Luteinizing Hormone-releasing Hormone System Research Articles

Is the metalloendopeptidase EC 3.4.24.15 (EP24.15), the enzyme that cleaves luteinizing hormone-releasing hormone (LHRH), an activating enzyme?

LHRH (GNRH) was first isolated in the mammalian hypothalamus and shown to be the primary regulator of the reproductive neuroendocrine axis comprising of the hypothalamus, pituitary and gonads. LHRH acts centrally through its initiation of pituitary gonadotrophin release. Since its discovery, this form of LHRH (LHRH-I) has been shown to be one of over 20 structural variants with a variety of roles in both the brain and peripheral tissues. LHRH-I is processed by a zinc metalloendopeptidase EC 3.4.24.15 (EP24.15) that cleaves the hormone at the fifth and sixth bond of the decapeptide (Tyr(5)-Gly(6)) to form LHRH-(1-5). We have previously reported that the auto-regulation of LHRH-I (GNRH1) gene expression and secretion can also be mediated by itself and its processed peptide, LHRH-(1-5), centrally and in peripheral tissues. In this review, we present the evidence that EP24.15 is the main enzyme of LHRH metabolism. Following this, we look at the metabolism of other neuropeptides where an active peptide fragments is formed during degradation and use this as a platform to postulate that EP24.15 may also produce an active peptide fragment in the process of breaking down LHRH. We close this review by the role EP24.15 may have in regulation of the complex LHRH system.

Central and Peripheral Administration of Kisspeptin‐10 Stimulates the Hypothalamic‐Pituitary‐Gonadal Axis

Kisspeptin is the peptide product of the KiSS-1 gene and the endogenous agonist for the GPR54 receptor. Recent evidence suggests the kisspeptin/GPR54 system is a key regulator of the reproductive system. We examined the effect of intracerebroventricular (i.c.v.) and peripheral administration of the active kisspeptin fragment, kisspeptin-10, on circulating gonadotrophins and total testosterone levels in adult male rats. The effect of kisspeptin-10 in vitro on the release of hypothalamic peptides from hypothalamic explants and gonadotrophins from anterior pituitary fragments was also determined. The i.c.v. administration of kisspeptin-10 dose-dependently increased plasma luteinizing hormone (LH) and increased plasma follicle stimulating hormone (FSH) and total testosterone at 60 min postinjection. In a separate study investigating the time course of this response, i.c.v. administered kisspeptin-10 (3 nmol) significantly increased plasma LH at 10, 20 and 60 min, FSH at 60 min and total testosterone at 20 and 60 min postinjection. Kisspeptin-10 stimulated the release of luteinizing hormone-releasing hormone (LHRH) from in vitro hypothalamic explants. Peripheral administration of kisspeptin-10 increased plasma LH, FSH and total testosterone. However, doses of 100-1000 nM kisspeptin-10 did not influence LH or FSH release from pituitary fragments in vitro. Kisspeptin therefore potently stimulates the hypothalamic-pituitary-gonadal axis. These effects are likely to be mediated via the hypothalamic LHRH system.

Development of Luteinizing Hormone Releasing Hormone Neurones

AbstractThis review concentrates on some of the recent discoveries and future questions relevant to the development of the neuroendocrine luteinizing hormone releasing hormone (LHRH) cells. Neuroendocrine LHRH cells originate outside the central nervous system, in the nasal placode, and thereafter migrate into the forebrain during prenatal development. It is this population of LHRH cells that is responsible for reproductive function, becoming integral members of the hypothalamo‐pituitary‐gonadal axis postnatally. Disruption of the development of this system results in reproductive dysfunction. Increasing our understanding of LHRH neuroendocrine cells establishes conditions where we can look with greater precision at the mechanisms controlling reproductive development, both activation and failure. In addition, the ability to manipulate the molecular and cellular biology of the LHRH system opens the route to understanding critical neurobiological issues such as phenotypic commitment, axonal path finding and mechanisms involved in neuronal migration. Each of the topics is discussed in turn and potential mechanisms controlling the development of the neuroendocrine LHRH system are indicated.

