Gonadotropin-releasing Hormone Cell Bodies Research Articles

Male minipuberty involves the gonad-independent activation of preoptic nNOS neurons.

The maturation of the hypothalamic-pituitary-gonadal (HPG) axis is crucial for the establishment of reproductive function. In female mice, neuronal nitric oxide synthase (nNOS) activity appears to be key for the first postnatal activation of the neural network promoting the release of gonadotropin-releasing hormone (GnRH), i.e. minipuberty. However, in males, the profile of minipuberty as well as the role of nNOS-expressing neurons remain unexplored. nNOS-deficient and wild-type mice were studied during postnatal development. The expression of androgen (AR) and estrogen receptor alpha (ERα) as well as nNOS phosphorylation were evaluated by immunohistochemistry in nNOS neurons in the median preoptic nucleus (MePO), where most GnRH neuronal cell bodies reside, and the hormonal profile of nNOS-deficient male mice was assessed using previously established radioimmunoassay and ELISA methods. Gonadectomy and pharmacological manipulation of ERα were used to elucidate the mechanism of minipubertal nNOS activation and the maturation of the HPG axis. In male mice, minipubertal FSH release occurred at P23, preceding the LH surge at P30, when balanopreputial separation occurs. Progesterone and testosterone remained low during minipuberty, increasing around puberty, whereas estrogen levels were high throughout postnatal development. nNOS neurons showed a sharp increase in Ser1412 phosphorylation of nNOS at P23, a phenomenon that occurred even in the absence of the gonads. In male mice, nNOS neurons did not appear to express AR, but abundantly expressed ERα throughout postnatal development. Selective pharmacological blockade of ERα during the infantile period blunted Ser1412 phosphorylation of nNOS at P23. Our results show that the timing of minipuberty differs in male mice when compared to females, but as in the latter, nNOS activity in the preoptic region plays a role in this process. Additionally, akin to male non-human primates, the profile of minipuberty in male mice is shaped by sex-independent mechanisms, and possibly involves extragonadal estrogen sources.

Morphometric and Myelin Basic Protein Expression Changes in Arcuate Nucleus Kisspeptin Neurons Underlie Activation of Hypothalamic Pituitary Gonadal-axis in Monkeys (Macaca Mulatta) during the Breeding Season

ABSTRACT Introduction Kisspeptin is involved in the hypothalamic pituitary gonadal-axis’ seasonal regulation in rodents and sheep. Studies of kisspeptin signaling in regulating the transition between breeding and nonbreeding seasons have focused on kisspeptin expression, myelin basic protein (MBP) expression around kisspeptin-ir cells, and quantifying the synaptic connections between kisspeptin and gonadotropin-releasing hormone (GnRH) neurons in various animal models; however, the role of kisspeptin in regulating the seasonal breeding of primates has not been explored yet. Objective This study investigated changes in kisspeptin signaling during breeding and a non-breeding season in a non-human primate model, the rhesus monkey. Methods Three adult male monkeys (n = 3) from the breeding season and two monkeys (n = 2) from the non-breeding season were used in this study. After measuring the testicular volume and collecting a single blood sample, all animals were humanely euthanized under controlled conditions, and their hypothalami were collected and processed. Two 20 µm thick hypothalamic sections (mediobasal hypothalamus) from each animal were processed for kisspeptin-MBP and kisspeptin-GnRH immunohistochemistry (IHC). One section from each animal was used as a primary antibody omitted control to check the nonspecific binding in each IHC. Results Compared to the non-breeding season, plasma testosterone levels and testicular volumes were significantly higher in monkeys during the breeding season. Furthermore, compared to the non-breeding season, increased kisspeptin expression and a higher number of synaptic contacts between kisspeptin fibers and GnRH cell bodies were observed in the arcuate nucleus of the breeding season monkeys. In contrast, enlarged kisspeptin soma and higher MBP expression were observed in non-breeding monkeys. Conclusion Our results indicated enhanced kisspeptin signaling in primate hypothalamus during the breeding season. These findings support the idea that kisspeptin acts as a mediator for the seasonal regulation of the reproductive axis in higher primates.

GnRH neurons recruit astrocytes in infancy to facilitate network integration and sexual maturation.

