Cellular expression of V1a vasopressin receptor mRNA in the female rat preoptic area: effects of oestrogen.

The preovulatory luteinizing hormone (LH) surge in female rats is dependent upon signals from the suprachiasmatic nucleus (SCN), the site of a dominant circadian pacemaker. Various lines of evidence indicate that arginine-vasopressin (AVP)-containing projections from the SCN to the preoptic area (POA) contribute to the production of the surge of LH-releasing hormone (LHRH). These actions may be mediated by V(1a) because the transcript for this AVP receptor subtype is predominant within the POA of the female rat. In this study, in situ hybridization histochemistry was used to examine V(1a) mRNA expression, either by itself or together with LHRH or glutamic acid decarboxylase 65 (GAD(65)) mRNA, within the POA of ovariectomized rats in the presence or absence of oestrogen. V(1a) mRNA was found in cells across the rostro-caudal axis of the POA; some were in close proximity to cells expressing LHRH mRNA. Coexpression of V(1a) and LHRH mRNAs was detected only very rarely. By contrast, cells with V(1a) mRNA commonly displayed GAD(65) mRNA. The density of V(1a) mRNA-expressing cells was particularly high within the anteroventral periventricular nucleus; at this site, V(1a) mRNA expression was elevated following oestrogen treatment. The present results indicate that V(1a)-mediated AVP actions may influence LHRH release via cells in the immediate vicinity of LHRH neurones and/or via oestrogen-regulated cells in the anteroventral periventricular nucleus, which is a site that lacks LHRH neurones but plays an essential role in initiating the preovulatory LH surge.

Central orexin A has site-specific effects on luteinizing hormone release in female rats.

Orexin A stimulates GnRH release from hypothalamic explants in vitro. The sites of action of orexin A in the regulation of LH release have been investigated in vivo in ovariectomized rats that were given vehicle or estradiol benzoate (EB), with or without an injection of progesterone 48 h later. Orexin A was administered intrahypothalamically under Saffan anesthesia, 50 h after the EB or vehicle; its effects on plasma LH levels were monitored in sequential blood samples. Orexin A (1.0 microg/side) injected into the rostral preoptic area (rPOA) at the level of the organum vasculosum of the lamina terminalis had a stimulatory effect on LH release in EB-treated ovariectomized rats. When orexin A was injected into the medial POA (mPOA) or the arcuate/median eminence, it had an inhibitory effect on the LH surge that occurs in ovariectomized rats primed with EB plus progesterone. Orexin A injected into the mPOA also reduced LH levels in ovariectomized rats untreated with ovarian steroids. Both the stimulatory and inhibitory effects of orexin A were antagonized by SB334867A, a selective orexin 1 receptor antagonist. Furthermore, when given alone into the rPOA, this antagonist attenuated the LH surge induced by EB plus progesterone. Thus, orexin appears to have a dual effect on LH release, being stimulatory in the rPOA and inhibitory in the mPOA or arcuate/median eminence. Both effects may be mediated, at least in part, by the orexin 1 receptor. Double label immunohistochemistry revealed close appositions between orexin A immunoreactive varicosities and a small proportion of GnRH cell bodies in the rPOA. It is suggested that the stimulatory effect of orexin A on LH release may involve direct actions on GnRH neurons.

Evidence in favour of a direct input from the ventromedial nucleus to gonadotropin-releasing hormone neurones in the ewe: an anterograde tracing study.

The mechanism by which oestrogen activates the gonadotropin releasing hormone (GnRH) neurones to induce the preovulatory luteinizing hormone (LH) surge is not understood. Previous work in the ewe has suggested that the primary site of action for oestradiol in stimulating the GnRH neurones was in the region of the ventromedial nucleus (VMN) within the mediobasal hypothalamus (MBH). In the present study, we used anterograde tracing techniques in the ewe to investigate whether direct neuronal projections may exist from neurones located in the region of the VMN to the GnRH neurones. Following the injection of biotinylated dextran amine into the VMN of four ewes, anterogradely labelled fibres were found located principally within the ipsilateral diagonal band of Broca (DBB), septum, preoptic and anterior hypothalamic areas, and periventricular, paraventricular, dorsomedial and arcuate nuclei of the MBH. Dual-labelling for GnRH revealed that fibres containing anterograde tracer were adjacent to the soma and/or dendrites of approximately 50% of all ipsilateral GnRH neurones located throughout the DBB and hypothalamus. Few anterogradely labelled fibres were detected within the median eminence. Although such studies cannot define the presence of direct synaptic connections between VMN neurones and the GnRH cells, these observations support further the hypothesis that oestrogen-sensitive VMN neurones represent a direct transsynaptic input to the GnRH cell bodies which are involved in the generation of the LH surge in the ewe.