Development of luteinizing hormone releasing hormone neurones.

This review concentrates on some of the recent discoveries and future questions relevant to the development of the neuroendocrine luteinizing hormone releasing hormone (LHRH) cells. Neuroendocrine LHRH cells originate outside the central nervous system, in the nasal placode, and thereafter migrate into the forebrain during prenatal development. It is this population of LHRH cells that is responsible for reproductive function, becoming integral members of the hypothalamo-pituitary-gonadal axis postnatally. Disruption of the development of this system results in reproductive dysfunction. Increasing our understanding of LHRH neuroendocrine cells establishes conditions where we can look with greater precision at the mechanisms controlling reproductive development, both activation and failure. In addition, the ability to manipulate the molecular and cellular biology of the LHRH system opens the route to understanding critical neurobiological issues such as phenotypic commitment, axonal path finding and mechanisms involved in neuronal migration. Each of the topics is discussed in turn and potential mechanisms controlling the development of the neuroendocrine LHRH system are indicated.

Effect of serotonin on the development of luteinizing hormone-releasing hormone system in Wistar rat embryos

The work studied effect of serotonin on development of the system of luteinizing hormone-realizing hormone (LHRH) in rats at the prenatal period. LHRH-neurons located along its migration pathway were revealed immunocytochemically at the 18th day of embryonal development (E18 and so on) after serotonin synthesis inhibition by daily intraperitoneal injections of n-chlorophenylalanine from E11 to E17 (experimental group). In control animal group, the saline was injected. The percent content of LHRH-neurons located in the forebrain rostral part was higher in the experimental group as compared to control. The opposite quantitative ratio of neurons was observed in the more caudal (hypothalamic) area. This indicates deceleration of the LHRH-neuronal migration under conditions of serotonin deficit. Study of the LHRH-neuron differentiation showed that in the experimental group the neuron optical density correlating with the LHRH content in the cell decreased as compared to the control group both in males and in females. The size of LHRH-neurons on n-chlorophenylalanine administration decreased, but only in females. Thus, serotonin affects both migration and differentiation of LHRH-neurons.

Novel gene expressed in nasal region influences outgrowth of olfactory axons and migration of luteinizing hormone-releasing hormone (LHRH) neurons

Although a variety of cues have been implicated in axonal targeting during embryogenesis and regeneration, the precise mechanisms guiding olfactory axons remain unclear. Appropriate olfactory axon pathfinding is essential for functional chemoreceptive and pheromone receptive systems. Olfactory axon pathfinding is also necessary for establishment of the neuroendocrine LHRH system, cells critical for reproductive function. LHRH cells exhibit neurophilic migration moving from the nasal region along olfactory axons into the brain. Factors involved in the migration of these neuroendocrine cells are as yet unresolved. We report identification of a novel factor termed nasal embryonic LHRH factor (NELF) that was discovered in a differential screen of migrating versus nonmigrating primary LHRH neurons. NELF is expressed in PNS and CNS tissues during embryonic development, including olfactory sensory cells and LHRH cells. NELF antisense experiments indicate that a reduction in NELF expression decreases olfactory axon outgrowth and the number of LHRH neurons that migrate out of the nasal tissue. These results demonstrate that NELF plays a role as a common guidance molecule for olfactory axon projections and subsequently, either directly or indirectly, in the neurophilic migration of LHRH cells.

Topography and associations of luteinizing hormone-releasing hormone and neuropeptide Y-immunoreactive neuronal systems in the human diencephalon.