Neurons that produce gonadotropin-releasing hormone (GnRH), which control fertility, complete their nose-to-brain migration by birth. However, their function depends on integration within a complex neuroglial network during postnatal development. Here, we show that rodent GnRH neurons use a prostaglandin D2 receptor DP1 signaling mechanism during infancy to recruit newborn astrocytes that 'escort' them into adulthood, and that the impairment of postnatal hypothalamic gliogenesis markedly alters sexual maturation by preventing this recruitment, a process mimicked by the endocrine disruptor bisphenol A. Inhibition of DP1 signaling in the infantile preoptic region, where GnRH cell bodies reside, disrupts the correct wiring and firing of GnRH neurons, alters minipuberty or the first activation of the hypothalamic-pituitary-gonadal axis during infancy, and delays the timely acquisition of reproductive capacity. These findings uncover a previously unknown neuron-to-neural-progenitor communication pathway and demonstrate that postnatal astrogenesis is a basic component of a complex set of mechanisms used by the neuroendocrine brain to control sexual maturation.

Chapter 18 - Kisspeptin and neurokinin B expression in the human hypothalamus: Relation to reproduction and gender identity

Gonadotropin-releasing hormone (GnRH) neurons in the hypothalamus are at the core of reproductive functioning. GnRH released into the median eminence regulates the secretion of the gonadotropins from the anterior pituitary, which in turn activates gametogenesis and steroid synthesis by the gonads. The GnRH system displays functional sex differences: GnRH is secreted in pulses at a constant frequency in men, whereas in women, pulse frequency varies over the menstrual cycle. In both sexes, GnRH release is regulated by sex steroid hormones, acting at the level of the hypothalamus and the anterior pituitary in a classic feedback loop. Because GnRH neurons do not express sex steroid receptors, hormone effects on GnRH release are presumed to be mediated indirectly through other steroid-sensitive neuronal systems, which then converge onto GnRH cell bodies and/or terminals. Human genetic studies demonstrated that kisspeptin (KP) as well as neurokinin B (NKB) signaling are both potent regulators of GNRH secretion. In humans, postmortem studies using immunohistochemistry have shown that women have higher KP and NKB expression in the infundibular nucleus than men. Sex differences in KP expression are present throughout life, which is from the infant/prepubertal into the elderly period, whereas sex differences in NKB expression do not emerge until adulthood. KP and NKB are often coexpressed together with dynorphin by the same population of neurons, also known as KDNy neurons in other species. Indeed, significant coexpression between KP and NKB but not with Dynorphin has been observed thereby challenging the KDNy concept in humans. Female-typical expression of both KP and NKB were observed in the infundibular nucleus of trans women (male sex assigned at birth and female gender identity). Taken together, sex differences in KP and NKB expression most likely reflect organizational actions of sex steroid hormones on the developing brain but they also remain sensitive to circulating sex steroids in adulthood. The female-dominant sex difference in infundibular KP and NKB expression suggests that this brain region is most likely involved in both the negative and positive feedback actions of estrogens on GnRH secretion. Finally, the sex-reversal observed in KP and NKB expression in trans women might reflect, at least partially, an atypical sexual differentiation of the brain.

Immunohistochemical study of gonadotropin-releasing hormone and somatolactin during induced spawning of Liza ramada

Introduction: Gonadotropin releasing hormone (GnRH) is a crucial regulator of gonadal development and reproduction in fish. In addition, it is a possible secretagogue of somatolactin (SL) in teleosts. Little is known about the possible association of GnRH and SL during reproduction of Liza ramada.Aim of the work: The present study was designed to investigate the GnRH and SL immunoreactivities during inducing the final stages of maturation, ovulation and spawning of Liza ramada; to assess the possible actions of these hormones on sexual maturation and spawning of teleosts.Materials and Methods: Histological and immunohistochemical techniques were generally performed to describe the immunoreactivity of both GnRH and SL during ovarian maturation and spawning of Liza ramada.Results: The mammalian GnRH (mGnRH) cell bodies were observed in the medulla oblongata. However, SL-immunoreactive (-IR) cells were detected in the pars intermedia (PI) of the pituitary gland. The GnRH-IR nerve terminals are in close contact with SL-IR pituitary cells. Importantly, the activity of both mGnRH-IR neurons and SL-IR pituitary cells were increased as reflected with the increased cell number and size during sexual maturation and spawning. The activity of these cells was more pronounced as indicated by the increased cells number and size with more immunoreactivity in pre-spawning female. During final maturation and spawning, mGnRH-IR neurons and SL cells showed an increase in the secretory activity as reflected by their small sizes, vacuolated appearance and weak immunoreactivity.Conclusions: Taken together, these findings suggest that the close contact of mGnRH-IR terminals with SL-IR cells in the pituitary, together with the concomitant changes of GnRH and SL immunoreactivity in response to progress in the reproductive events suggest that GnRH may regulate SL release and the regulation of reproductive activity in L. ramada.

