Pulsatile release of luteinizing hormone-releasing hormone (LHRH) in cultured LHRH neurons derived from the embryonic olfactory placode of the rhesus monkey

To study the mechanism of LH-releasing hormone (LHRH) pulse generation, the olfactory pit/placode and the migratory pathway of LHRH neurons from monkey embryos at embryonic age 35-37 were dissected out, under the microscope, and cultured on plastic coverslips coated with collagen in a defined medium for 2-5 weeks. First, we examined whether cultured neurons release the decapeptide into media. It was found that LHRH cells release LHRH in a pulsatile manner at approximately 50-min intervals. Further, LHRH release was stimulated by depolarization with high K+ and the Na+ channel opener, veratridine. However, whereas the Na+ channel blocker, tetrodotoxin suppressed the effects of veratridine, tetrodotoxin did not alter the effects of high K+. Subsequently, the role of extracellular and intracellular Ca2+ in LHRH release was examined. The results are summarized as follows: 1) exposing the cells to a low Ca2+ (20 nM) buffer solution suppressed LHRH release, whereas exposure to a normal Ca2+ solution (1.25 mM) maintained pulsatile LHRH release; 2) LHRH release from cultured LHRH cells was stimulated by the voltage-sensitive L-type Ca2+ channel agonist, Bay K 8644 (10 microM), whereas it was suppressed by the L-type Ca2+ channel blocker, nifedipine (1 microM), but not by the N-type channel blocker, omega-conotoxin GVIA (1 microM); 3) the intracellular Ca2+ stimulant, ryanodine (1 microM), stimulated LHRH release, whereas the intracellular Ca2+ transporting adenosine triphosphatase antagonist, thapsigargin (1 and 10 microM), did not yield consistent results; and 4) carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone (1 microM), a mitochondrial Ca2+ mobilizer, stimulated LHRH release, whereas ruthenium red, a mitochondrial Ca2+ uptake inhibitor, did not induce consistent results. These results indicate that: 1) the presence of extracellular Ca2+ is essential for LHRH neurosecretion; 2) Ca2+ enters the cell via L-type channels but not N-type channels; and 3) mobilization of intracellular Ca2+ from inositol 1,4,5-triphosphate-sensitive stores, as well as mitochondrial stores, seem to contribute to LHRH release in these cells.     

Pulsatile gonadotropin secretion after discontinuation of long term gonadotropin-releasing hormone (GnRH) administration in a subset of GnRH-deficient men

Normal pituitary and gonadal function can be maintained with long term pulsatile GnRH administration in men with idiopathic hypogonadotropic hypogonadism (IHH), and both pituitary and gonadal priming occur during the process of GnRH-induced sexual maturation. Still, the long term effects of discontinuing GnRH therapy in IHH men have not been examined. Therefore, we evaluated the patterns of gonadotropin and alpha-subunit secretion before and after a prolonged period of pulsatile GnRH administration in 10 IHH men. Before exogenous GnRH stimulation, no patient had any detectable LH pulsations. In 6 of these men, who were typical of most of our IHH patients (group I), no LH pulsations were detectable after cessation of GnRH administration. However, in the other 4 men (group II), LH pulsations were easily detectable despite cessation of exogenous GnRH stimulation, and the amplitude (9.3 +/- 3.5 IU/L) and frequency (13.8 +/- 1.7 pulses/day) of these LH pulses were similar to those in 20 normal men (10.6 +/- 0.7 IU/L and 11.0 +/- 0.7 pulses/day). Three of these 4 men in group II maintained normal serum testosterone levels after discontinuation of GnRH delivery. To determine if there were any characteristics that might be useful in predicting which IHH men could maintain normal pituitary-gonadal function after long term GnRH administration, we evaluated various clinical and hormonal parameters at the time of initial presentation. Mean alpha-subunit levels (P less than 0.01) and alpha-subunit pulse amplitude (P less than 0.02) were significantly higher in the group II than the group I men, suggesting that the group II patients had partial, rather than complete, deficiency of endogenous GnRH secretion. None of the other parameters that were assessed distinguished the two groups. We conclude that gonadotropin and sex steroid levels return to their pretreatment state in the majority of IHH men when long term GnRH administration is discontinued. Normal pituitary-gonadal function can be maintained after discontinuation of long term GnRH administration in a rare subset of IHH men who present with higher levels of alpha-subunit. We hypothesize that these latter IHH men have an incomplete GnRH deficiency and that long term exogenous GnRH administration induces pituitary and gonadal priming, which subsequently enables them to sustain normal pituitary and gonadal function in response to their own enfeebled GnRH secretion.     

