Effects of GABA(A) receptor modulation on the expression of GnRH gene and GnRH receptor (GnRH-R) gene in the hypothalamus and GnRH-R gene in the anterior pituitary gland of follicular-phase ewes

The effect of prolonged, intermittent infusion of GABA(A) receptor agonist (muscimol) or GABA(A) receptor antagonist (bicuculline) into the third cerebral ventricle on the expression of GnRH gene and GnRH-R gene in the hypothalamus and GnRH-R gene in the anterior pituitary gland was examined in follicular-phase ewes by real-time PCR. The activation or inhibition of GABA(A) receptors in the hypothalamus decreased or increased the expression of GnRH and GnRH-R genes and LH secretion, respectively. The present results indicate that the GABAergic system in the hypothalamus of follicular-phase ewes may suppress, via hypothalamic GABA(A) receptors, the expression of GnRH and GnRH-R genes in this structure. The decrease or increase of GnRH-R mRNA in the anterior pituitary gland and LH secretion in the muscimol- or bicuculline-treated ewes, respectively, is probably a consequence of parallel changes in the release of GnRH from the hypothalamus activating GnRH-R gene expression. It is suggested that GABA acting through the GABA(A) receptor mechanism on the expression of GnRH gene and GnRH-R gene in the hypothalamus may be involved in two processes: the biosynthesis of GnRH and the release of this neurohormone in the hypothalamus.     

Pituitary gonadotropin-releasing hormone (GnRH) receptor: structure, distribution and regulation of expression

Reproduction in mammals is controlled by interactions between the hypothalamus, anterior pituitary and gonads. Interaction of GnRH with its cognate receptor is essential to regulating reproduction. Characterization of the structure, distribution and expression of GnRH receptors (GnRH-R) has furthered our understanding of the physiological consequences of GnRH stimulation of pituitary gonadotropes. Based on the putative topology of the amino acid sequence of the GnRH-R and point mutation studies, key elements of the GnRH-R have been identified to play a role in ligand recognition and binding, G-protein activation and internalization. Normally, reproductive function is mediated by GnRH-R expressed only on the membranes of pituitary gonadotropes. The density of GnRH-R on gonadotropes determines their ability to respond to GnRH. This density is highest just prior to ovulation and likely is important for complete expression of the pre-ovulatory surge of LH. Therefore, knowledge regarding what regulates the density of GnRH-R is essential to understanding changes in pituitary sensitivity to GnRH and ultimately, to expression of the LH surge. Regulation of GnRH-R gene expression is influenced by a multitude of factors including gonadal steroid hormones, inhibin, activin and perhaps most importantly GnRH itself.     

Cloning and expression of gonadotropin-releasing hormone receptor in the brain and pituitary of the European sea bass: an in situ hybridization study

A full-length cDNA encoding a GnRH receptor (GnRH-R) has been obtained from the pituitary of the European sea bass, Dicentrarchus labrax. The complete cDNA is 1814 base pairs (bp) in length and encodes a protein of 416 amino acids. The 5' UTR and 3' UTR are 239 bp and 324 bp in size, respectively. The expression sites of this GnRH-R were studied in the brain and pituitary of sea bass by means of in situ hybridization. A quantitative analysis of the expression of the GnRH-R gene along the reproductive cycle was also performed. The GnRH-R brain expression was especially relevant in the ventral telencephalon and rostral preoptic area. Some GnRH-R messenger-expressing cells were also evident in the dorsal telencephalon, caudal preoptic area, ventral thalamus, and periventricular hypothalamus. A conspicuous and specific GnRH-R expression was detected in the pineal gland. The highest expression of the GnRH-R gene was observed in the proximal pars distalis of the pituitary. This expression was evident in all LH cells and some FSH cells but not in somatotrophs. In the pituitary, the quantitative analysis revealed a higher expression of GnRH-R gene during late vitellogenesis in comparison with maturation, spawning, and postspawning/resting periods. However, in the brain, the highest GnRH-R expression was evident at spawning or postspawning/ resting periods. These results suggest that the expression of this GnRH-R is regulated in a different manner in the brain and the pituitary of sea bass.     