Leptin Responsive and GABAergic Projections to the Rostral Preoptic Area in Mice.

The adipocyte-derived hormone leptin plays a critical role in the control of reproduction via signalling in hypothalamic neurones. The drivers of the hypothalamic-pituitary-gonadal axis, the gonadotrophin-releasing hormone (GnRH) neurones, do not have the receptors for leptin. Therefore, intermediate leptin responsive neurones must provide leptin-to-GnRH signalling. We investigated the populations of leptin responsive neurones that provide input to the rostral preoptic area (rPOA) where GnRH cell bodies reside. Fluorescent retrograde tracer beads (RetroBeads; Lumafluor Inc., Naples, FL, USA) were injected into the rPOA of transgenic leptin receptor enhanced green fluorescent protein (Lepr-eGFP) reporter mice. Uptake of the RetroBeads by Lepr-eGFP neurones was assessed throughout the hypothalamus. RetroBead uptake was most evident in the medial arcuate nucleus (ARC), the dorsomedial nucleus (DMN) and the ventral premammillary nucleus (PMV) of the hypothalamus. The uptake of RetroBeads specifically by Lepr-eGFP neurones was highest in the medial ARC (18% of tracer-labelled neurones Lepr-eGFP-positive). Because neurones that are both leptin responsive and GABAergic play a critical role in the regulation of fertility by leptin, we next focussed on the location of these populations. To address whether GABAergic neurones in leptin-responsive hypothalamic regions project to the rPOA, the experiment was repeated in GABA neurone reporter mice (Vgat-tdTomato). Between 10% and 45% of RetroBead-labelled neurones in the ARC were GABAergic, whereas uptake of tracer by GABAergic neurones in the DMN and PMV was very low (<5%). These results show that both leptin responsive and GABAergic neurones from the ARC project to the region of the GnRH cell bodies. Our findings suggest that LEPR-expressing GABA neurones from the ARC may be mediators of leptin-to-GnRH signalling.

Physiology of the gonadotrophin-releasing hormone (GnRH) neurone: studies from embryonic GnRH neurones.

Gonadotrophin-releasing hormone (GnRH)-secreting neurones are the final output of the central nervous system driving fertility in all mammals. Although it has been known for decades that the efficiency of communication between the hypothalamus and the pituitary depends on the pulsatile profile of GnRH secretion, how GnRH neuronal activity is patterned to generate pulses at the median eminence is unknown. To date, the scattered distribution of the GnRH cell bodies remains the main limitation to assessing the cellular events that could lead to pulsatile GnRH secretion. Taking advantage of the unique developmental feature of GnRH neurones, the nasal explant model allows primary GnRH neurones to be maintained within a micro-network where pulsatile secretion is preserved and where individual cellular activity can be monitored simultaneously across the cell population. This review summarises the data obtained from work using this in vitro model, and brings some insights into GnRH cellular physiology.

Expression of Kv4.3 Voltage-Gated Potassium Channels in Rat Gonadotrophin-Releasing Hormone (GnRH) Neurons During the Estrous Cycle

Regular and timely electrical activity of gonadotrophin-releasing hormone (GnRH) neurons accompanies the pulsatile release of GnRH that plays a central role in regulating fertility. Although transient outward A-type currents (I(A)) have been electrophysiologically identified in GnRH neurons, the molecular identity of the channels that underlie these currents are unknown. Several families of voltage-gated potassium channels can underlie I(A). However, the biophysical properties of I(A) described in previous electrophysiological studies are strongly characteristic of members of the Kv4 family of voltage-gated channels. We, therefore, sought to determine the presence of Kv4 channels in GnRH neurons. We used reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis to determine whether Kv4 messenger RNA (mRNA) and protein are present in the rat medial preoptic area (MPOA) and median eminence (ME). We used double-label immunohistochemistry to determine whether Kv4 colocalized with GnRH cell bodies in the MPOA and GnRH axons in the ME. Kv4.3 channels co-localized with GnRH in the MPOA but not in the ME. Neither Kv4.2 nor Kv4.1 co-localized with GnRH in either the MPOA or the ME. The electrical activity of GnRH neurons changes dramatically during the estrous cycle. We, therefore, studied the change in Kv4.3 expression in GnRH neurons during the estrous cycle. In the estrus phase, 58.05% of GnRH neurons expressed Kv4.3 compared to 74.48% in diestrus-proestrus rats (P < .05). Our data suggest that Kv4.3 is the major molecular component of I(A) in GnRH neurons, and furthermore that the expression of Kv4.3 changes significantly during the rat estrous cycle.