Spatially selective, testosterone-independent remodeling of dendrites in gonadotropin-releasing hormone (GnRH) neurons prepubertally in male rats

Adult GnRH neurons exhibit a stereotypic morphology with a small soma, single axon, and single dendrite arising from the soma with little branching. The adult morphology of GnRH neurons in mice reflects an anatomical consolidation of dendrites over postnatal development. We examined this issue in rat GnRH neurons with biocytin filling in live hypothalamic slices from infant males, as adult littermates and in gonad-intact males, castrated males, and in males with one of three levels of testosterone (T) treatment. Somatic area and total dendritic length were significantly greater in infant males than in adults. Moreover, total numbers of dendrite branches were greater in infant males as compared with adults. The number of higher order branches and the lengths of higher order branches were also greater in infant males than in adults. Most interestingly, in adults a single dendrite arose from the somata, consistently at 180° from the axon. In contrast, prepubertal animals had an average of 2.2 ± 0.2 primary dendrites arising from somata (range, one to seven primary dendrites). Angles relative to the axon at which dendrites in prepubertal males emanated from GnRH somata were highly variable. Castration at 25 d of age and castration at 25 d of age with one of three levels of T treatment did not influence morphological parameters when GnRH neurons were examined between 40 d and 48 d of age. Thus, a spatially selective remodeling of primary dendrites and consolidation of distal GnRH dendritic arbors occurs during postnatal development and is largely independent of T.     

Differential regulation of luteinizing hormone, follicle-stimulating hormone, and free alpha-subunit secretion from the gonadotrope by gonadotropin-releasing hormone (GnRH): evidence from the use of two GnRH antagonists

To examine the differential regulation of glycoprotein hormone secretion from the gonadotrope by GnRH, the Nal-Glu GnRH antagonist was administered to euthyroid women in the early follicular phase (days 1-5) of the menstrual cycle, and the results compared to previous studies with the Nal-Arg GnRH antagonist. After a 4-h period of baseline sampling at a frequency of every 10 min, a single sc dose of the GnRH antagonist was administered to each subject. Frequent sampling continued for 8 h, followed by hourly sampling for a further 16 h. LH, FSH, and free alpha-subunit were measured serially in assays with high specificity. There was a 90% concordance of LH and free alpha-subunit pulses during the baseline sampling period. Pulsatile secretion of LH and free alpha-subunit was immediately abolished at the highest dose of the Nal-Glu antagonist for at least 8 h. The maximum percent suppression of LH after administration of the Nal-Glu GnRH antagonist was 70 +/- 4%, 80 +/- 4%, and 83 +/- 1% at doses of 15, 50, and 150 micrograms/kg, respectively, compared to 51 +/- 10%, 70 +/- 5%, and 69 +/- 5% at doses of 50, 150, and 500 micrograms/kg Nal-Arg antagonist. Decreases in FSH were 28 +/- 2%, 32 +/- 7%, and 39 +/- 2%, with increasing doses of the Nal-Glu antagonist compared with 25 +/- 6%, 17 +/- 6%, and 28 +/- 4% reductions at increasing doses of the Nal-Arg antagonist. Free alpha-subunit decreased 22 +/- 4%, 23 +/- 4%, and 28 +/- 3% at increasing doses of the Nal-Glu antagonist and 12 +/- 4%, 27 +/- 4%, and 30 +/- 7% with increasing doses of the Nal-Arg antagonist. For the Nal-Glu antagonist, suppression of LH was greater than that of FSH and free alpha-subunit at all doses (P less than 0.001), while FSH suppression was greater than that of free alpha-subunit at the highest dose only (P less than 0.05). For the Nal-Arg antagonist, LH suppression was greater than that of FSH or free alpha-subunit at all doses (P greater than 0.01), and FSH suppression exceeded that of free alpha-subunit at the 50 micrograms/kg dose. Suppression of LH was greater with the Nal-Glu antagonist than with the Nal-Arg antagonist at doses of 50 and 150 micrograms/kg (P less than 0.05), and FSH suppression was greater with the Nal-Glu antagonist at 150 micrograms/kg (P less than 0.01), while the degrees of maximum suppression were similar for the two different GnRH antagonists for free alpha-subunit.(ABSTRACT TRUNCATED AT 400 WORDS)     