Neuropeptide Y (NPY) potentiates the effect of luteinizing hormone-releasing hormone (LHRH) on luteinizing hormone secretion in several species, including human. In addition to the pituitary sites, the interactions of the NPY and LHRH systems may involve diencephalic loci. However, the morphologic basis of this putative communication has not yet been elucidated in the human brain. To discover interaction sites, the distribution and connections of LHRH and NPY-immunoreactive (IR) neuronal elements in the human hypothalamus were investigated by means of light microscopic single- and double-label immunocytochemistry. NPY-IR perikarya and fibers were found to be widely distributed in the ventral diencephalon, with high densities in the preopticoseptal, periventricular, and tuberal regions. Small neuronal cell groups were infiltrated with a dense network of varicose NPY-IR fibers in the lateral preoptic area. The LHRH-IR perikarya were located mainly in the preopticoseptal region, diagonal band of Broca, lamina terminalis, and periventricular and infundibular nuclei. A few LHRH-IR neurons and fibers were scattered in the mamillary region. The overlap between the NPY and LHRH systems was apparent in the periventricular, paraventricular, and infundibular nuclei. Double-labeling immunohistochemistry showed NPY-IR axon varicosities in contact with LHRH-IR perikarya and main dendrites. The putative innervation of LHRH neurons by NPY-IR fibers was also seen in 1-microm-thick plastic sections and with confocal laser scanning microscope, thus further supporting the functional impact of NPY-IR terminals on LHRH-IR neurons. The present findings suggest that the hypophysiotropic LHRH-synthesizing neurons may be innervated by intrahypothalamic NPY-IR fibers. Confirmation by ultrastructural analysis would demonstrate that the LHRH system in the human hypothalamus is regulated by NPY, as has been demonstrated in nonhuman species.

Luteinizing hormone releasing hormone (LHRH) neurons maintained in nasal explants decrease LHRH messenger ribonucleic acid levels after activation of GABA(A) receptors.

Inhibition of the LHRH system appears to play an important role in preventing precocious activation of the hypothalamic-pituitary-gonadal axis. Evidence points to gamma-aminobutyric acid (GABA) as the major negative regulator of postnatal LHRH neuronal activity. Changes in LHRH messenger RNA (mRNA) levels after alterations of GABAergic activity have been reported in vivo. However, the extent to which GABA acts directly on LHRH neurons to effect LHRH mRNA levels has been difficult to ascertain. The present work evaluates the effect of GABAergic activity, via GABA(A) receptors, on LHRH neuropeptide gene expression in LHRH neurons maintained in olfactory explants generated from E11.5 mouse embryos. These explants maintain large numbers of primary LHRH neurons that migrate from bilateral olfactory pits in a directed manner. Using in situ hybridization histochemistry and single cell analysis, we report dramatic alterations in LHRH mRNA levels. Inhibition of spontaneous synaptic activity by GABA(A) antagonists, bicuculline (10(-5) M) or picrotoxin (10(-4) M), or of electrical activity by tetrodotoxin (TTX, 10(-6) M) significantly increased LHRH mRNA levels. In contrast, LHRH mRNA levels decreased in explants cultured with the GABA(A) receptor agonist, muscimol (10(-4) M), or KCl (50 mM). The observed responses suggest that LHRH neurons possess functional pathways linking GABA(A) receptors to repression of neuropeptide gene expression and indicate that gene expression in embryonic LHRH neurons, outside the CNS, is highly responsive to alterations in neuronal activity.

The temporal pattern of mating-induced immediate-early gene product immunoreactivity in LHRH and non-LHRH neurons of the estrous ferret forebrain.

