Dibutyryl cyclic adenosine monophosphate stimulates in vitro luteinizing hormone-releasing hormone release only from median eminence derived from ovariectomized, estradiol-primed rats

The present study examined the effect of intermittent infusion of dibutyryl cyclic AMP (dbcAMP; 10(-7) M; 10 min on, 20 min off) on in vitro luteinizing hormone-releasing hormone (LH-RH) release from the rat median eminence (ME) derived from immature rats: intact females, intact males, ovariectomized (OVX) females, castrated (CAST) males, ovariectomized, estradiol primed (OVX + E2) females and castrated, estradiol primed (CAST + E2) males. In intact, OVX and CAST conditions, spontaneous LH-RH release from MEs was not modified by dbcAMP infusion. However, E2 implants in OVX and CAST rats selectively affected the responsiveness of MEs to dbcAMP: ME from OVX + E2 were highly responsive to dbcAMP; contrarily, MEs from CAST + E2 were unresponsive to this nucleotide. Therefore, these differences in MEs responsiveness to dbcAMP-induced LH-RH release appear to be dependent upon a critical effect of E2 priming on this tissue in female but not in male rats.     

Localization of luteinizing hormone-releasing hormone (LHRH) neurons that project to the median eminence

The neuropeptide, luteinizing hormone-releasing hormone (LHRH), is released from nerve terminals in the median eminence and carried via the hypophysial portal system to the anterior pituitary, where it stimulates the release of gonadotropins. LHRH-containing neurons are located in many different regions of the rodent brain, including olfactory, septal, preoptic, and hypothalamic structures. Since those LHRH neurons that project to the median eminence form the final common pathway for the regulation of the pituitary/gonadal axis, we wished to determine which of these cell groups are afferent to this structure. A retrograde tracer, the lectin wheat germ agglutinin (WGA), was placed directly on the exposed surface of median eminence. Following survival times of 8-13 hr, brains were prepared for the dual immunocytochemical detection of WGA and LHRH. Approximately 50% of the LHRH neurons from the level of the septal nuclei caudalward were found to contain WGA immunoreactivity and therefore to project to the median eminence. The remaining single-labeled LHRH neurons were intermingled with the double-labeled cells. The 2 populations were not distinguishable from each other on either cytological or cytoarchitectonic criteria. Those LHRH neurons that were not retrogradely labeled following an injection of tracer into the median eminence are presumed to project to other regions of the central nervous system. We conclude that the LHRH neurons that are directly involved in the regulation of reproductive function are very heterogeneous, widely scattered in telencephalic and diencephalic regions.     

A putative role for extracellular ATP: Facilitation ofcopper uptake and of copper stimulation of the release of luteinizing hormone-releasing hormone from median eminence explants

We have previously shown that extracellular copper stimulates the release of the luteinizing hormone-releasing hormone (LH-RH) from explants of the median eminence area (MEA), that chelated copper (Cu) but not ionic Cu is the active form of the metal, and that there is a direct correlation between the ligand specificity for⁶⁷Cu uptake and Cu action. In this study, we examined the possibility that extracellular ATP can serve as a ligand facilitating Cu action on the LH-RH neuron. Hypothalamic slices or MEA explants of adult male rats were used. It was found that ATP facilitates⁶⁷Cu uptake by hypothalamic slices when Cu:ATP molar ratio was 1:2000 but not 1:2. Keeping the [Cu] constant (150 μM) and varying [ATP], ATP facilitation of Cu stimulation of LH-RH release from MEA explants was found to be a saturable function of [ATP]; maximal facilitation occurred with 2.5 mM ATP. When the nucleotide phosphate specificity for facilitation of Cu action was assessed, ADP, ATP, α,β-methylene-ATP (the non-hydrolyzable analogue of ATP) and GTP were equally effective, whereas AMP and adenosine were ineffective. These results indicate that extracellular ATP can facilitate Cu action on the LH-RH neuron and they are consistent with two mechanisms: (1) ATP facilitating Cu uptake and hence, Cu action and (2) ATP facilitating Cu action via an interaction with a purinergic receptor.     