Different modes of gonadotropin-releasing hormone (GnRH) release from multiple GnRH systems as revealed by radioimmunoassay using brain slices of a teleost, the dwarf gourami (Colisa lalia)

It has become a general notion that there are multiple GnRH systems in the vertebrate brains. To measure GnRH release activities from different GnRH systems, we conducted a static incubation of brain-pituitary slices under various conditions, and GnRH released into the incubation medium was measured by RIA. The slices were divided into two parts, one containing GnRH neurons in the preoptic area and axon terminals in the pituitary (POA-GnRH slices), and the other containing the cell bodies and fibers of terminal nerve-GnRH neurons and midbrain tegmentum-GnRH neurons (TN-TEG-GnRH slices). We demonstrated that GnRH release was evoked by high [K(+)](o) depolarizing stimuli (in both POA-GnRH and TN-TEG-GnRH slices) via Ca(2+) influx through voltage-gated Ca(2+) channels. The most prominent result was the presence of conspicuous sexual difference in the amount of GnRH release in the POA-GnRH slices. The GnRH release from TN-TEG-GnRH slices also showed a small sexual difference, which was by far more inconspicuous than that of POA-GnRH slices. Immunohistochemical analysis using an antiserum specific to the seabream GnRH (sbGnRH; suggested to be specific to POA-GnRH neurons) revealed the presence of a much larger number of POA-GnRH neurons in males than in females. This clear morphological sexual difference is suggested to underlie that of GnRH release in the POA-GnRH slices.     

Evidence of differential control of FSH and LH secretion by gonadotropin-releasing hormone (GnRH) from the use of a GnRH antagonist

The differential regulation of immunoactive FSH and LH secretion by endogenous GnRH was studied using a GnRH antagonist, [Ac-D2Nal1,D4FPhe2,DTrp3,DArg6]GnRH (the NAL-ARG antagonist), in normal women in the early follicular phase of the menstrual cycle, and their responses were compared to those in two groups of control women. Pulsatile LH secretion was examined as an index of the completeness of blockade of endogenous GnRH secretion. There was a dose-dependent decrease in both the frequency and amplitude of LH pulses. At the highest dose, LH pulses were completely abolished within 20 min after sc administration of the GnRH antagonist and for a minimum of 8 h in all women. The mean plasma LH levels were reduced within the first 4 h after antagonist administration at all doses (P less than 0.001). The duration of LH suppression was influenced by antagonist dose, with a continued effect 24 h after administration of the 500 micrograms/kg dose only. The maximum degree of LH suppression was 40% after 50 micrograms/kg (n = 6), 60% after 150 micrograms/kg (n = 6), and 59% after 500 micrograms/kg (n = 5). In contrast, plasma immunoreactive FSH levels did not change after these doses of the NAL-ARG GnRH antagonist. The maximum degree of FSH suppression was 16%, and the changes in plasma FSH concentrations were not dose dependent. Serum antagonist concentrations rose within 30 min after its administration to mean peak levels of 7.5 +/- 2.1 (+/- SE), 20.4 +/- 6.1, and 151 +/- 21 ng/mL after the 50, 150, and 500 micrograms/kg doses, respectively. The half-time of the disappearance of the NAL-ARG GnRH antagonist from plasma was 8.8 +/- 1.5 h. While there were no effects of antagonist administration on hematological, hepatic, or renal function, three women developed urticaria distant from the site of injection when administered the highest dose. We conclude that blockade of GnRH receptors by a GnRH antagonist 1) effectively antagonizes the action of GnRH, as assessed by its ability to block pulsatile LH secretion and reduce mean plasma LH levels; and 2) inhibits LH release to a considerably greater degree than FSH release, providing further evidence of possible GnRH-independent FSH secretion.     

Pulsatile release of gonadotropin-releasing hormone (GnRH) from the rat hypothalamus in vitro: calcium and glucose dependency and inhibition by superactive GnRH analogs