The mating-induced preovulatory surge of luteinizing hormone (LH) lasts for at least 12 h in the female ferret. This prolonged increase in circulating LH is presumably accompanied by a corresponding elevation in the activity and output of luteinizing hormone-releasing hormone (LHRH) neurons projecting to the hypothalamic-hypophyseal portal blood vessels and adenohypophysis. We used the protein products of the immediate early genes (IEGs) c-fos, and c-jun as markers of neural activation in order to determine whether a sub-population of LHRH neurons is differentially activated by mating and whether non-LHRH neurons in specific forebrain regions are selectively activated at different times during the mating-induced preovulatory LH surge. In Experiment 1, estrous female ferrets were perfused 0.5, 1.5, 3.0, 6.0 or 12.0 h after receiving one 5-min intromission from a male or after being placed alone in a testing cage for 20 min. Fos-like immunoreactivity (Fos-IR; Oncogene Ab-2 antiserum) and LHRH-like immunoreactivity (LHRH-IR; LR-1 antiserum) were visualized. The percentage of Fos-IR LHRH neurons was significantly augmented 1.5 h after mating but had returned to basal levels by 3.0 h. The double-labeled LHRH neurons were concentrated in the caudal medio-basal hypothalamus. In non-LHRH neurons the number of Fos-IR neural nuclei was significantly increased by mating in the medial preoptic area (MPOA), bed nucleus of the stria terminalis (BNST), medial amygdala (MA), ventrolateral hypothalamus (VLH), and midbrain central tegmental field (CTF) 1.5 h after mating but, as in LHRH neurons, had returned to basal levels by 3.0 h. In Experiment 2, estrous females were perfused 1.5 h or 8.0 h after either receiving one 5-min intromission or being placed alone in a testing cage, and the brains were processed for LHRH and c-Fos-like (DCH-1, Dr Gerard Evan), c-Jun-like (Jun-IR; Oncogene Ab-2) or Egr-1-like (Egr-IR; Santa Cruz) immunoreactivity. The percentage of LHRH neurons colabeled with both Fos-IR and Jun-IR was significantly greater in the 1.5 h group than in the unpaired group. Again, the induction of these IEG products occurred in LHRH neurons in the caudal medio-basal hypothalamus. Mating significantly increased the number of Fos-IR non-LHRH neural nuclei in the MPOA, BNST, MA, VMH and CTF, as well as the number of Egr-IR nuclei in the MPOA, BNST and MA in the 1.5 h group. By contrast, the number of Jun-IR non-LHRH neurons was unaffected by mating. In these Experiments we have identified a sub-population of LHRH neurons which, using Fos and Jun as markers of neural activation, is activated by mating and may be differentially involved in the generation of the preovulatory LH surge. Although the LHRH system is presumably activated throughout the duration of the 12 h preovulatory LH surge, c-Fos and c-Jun immunoreactivity in LHRH neurons is augmented only transiently. Fos-IR and Egr-IR in non-LHRH neurons show a similar time-course. Together, these results suggest that the presence of augmented levels of these proteins is not required for the maintenance or termination of the preovulatory output of LHRH.

Luteinizing hormone-releasing hormone (LHRH) receptors in the neuroendocrine-immune network. Biochemical bases and implications for reproductive physiopathology.