Oestrogen receptor beta-immunoreactivity in gonadotropin releasing hormone-expressing neurones: regulation by oestrogen.

Double-label immunohistochemistry was employed to establish whether immunoreactivity for the beta subtype of the oestrogen receptor (ER beta-IR) is present in gonadotropin releasing hormone (GnRH)-containing cells. In the immortalized GnRH cell line, GT1-7, almost all nuclei were immunoreactive for ER beta. In the preoptic area of ovariectomized rats, more than one-half of the GnRH neurones (52.0-63.5%) contained ER beta-IR within the nucleus; a smaller proportion of these neurones (5-10%) displayed a particularly intense nuclear signal for ER beta. The presence of ER beta-IR in the nuclei of GT1-7 cells and GnRH neurones is consistent with recent reports of ER beta mRNA in these cells. Oestrogen treatment reduced the percentage of GnRH neurones with detectable ER beta-IR. The range of signal intensity for ER beta and the incidence of the ER beta signal in GnRH neurones were comparable following double-label immunohistochemistry involving either bright field or fluorescent techniques. These findings raise the possibility that ER beta receptors mediate direct effects of oestrogen on GnRH neurones.

Identification of neurokinin B-expressing neurons as an highly estrogen-receptive, sexually dimorphic cell group in the ovine arcuate nucleus.

Studies were undertaken to examine the hypothesis that neurons expressing neurokinin B (NKB) may represent an estrogen-receptive input to GnRH neurons in the sheep. Cells immunoreactive for NKB were located almost exclusively within the arcuate nucleus of the ovine hypothalamus. Dual labeling experiments revealed that essentially all NKB neurons (97%) were immunoreactive for estrogen receptor alpha and that NKB-immunoreactive fibers were found in close proximity to approximately 40% of GnRH neurons located in the rostral preoptic area as well as intermingled with GnRH fibers in the median eminence. The analysis of male and female brains revealed a marked female-dominant sex difference in the numbers of NKB neurons, and sections obtained from in utero androgen-treated females indicated that this sex difference resulted from an organizational influence of testosterone during neural development. In adult ovariectomized ewes, in situ hybridization studies failed to detect any significant effect of 8- to 26-h exposure of estrogen on cellular NKB messenger RNA levels. Together, these studies identify the first sexually differentiated neuronal cell population in the ovine hypothalamus and, remarkably, show that essentially all of these female-dominant NKB neurons express estrogen receptors. Although these neurons may be involved in any number of steroid-dependent, sexually differentiated functions in the sheep, the neuroanatomical evidence for potential NKB inputs to GnRH neurons suggests a role for this novel population in the regulation of reproductive function.

Ketamine-induced general anesthesia is compatible with gonadotropin-releasing hormone pulse generator activity in gonadectomized rats: prospects for detailed electrophysiological studies in vivo.

Previous studies on the mediobasal hypothalamus (MBH) of rats, rhesus monkeys and goats have detected episodic increases in multiunit activity (MUA volleys) which immediately precede a pulse of luteinizing hormone (LH). These volleys are considered to reflect the activity of the gonadotropin-releasing hormone (GnRH) pulse generator. Our understanding of this system would be greatly enhanced if it were possible to study the electrophysiological aspects of this system at the single cell level; such an approach requires anesthesia of the animals (as in the classic studies on the oxytocin and vasopressin systems). Although it is widely held that anesthetic agents disrupt the processes regulating LH release, little is known about their specific effects on the dynamics of this system. Using on-line electrophysiological techniques in addition to subsequent radioimmunoassay for LH, we have found that anesthesia by ketamine is compatible with the continued production of MUA volleys and LH pulses in gonadectomized rats. In contrast to the hypothermia induced by the LH pulse-suppressing anesthetic sodium pentobarbitone, a small rise in core temperature occurs following ketamine. The present findings offer the prospect of detailed electrophysiological analysis of GnRH pulse generator activity in rats maintained under general anesthesia.