We have shown previously that the rat hypothalamus retains in vitro its capacity of generating pulsatile release of GnRH. The present study evaluated if pulsatile release of GnRH in vitro was influenced by metabolic conditions (calcium and glucose availability) and the possible self-regulatory role of GnRH in its pulsatile secretion. In the presence of a calcium-chelating agent (EGTA, 20 mM) or a calcium-channel blocker (D-600, 0.1 mM), the release of GnRH induced by a depolarization (veratridine, 50 microM) was markedly and reversibly decreased. In addition, frequency and amplitude of GnRH secretory pulses were significantly reduced (P less than 0.05). When glucose use was inhibited using 2-deoxyglucose (5.6 mM) the release of GnRH induced by veratridine and the frequency of GnRH pulses were also blunted (P less than 0.05). Superactive agonists of GnRH (Buserelin and D-TRP6-PRO9-N-ET, 10 nM) caused a prompt decrease of GnRH release in basal conditions and in the presence of veratridine. A significant inhibition (P less than 0.05) was observed using buserelin concentrations greater than 0.01 nM, whereas two GnRH analogs without biopotency (Leu8-GnRH, Des-gly10-N picolylamide GnRH, 100 nM) did not affect GnRH release. The two agonists of GnRH reduced by 43% to 66% (P less than 0.05) the occurrence of significant GnRH pulses. We conclude that, in vitro, the hypothalamic neuronal circuitry resulting in GnRH pulsatile secretion is dependent on calcium and glucose availability and is sensitive to an ultrashort-loop inhibitory feedback mechanism.     

A subset of gonadotropin-releasing hormone neurons in the ovine medial basal hypothalamus is activated during increased pulsatile luteinizing hormone secretion

GnRH neurons active in the preovulatory LH surge have been identified in several species using the early intermediate gene product, Fos, but the GnRH neurons active during episodic LH secretion remain unknown. In this study, we have used Fos and Fos-related antigens (FRA) to determine whether a subset of GnRH neurons is active when pulsatile LH secretion is acutely stimulated in sheep. In experiment 1, episodic LH secretion was stimulated in five of six ewes by injection of an opioid antagonist to luteal phase ewes. These five ewes had a 6-fold increase in the percentage of GnRH neurons in the medial basal hypothalamus (MBH) expressing Fos/FRA, compared with control ewes that had no LH pulses before death. Fos/FRA expression was not increased in GnRH neurons found in any other area. In experiment 2, episodic LH secretion was induced in rams by introduction of estrous ewes. This treatment increased Fos/FRA expression in MBH GnRH neurons approximately 10-fold compared with control rams. Again, this increase in Fos/FRA expression in GnRH neurons was limited to the MBH. This selective activation of MBH GnRH neurons could reflect the preferential inhibition of these perikarya by endogenous opioid peptides. It also raises the possibility that a subset of GnRH neurons in the MBH may be responsible for episodic GnRH secretion in sheep.     

A role for hypothalamic astrocytes in dehydroepiandrosterone and estradiol regulation of gonadotropin-releasing hormone (GnRH) release by GnRH neurons

Molecules of astrocyte origin influence gonadotropin-releasing hormone (GnRH) release and GnRH neuronal growth and differentiation. Furthermore, type 1 astrocytes express steroid receptors, presenting the possibility that steroid actions on GnRH neurons might occur via astrocytes. Utilizing GT1-7 cells, a GnRH-secreting cell line, the present study demonstrates that astrocytes mediate dehydroepiandrosterone (DHEA) or estradiol (E2) stimulated GnRH secretion. Conditioned media (CM) from astrocytes cultured for 48 h alone, with DHEA (DHEA-CM), or with E2 (E2-CM) were collected, treated with charcoal to remove steroids, and added to GT1-7 cells in culture for 12 h to test the effect on GnRH secretion. DHEA-CM and E2-CM stimulated GnRH secretion by GT1-7 cells by 4- and 3-fold, respectively. The effect of DHEA-CM on GnRH secretion by GT1-7 cells appears to be related to both DHEA and its metabolite, E2, since blocking the metabolism of DHEA into estrogen in the DHEA-treated astrocytes partially reversed the stimulatory effect of DHEA-CM. Addition of transforming growth factor (TGF)-beta1-neutralizing antibody to the astrocyte cultures reversed the stimulatory effects of both DHEA-CM and E2-CM on GnRH secretion by GT1-7 cells, suggesting that TGF-beta1 derived from astrocytes may be the principle mediator of E2 and DHEA effects. These data provide evidence for a novel mechanism by which circulating steroids and/or neurosteroids may modulate GnRH secretion.     

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.     