Amplification of prostaglandin E2 stimulation of luteinizing hormone-releasing hormone release from median eminence explants: a metal(II)-specific effect of chelated copper

We have previously demonstrated that extracellular copper amplifies prostaglandin (PG) E2 stimulation of the release of luteinizing hormone-releasing hormone (LH-RH) from explants of the median eminence area (MEA). Two questions were addressed: what is the active form of copper and the metal(II) specificity for copper action? MEA explants were incubated for 5 min in the presence of CuCl2 (ionic) or copper complexed to histidine (CuHis) at a concentration of 200 microM each and then for 15 min in the presence of 10 microM PGE2. It was found that chelated copper but not ionic amplified PGE2 action, and that the magnitude of PGE2 stimulation of LH-RH release was 3-4-fold in copper-treated than untreated tissue. Moreover, PGE2-stimulated release was directly related to the dose of CuHis. To test the metal specificity, MEA explants were incubated for 5 min with one of the following metal(II) complexes: CuHis, NiHis, FeHis, ZnHis, CdHis, MnHis, or BaHis (200 microM each) and then for 15 min with 10 microM PGE2. Controls were incubated with metal(II) complex or PGE2. Of these complexes, only CuHis and to a lesser extent NiHis stimulated LH-RH release. However, CuHis was the only complex that amplified PGE2 stimulation of LH-RH release. Thus, amplification is specific for copper. The finding that chelated copper but not ionic copper amplifies PGE2 is suggestive that the copper-interactive sites on the LH-RH neurons are not exposed to the extracellular space but that they are either embedded in the plasma membrane, facing the intracellular space, or in the cytoplasm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes of melanin-concentrating hormone related to LHRH release in the median eminence of rats

The present research was carried out to study the distribution of melanin-concentrating hormone (MCH) fibers in the median eminence of rats and to evaluate if changes in the MCH content of the median eminence could be related to the release of LHRH. Immunocytochemical studies in the median eminence of males and estrous females showed the presence of MCH fibers, mainly in its internal layer. Diestrous and proestrous animals displayed MCH immunoreactivity in both the internal and external layers of the median eminence. Longitudinal sections of the median eminence in proestrous animals showed that MCH-immunoreactive (ir) density is higher at 12 than at 9 h in both layers of the median eminence. MCH was assayed by radioimmunoassay in median eminences of males and in females in all stages of the estrous cycle at 10 h. It was observed that the content of MCH at diestrus-1 and -2 was higher than in estrus and in male rats. In the day of proestrus, MCH and LHRH were assayed at 10, 12, 13, 14, 15 and 17 h. At 12 h, the content of MCH and LHRH showed the maximal values. At 13 h, MCH content showed a decline, while LHRH was still high. At 14 h, the LHRH content started to decrease. The present results suggest that MCH is involved in the regulation of LHRH release in the female rat.     

Peripubertal development of noradrenergic stimulation of luteinizing hormone-releasing hormone neurosecretion in vitro

The effect of age on norepinephrine (NE) stimulation of luteinizing hormone-releasing hormone (LH-RH) secretion from preoptic area-mediobasal hypothalamic (POA-MBH) explants was examined in the present study. Explants were obtained from juvenile (9-day-old), prepubertal (29-day-old) and adult female rats. Following decapitation and surgical isolation, POA-MBH explants were individually perifused with culture medium which was collected for radioimmunoassay of LH-RH. Explants were exposed to two pulses of medium containing NE (5 x 10(-4) M, peak concentration) and a terminal pulse of medium containing KCl (45 mM, peak concentration) for assessment of viability. POA-MBH explants obtained from prepubertal female rats exhibited increased LH-RH release in response to the two pulses of NE and subsequent KCl pulse (P less than 0.05). NE was without effect in stimulating LH-RH neurosecretion from POA-MBH explants obtained from 9-day-old female rats although these explants were responsive to KCl (P less than 0.01). Two-day pretreatment of 9-day-old, and prepubertal rats with estradiol benzoate (EB) did not alter the LH-RH response to this dose of NE or KCl (no group or interaction effects) in prepubertal female rat explants and did not render the explants from 9-day-old rats responsive to NE. Furthermore, NE was equally effective in stimulating LH-RH release from explants obtained from estradiol-treated or control ovariectomized adult rats. These observations demonstrate a peripubertal activation of the stimulatory effect of NE on LH-RH release from POA-MBH explants in vitro. Although these data also suggest that estrogen is not obligatory for NE stimulation of LH-RH release from POA-MBH explants, further investigation is required to determine the developmental time course and estrogen dependency of the noradrenergic stimulation of LH-RH neurosecretion and to evaluate whether estrogen alters the sensitivity of POA-MBH explants to lower concentrations of NE as has been previously reported for median eminence fragments.     