Differences in gonadotrophin concentrations and pituitary responsiveness to GnRH between Booroola ewes which were homozygous (FF), heterozygous (F+) and non-carriers (++) of a major gene influencing their ovulation rate

The mean plasma concentrations of FSH and LH were significantly higher in FF ewes than in ++ ewes with those F+ animals being consistently in between. These gene-specific differences were found during anoestrus, the luteal phase and during a cloprostenol-induced follicular phase, suggesting that the ovaries of ewes with the F-gene are more often exposed to elevated concentrations of FSH and LH than are the ovaries of ewes without the gene. The gene-specific differences in LH secretion arose because the mean LH amplitudes were 2-3 times greater in FF compared to ++ ewes with the LH amplitudes for F+ ewes being in between. The LH pulse frequencies were similar. In these studies the pulsatile nature of FSH secretion was not defined. The pituitary contents of LH during the luteal phase, were similar in all genotypes whereas for FSH they were significantly higher in the F-gene carriers compared to ++ ewes. The pituitary sensitivity to exogenous GnRH (0.1, 0.5 and 25 micrograms i.v.) was related to genotype. Overall the LH responses to GnRH were lower in FF ewes than in ++ ewes with the results for the F+ ewes being in between. The FSH responses to all GnRH doses in the FF genotype were minimal (i.e. less than 2-fold). In the other genotypes a greater than 2-fold response was noted only at the highest GnRH dose (i.e. 25 micrograms). Treatment of FF and F+ but not ++ ewes with GnRH eventually led to a reduced FSH output, suggesting that the pituitary responses to endogenous GnRH were being down-regulated in the F-gene carriers whereas this was not the case in the non-carriers. Collectively these data confirm that peripheral plasma and the pituitary together with the ovary are compartments in which F-gene differences can be observed. In conclusion, these findings raise the possibility that F-gene-specific differences may also extend to the hypothalamus and/or other regions of the brain.     

No evidence for pituitary priming to gonadotropin-releasing hormone in relation to luteinizing hormone (LH) secretion prior to the preovulatory LH surge in ewes

The purpose of this study was to determine the occurrence of and the regulatory mechanisms involved in priming of the pituitary to GnRH before the preovulatory LH surge in sheep. Experiment 1: Forty-two ewes had progestagen devices removed after 14 days and were assigned to luteal (Lut) or follicular (Foll) groups. Fifteen days later, blood sampling was initiated either immediately or 36 h after induced luteolysis in groups Lut and Foll, respectively. After 4 h, ewes were administered either saline (n = 5) or 250 ng (n = 8) or 10 microg (n = 8) of GnRH. Five ewes per treatment group were killed 1 h later, while remaining animals were blood sampled for a further 7 h. Experiment 2: Eighteen ewes were allocated to Lut and Foll groups (described above). Blood samples were collected from 2 h before GnRH (10 microg) treatment until 7 h after. Despite up-regulated GnRH-R mRNA levels in Foll ewes, pituitary content and plasma levels of LH and LHbeta mRNA levels were similar between groups. Mean FSHbeta mRNA and plasma FSH levels were elevated in Lut ewes but declined after GnRH treatment. Inversely, plasma estradiol and inhibin-A concentrations were higher in Foll ewes and declined after GnRH treatment. Fewer LH(+ve)/secretogranin II(-ve) (SgII(-ve)) granules were present in gonadotropes of Foll ewes, coincident with increased basal LH levels. Fewer smaller sized granules were present after GnRH treatment. In conclusion, there was no evidence of self-priming before onset of the preovulatory LH surge. Constitutive release of LH(+ve)/SgII(-ve) granules may maintain basal LH levels while smaller sized, presumably mature granules may be preferentially released after GnRH stimulation.     

Effect of neuropeptide Y on GnRH-induced LH release from bovine anterior pituitary cell cultures derived from heifers in a follicular,luteal or ovariectomized state

Objectives were to determine if neuropeptide Y (NPY) had direct effects GnRH induced secretion of LH from the anterior pituitary gland, and if endogenous steroids modulated the effect of NPY. To accomplish these objectives, 15 Hereford heifers were assigned to one of three ovarian status groups: follicular, luteal, or ovariectomized. One animal from each of the three ovarian status groups was slaughtered on each of 5 days and anterior pituitary gland harvested. Anterior pituitary gland cells within ovarian status were equally distributed and randomly assigned to one of three cell culture treatments: no NPY or GnRH (control), 10 nM GnRH, or 100 nM NPY+10 nM GnRH. Anterior pituitary cell cultures were incubated with or without NPY for 4 h and further incubated for an additional 2 h with or without GnRH and supernatant collected for quantification of LH. Treatment of anterior pituitary cell cultures with GnRH or GnRH+NPY did not affect LH release in cultures obtained from follicular (S.E.=5%; P=0.58) or ovariectomized (S.E.=7%; P=0.22) heifers. Both GnRH and GnRH+NPY increased LH release from anterior pituitary cell cultures from heifers in the luteal phase (S.E.=14%; P < or = 0.05) compared to control cultures. Cultures from luteal phase heifers treated with GnRH did not differ from those treated with GnRH+NPY (P=0.34). These data provide evidence to suggest that effects of NPY on LH release may occur primarily at the level of the hypothalamus.     