Integration Between Different Hypothalamic Nuclei Involved in Stress and GnRH Secretion in the Ewe

This study investigated possible integrated links in the neuroanatomical pathways through which the activity of neurones in the paraventricular nucleus and arcuate nucleus may modulate suppression of gonadotrophin-releasing hormone (GnRH) secretion during stressful situations. Double-label immunofluorescence and laser scanning confocal microscopy were used to examine the hypothalamic sections from the follicular phase ewes. Noradrenergic terminals were in close contact with 65.7 ± 6.1% corticotrophin-releasing hormone (CRH) and 84.6 ± 3.2% arginine vasopressin (AVP) cell bodies in the paraventricular nucleus but not with β-endorphin cell bodies in the arcuate nucleus. Furthermore, γ-amino butyric acid (GABA) terminals were close to 80.9 ± 3.5% CRH but no AVP cell bodies in the paraventricular nucleus, as well as 60.8 ± 4.1%β-endorphin cell bodies in the arcuate nucleus. Although CRH, AVP and β-endorphin cell terminals were identified in the medial pre-optic area, no direct contacts with GnRH cell bodies were observed. Within the median eminence, abundant CRH but not AVP terminals were close to GnRH cell terminals in the external zone; whereas, β-endorphin cells and terminals were in the internal zone. In conclusion, neuroanatomical evidence is provided for the ewe supporting the hypothesis that brainstem noradrenergic and hypothalamic GABA neurones are important in modulating the activity of CRH and AVP neurones in the paraventricular nucleus, as well as β-endorphin neurones in the arcuate nucleus. These paraventricular and arcuate neurones may also involve interneurones to influence GnRH cell bodies in medial pre-optic area, whereas the median eminence may provide a major site for direct modulation of GnRH release by CRH terminals.

Kisspeptin Signaling Is Required for Peripheral But Not Central Stimulation of Gonadotropin-Releasing Hormone Neurons by NMDA

NMDA and kisspeptins can stimulate gonadotropin-releasing hormone (GnRH) release after peripheral or central administration in mice. To determine whether these agonists act independently or through a common pathway, we have examined their ability to stimulate GnRH/luteinizing hormone (LH) release after peripheral or central administration in Kiss1- or Gpr54 (Kiss1r)-null mutant mice. Peripheral injection of NMDA failed to stimulate GnRH/LH release in prepubertal or gonadally intact mutant male mice. Dual-labeling experiments indicated a direct activation of Kiss1-expressing neurons in the arcuate nucleus. In contrast, central injection of NMDA into the lateral ventricle increased plasma LH levels in both Kiss1 and Gpr54 mutant male mice similar to the responses in wild-type mice. Central injection of NMDA stimulated c-Fos expression throughout the hypothalamus but not in GnRH neurons, suggesting an action at the nerve terminals only. In contrast, kisspeptin-10 stimulated LH release after both central and peripheral injection but induced c-Fos expression in GnRH neurons only after central administration. Finally, central injection of NMDA induces c-Fos expression in catecholamine- and nitric oxide-producing neurons in the hypothalamus of mutant mice, indicating a possible kisspeptin-independent GnRH/LH release by NMDA through activation of these neurons. Thus, NMDA may act at both GnRH cell bodies (kisspeptin-independent) and nerve terminals (kisspeptin-dependent) in a dual way to participate in the GnRH/LH secretion in the male mouse.

