Intravenous injections of nicotine decrease the pulsatile secretion of LH by inhibiting the gonadotropin-releasing hormone (GnRH) pulse generator activity in female rats.

Whether nicotine inhibits the electrical activity of the gonadotropin-releasing hormone (GnRH) pulse generator to suppress pulsatile LH secretion, and whether this suppression of LH secretion by nicotine is mediated by opioid neurons, were studied in ovariectomized rats by examining changes in LH secretion and the multiunit activity (MUA) of the medial basal hypothalamus. Intravenous (i.v.) injection of nicotine (nicotine bitartrate, 100 micrograms) significantly increased the interval between characteristic increases (volleys) in MUA and LH pulses. This inhibitory effect of nicotine on the GnRH pulse generator activity was not blocked by the prior injection of an opiate receptor antagonist naloxone (naloxone hydrochlolide, 2 mg/kg bw), which was effective in significantly decreasing the interval between MUA volleys. The results suggest that nicotine alters the activity of the GnRH pulse generator, and that cholinergic neurons appear to be directly involved in suppressing pulsatile secretion of LH.     

High postovariectomy LH levels are not due to decreased opioid inhibition of GnRH

It has been proposed that endogenous opioid peptide (EOP) inhibition of hypothalamic GnRH secretion mediates and is dependent upon gonadal steroid feedback of LH secretion, although considerable conflicting data have been reported. Accordingly, a well-characterized replacement regimen was used to approximate physiological stimulation by estradiol (E2) and progesterone (P4) in adult rats 10 days after ovariectomy (OVX), followed by in vitro incubation of the isolated median eminence to evaluate the role of E2 and P4 in modifying GnRH release in response to the opiate receptor antagonist, naloxone (NAL). Basal (control) GnRH release from median eminences of OVX, OVX + E2, and OVX + E2 + P4 rats was similar, and NAL treatment elicited a comparable increase in GnRH release under all three gonadal steroid conditions. Thus, EOP suppression of median eminence GnRH secretion does not appear to mediate or be dependent upon negative feedback regulation of LH secretion by physiological concentrations of gonadal steroids.     

Pentobarbital stimulates the activity of the GnRH pulse generator interacting with opioid neurons in rats in proestrus

In order to investigate the possibility that i.p. injection of pentobarbital sodium (PB, 32 mg/kg bw) potentiates the GnRH pulse generator activity, effects of i.v. infusions of an opiate receptor antagonist naloxone (NAL, 2 mg/h) on the pulsatile LH secretion were compared in saline (SAL)- and PB-injected rats in proestrus and diestrus 1. In SAL-injected rats in proestrus, NAL infusions significantly increased both the frequency and amplitude of LH pulses, and also the overall mean LH concentration. In PB-injected rats in proestrus, all the parameters of the pulsatile LH secretion were similar to those in SAL-injected rats in proestrus. The NAL infusion in PB-injected rats caused an increase in the frequency, but it was similar to that in SAL-injected rats. But, increases in the amplitude and the overall mean LH observed during NAL infusions in PB-injected rats were greater than in SAL-injected rats. In SAL-injected rats in diestrus 1, NAL infusions increased all the parameters, as in rats in proestrus. In PB-injected rats in diestrus 1, LH secretion was severely suppressed. NAL infusions recovered the pulsatile LH secretion, but the frequency and the overall mean LH of the secretion were smaller than those obtained during NAL infusions in SAL-injected rats. In addition, characteristic increases in the MUA (volleys), which occur in association with the initiation of an LH pulse and thus are considered to represent an increased activity of the GnRH pulse generator, appeared more frequently during NAL infusions in PB-injected rats in proestrus than in SAL-injected rats. These results suggest that the GnRH pulse generator in rats in proestrus, but not in rats in diestrus 1, is refractory to PB and further is potentiated by PB in the response to NAL. Together with the fact that this dosage of PB blocks the surge of LH secretion in rats in proestrus, the concept of the existence of separate neuronal mechanisms responsible for the surge and pulsatile secretion of LH are supported.     