Effects of cytokines on gonadotropin-releasing hormone (GnRH) gene expression in primary hypothalamic neurons and in GnRH neurons immortalized conditionally

Various cytokines produced during the immune reaction can modulate the neuroendocrine reproductive axis, probably by inducing changes in the activity of hypothalamic GnRH neurons. However, the precise cellular and molecular effects of cytokines on these neurons have not been reported yet. To gain a better insight into these regulations, we first examined the pattern of expression of cytokine receptors in a novel neuronal cell line expressing GnRH (Gnv-4 cells). Among others, gp130 is expressed in Gnv-4 cells, together with the ligand receptor subunits specific for IL-6 as well as oncostatin M (OSM). Consistent with the latter observation, we show that OSM stimulates the expression of the immediate early genes c-fos and early growth response-1 in Gnv-4 cells, an effect dependent upon the activation of the MAPK Erk1/2 intracellular signaling pathway. Functional studies performed in parallel in Gnv-4 cells and in primary hypothalamic neuronal cell cultures show that OSM, although devoid of any effect of its own on GnRH gene expression, can inhibit dose-dependently the stimulation of GnRH expression by N-methyl-d-aspartic acid. In conclusion, these data demonstrate that a GnRH-expressing neuronal cell line can be modulated in vitro by cytokines implicated in the regulation of the reproductive axis. Moreover, they provide the first evidence of an involvement of OSM in these regulations.     

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.     

Pulsatile release of gonadotropin-releasing hormone from hypothalamic explants is restrained by blockade of N-methyl-D,L-aspartate receptors

We have shown previously that N-methyl-D,L-aspartate (NMDA) and kainate, two neuroexcitatory amino acids acting through distinct receptors, may induce the release of GnRH from hypothalamic explants. However, that effect could have no physiological significance, since very high concentrations (50 mM) of NMDA and kainate were required. Here, using agents blocking the activation of receptors to neuroexcitatory amino acids, we evaluated their possible physiological involvement in the pulsatile release of GnRH from the hypothalamus of 50-day-old male rats in vitro. In control conditions (10 nM glycine and 1 mM mg2+), the release of GnRH in 7.5-min fractions collected for 2-4 h showed an obvious pulsatile pattern. The mean (+/- 1 SD) interval between pulses, identified by PULSAR program, was 34.3 +/- 11.4 min. The stimulation of GnRH release by NMDA (50 mM) added to the medium for 7.5 min could be blocked reversibly in the presence of MK-801 (100 microM) using medium without glycine or enriched with Mg2+ (2 mM). The endogenous pulses of GnRH secretion were abolished in the presence of MK-801 or using increased Mg2+ concentrations as well as in the absence of glycine. In contrast, pulsatile release of GnRH was not affected in the presence of 6,7-dinitroquinoxaline-2,3-dione (0.1 mM), a selective inhibitor of kainate and quisqualate receptors which suppressed the increase in GnRH release induced by kainate (50 mM) without affecting the response to NMDA. These data indicate that the physiological mechanism of pulsatile GnRH secretion in the hypothalamus may involve endogenous neuroexcitatory factors acting through NMDA-sensitive receptors.     

Dynamics of gonadotropin-releasing hormone (GnRH) secretion during the GnRH surge: insights into the mechanism of GnRH surge induction.

Recent studies demonstrate unequivocally that a preovulatory surge of GnRH is secreted into pituitary portal blood during the estrous cycle of the ewe and that this surge is induced by the follicular phase rise in estradiol. These data, obtained at 10-min intervals, suggested the surge results from a continuous elevation of GnRH rather than from a sequence of discrete pulses. This study examines the dynamics of GnRH secretion in more detail to determine if the surge results from strictly episodic release of the decapeptide. Our approach was to monitor GnRH secretion into pituitary portal blood at very frequent intervals during several "windows" of the GnRH surge induced using a physiological model for the estrous cycle. Samples of portal blood were obtained at either 2-min intervals (6 ewes), or 30-sec intervals (12 ewes) at several times during the surge; at other times portal blood was sampled less often to monitor progression of the GnRH surge. All ewes had an unambiguous GnRH surge; amplitude ranged from 100- to 500-fold over pressure levels. Regardless of sampling interval, our results provide no convincing evidence to indicate the enhanced secretion of GnRH is strictly episodic; values remained continuously elevated in portal blood. Our findings are consistent with the hypothesis that the GnRH surge is not composed entirely of discrete synchronous secretory events, and they raise the possibility that one action of estradiol in inducing the GnRH surge may be to switch the pattern of GnRH secretion into portal blood from episodic to continuous.     