Attenuation of the estradiol-induced surge release of luteinizing hormone by antihistamine in ovariectomized ewes

Ovariectomized ewes received intramuscular (i.m.) injections of an H₁-histamine receptor antagonist, diphenhydramine, or saline during the anestrous and breeding seasons to determine if histamine may regulate the estradiol-induced surge release of LH in ewes. In addition, concentrations of histamine and GnRH in hypothalamic regions and histamine and LH in the pituitary gland were determined during the estradiol-induced surge of LH. Pretreatment mean, basal, and estradiol-induced secretion of LH did not differ (P > 0.05) among seasons. However, the quantity of LH (ng) measured during the estradiol-induced surge of LH was less (P < 0.05) in ewes treated with diphenhydramine (411 ± 104) than saline (747 ± 133). Treatment with diphenhydramine did not (P > 0.05) influence steady-state concentrations of histamine in hypothalamic or pituitary gland tissues, hypothalamic concentrations of GnRH, or anterior pituitary concentrations of LH during the estradiol-induced surge of LH. It is concluded that histamine may modulate the estradiol-induced surge release of LH in ewes by affecting the secretion of GnRH.     

Prepubertal changes in the hypothalamic-pituitary axis of Holstein bulls

We investigated the nature and sites of changes in the hypothalamic-pituitary axis associated with the onset of high-frequency, high-amplitude discharges of luteinizing hormone (LH) in young bulls during the transition from the infantile to the prepubertal phase of development. Blood serum and neuroendocrine tissues from bulls killed at 1, 6, 10, 14, or 18 wk of age were evaluated. Concentrations of LH in serum from bulls 1 or 6 wk old averaged less than 0.25 ng/ml and only one episodic discharge of LH was detected for 10 bulls. At 10, 14, or 18 wk, 14 of 15 bulls had episodic discharges of LH. Concentrations of testosterone in serum were progressively higher at 10, 14, and 18 wk, but the concentration of estradiol was maximal at 6 wk. The concentrations of gonadotropin-releasing hormone (GnRH) in the anterior hypothalamus, posterior hypothalamus, or median eminence were not influenced by age. However, concentration of GnRH receptors in the anterior pituitary gland increased 314% between 6 and 10 wk and the concentration of LH increased 67%. Between 6 and 10 wk, concentrations of estradiol receptors in the anterior and posterior hypothalamus declined by 68% and 46%, but the concentration of estradiol receptors in the anterior pituitary gland increased by 103%. For most characteristics, there was no major change between 10 and 18 wk. We postulate that between 6 and 10 wk of age, there is 1) removal of an estradiol-mediated block of GnRH secretion and 2) an estradiol-mediated, and possibly GnRH-mediated, increase in pituitary GnRH receptors. Together, these changes result in greatly increased stimulation of the anterior pituitary gland by GnRH between 6 and 10 wk of age and stimulation of the discharges of LH characteristic of bulls in the early prepubertal phase of development.     

Changes in the hypothalamic-hypophyseal axis of mares in relation to the winter solstice.

In mares, the amount of gonadotrophin-releasing hormone (GnRH) is low in the hypothalamus during seasonal anoestrus, but by early spring, concentrations of GnRH are high. The timing of this response was characterized more precisely by determining concentrations of GnRH in hypothalamic tissue collected immediately before and at various times after the winter solstice (22 December 1986). Ovaries, pituitary gland, hypothalamus and a blood sample were collected from six groups of mares (6-12 mares per group) at death, 1 week before day of the winter solstice and 1, 2, 3 and 12 weeks afterwards. No significant changes in weight of the anterior pituitary gland or concentrations of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were observed in the anterior pituitary gland (P > 0.1). Mean diameter of the largest follicle, number of follicles > or = 20 mm in diameter and concentrations of LH and FSH in serum remained unchanged for weeks -1 to +3 (P < 0.05), then increased significantly by week 12 (P < 0.001). Content and concentration of GnRH in the median eminence was low at -1 week, increased gradually (P < 0.05) to a maximum by +1 week, then decreased gradually (P < 0.05) to low values at 12 weeks. Means (+/- SEM) for -1, +1 and +12 weeks were 33.5 +/- 5.5, 117.7 +/- 18.6 and 29.8 +/- 3.7 ng GnRH, respectively. Mean content of GnRH in the preoptic area of the hypothalamus showed a reciprocal pattern.(ABSTRACT TRUNCATED AT 250 WORDS)     