Effects of Ethane Dimethane Sulphonate and Orchidectomy on Luteinizing Hormone Secretion

The aim of this study was to determine whether testicular products of non-Leydig cell origin modulate rat luteinizing hormone (LH) secretion in vivo. We therefore compared the effects of ethane dimethane sulphonate (EDS), a toxin regarded as highly selective for Leydig cells, with that of bilateral orchidectomy on LH secretion in mature male Wistar rats. The intention was thereby to compare the effects of selective removal of Leydig cells with that of removing both Leydig cells and seminiferous tubules, respectively. Following a single dose of EDS (75 mg/kg, ip), plasma LH concentrations rose equally with those of castrated rats for the first 3 days. After that time, however, plasma LH concentrations in the EDS-treated rats fell progressively below those of the orchidectomized rats despite the continuing castrate level of circulating testosterone until Day 17. The effects of EDS treatment or orchidectomy on pulsatile LH secretion were then compared after 11 days to castrate levels of testosterone. EDS-treated rats demonstrated reduced LH pulse amplitude, mean plasma LH levels and net LH secretion compared with castrate rats, although LH pulse frequency was unaltered. However, a further group of rats treated with EDS and orchidectomized failed to demonstrate that these changes were fully reversed by the castration and therefore EDS may have direct effects upon pituitary LH secretion. In order to determine the mechanism of the reduced LH pulse amplitude after EDS treatment, a further study was conducted to determine whether EDS treatment resulted in reduced pituitary sensitivity to gonadotropin-releasing hormone (GnRH). Responsiveness of pituitary LH to exogenous GnRH (0.01 to 10 μg/kg body wt) was studied 11 days after removal of testicular testosterone feedback by either EDS or castration. Plasma LH response was linearly related to the log of the GnRH dose. At 10 min after GnRH administration, the plasma LH response in EDS-treated rats was less sensitive than in castrate rats. We conclude that the lesser augmentation of LH secretion between Days 3 and 17 after EDS treatment compared with castrate rats cannot be explained solely by changes in Leydig cell secretion but may involve direct effects of EDS on pituitary LH secretion or non-Leydig cell testicular products. Dampening of LH pulse amplitude without change in LH pulse frequency together with the reduced sensitivity to GnRH in EDS-treated rats suggests that this toxin may have direct effects on pituitary LH secretion independent of its effects on Leydig cell function.     

The negative feedback action of progesterone on luteinizing hormone release is not associated with changes in GnRH mRNA expression in the Ewe

Progesterone is the ovarian hormone that times events in the ovine reproductive cycle. When elevated, this ovarian hormone acts centrally to inhibit both the tonic and surge modes of gonadotrophin releasing hormone (GnRH) release. Two studies were performed to address the underlying neural mechanisms. The first tested the hypothesis that the rapid rise in GnRH release, that results from an acute fall in progesterone concentrations (such as occurs following luteolysis), is temporally associated with a rapid rise in the cellular content of GnRH mRNA. Three groups of ovariectomised (OVX) ewes were treated with exogenous progesterone for 10 days, while one remained steroid free (OVX, n=7). To determine the effects of acute progesterone (P) withdrawal, ewes were killed on day 10 while implants were still in place (OVX+P, n=6) or 4 (OVX-P4, n=7) or 12 h (OVX-P12, n=7) after progesterone removal. Coronal sections through the rostral portion of the medial preoptic area (rPOA) were processed for cellular in-situ hybridization for GnRH mRNA. An increase in progesterone concentrations markedly suppressed luteinizing hormone (LH) release, while removal of the implants caused progesterone concentrations to fall (P<0.01) within 1 h and LH pulse frequency to increase (P<0.05) within 4 h. Despite these progesterone-induced changes in LH/GnRH release there were no differences in the cellular content of GnRH mRNA among the four groups. In the second study, three groups of ovariectomised ewes were used to determined whether the inhibitory actions of early (EL; n=8) and mid-luteal (ML; n=8) phase concentrations of progesterone on LH release are accompanied by a decrease in GnRH mRNA expression. P inhibited the secretion of LH in a dose dependant manner; pulses of LH were virtually absent in the ML group. Despite this marked inhibitory steroid action, there was no significant difference in the cellular content of GnRH mRNA among the OVX, OVX (EL) and OVX (ML) groups. Thus, both the negative feedback actions of physiological concentrations of progesterone on GnRH release and the rapid escape from progesterone-inhibition are independent of changes in the cellular content of GnRH mRNA. These data suggest that the mechanism by which progesterone controls the timing of events in the ovine oestrous cycle is primarily by altering the secretion of GnRH rather than GnRH biosynthesis.     