It seems apparent that the brain-pituitary-reproductive axis and the brain-thymus-lymphoid axis are linked by an array of internal mechanisms of communication that use similar signals (neurotransmitters, peptides, growth factors, hormones) acting on similar recognition targets. Moreover, such communication networks form the basis and control of each step and every level of reproductive physiology. This work has focused on the LHRH system, a primary central and peripheral clock of both neuroendocrine and immune functions. From the initiation of a sexually organized response, the detection of sexual odors, and the induction of mating behavior, extrahypothalamic and hypothalamic LHRH orchestrates the neuroendocrine modulation of gonadotropin secretion, while its expression within the ovary directly controls specific events such as follicular atresia. The presence of LHRH receptors in oocytes clearly anticipates a potential action of the decapeptide during the process of fertilization and/or implantation. Within the thymus and other peripheral immune organs, LHRH plays a unique role of immunomodulator, contributing to the sex-dependent changes in immune responsiveness during the estrous-menstrual cycle as well as pregnancy. The reciprocity of the neuroendocrine-immune signaling systems is further supported by the ability of sex steroids to modulate thymus-dependent immune functions via direct effects on specific target genes involved in the development of sex dimorphism and sex-dimorphic immune responses, including the downregulation of immune response observed during pregnancy. Such cyclic changes in immune responsiveness could have a physiological implication, such as the decrease or suppression in cell-mediated immunity observed in the postovulatory phase of the cycle and in pregnancy, respectively, and might play a role during the implantation process and the establishment of pregnancy. In this context, the ability of corticosterone to directly inhibit both GR transcript levels as well as a cell-mediated immune response within the thymus, and the modulation of such an inhibitory effect by the sex steroid hormone milieu, may offer an explanation and a molecular mechanism whereby stress may be deleterious for reproduction, also via immunomodulation. On the other hand, hormonally mediated alterations in immunity might also have a pathological implication in sexually related immune diseases. For example, in mouse and humans, lupus erythematosus is more prevalent in females and estrogen accelerates the disease process, while menstruation is known to exacerbate idiopathic thrombocytopenia purpura. Sex steroid hormone milieu might also have a role in controlling the stress response through immunomodulation. Within the placenta, an intricate network of signaling systems controls a delicate interplay between the neuroendocrine hormones, growth factors, and cytokines that are susceptible to play a major local role in the processes of implantation and the establishment and completion of pregnancy. The neuroendocrine and immunomodulatory role of LHRH continues well after parturition because the presence of LHRH-like material within the mammary gland and milk participates in the physiological modulation of hypophyseal, gonadal, and immune functions of the pups. Such a significant role played by the hypothalamic peptide in the modulation of immune responsiveness would indicate LHRH as the signal conveying information to both neuroendocrine and immune cells, with the role of informing and then transducing the messages into appropriate biological responses.(ABSTRACT TRUNCATED)

Discrete-time stimulation of the oscillatory and excitable forms of a FitzHugh-Nagumo model applied to the pulsatile release of luteinizing hormone releasing hormone.

A model for the pulsatile release of luteinizing hormone releasing hormone (LHRH) can be reduced to a FitzHugh-Nagumo model subject to regular and quasiregular (i.e., with slight random variation in the interstimulus interval), discrete-time stimulation. The relationship of output pulse frequency (OPF) to stimulus frequency is compared between the excitable and oscillatory forms of the model and discussed in the context of results from other pulse-driven model systems. Some examples of the changes in OPF caused by quasiregular and purely Poissonian stimuli are given for the excitable case. The unstimulated system frequently interacts with the stimulation in such a complex manner that the OPF bears little resemblance to the frequency of stimulation or of the unstimulated system. Furthermore, the inability of the oscillatory form of the model to allow complete suppression of output pulses for moderate stimulation frequencies suggests that the LHRH system can be more appropriately described by the excitable form of the model. (c) 1995 American Institute of Physics.

Acute stress suppresses the N-methyl-D-aspartate-induced luteinizing hormone release in the ovariectomized estrogen-primed rat.

The effects of N-methyl-D-aspartate (NMDA) and luteinizing hormone releasing hormone (LH-RH) on luteinizing hormone (LH) secretion were examined in ovariectomized estrogen-primed rats under nonstressed and acutely stressed conditions. The basal LH levels were significantly elevated 15 min after the onset of acute immobilization stress, but were not altered in emotionally stressed or nonstressed rats. Intravenous injections of 10 and 40 mg/kg NMDA significantly elevated serum LH levels by 161 and 212%, respectively, from baseline within 10 min in nonstressed animals. However, the NMDA-induced LH release was significantly reduced when tested 30 min after the onset of acute immobilization stress. Acute emotional stress, which did not affect the baseline LH, also suppressed the LH release response to NMDA, suggesting that the reduced LH responses to NMDA in stressed animals was not due to the elevated baseline level. Pituitary LH release responses to LH-RH were not affected by acute immobilization. We conclude from these results: (1) acute immobilization stress exerts both stimulatory and inhibitory effects on LH release, while acute emotional stress has only an inhibitory effect in estrogen-primed ovariectomized rats; (2) this inhibition occurs at the suprapituitary level, and (3) it involves a suppression of the responsiveness of the hypothalamic LH-RH neuronal system to the excitatory amino acid input.