The effect of D-aspartate on luteinizing hormone-releasing hormone,alpha-melanocyte-stimulating hormone,GABA and dopamine release

Since D-aspartate stimulates prolactin and LH release, our objective was to determine whether D-aspartate modifies the release of hypothalamic and posterior pituitary factors involved in the control of their secretion and whether its effects on these tissues are exerted through NMDA receptors and mediated by nitric oxide. In the hypothalamus, D-aspartate stimulated luteinizing hormone-releasing hormone (LHRH), alpha-melanocyte-stimulating hormone (alpha-MSH) and GABA release and inhibited dopamine release through interaction with NMDA receptors. It increased nitric oxide synthase (NOS) activity, and its effects on LHRH and hypothalamic GABA release were blunted when NOS was inhibited. In the posterior pituitary gland, D-aspartate inhibited GABA release but had no effect on dopamine or alpha-MSH release. We report that D-aspartate differentially affects the release of hypothalamic and posterior pituitary factors involved in the regulation of pituitary hormone secretion.     

Autoradiography of GABA in the rat hypothalamic median eminence

Light microscopy autoradiographs of the rat hypothalamic median eminence were prepared after injection of high specific activity tritiated GABA and GABA structural analogs.Following intracardiac injection of labeled GABA with short (15 min) survival time, a dense accumulation of silver grains was observed over the external layer of the median eminence. The silver grains appeared much less numerous and randomly scattered over the internal layer. No conspicuously labeled cells could be detected in the median eminence.A similar pattern of labeling was observed after 10 min in vitro incubation of the median eminence with a low concentration(2.5 × 10⁻⁷M) of labeled GABA. Clusters of silver grains were also visible over the external layer following intraventicular injection of labeled GABA. In this latter case, however, other sites of labeling were revealed over the internal and ependymal layers.The dense labeling over the external layer with tritiated GABA was partially reduced by a simultaneous intracardiac injection of a 50-fold excess of non-radioactive cis-aminocyclohexane car☐ylic acid — a reported preferential substrate for GABA neuronal uptake — but it was not displaced by a 2000-fold excess of non-radioactive β-alanine — a reported specific substrate for GABA glial uptake.Intracardiac injection of triated β-alanine led to a faint and even labeling over the entire median eminence with no preferential accumulation of silver grains over the various layers. Following intraventricular injection of labeled β-alanine the tanycytes and their processes as well as numerous glial cells appeared heavily labeled.These results suggested that there exist cell elements in the external layer of the hypothalamic median eminence which are capable of accumulating exogenous GABA according to its neuronal uptake characteristics. Although the exact nature of these cells is not readily apparent at this stage of our investigations, these findings led us to speculate that there might be a subpopulation of GABAergic nerve endings in the vicinity of the primary plexus capillaries.     

Intracerebroventricular administration of melanin-concentrating hormone suppresses pulsatile luteinizing hormone release in the female rat