LH release in ovariectomized rats is maintained without noradrenergic neurotransmission in the preoptic/anterior hypothalamic area: extreme functional plasticity of the GnRH pulse generator.

Norepinephrine (NE) in the preoptic/anterior hypothalamic area (PO/AH) is known to be involved in the regulation of luteinizing hormone (LH) secretion. The effects of selective and complete depletion of NE in the PO/AH of ovariectomized (ovx) rats on LH secretion were studied. PO/AH concentrations of NE were reduced by 90% within 6 h and were undetectable (more than 98% depletion) 52 h after bilateral stereotaxic microinjections of 50 micrograms of 5-amino-2,4-dihydroxy-alpha-methylphenylethylamine (5-ADMP). LH levels in the blood were significantly reduced within 60 min after NE depletion but remained low only for several hours. Despite continuously low preoptic NE concentrations episodic LH secretion reoccurred within 4-6 h such that normal blood LH levels were present 6 and 52 h after selective NE depletion. While the alpha 1-adrenoreceptor antagonist prazosin was inhibitory to LH secretion in control rats the drug was totally ineffective in the NE depleted animals. NE may be inhibitory to LH secretion via a beta-adrenergic receptor mechanism. It was therefore also tested whether 5-ADMP causes a massive NE release which might be inhibitory to LH secretion. Propranolol (PROP), a beta-adrenoreceptor blocking drug, was given 30 min prior to preoptic injection of 5-ADMP. Blockade of beta-receptors did not prevent the transient inhibition of LH release. These results indicate that under physiologic conditions the GnRH pulse generator functions only properly when NE is present in the PO/AH and that the stimulatory effect of NE is mediated via an alpha 1-adrenoreceptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

'Hamster-like' Cycles in Testicular Size in the Absence of Gonadotrophin Secretion in HPD Rams Exposed to Long-term Changes in Photoperiod and Treatment with Melatonin