Early pituitary LH response to injections of GnRH in ewes

In the deep anoestrous period (June), five intact ewes and five ovariectomized ewes received 50 ug synthetic gonadotrophin-releasing hormone (GnRH). In the mid-breeding season (October), the GnRH administrations were repeated in five intact and four ovariectomized ewes; the former were in the luteal phase of the cycle. Blood samples were collected every 30 sec for 15 min, then at 15-min intervals. Release of luteinizing hormone (LH) occurred as soon as the second minute after injection in all ewes. This early response was earlier and more abrupt in the ovariectomized ewes than in the intact animals. In a second experiment three intact ewes that were in deep anoestrus received 50 ug GnRH followed 5 h 20 min later by a second identical injection. Another three intact ewes in deep anoestrus received two injections of 1 ug GnRH. Blood samples were taken every 15 sec for 15 min, then every 20 min until the next injection, and for a further 5 h after the second injection. This regimen was repeated in mid-breeding season during the luteal phase. There was again a very early release of LH; the magnitude of response was similar after the first injection of either 50 ug or 1 ug GnRH to intact ewes either in the breeding season or during deep anoestrus. However, a greater early release of LH was obtained at the lower dose only after the second injection of GnRH. Apart from this exception, the similar early release of LH occurred in spite of different amounts of LH released thereafter in response to the two doses of GnRH. It is suggested that the early response to GnRH consists of LH stored in a "readily releasable" pool in the pituitary, whereas the main release of LH may be a result of increased synthesis and/or release of a more stable pool.     

Interactions between 17 beta-estradiol and the hypothalamo-pituitary beta-endorphin system in the regulation of the cyclic LH secretion

This paper further substantiates the physiological role of beta-endorphin (beta-END) in the control of the cyclic LH secretion and provides new data on the interactions between 17 beta-estradiol (17 beta-E2) and beta-END at both the hypothalamic and pituitary levels. At the hypothalamic level, during the estrous cycle in rats, beta-END concentrations were highest on diestrus I in the arcuate nucleus, median preoptic area and median eminence and lowest at the time of the preovulatory 17 beta-E2 surge on proestrus, before the subsequent preovulatory hypothalamic GnRH and plasma LH surges. Data obtained in ovariectomized 17 beta-E2-treated ewes support the direct involvement of 17 beta-E2 in changes in beta-END and GnRH concentrations in these hypothalamic areas. At the anterior pituitary level, in vitro results obtained using anterior pituitaries from the proestrus morning cycling female rat have shown that 17 beta-E2 strongly suppresses beta-END secretion and that GnRH stimulates the release of beta-END. Furthermore, marked fluctuations were observed for plasma beta-END throughout the menstrual cycle in the woman. Low beta-END concentrations were observed in the period preceding the LH preovulatory surge. Taken together, these results show that: (1) decreases in hypothalamic beta-END concentrations, which are controlled at least by circulating levels of 17 beta-E2, modulate GnRH synthesis and/or release and contribute to the mechanisms which initiate the LH surge; (2) anterior pituitary beta-END might be involved in the mechanisms which terminate the LH surge.     

Progesterone exposure of the preovulatory follicle in the seasonally anestrous ewe alters the expression of angiogenic growth factors in the early corpus luteum

