in a derivative which stimulated LH release with full efficacy. At concentrations of immobilized releasing hormone analog sufficient to evoke gonadotropin release, the quantity of LH release was restricted by the number of beads added. This finding was interpreted as evidence that the attachment of immobilized agonist was stable during the bioassay and indicated that LH release could be stimulated with full efficacy without the requirement for GnRH internalization. 3) Comparative studies using image-intensified microscopy and the cell culture bioassay showed that 100 microM vinblastin markedly inhibited large scale patching and capping of the GnRH receptor (viewed by image-intensified microscopy), but did not alter the EC50 or efficacy of LH release stimulated by GnRH or the agonist described above. These observations indicated that internalization as well as large scale patching and capping of the GnRH receptor are not required for LH release.     

Hyperprolactinemia inhibits gonadotropin-releasing hormone (GnRH) stimulation of the number of pituitary GnRH receptors

Gonadotropin secretion is diminished in the presence of hyperprolactinemia, and previous studies have shown that PRL can reduce GnRH secretion and impair LH responses to GnRH. To investigate the mechanisms of the inhibitory effects of PRL on the pituitary, we administered intraarterial pulse injections of GnRH (25 ng/pulse every 30 min) to castrate testosterone-implanted male rats placed in restraint cages. Serum PRL, GnRH receptor (GnRH-R), and LH responses to GnRH were measured at intervals over 72 h. In control animals which received saline pulses, serum PRL was transiently elevated to the range of 100-150 ng/ml during the first 24 h, GnRH-R remained stable (approximately 300 fmol/mg protein) and serum LH was low (less than 10 ng/ml) throughout the 72 h. GnRH pulses in castrate testosterone-implanted animals increased GnRH-R to values (approximately 600 fmol/mg) similar to those in castrate controls (no testosterone implant, saline pulses) through 48 h, but GnRH-R declined to baseline values by 72 h in both groups. Serum LH responses to GnRH pulses were only present at 24 h. Administration of bromocriptine throughout the 72 h to immobilized castrate rats or to castrate testosterone-replaced animals treated with GnRH pulses suppressed serum PRL, and GnRH-R concentrations remained elevated through 72 h. Serum LH responses to GnRH pulses were 5- to 20-fold higher in bromocriptine-treated rats, and responses were present throughout the 72 h of the experiment. Delaying the start of bromocriptine treatment until 36 h (after the spontaneous PRL peak) resulted in reduced GnRH-R and LH responses at 72 h. Similarly, administration of ovine PRL (during the first 48 h) to bromocriptine-treated rats produced low GnRH-R concentrations at 72 h. Thus, the transient elevation of PRL seen in immobilized rats can inhibit the GnRH-stimulated increase in GnRH-R and is associated with reduced LH responses to GnRH. These results indicate that PRL has a direct inhibitory effect on the gonadotrope and suggest that impaired GnRH-R responses to GnRH are one of the mechanisms involved in the diminished gonadotropin secretion seen in hyperprolactinemia.     

Autosomal recessive idiopathic hypogonadotropic hypogonadism: genetic analysis excludes mutations in the gonadotropin-releasing hormone (GnRH) and GnRH receptor genes

Failure of the normal pattern of episodic secretion of GnRH from the hypothalamus results in the clinical syndrome of idiopathic hypogonadotropic hypogonadism (IHH), with failure of pubertal development and infertility. The only gene that has been implicated in normosmic IHH is the GnRH receptor gene (GNRHR), which accounts for 10% of cases. This report presents four families with autosomal recessive IHH, including a consanguineous pedigree from the Middle East. Defects within the genomic coding sequence of the GNRHR, and the GnRH gene itself, GNRH1, were excluded by temperature gradient gel electrophoresis, direct sequencing, and haplotypes created from simple sequence polymorphisms flanking the GNRH1 and GNRHR loci. We concluded that: 1) genetic analysis has excluded sequence variations in GNRH1 and GNRHR in four families with recessive IHH, suggesting the existence of a novel, as-yet-undiscovered gene for this condition, and 2) because mutation analysis of genomic coding sequence will fail to detect mutations deep within introns or regulatory regions, haplotype analysis is the preferred genetic methodology to eliminate the role of specific candidate genes.     

Influence of continuous gonadotropin-releasing hormone (GnRH) agonist treatment on luteinizing hormone and testosterone secretion, the response to GnRH, and the testicular response to human chorionic gonadotropin in male rhesus monkeys

Five adult male rhesus monkeys were continuously infused for 56 days with 25 micrograms/day of GnRH agonist (Wy-40972; Ag) using an implanted osmotic pump. Bioassayable serum levels of LH were elevated 8-fold on the second day of Ag treatment and then declined precipitously to below pretreatment levels by day 15. Serum levels of testosterone (T) changed similarly during Ag treatment, except that the fall in serum LH levels preceded the decline in serum levels of T by at least 2 days. Ag administration also eliminated the diurnal variation in serum LH and T. GnRH administration (50 micrograms) induced a 13- to 20-fold rise in serum LH and a 3- to 7-fold increase in serum T in control monkeys. After 4 weeks of Ag administration , none of the animals responded to GnRH. Both control and experimental monkeys had a rise in serum T in response to hCG after 7 weeks of Ag treatment. Basal levels of LH and T returned to normal by 12 days posttreatment, and the serum LH and T responses to GnRH were normal 19 days posttreatment. These results indicate that 1) continuous administration of Ag is an effective method of inducing antiferility effects in male rhesus monkeys; 2) pituitary desensitization is a major factor involved in Ag-induced gonadal dysfunction in this species; and 3) the method of administration may be the critical factor in determining the effectiveness of GnRH agonists.