Gonadotrophin‐Releasing Hormone (GnRH) Exerts Stimulatory Effects on GnRH Neurones in Intact Adult Male and Female Mice

There is substantial evidence for a role of the neuropeptide gonadotrophin-releasing hormone (GnRH) in the regulation of GnRH neurone secretion but how this is achieved is not understood. We examined here the effects of GnRH on the electrical excitability and intracellular calcium concentration ([Ca2+](i)) of GnRH neurones in intact adult male and female mice. Perforated-patch electrophysiological recordings from GnRH-green fluorescent protein-tagged GnRH neurones revealed that 3 nm-3 mum GnRH evoked gradual approximately 3 mV depolarisations in membrane potential from up to 50% of GnRH neurones in male and female mice. The depolarising effect of GnRH was observed on approximately 50% of GnRH neurones throughout the oestrous cycle. However, at pro-oestrus alone, GnRH was also found to transiently hyperpolarise approximately 30% of GnRH neurones. Both hyperpolarising and depolarising responses were maintained in the presence of tetrodotoxin. Calcium imaging studies undertaken in transgenic GnRH-pericam mice showed that GnRH suppressed [Ca2+](i) in approximately 50% of GnRH neurones in dioestrous and oestrous mice. At pro-oestrus, 25% of GnRH neurones exhibited a suppressive [Ca2+](i) response to GnRH, whereas 17% were stimulated. These results demonstrate that nm to mum concentrations of GnRH exert depolarising actions on approximately 50% of GnRH neurones in males and females throughout the oestrous cycle. This is associated with a reduction in [Ca2+](i). At pro-oestrus, however, a further population of GnRH neurones exhibit a hyperpolarising response to GnRH. Taken together, these studies indicate that GnRH acts predominantly as a neuromodulator at the level of the GnRH cell bodies to exert a predominant excitatory influence upon GnRH neurones in intact adult male and female mice.

329. IS THERE VARIATION IN THE LEVEL OF INPUT OF KISSPEPTIN TERMINALS ONTO GnRH NEURONS ACROSS THE EQUINE OESTROUS CYCLE?

Kisspeptin (KP) plays a key role in reproductive function including the regulation of gonadotrophin releasing hormone (GnRH) and luteinising hormone (LH) secretion in many species but little is known about its role in the mare. In this study, we examined the location of KP-producing neurons in the brain of the mare, their potential interactions with GnRH neurons, and temporal changes in their expression across the oestrous cycle. Mares (n = 3/group) were killed at oestrus (just prior to ovulation), mid-dioestrus, and late dioestrus and the head was perfusion fixed with paraformaldehyde, and hypothalamus collected. Coronal sections (40 μm) were used for dual-label immuno-stained for KP &amp; GnRH. The majority of KP-immunoreactive (-ir) neurons were located in the arcuate nucleus/median eminence (especially mid and caudal regions), and periventricular nucleus. There was a trend (P = 0.09) towards increasing numbers of KP-ir neurons across the cycle. GnRH-ir neurons, located primarily in the arcuate nucleus (especially mid arcuate), as well as the preoptic area, did not change in number across the cycle. Numerous interactions between KP and GnRH neurons were observed, primarily in the arcuate nucleus; KP fibres interacting with GnRH cell bodies, fibre-fibre interactions between KP and GnRH, and GnRH fibres interacting with KP cell bodies. Overall we found KP inputs to 32% of GnRH-ir cells, but the number of these interactions did not vary across the oestrous cycle. This study has confirmed the reciprocal innervation between KP &amp; GnRH neurons in the mare. Although we did not detect variation in the degree across the oestrous cycle this may reflect the sample size issues inherent to equine research.

Gonadotrophin‐Releasing Hormone Pulse Generator Activity in the Hypothalamus of the Goat

Pulsatile release of gonadotrophin-releasing hormone (GnRH) is indispensable to maintain normal gonadotrophin secretion. The pulsatile secretion of GnRH is associated with synchronised electrical activity in the mediobasal hypothalamus (i.e. multiple unit activity; MUA), which is considered to reflect the rhythmic oscillations in the activity of the neuronal network that drives pulsatile GnRH secretion. However, the cellular source of this ultradian rhythm in GnRH activity is unknown. Direct input from kisspeptin neurones in the arcuate nucleus (ARC) to GnRH cell bodies in the medial preoptic area or their terminals in the median eminence could be the intrinsic source for driving the GnRH pulse generator. To determine whether kisspeptin signalling could be responsible for producing pulsatile GnRH secretion, we studied goats, measured plasma levels of luteinising hormone (LH) and recorded MUA in the posterior ARC, where the majority of kisspeptin neuronal cell bodies are located. Rhythmic volleys of MUA were found to be accompanied by LH pulses with regular intervals in the ARC, where kisspeptin neuronal cell bodies were found. Exogenous administration of kisspeptin stimulated a sustained increase in LH secretion, without influencing MUA, suggesting that the GnRH pulse generator, as reflected by MUA, originated from outside of the network of GnRH neurones, and could plausibly reflect the pacemaker activity of kisspeptin neurones, whose projections reach the median eminence where GnRH fibres project. These observations suggest that the kisspeptin neurones in the ARC may be the intrinsic source of the GnRH pulse generator.