Melanin-concentrating hormone (MCH) has been reported to be involved in the regulation of feeding behaviour in rats and mice. Because many neuropeptides that influence ingestive behaviour also regulate reproductive function, the present study was designed to determine if central administration of MCH changes pulsatile secretion of luteinizing hormone (LH) in the rats. Wistar-Imamichi strain female rats were ovariectomized and implanted with oestradiol to produce a moderate inhibitory feedback effect on LH release. The effects of i. c.v. injections of MCH on LH release were examined in freely moving animals. Blood samples were collected every 6 min for 3 h through an indwelling cannula. After 1 h of sampling, MCH (0.1, 1 or 10 microg/animal) or vehicle (saline) was injected into the third cerebroventricle. Because MCH is also reported to affect the hypothalamo-pituitary-adrenal (HPA) axis, which in turn, can influence reproductive function, plasma corticosterone concentrations were determined in the same animals at 30-min intervals during the first and last hours and every 12 min during the second hour of the 3-h sampling period. When expressed as per cent changes, mean plasma LH concentrations after MCH administration were significantly lower in the animals injected with all doses of the peptide compared with vehicle-treated animals; LH pulse frequency was significantly lowered by 1 microg of MCH. Per cent changes in mean plasma corticosterone levels were not significantly affected by MCH administration. These results in oestradiol-treated ovariectomized rats indicate that central MCH is capable of inhibiting pulsatile LH secretion. We have previously shown that 48-h fasting suppresses pulsatile LH release in the presence of oestrogen. Take together, these results raise the possibility that MCH could play a role in mediating the suppression of LH secretion during periods of reduced nutrition.     

The luteinizing hormone-releasing hormone (LH-RH) neuronal networks of the guinea pig brain. II. The regulation on gonadotropin secretion and the origin of terminals in the median eminence.

Cell bodies synthesizing LH-RH are located throughout the central nervous system including the hypothalamic arcuate nucleus, the medial preoptic area and medial septal nucleus. The contribution of each of these cell groups to the LH-RH terminals in the median eminence was assessed by immunocytochemistry following placement of radiofrequency lesions in male guinea pigs. Lesions in the arcuate nucleus resulted in an almost complete absence of LH-RH fibers in the median eminence. Lesions in the medial preoptic area or suprachiasmatic nucleus produced a decrease in the amount of immunoreactivity throughout the median eminence; but there was only a small decrease in the numbers of labeled fibers, which was localized to the internal zone of the median eminence and the external zone on the ventral and lateral surfaces of the infundibular stalk. Lesions of the mammillary bodies, medial amygdaloid nucleus, septal nucleus and fornix had no effect. The effects of these lesions on pituitary gonadotropin secretion was also assessed. Only lesions in the arcuate nucleus diminished plasma concentrations of luteinizing hormone (LH) and testosterone in the male guinea pigs. Similary only arcuate lesions prevented the postcastration rise in plasma LH in ovariectomized female guinea pigs. These data strongly suggest that the arcuate nucleus is the major but not the sole source of LH-RH terminals in the median eminence, and these are responsible for the neural regulation of tonic gonadotropin secretion.     

Electrophysiological actions of luteinizing hormone-releasing hormone: intracellular studies in the rat hippocampal slice preparation

The electrophysiological effects of luteinizing hormone-releasing hormone (LHRH) on CA1 pyramidal cells were investigated utilizing intracellular recordings from the in vitro rat hippocampal slice preparation. Bath application of LHRH (10(-7) - 10(-12) M) resulted in several changes in the electrophysiological properties of CA1 neurons. LHRH induced a long-lasting depolarization associated with increased input resistance, a decrease in the afterhyperpolarization (AHP) following a train of action potentials, and a reduction in accommodation of repetitive cell discharge. These effects were blocked by the synthetic LHRH antagonist [Ac-delta-Pro1,pCl-D-Phe2,D-Trp3,6]-LHRH. These findings provide electrophysiological evidence for the role of LHRH as a neurotransmitter/neuromodulator in the hippocampus.     

In vivo and in vitro studies on the effect of the serotoninergic system on luteinizing hormone and luteinizing hormone-releasing hormone secretion in prepubertal and peripubertal female rats