Gonadotrophin releasing hormone (GnRH)-induced ovulation in seasonally anestrous ewes is associated with a high incidence of defective corpora lutea (CL), which can be completely eliminated by priming ewes with progesterone before GnRH treatment, but the physiological basis of this has remained elusive. This study tested the hypothesis that progesterone priming eliminates defective luteal function by altering the expression of Vascular Endothelial Growth Factor (VEGF), its receptor VEGFR-2, and angiopoietin (ANG)-1, ANG-2 and their receptorTIE-2 in the early CL. Fifteen seasonally anestrous ewes were treated by i.m. injection with 20 mg of progesterone 3 days before the start of GnRH treatment, while another 15 animals served as controls. Intravenous injections of 500 ng GnRH were given to all the ewes every 2 h for 28 h, followed by a 300 μg GnRH bolus injection to synchronize the preovulatory luteinizing hormone (LH) surge. Corpora lutea were collected 1, 2 and 4 days after ovulation and analyzed for protein and mRNA expression of VEGF, VEGFR-2, ANG-1, ANG-2 and Tie-2 using Western Immunoblotting and in situ hybridization. VEGF, VEGFR-2 and ANG-1 expression was significantly higher (P ≤ 0.05) in the CL of progesterone-primed animals compared to non-primed ones. However, no differences were observed in the ANG-2 or Tie-2 expression levels between the two treatment groups. These data suggest that progesterone priming of the preovulatory follicle alters the expression of some angiogenic growth factors in the early CL, leading to greater vascular stability and thereby normal luteal function.     

Does the seasonal increase in estradiol negative feedback prevent luteinizing hormone surges in anestrous ewes by suppressing hypothalamic gonadotropin-releasing hormone pulse frequency?

To test the hypothesis that the anestrous increase in estradiol negative feedback prevents estrous cycles by suppressing hypothalamic gonadotropin-releasing hormone (GnRH) pulse frequency, a variety of regimens of increasing GnRH pulse frequency were administered to anestrous ewes for 3 days. A luteinizing hormone (LH) surge was induced in 45 of 46 ewes regardless of amplitude or frequency of GnRH pulses, but only 19 had luteal phases. Estradiol administration induced LH surges in 6 of 6 ewes, only 3 having luteal phases. Anestrous luteal phase progesterone profiles were similar in incidence, time course, and amplitude to those of the first luteal phases of the breeding season, which in turn had lower progesterone maxima than late breeding season luteal phases. In the remaining ewes, progesterone increased briefly or not at all, the increases being similar to the transient rises in progesterone occurring in most ewes at the onset of the breeding season. These results demonstrate that increasing GnRH pulse frequency induces LH surges in anestrus and that the subsequent events are similar to those at the beginning of the breeding season. Finally, they support the hypothesis that the negative feedback action of estradiol prevents cycles in anestrus by suppressing the frequency of the hypothalamic pulse generator.     

Immunoassay of gonadotrophin-releasing hormone in brain tissue of Booroola Merino ewes

The presence of a fecundity gene (F) in Booroola Merino ewes increases the ovulation rate. To test how F gene expression affects the gonadotrophin-releasing hormone (GnRH) concentration in hypothalamic or extrahypothalamic regions of the brain, GnRH was measured by radioimmunoassay in acetic acid extracts of various brain tissues from Booroola ewes which were homozygous (FF), heterozygous (F+) or non-carriers (++) of the F gene. The GnRH concentration in brain tissues from FF, F+ and ++ animals which had been ovariectomized 5 months previously was also evaluated. No significant F gene-specific differences were noted in any of the brain areas tested, in intact or ovariectomized animals. However, in ovariectomized ewes, the concentrations of GnRH increased about 2-fold in the median eminence of the hypothalamus, remained unchanged in the medial basal hypothalamus and dropped to less than 10% of the values in intact ++ animals in the preoptic area. These studies suggest that the changed pituitary sensitivity and increased gonadotrophin release in Booroolas carrying the F gene(s) is not attributable to increased hypothalamic GnRH concentrations in these animals.     

Modulation of hypothalamic galanin gene expression by estrogen in peripubertal rats.

Hypothalamic galanin gene expression was investigated during reproductive maturation in peripubertal rats. Rat galanin-like immunoreactivity (rGAL-LI) increased in the median eminence and anterior pituitary during the extended first estrous and diestrous phases relative to anestrous phase female or male rats. In the neurointermediate lobe, rGAL-LI was elevated in first diestrous phase compared to anestrous phase females or males. In a second study, hypothalamic tissue was divided into quadrants for analysis of rat galanin (rGAL) mRNA by Northern blot hybridization. Two days after the injection of pregnant mare's serum gonadotropin (PMSG), rGAL mRNA increased approximately twofold in the paraventricular area and preoptic area quadrants. No effects of PMSG on galanin gene expression were found in medial basal or supraoptic hypothalamic quadrants. Because PMSG acts through the stimulation of ovarian estrogen secretion, these studies conclude that galanin gene expression in dorsal hypothalamic nuclei is under the stimulatory influence of estrogen and suggest that galanin may be a mediator of central ovarian steroid feedback.