Glutamatergic Stimulation of Luteinising Hormone Secretion in Relatively Refractory Male Songbirds

Seasonal breeding in two Sonoran desert passerines, the Cassin's (Aimophila cassinii) and Rufous-crowned (Aimophila ruficeps) Sparrows is thought to be terminated by the development of a decrease in responsiveness to photostimulation, a condition known as relative photorefractoriness. It was predicted that the development of relative refractoriness is a consequence of a decrease in gonadotrophin-releasing hormone (GnRH) synthesis and associated stores of releasable GnRH. This hypothesis was tested by determining the luteinising hormone (LH) responses to the excitatory amino acid glutamate agonist N-methyl-D,L-aspartate (NMA) in males of the two species subjected to photomanipulations aimed at generating five groups: Fully photosensitive with undeveloped testes on short days (8L : 16D); fully photosensitive with developed testes on 13L : 11D; relatively photorefractory with regressed testes on 13L : 11D, and groups with developed testes held on 15L : 9D or 16L : 8D. LH release was stimulated in the Cassin's Sparrow by NMA most in the 8L group; to a lesser, but similar extent in the two 13L groups; and not at all in the 15L and 16L groups. LH release was not stimulated by NMA in any of the photoperiodic regimes in the Rufous-crowned Sparrow. In both species, NMA induced Fos-like immunoreactivity in the anterior and basal hypothalamus, but not in GnRH cell bodies. It is concluded that the development of relative photorefractoriness in Cassin's Sparrows is a consequence of reduced GnRH synthesis, reflected in a reduction in releasable GnRH. The lack of LH response of the Rufous-crowned Sparrows to NMA administration may be a consequence of high responsiveness to handling stress.

Transcriptional regulation of PSA-NCAM mediated neuron–glial plasticity in the adult hypothalamus

Gonadotropin releasing hormone (GnRH)-secreting neurons in mammal's project principally to the median eminence-arcuate (ME-ARC) region where they make contact with basal lamina and open into the pericapillary space of the primary hypophyseal portal plexus. In the present study we report the expression of polysialylated form of neural cell adhesion molecule (PSA-NCAM) on the cell bodies of GnRH-secreting neurons and on glial cells in the medial preoptic area (mPOA) of the hypothalamus in both the proestrous and diestrous phases of cycling rats, using dual immunohistofluorescent staining. Regulation of PSA-NCAM occurs via regulation of PSA biosynthesis by the polysialyltransferase enzyme (PST-1). Therefore, we studied the expression of PST mRNA and GnRH in GnRH-releasing cell bodies by combining fluorescent in situ hybridization with immunohistofluorescence, and expression of PST mRNA in the mPOA using northern blotting. We observed dynamic upregulation of PSA-NCAM on GnRH cell bodies in the proestrous phase, accompanied by enhanced expression of PST-1 mRNA. The present results indicate that PSA-NCAM has a permissive role in the structural remodeling of GnRH neurons. Enhanced expression of mRNA encoding PST-1 in the proestrous phase indicates that the biosynthesis of PSA and, thus, PSA-NCAM is regulated at the transcriptional level.

Mechanisms mediating the effects of cytokines on neuroendocrine functions in the rat.