Localized Changes in LHRH mRNA Levels as Cellular Correlates of the Positive Feedback Effects of Estrogen on LHRH Neurons

SYNOPSIS. The luteinizing hormone-releasing hormone (LHRH) system appears to be the final common pathway for integrating multiple estrogeninduced signals into a trigger for the preovulatory LH surge. Because LHRH neurons do not contain estrogen receptors, we have had no cellular marker to identify LHRH neurons involved in triggering the LH surge. Using an estrogenized ovariectomized rat model, we selectively deprived brain regions of estrogen action with microimplants of antiestrogen. We showed that the rostral medial preoptic area (MPOA) is required for estrogen-dependent LH surges and that LHRH neurons in this region are involved in the surge. Using in situ hybridization histochemistry, we compared temporal changes in LHRH mRNA levels in MPOA neurons in animals exhibiting LH surges with those in which LH surges were absent. We defined a characteristic pattern of changes in LHRH mRNA seen only in animals exhibiting the surge and, therefore, believe that these changes in LHRH mRNA levels are cellular correlates of the positive feedback effects of estrogen on LHRH neurons. These findings are exciting because we now have a cellular marker for detecting changes in the neuronal activity of subpopulations of the anatomically and functionally diverse LHRH system. We can use this marker in future studies to definethe complex neurocircuitry which mediates the effects of estrogen on LHRH neurons. In addition, we are currently examining temporal changes in LHRH transcription rate, LHRH mRNA stability and LHRH translation to determine the significance of changes in LHRH mRNA levels in terms of biosynthesis

Detecting steroidal effects on immediate early gene expression in the hypothalamus

Immediate early genes are rapidly and transiently expressed within neurons following stimulation. While it is likely immediate early gene products alter gene expression in neurons, the products of the Immediate early genes also serve as excellent markers for Identifying activated neurons. The immunocytochemical localization of immediate early gene products enables the analysis of changes In activation of individual neurons in the brain in response to gonadal steroids or other stimuli. The technique presented in this article outlines the strategies for using immediate early gene proteins in the fos family as markers for neuronal activity and focuses principally on the examination of one chemically identified neuron population, the luteinizing hormone-releasing hormone system. Included are comparisons of the staining of brain tissue for c-fos and related antigens following use of different tissue fixatives. Because some of the c-fos antibodies also recognize other proteins in the fos family, we have presented data comparing staining patterns obtained with a number of available antisera. We also describe some of the approaches we use to analyze c-fos expression.

Further Evidence that Most Luteinizing Hormone‐Releasing Hormone Neurons are not Directly Estrogen‐Responsive: Simultaneous Localization of Luteinizing Hormone‐Releasing Hormone and Estrogen Receptor Immunoreactivity in the Guinea‐Pig Brain

Gonadotropin secretion from the pituitary is regulated in large part by steroid action on the brain. An important question concerns whether luteinizing hormone-releasing hormone (LHRH) neurons themselves transduce steroid signals, or whether, alternatively, steroid-sensitive interneuronal populations regulate their activity. A previous study in the rat employing steroid autoradiography combined with LHRH immunocytochemistry revealed that only an exceedingly small percentage of LHRH-immunoreactive (ir) neurons was estrogen concentrating. This study has examined the relationship of estrogen receptive and LHRH-ir cells in the male and female guinea-pig brain with double label immunocytochemistry. Since estrogen receptor-ir, as demonstrated with antibody H222, is known to be confined predominantly to the cell nucleus, whereas LHRH-ir is localized mainly in the cytoplasm, single cells can be double-labeled. Diaminobenzidine tetrahydrochloride was used for localization of LHRH-ir while nickel-enhanced diaminobenzidine tetrahydrochloride was used for localization of estrogen receptor-ir. The results revealed that there were many brain nuclei that contained both LHRH and estrogen receptor-positive cells, including the preventricular periventricular nucleus, the anterior subcompact nucleus of the medial preoptic nucleus (MPNa), the remainder of the medial preoptic nucleus, the retrochiasmatic area, and the anterior, dorsomedial, ventrolateral and arcuate nuclei. However, of a total of 2,604 LHRH-ir cells that were examined, we observed only 5 double-labeled cells (<0.2%). The double-labeled cells were not restricted to a single nucleus; they were present in the MPNa, the retrochiasmatic area and the arcuate nucleus. Moreover, at the light microscopic level, LHRH cells quite frequently appeared to be apposed to estrogen receptor-positive cells (8.8% in the female), especially in the MPNa. These results lend further support to the notion that estrogen-mediated regulation of the LHRH system is achieved primarily through estrogen receptive interneurons. However, due to the existence of LHRH-LHRH synaptic interactions, the possibility also exists that a small population of estrogen-sensitive LHRH neurons could contribute to generalized activation of the LHRH system.