Adrenomedullectomy prevents the suppression of pulsatile luteinising hormone release during fasting in female rats.

Fasting inhibits the pulsatile secretion of luteinising hormone (LH) in female rats, an effect which is potentiated by the presence of oestradiol (E2). We have previously described various pharmacological or surgical treatments that can rapidly restore the pulses in a fasting animal. Nevertheless, the central and peripheral mechanisms that mediate this suppression of the pulses remain unclear. We have recently shown that adrenomedullectomy prevents the suppression of LH pulses by insulin-induced hypoglycaemia, a state which activates the sympathoadrenal axis. The present study was undertaken to establish whether this axis might contribute to the loss of the pulses that occurs in ovariectomised E2-treated rats that have been fasted for 48 h. Following sham adrenomedullectomy LH pulses were observed in animals fed ad libitum; after 48 h of fasting the animals that had received this sham procedure showed a significant suppression of LH levels and LH pulse frequency. In contrast, adrenomedullectomy prevented the inhibition of the pulses by 48 h of fasting; it had no effect on the pulses in the absence of fasting. These results suggest that adrenomedullary activity plays a significant role in the fasting-induced suppression of LH pulses in rats.

[Importance of ultradian rhythms in endocrinology]. / Importance des rythmes ultradiens en endocrinologie.

Biological rhythms have been reported for a long time in endocrinology. However, their real study started in the late 60's when the possibility of repeatedly measuring the hormonal signal directly in blood over extended periods of time became available. Although episodic secretory fluctuations have been reported for the majority of hormones, and more specifically peptidic hormones, in mammals, organised secretory episodes on a regular or quasi-regular ultradian basis are less common. Their importance, which is directly connected to the number and the processing of the corresponding receptors on the target cells, is illustrated by the paradigm of the pulsatile release of the gonadotropin hormones. Hormonal ultradian rhythms should be considered both from the nosological and therapeutical point of view.

Insulin-induced hypoglycemia decreases luteinizing hormone secretion in the castrated male rat: involvement of opiate peptides.

Insulin-induced hypoglycemia (IIH) has been reported to inhibit luteinizing hormone (LH) secretion in ovariectomized ewes as well as in intact or gonadectomized female rhesus monkeys. An infusion of naloxone, a specific opiate receptor antagonist, while able to prevent this inhibitory effect in ewes, was ineffective in monkeys, suggesting a difference in the mechanisms involved in the response to IIH in these two species. The present study was designed to investigate the influence of this stimulus on the dynamics of LH release and the possible activation of an opioidergic pathway in the long-term castrated male rat. Blood samples were collected every 7 min, for several hours, in freely-moving rats bearing a jugular cannula. The intravenous injection of 0.4 IU of insulin triggered an acute hypoglycemia and reduced LH release. In contrast, during a continuous intravenous naloxone infusion (0.5 mg/kg/h), insulin, while still lowering the glycemia, failed to affect the LH secretory pattern. In a control experiment, naloxone infusion alone had no significant effect on mean LH concentration and pulse intervals. These experiments indicate that, in long-term gonadectomized male rats, IIH impairs the LH secretory pattern, and that endogenous opioid peptides are involved in this response.

Hypothalamic multiunit activity and pulsatile luteinizing hormone release in the castrated male rat.

Using chronically implanted microelectrodes, multiunit electrical activity (MUA) was recorded from the arcuate nucleus of freely moving gonadectomized male rats. Intermittent increases in MUA activity (MUA volleys) closely associated with luteinizing hormone pulses measured in the peripheral circulation were observed, which confirms that this experimental approach can be used for monitoring the activity of the gonadotropin-releasing hormone-associated hypothalamic pulse generator in the male rat. The mean MUA volley frequency was 22.2 min (range 13-38 min), whereas the mean MUA volley duration was 2.7 +/- 0.8 min (standard deviation). In addition to a large inter-individual variability. MUA volley intervals also showed an important intra-individual variability. This observation suggests that, beside the mean frequency of pulse generator activation, the degree of variability in gonadotropin-releasing hormone-associated pulse generator activity might be an additional relevant parameter in the characterization of the reproductive function in the male rat.