The present investigations were designed to assess the effect of the serotoninergic system on luteinizing hormone (LH) and LH-releasing hormone (LH-RH) secretion in female rats aged 14 and 30 days. The administration of 5-hydroxytryptophan (5-HTP; 75 mg/kg i.p.) increased hypothalamic serotonin (5-HT) concentrations in both age groups, and did not affect hypothalamic norepinephrine (NE) concentrations or release. Serum LH levels were raised by 5-HTP in 14-day-old, but not in 30-day-old rats. Basal and KCl- (28 mM) stimulated LH-RH release by incubated hypothalamic fragments was significantly enhanced when 5-HTP was injected previously to 14-day-old animals. In 30-day-old rats, 5-HTP treatment did not modify basal LH-RH release, and decreased the KCl-stimulated LH-RH output. Similarly, the addition of 5-HT (10(-7) M) to superfused hypothalamic fragments enhanced basal LH-RH release in 14-day-old rats and blocked the increment in LH-RH release evoked by KCl in 30-day-old rats. The present results show that in 14-day-old female rats, the serotoninergic system (activated in vivo by 5-HTP treatment, or in vitro by 5-HT addition) exerts a stimulatory effect on LH-RH, and thus, on LH release. On the contrary, in 30-day-old animals, stimulated LH-RH secretion was inhibited by 5-HT. Apparently, the hypothalamic NE system is not implicated in this response. The participation of this changing effect of 5-HT on LH-RH/LH release at the onset of puberty is postulated.     

Effect of aging on in vitro dopamine biosynthesis in the median eminence of rat hypothalamic slices

The rates of basal and cyclic AMP-dependent DOPA accumulation in the median eminence of hypothalamic slices were not different between ovariectomized young and aged rats. However, the rate of Ca2+-dependent, depolarization-induced DOPA accumulation was smaller in aged rats, suggesting that the Ca2+ system in the regulation of dopamine biosynthesis in tuberoinfundibular dopaminergic neurons is altered by aging.     

Targeting of gonadotropin-releasing hormone axons from preoptic area grafts to the median eminence.

Implantation of normal GnRH neurons can reverse many of the reproductive deficiencies that characterize hypogonadal (hpg) mice. Since the GnRH axons follow a stereotyped trajectory to their target we investigated the possibility that host brain regions adjacent to the graft might provide signals that induced this directional growth. The role of the adenohypophysis in GnRH axonal outgrowth was studied in mice with co-grafts of fetal preoptic area (POA) and pituitary and in hypophysectomized hosts. When fetal pituitaries were grafted together with the POA, immunoreactive GnRH fibers did enter the glandular tissue but they also grew into the host median eminence. Surgical removal of the pituitary of hpg hosts prior to POA graft placement was also compatible with GnRH innervation of the host median eminence although in some individuals that innervation pattern was confined to the more caudal aspects. The results of these two experiments suggest that the anterior pituitary gland may be an attractive target for GnRH axons but that this tissue is not essential for directed GnRH axonal outgrowth to its target. To determine if the median eminence itself could direct the growth of GnRH axons, co-grafts of POA and a fetal medial basal hypothalamic (MBH) block, which was predominantly median eminence, were made. Immunocytochemistry showed that an intragraft mini-median eminence was formed with a highly organized and robust GnRH innervation. Ultrastructural analysis indicated that these axons terminated near fenestrated capillaries. However, even under these conditions some GnRH axons exited into the host median eminence. It now seems likely that a cellular component of the median eminence can provide a signal to attract GnRH axons. Whether this signal is produced by the specialized ependymal cells, by the endothelia, or by meningeal (pial) components must now be tested.     

Involvement of metallo-endopeptidase in degradation of luteinizing hormone-releasing hormone by neuronal and glial cells cultured from rat fetal brain

Luteinizing hormone-releasing hormone (LHRH) was degraded by neuronal and glial cells cultured from fetal rat brain. The degradation of LHRH by neuronal cells was strongly inhibited by a metal chelator. Captopril only inhibited by generation of fragment (1-3) from fragment (1-5). In the presence of captopril, fragment (1-5) accumulated in the highest amount among the N-terminal fragments identified. The initial cleavage of LHRH, as determined by following the loss of the LHRH peak, was strongly inhibited by thiol-blocking reagents, as well as metal chelators. The results with glial cells were almost the same as those seen with neuronal cells. Thus, we propose that a thiol-dependent membrane-bound metallo-endopeptidase plays a major role in the initial stage of degradation of LHRH at the Tyr5-Gly6 bond in both neurons and glia. Angiotensin-converting enzyme is involved in the secondary process of the LHRH degradation in both cells.     