The luteinizing hormone-releasing hormone (LH-RH) system in neonatally estradiol-feminized male gray short-tailed opossums ( Monodelphis domestica)

In this study, the luteinizing hormone-releasing hormone (LH-RH) system was examined in adult gray short-tailed opossums that had been treated with estradiol benzoate (EB) on days 1 and 3 of postnatal life, a treatment which results in complete block of testicular development. The finding that the organization of the LH-RH system in neonatally EB-treated males did not differ from that of neonatally untreated animals indicates that the LH-RH system can develop normally despite the absence of gonads throughout perinatal life.

Evidence that cells expressing luteinizing hormone-releasing hormone mRNA in the mouse are derived from progenitor cells in the olfactory placode.

In situ hybridization histochemistry and immunocytochemistry were used to study the prenatal expression of luteinizing hormone-releasing hormone (LHRH) cells in the mouse. Cells expressing LHRH mRNA and peptide product were first detected on embryonic day 11.5 (E11.5) in the olfactory pit. On E12.5, the majority of LHRH cells were located on "tracks" extending from the olfactory pit to the base of the telencephalon. From E12.5 to E15.5, LHRH cells were detected in a rostral-to-caudal gradient in forebrain areas. Prior to E12.5, cells expressing LHRH mRNA were not detected in forebrain areas known to contain LHRH cells in postnatal animals. Quantitation of cells expressing LHRH mRNA showed that the number of labeled cells on E12.5 (approximately 800) equaled the number of LHRH cells in postnatal animals, but more than 90% of these cells were located in nasal regions. Between E12.5 and E15.5, the location of LHRH cells shifted. The number of LHRH cells in the forebrain increased, while the number of LHRH cells in nasal regions decreased over this same period. These findings establish that cells first found in the olfactory pit and thereafter in forebrain areas express the LHRH gene and correspond to the position of LHRH immunopositive cells found at these developmental times. To further examine the ontogeny of the LHRH system, immunocytochemistry in combination with [3H]thymidine autoradiography was used to determine when LHRH cells left the mitotic cycle. We show that LHRH neurons exhibit a discrete time of birth, suggesting that they arise as a single neuronal population between E10.0 and E11.0. Postnatal LHRH neurons were "birth-dated" shortly after differentiation of the olfactory placode and before LHRH mRNA was expressed in cells in the olfactory pit. Taken together, these studies support the hypothesis that all LHRH cells in the central nervous system arise from a discrete group of progenitor cells in the olfactory placode and that a subpopulation of these cells migrate into forebrain areas where they subsequently establish an adult-like distribution.