Long term changes in the secretion of FSH, LH and testosterone, and the size of the testis were measured in groups of hypothalamo-pituitary disconnected Soay rams (HPD rams, n=8) and control Soay rams (HPD-sham operated and unoperated, total n=8) while exposed to an artificial lighting regimen of alternating 16-weekly periods of long days (16L:8D) and short days (8L:16D), and when treated with a constant-release implant of melatonin given under long days (total study: 136 weeks). Short term provocation tests using NMDA (glutamate receptor agonist), GnRH and LH were used to assess functionality of the hypothalamus, pituitary gland and testis, respectively. Control rams expressed normal photoperiod-induced cycles in the reproductive axis. Blood concentrations of FSH, LH and testosterone were significantly increased under short days, and decreased under long days with parallel changes in testicular diameter. Treatment with implants of melatonin under long days mimicked the effect of short days and induced rapid reactivation of the reproductive axis. In the HPD rams the blood concentrations of FSH, LH and testosterone declined immediately after the HPD surgery and values remained close to the lower limit of detection of the radioimmunoassays throughout the experiment. LH pulses were absent in the HPD rams and NMDA failed to induce LH secretion consistent with functional disconnection of the pituitary gland from the hypothalamus. The testes regressed to a significantly smaller size in the HPD rams compared with controls even at the nadir of the sexual cycle (testis diameter: 30.2±0.7 vs 41.3±0.8 mm, HPD vs control rams, respectively). A low amplitude cycle in testicular diameter (peak to nadir: 5.0±0.7 mm) persisted in the HPD rams with a temporal pattern opposite to the controls (growth under long days instead of short days; ‘hamster-like’). In the HPD rams, the treatment with melatonin blocked the long day-associated increase in testicular size, without effects on FSH, LH and testosterone secretion, pituitary responsiveness to GnRH (LH increment) or testicular responsiveness to LH (testosterone increment). This was in contrast to the cyclical changes in all parameters induced by melatonin in the control rams. At post-mortem, the reproductive tract in HPD rams was markedly regressed compared with the controls. The efficiency of spermatogenesis was reduced with few germ cells maturing beyond primary spermatocytes. Immunocytochemical staining, however, revealed the maintenance of androgen receptor expression in Sertoli cells, peritubular cells and Leydig cells, and steroid activity as measured by 17α-hydroxylase expression in Leydig cells. Overall, the absence of photoperiod-induced changes in gonadotrophin secretion in the HPD rams illustrates the dependence on regulation by the hypothalamus, presumably through the secretion of GnRH. The residual cycle in the size of the testes in the HPD rams was closely correlated with the photoperiod-induced changes in prolactin secretion which persisted in these animals (summary of previous published data included). The combined results support the view that melatonin acts in the hypothalamus to mediate effects of photoperiod on gonadotrophin secretion and in the pituitary gland to mediate effects on prolactin secretion (dual site hypothesis), and that FSH, LH and prolactin act synergistically to regulate the long-term cycle in testicular activity in the ram.     

Oestradiol Microimplants in the Ventromedial Preoptic Area Inhibit Secretion of Luteinizing Hormone via Dopamine Neurones in Anoestrous Ewes

Oestradiol exerts a season-specific negative feedback effect on the GnRH/LH neurosecretory system of the Suffolk ewe. This neuroendocrine suppression is mediated in part by dopamine A15 neurones, but these neurones do not possess the oestrogen receptor. Based on indirect evidence, we hypothesized that oestrogen receptor-containing neurones in the ventromedial preoptic area (vmPOA) may be the initial step in a neuronal system whereby oestradiol suppresses GnRH secretion during the non-breeding season. To test this, three experiments were conducted using ovariectomized ewes receiving either empty or oestradiol-containing bilateral microimplants directed at the vmPOA or s.c. subcutaneous oestradiol-containing implants. In the first experiment, LH pulse frequency was measured on days 0, 1, 7 and 14 of treatment during seasonal anoestrus. In vmPOA oestradiol and s.c. oestradiol groups only, LH pulse frequency was suppressed on days 7 and 14, with maximal suppression evident by day 7. In the second experiment, this protocol was repeated during the breeding season, with LH pulses examined on days 0 and 7; LH pulse frequency did not change in any group. The third experiment tested if the effect of vmPOA oestradiol during anoestrus could be overcome by an injection of the dopamine-D2 receptor antagonist (-)-sulpiride. The vmPOA microimplants and s.c. oestradiol implants again suppressed LH pulse frequency and this was reversed by sulpiride in vmPOA oestradiol ewes. We conclude that oestradiol acts on cells in the vmPOA to stimulate a system involving dopamine neurones that inhibits GnRH/LH pulsatility in the anoestrous ewe.     