Exposure to an antigen causes significant endocrine changes, some of which in turn affect immune functioning. Proteins produced by activated immune cells, cytokines, act as messengers between the immune and the endocrine systems, and convey to the brain the occurrence of immune activation. We have investigated the ability of interleukin 1 (IL-1) alpha and beta to alter endocrine functioning in the adult rat. Acute peripheral injection of IL-1 alpha or beta causes dose-dependent increases in plasma adrenocorticotropic hormone (ACTH) and corticosterone secretion. These changes are primarily dependent upon increased release of corticotropin-releasing factor (CRF) into the portal circulation, and recent studies have indicated that the paraventricular nucleus (PVN) of the hypothalamus is the main source of this CRF. This conclusion is based on our finding that intravenous injection of IL-1 increases CRF biosynthesis in the PVN, and that lesion of this hypothalamic area interferes with IL-1's stimulatory action on ACTH secretion. Indomethacin partially reverses the effect of IL-1, suggesting that increased prostaglandin synthesis plays some part in this activation. Administration of IL-1 beta into the brain, but not into the general circulation, interferes with secretion of luteinizing hormone (LH) and ovulation through mechanisms involving endogenous opiates. Because neither CRF antagonists, nor lesions of the PVN, alter the inhibitory effect of IL-1 on LH release, CRF perikarya in the PVN do not appear to be involved in this phenomenon. Central administration of IL-1 beta strongly increases c-Fos immunoreactivity in the PVN, mainly within CRF neurons. Infusion of IL-1 beta into the PVN does not induce measurable changes in release of gonadotropin-releasing hormone (GnRH), but infusion of IL-1 directly into the median preoptic area (MPOA), a region rich in GnRH perikarya, markedly decreases GnRH secretion in rats bearing a push-pull cannula in the median eminence. Furthermore, central administration of IL-1 beta during the critical phase of pro-oestrus (1600-1930) also inhibits the expression of c-fos in GnRH cell bodies in the MPOA. Thus, we suggest that IL-1 interferes with reproductive functioning through a direct action at the level of the MPOA. These results indicate that circulating cytokines can alter the activity of the hypothalamo-pituitary-adrenal axis by increasing CRF release, probably through both immediate stimulation of CRF terminals within the median eminence and stimulation of CRF synthesis in the PVN. In contrast, cytokine-induced changes in LH and GnRH secretion are mediated through pathways lying primarily beyond the blood-brain barrier.

Discovery of gonadotropin-inhibitory hormone in a domesticated bird, its mode of action and functional significance

Neuropeptide control of gonadotropin secretion at the level of the anterior pituitary gland is primarily through the stimulatory action of the hypothalamic decapeptide gonadotropin-releasing hormone (GnRH) in vertebrates. Until recently, no neuropeptide that directly inhibits gonadotropin secretion had been identified. In 2000, we discovered a novel hypothalamic dodecapeptide that directly inhibits gonadotropin release in quail and termed it gonadotropin-inhibitory hormone (GnIH). A gonadotropin inhibitory system is an intriguing concept and provides us with an unprecedented opportunity to study the regulation of avian reproduction from an entirely novel standpoint. To elucidate the mode of action of GnIH, we then identified a novel G protein-coupled receptor for GnIH in quail. The receptor possessed seven transmembrane domains and specifically bound to GnIH in a concentration-dependent manner. The GnIH receptor was found to be expressed in the pituitary and several brain regions including the hypothalamus. These results indicate that GnIH acts directly on the pituitary via GnIH receptor to inhibit gonadotropin release. GnIH may also act on the hypothalamus to inhibit GnRH release, because GnIH fibers were observed to contact GnRH cell bodies. To demonstrate the functional significance of GnIH and its potential role as a key neuropeptide involved in avian reproduction, we further investigated the action of GnIH on gonadal development and maintenance in quail. Chronic treatment with GnIH inhibited photoinduced testicular development and maintenance by decreasing gonadotropin synthesis and release. Melatonin is a key factor involved in GnIH neural function. Quail GnIH neurons were found to contain melatonin receptor and melatonin stimulated expression of GnIH mRNA and mature GnIH peptide in a dose-dependent manner. It is concluded that GnIH is capable of transducing photoperiodic information via changes in the melatonin signal and of influencing the reproductive axes of birds.

Potential of PSA-NCAM in neuron-glial plasticity in the adult hypothalamus: Role of noradrenergic and GABAergic neurotransmitters