Localization of Proopiomelanocortin (POMC) Immunoreactive Neurons in the Forebrain of the Pig13

The distribution of proopiomelanocortin (POMC)-immunoreactive neurons was examined in the forebrains of nine sexually mature female pigs by indirect biotin-avidin horseradish peroxidase immunocytochemistry. Primary antiserum against ovine beta-endorphin (Bioflex #BF-EP-3-1) yielded positive staining of neuronal perikarya and processes. Adjacent control sections treated either with primary antiserum preabsorbed with beta-endorphin or substituted with normal rabbit serum lacked specific staining. POMC-immunoreactive cells were located in the anterior and intermediate lobe of the pituitary gland. POMC-immunoreactive perikarya were located in the arcuate nucleus and periarcuate area. The pituitary stalk/median eminence contained sparsely distributed POMC-immunoreactive fibers, which were confined to the zona interna. POMC-immunoreactive fibers were located in the arcuate nucleus and extended rostrally from the arcuate nucleus into the telencephalon coursing adjacent to the wall of the third ventricle as well as through the anterior hypothalamus, suprachiasmatic, supraoptic nuclei and preoptic areas to the nucleus accumbens, diagonal band of Broca, olfactory tubercle, bed nucleus of the stria terminalis and the ventro-lateral aspect of the septum. Caudal projections extended along the wall of the third ventricle to the level of the mammillary bodies and also coursed dorsally, passing through the periventricular, paraventricular, and dorsal medial nuclei of the hypothalamus to the midline thalamic nuclei and habenular nucleus. Lateral projections extended from the arcuate nucleus along the dorsal aspect of the optic tract and terminated in the amygdaloid complex. The distribution of POMC-immunoreactive perikarya and fibers is similar to that of the luteinizing hormone-releasing hormone (LHRH) fiber network. Therefore the opportunities exist, anatomically, for interactions between the POMC and the LHRH systems.

Slice cultures of LHRH neurons in the presence and absence of brainstem and pituitary.

Luteinizing hormone releasing hormone (LHRH) neurons from the preoptic area (POA)/hypothalamus of the postnatal rat were cultured for up to 7 weeks using a slice explant roller culture technique. The slices thinned to quasi-monolayers, but maintained organotypic distributions of large numbers of immunocytochemically identifiable LHRH, neurotensin, tyrosine hydroxylase, neurophysin and corticotropin releasing hormone-containing neurons. The distribution, survival and morphology of LHRH cells in co-cultures with brainstem and anterior pituitary was quantitated, and found to be similar to that observed in single cultures. LHRH fibers grew into either pituitary or brainstem tissue, however when all three tissues were co-cultured, LHRH fibers preferentially invaded the pituitary. LH immunoreactive anterior pituitary gonadotropes were maintained only in co-cultures containing POA/hypothalamic slices, and addition of an LHRH antagonist in such cultures, inhibited LH immunoreactivity in the gonadotropes. This slice explant roller culture method effectively maintains the cyto- and chemoarchitecture and functional properties of the LHRH system for long periods in vitro and should provide excellent models for studying the interactive and molecular characteristics of postnatal LHRH neurons.

Immunocytochemical studies on the LHRH system of the Japanese quail: influence by photoperiod and aspects of sexual differentiation.

Immunocytochemistry was used to determine if photoperiod and/or sex have any effect on the pattern of the luteinizing hormone-releasing hormone (LHRH) system in the brain of the Japanese quail. Immunopositive perikarya were found within three major areas of the brain: the rostral paraolfactory lobe, the preoptic, and the septal region. A quantitative analysis of LHRH cell numbers was performed on male and female quail after two photoperiodic treatments: sexually mature birds exposed to 24 weeks of 20 h light: 4 h darkness (20L:4D), and birds with a regressed reproductive system (induced by transfer from a photoregime of 20L:4D to 25 short days of 8L:16D). Two-way analysis of variance showed that short-day males display significantly (p less than 0.05) more immunopositive perikarya (607 +/- 134) than long-day males (291 +/- 114), short-day females (293 +/- 103) or long-day females (330 +/- 92). The density of LHRH-immunoreactive nerve fibres and the intensity of the immunostaining in the median eminence were always greater in long-day sexually mature quail (male and female) than in animals exposed to 25 days of 8L:16D. These results demonstrate that the LHRH system of the quail is influenced by photoperiod and mirrors sexual differentiation.