Prenatal Testosterone Excess Decreases Neurokinin 3 Receptor Immunoreactivity within the Arcuate Nucleus KNDy Cell Population

Prenatal exposure of the female ovine foetus to excess testosterone leads to neuroendocrine disruptions in adulthood, as demonstrated by defects in responsiveness with respect to the ability of gonadal steroids to regulate gonadotrophin‐releasing hormone (GnRH) secretion. In the ewe, neurones of the arcuate nucleus (ARC), which co‐expresses kisspeptin, neurokinin B (NKB) and dynorphin (termed KNDy cells), play a key role in steroid feedback control of GnRH and show altered peptide expression after prenatal testosterone treatment. KNDy cells also co‐localise NKB receptors (NK3R), and it has been proposed that NKB may act as an autoregulatory transmitter in KNDy cells where it participates in the mechanisms underlying steroid negative‐feedback. In addition, recent evidence suggests that NKB/NK3R signalling may be involved in the positive‐feedback actions of oestradiol leading to the GnRH/luteinising hormone (LH) surge in the ewe. Thus, we hypothesise that decreased expression of NK3R in KNDy cells may be present in the brains of prenatal testosterone‐treated animals, potentially contributing to reproductive defects. Using single‐ and dual‐label immunohistochemistry we found NK3R‐positive cells in diverse areas of the hypothalamus; however, after prenatal testosterone treatment, decreased numbers of NK3R immunoreactive (‐IR) cells were seen only in the ARC. Moreover, dual‐label confocal analyses revealed a significant decrease in the percentage of KNDy cells (using kisspeptin as a marker) that co‐localised NK3R. To investigate how NKB ultimately affects GnRH secretion in the ewe, we examined GnRH neurones in the preoptic area (POA) and mediobasal hypothalamus (MBH) for the presence of NK3R. Although, consistent with earlier findings, we found no instances of NK3R co‐localisation in GnRH neurones in either the POA or MBH; in addition, > 70% GnRH neurones in both areas were contacted by NK3R‐IR presynaptic terminals suggesting that, in addition to its role at KNDy cell bodies, NKB may regulate GnRH neurones by presynaptic actions. In summary, the finding of decreased NK3R within KNDy cells in prenatal testosterone‐treated sheep complements previous observations of decreased NKB and dynorphin in the same population, and may contribute to deficits in the feedback control of GnRH/LH secretion in this animal model.     

Sexual Differentiation of the Surge Mode of Gonadotropin Secretion: Prenatal Androgens Abolish the Gonadotropin-Releasing Hormone Surge in the Sheep

In sheep, the surge mode of gonadotropin secretion is sexually differentiated, i.e. the LH surge is present in the female, but not in the male. The present study tested the hypothesis that sexual differentiation of the LH surge mechanism reflects a sex difference in the pattern of GnRH, and that prenatal androgens abolish the surge mode of GnRH secretion. We monitored the pattern of GnRH secretion in pituitary portal blood after acute treatment with estradiol in gonadectomized postpubertal males (n=6), females (n=6), and androgenized females (exposed prenatally to testosterone from day 30–90 in gestation, n=7). Four capsules, each containing a 30-mm column of estradiol were implanted s.c. into each lamb to produce high physiologic concentrations of the hormone. Beginning 7 h later, portal and peripheral blood samples were collected hourly for 48 h for measurement of GnRH and LH, respectively. All females exhibited a GnRH surge beginning 13.0±0.4 h after estradiol treatment; this was accompanied by an LH surge. By contrast, only one male produced a small surge in GnRH (1.7 pg/min) with a latency of 32 h; a corresponding increase in LH occurred in this male. Likewise, among the androgenized females, only one exhibited GnRH and LH surges which began at about 22 h after estradiol treatment. Some of the androgenized females had sporadic increases in GnRH which were of lower amplitude than in the control females, and were unaccompanied by rises in LH. These findings provide the first direct evidence that the sex difference in the surge mode of LH secretion results from the sexual differentiation of the pattern of GnRH release. The study also suggests that androgens during prenatal development abolish the GnRH surge and subsequently, the generation of the LH surge.