The present study was designed to establish the dynamic regulation of polysialylated form of neural cell adhesion molecule (PSA-NCAM) expression by neurotransmitters controlling gonadotropin releasing hormone (GnRH) secretion. The expression of PSA-NCAM and glial fibrillary acidic protein (GFAP) on GnRH cell bodies and axon terminals in the medial preoptic area (mPOA) and median eminence-arcuate (ME-ARC) region of hypothalamus was studied in the proestrous phase of cycling rats treated with α-adrenergic receptor blocker phenoxybenzamine (PBZ) and γ-aminobutyric acid (GABA) by using dual immunohistofluorescent staining and Western blot hybridization. To further elucidate whether activity mediated regulation of PSA-NCAM in GnRH neuron is via regulation of PSA biosynthesis by polysialytransferase (PST) enzyme, the expression of PST-1 enzyme was studied by using fluorescent in situ hybridization (FISH). Both GnRH and PSA-NCAM immunostaining was much higher in the mPOA and ME-ARC region from proestrous phase rats, whereas, PBZ and GABA treatments significantly reduced their expression, GFAP-ir and its content were increased in the PBZ and GABA treated proestrous rats. Taken together, our observations add to the growing evidence that PSA-NCAM plays permissive role for neuronal-glial remodeling and further suggest a functional link between activity dependent structural remodeling in GnRH neurons. Further, enhanced mRNA expression of PST suggests that the biosynthesis of PSA on NCAM is regulated at the transcriptional level.

Morphological Evidence for Direct Interaction Between Gonadotrophin‐Releasing Hormone Neurones and Astroglial Cells in the Human Hypothalamus

In rodents, there is compelling evidence indicating that dynamic cell-to-cell communications involving cross talk between astroglial cells (such as astrocytes and specialised ependymoglial cells known as tanycytes) and neurones are important in regulating the secretion of gonadotrophin-releasing hormone (GnRH), the neurohormone that controls both sexual maturation and adult reproductive function. However, whether such astroglial cell-GnRH neurone interactions occur in the human brain is not known. In the present study, we used immunofluorescence to examine the anatomical relationship between GnRH neurones and glial cells within the hypothalamus of five women. Double-staining experiments demonstrated the ensheathment of GnRH neurone perikarya by glial fibrillary acidic protein (GFAP)-immunoreactive astrocyte processes in the periventricular zone of the tuberal region of the hypothalamus. GFAP immunoreactivity did not overlap that of GnRH at the GnRH neurone's projection site (i.e. the median eminence of the hypothalamus). Rather, human GnRH neuroendocrine fibres were found to be closely associated with vimentin or nestin-immunopositive radial glial processes likely belonging to tanycytes. In line with these light microscopy data, ultrastructural examination of GnRH-immunoreactive neurones showed numerous glial cells in direct apposition to pre-embedding-labelled GnRH cell bodies and/or dendrites in the infundibular nucleus, whereas postembedding immunogold-labelled GnRH nerve terminals were often seen to be enwrapped by glial cell processes in the median eminence. GnRH nerve button were sometimes visualised in close proximity to fenestrated pituitary portal blood capillaries and/or evaginations of the basal lamina that delineate the pericapillary space. In summary, these data demonstrate that GnRH neurones morphologically interact with astrocytes and tanycytes in the human brain and provide evidence that glial cells may contribute physiologically to the process by which the neuroendocrine brain controls the function of GnRH neurones in humans.

Hypothalamic Gonadotrophin‐Releasing Hormone Expression in Female Monkeys with Different Sensitivity to Stress

Psychosocial stress, combined with mild dieting and moderate exercise, are observed in women seeking treatment for hypothalamic amenorrhea. Using female cynomolgus macaques, we previously reported that the same combination of mild stresses suppressed reproductive hormone secretion and menstrual cycles in some individuals (stress-sensitive, SS), but not in others (highly stress-resilient, HSR). Compared to HSR monkeys, SS monkeys exhibited lower oestradiol and progesterone levels at the midcycle peak and decreased gene expression in the central serotonergic system during nonstressed cycles. Because steroids and serotonin impinge upon the hypothalamic-pituitary-gonadal (HPG) axis, we hypothesised that the differences between SS and HSR monkeys in the sensitivity of the HPG axis to stress may ultimately manifest in differences in the gonadotrophin-releasing hormone (GnRH) system. GnRH in situ hybridisation and immunohistochemistry were performed with hypothalamic sections from SS and HSR animals, euthanised in the early follicular phase of a nonstressed menstrual cycle. Compared to HSR monkeys, SS monkeys exhibited a significantly higher number and density of GnRH cell bodies, as well as a higher number of soma with extremely robust expression of GnRH mRNA, but SS monkeys exhibited a lower density of immunostained GnRH fibres in the median eminence. We suggest that neuronal mechanisms involved in the control of GnRH synthesis, transport and release differ in SS compared to HSR animals.