Morphine and anandamide coupling to nitric oxide stimulates GnRH and CRF release from rat median eminence: neurovascular regulation

Nitric oxide (NO) is involved in neurohormonal secretion from median eminence neuroendocrine nerve terminals. We report that stimulation of NO release from median eminence fragments including vascular tissues occurs by μ₃ receptor activation by morphine, or by cannabinoid type 1 receptor activation by anandamide. The released levels of NO are lower after anandamide than after morphine stimulation. These processes can be blocked by L-NAME, a specific nitric oxide synthase inhibitor, by naloxone for the morphine-stimulated NO release, or SR 141716A, a specific CB1 receptor inhibitor, for the anandamide-stimulated NO release. Furthermore, morphine and anandamide, by this NO dependent process, influences neurohormonal release from median eminence nerve terminals within 10 min. Via this NO dependent process, morphine stimulates both GnRH and CRF release, whereas anandamide selectively stimulates GnRH release. These observations together with previous data suggest that morphine and the anandamide-stimulated NO originates from the vascular endothelium of the portal plexus. These results indicate that endothelial cells of the median eminence may be involved in the release of neurohormones.     

Presence of luteinizing hormone-releasing hormone fragments in the rhesus monkey forebrain

Previously, we have shown that two types of luteinizing hormone-releasing hormone (LHRH) -like neurons, "early" and "late" cells, were discernible in the forebrain of rhesus monkey fetuses by using antiserum GF-6, which cross-reacts with several forms of LHRH. The "late" cells that arose from the olfactory placode of monkey fetuses at embryonic days (E) 32-E36, are bona fide LHRH neurons. The "early" cells were found in the forebrain at E32-E34 and settled in the extrahypothalamic area. The molecular form of LHRH in "early" cells differs from "late" cells, because "early" cells were not immunopositive with any specific antisera against known forms of LHRH. In this study, we investigated the molecular form of LHRH in the "early" cells in the nasal regions and brains of 13 monkey fetuses at E35 to E78. In situ hybridization studies suggested that both "early" and "late" LHRH cells expressed mammalian LHRH mRNA. Furthermore, "early" cells predominantly contain LHRH1-5-like peptide and its cleavage enzyme, metalloendopeptidase E.C.3.4.24.15 (EP24.15), which cleaves LHRH at the Tyr5-Gly6 position. This conclusion was based on immunocytochemical labeling with various antisera, including those against LHRH1-5, LHRH4-10, or EP24.15, and on preabsorption tests. Therefore, in primates, a group of neurons containing mammalian LHRH mRNA arises at an early embryonic stage before the migration of bona fide LHRH neurons, and is ultimately distributed in the extrahypothalamic region. These extrahypothalamic neurons contain LHRH fragments, rather than fully mature mammalian LHRH. The origin and function of these neurons remain to be determined.     

Ontogenesis of neurons producing luteinizing hormone-releasing hormone (LHRH) in the nervus terminalis of the rat

Immunoreactive luteinizing hormone-releasing hormone (LHRH) was first detected at 15 days of gestation in ganglion cells associated with the peripheral, intracranial, and central parts of the nervus terminalis of the rat. LHRH was not detected in any other structure of the central nervous system at this age. In the 17-day-old fetal rat, 62% of the total LHRH-reactive neuronal population was found in ganglion cells of the nervus terminalis. At this same age, immunoreactive beta-luteinizing hormone (beta-LH) was first seen in gonadotropes of the anterior pituitary gland. At 19 days of gestation, 31% of the total number of LHRH-reactive neurons observed in the rat brain was found in the nervus terminalis, and immunoreactive processes were first seen in the organum vasculosum of the lamina terminalis and in the median eminence. Our data indicate that from 15 to 19 days of gestation the nervus terminalis is a principal source of LHRH in the fetal rat. Presence of the decapeptide in the nervus terminalis prior to appearance of beta-LH in the anterior pituitary suggests a possible role for LHRH in this system on maturation of the gonadotropes and differentiation of the brain-pituitary-gonadal axis.