Prenatal androgenization of female mice programs an increase in firing activity of gonadotropin-releasing hormone (GnRH) neurons that is reversed by metformin treatment in adulthood

Prenatal androgenization (PNA) of female mice with dihydrotestosterone programs reproductive dysfunction in adulthood, characterized by elevated luteinizing hormone levels, irregular estrous cycles, and central abnormalities. Here, we evaluated activity of GnRH neurons from PNA mice and the effects of in vivo treatment with metformin, an activator of AMP-activated protein kinase (AMPK) that is commonly used to treat the fertility disorder polycystic ovary syndrome. Estrous cycles were monitored in PNA and control mice before and after metformin administration. Before metformin, cycles were longer in PNA mice and percent time in estrus lower; metformin normalized cycles in PNA mice. Extracellular recordings were used to monitor GnRH neuron firing activity in brain slices from diestrous mice. Firing rate was higher and quiescence lower in GnRH neurons from PNA mice, demonstrating increased GnRH neuron activity. Metformin treatment of PNA mice restored firing activity and LH to control levels. To assess whether AMPK activation contributed to the metformin-induced reduction in GnRH neuron activity, the AMPK antagonist compound C was acutely applied to cells. Compound C stimulated cells from metformin-treated, but not untreated, mice, suggesting that AMPK was activated in GnRH neurons, or afferent neurons, in the former group. GnRH neurons from metformin-treated mice also showed a reduced inhibitory response to low glucose. These studies indicate that PNA causes enhanced firing activity of GnRH neurons and elevated LH that are reversible by metformin, raising the possibility that central AMPK activation by metformin may play a role in its restoration of reproductive cycles in polycystic ovary syndrome.     

Regulation of pituitary gonadotropin-releasing hormone (GnRH) receptors by pulsatile GnRH in female rats: effects of estradiol and prolactin

GnRH has been shown to modulate the concentration of its own pituitary receptors (GnRH-R), and changes in GnRH-R during the rat estrous cycle may reflect changes in GnRH secretion. To examine the relationship between GnRH and GnRH-R in female rats, we measured GnRH-R and serum gonadotropin responses to pulsatile GnRH in restrained ovariectomized (OVX) and ovariectomized estradiol-implanted (OVX-E2) rats. In addition, we examined the effects of suppression of serum PRL. Pulsatile injections of GnRH (10-250 ng/pulse) given every 30 min for 24 or 48 h did not increase GnRH-R in OVX or OVX-E2 rats compared to that in saline controls (246 +/- 27 fmol/mg). Bromocriptine treatment (2 mg/day) had no effect on GnRH-R in OVX animals. In contrast, OVX-E2 rats treated with bromocriptine showed significantly increased GnRH-R (500 +/- 43 fmol/mg) in response to GnRH injections. When ovine PRL was administered to bromocriptine-treated OVX-E2 rats, the GnRH induced rise in GnRH-R was abolished. Gonadotropin responses to GnRH were not correlated with changes in GnRH-R. In OVX animals, LH was only elevated in response to 250-ng pulses of GnRH. In OVX-E2 animals, basal LH was increased by all doses of GnRH, and acute responses to 50- and 250-ng pulses were observed. Bromocriptine treatment resulted in increased LH sensitivity to GnRH in OVX rats, but did not further enhance the responses in OVX-E2 animals. We conclude that in female rats, the presence of both E2 and a low serum PRL level is necessary for GnRH to increase GnRH-R, and the interaction of these factors may be involved in the regulation of GnRH-R during the estrous cycle.     

Effects of gonadotropin-releasing hormone (GnRH) on gastric secretion and gastrin release in the dog

The effects of GnRH on gastric secretion and gastrin release from dogs provided with gastric fistulae and Heidenhain pouches have been investigated. A transient yet significant inhibition of pentagastrin-stimulated secretion from gastric fistulae was observed, while secretion from Heidenhain pouches was unchanged. The maximal inhibitory effect of GnRH on both acid and pepsin secretion stimulated by 2-deoxy-D-glucose was obtained from gastric fistulae. On the contrary, GnRH failed to affect either acid secretion stimulated by bethanechol or acid secretion and gastrin release induced by bombesin. The present results indicate that GnRH possesses an inhibitory action on gastric secretion from the vagally innervated stomach of the dog. The most likely inhibitory mechanism seems to be represented by a decrease of the vagal activity.     

Antibodies against gonadotropin-releasing hormone (GnRH) and destruction of enteric neurons in 3 patients suffering from gastrointestinal dysfunction

Background  

Antibodies against gonadotropin-releasing hormone (GnRH) and gastrointestinal dysmotility have been found after treatment with GnRH analogues. The aim of this study was to examine the presence of such antibodies in patients with dysmotility not subjected to GnRH treatment and study the anti-GnRH antibody effect on enteric neurons viability in vitro.