Hypothalamic Overexpression of Neurosecretory Protein GL Leads to Obesity in Male C57BL/6J Mice.

INTRODUCTION: The mechanisms underlying obesity are not fully understood, necessitating the creation of novel animal models for the investigation of metabolic disorders. We have previously found that neurosecretory protein GL (NPGL), a newly identified hypothalamic neuropeptide, is involved in feeding behavior and fat accumulation in rats. However, the impact of NPGL on obesity remains unclear in any animal model. The present investigation sought to elucidate whether NPGL causes obesity in the obesity-prone mouse strain C57BL/6J. METHODS: We overexpressed the NPGL-precursor gene (Npgl) in the hypothalamus using adeno-associated virus in male C57BL/6J mice fed normal chow (NC) or a high-calorie diet (HCD). After 9 weeks of Npgl overexpression, we measured adipose tissues, muscle, and several organ masses in addition to food intake and body mass. To assess the effects of Npgl overexpression on peripheral tissues, we analyzed mRNA expression of lipid metabolism-related genes by quantitative RT-PCR. Whole body energy consumption was assessed using an O2/CO2 metabolism measurement before an apparent increase in body mass. RESULTS: Npgl overexpression increased food intake, body mass, adipose tissues and liver masses, and food efficiency under both NC and HCD, resulting in obesity observable within 8 weeks. Furthermore, we observed fat accumulation in adipose tissues and liver. Additionally, mRNA expression of lipid metabolism-related factors was increased in white adipose tissue and the liver after Npgl overexpression. Npgl overexpression inhibited energy expenditure during a dark period. CONCLUSION: Taken together, the present study suggests that NPGL can act as an obesogenic factor that acts within a short period of time in mice. As a result, this Npgl overexpression-induced obesity can be widely applied to study the etiology of obesity from genes to behavior.

Advancing reproductive neuroendocrinology through research on the regulation of GnIH and on its diverse actions on reproductive physiology and behavior.

The discovery of gonadotropin-inhibitory hormone (GnIH) in 2000 has led to a new research era of reproductive neuroendocrinology because, for a long time, researchers believed that only gonadotropin-releasing hormone (GnRH) regulated reproduction as a neurohormone. Later studies on GnIH demonstrated that it acts as a new key neurohormone inhibiting reproduction in vertebrates. GnIH reduces gonadotropin release andsynthesis via the GnIH receptor GPR147 on gonadotropes and GnRH neurons. Furthermore, GnIH inhibits reproductive behavior, in addition to reproductive neuroendocrine function. The modification of the synthesis of GnIH and its release by the neuroendocrine integration of environmental and internal factors has also been demonstrated. Thus, the discovery of GnIH has facilitated advances in reproductive neuroendocrinology. Here, we describe the advances in reproductive neuroendocrinology driven by the discovery of GnIH, research on the effects of GnIH on reproductive physiology and behavior, and the regulatory mechanisms underlying GnIH synthesis and release.

Adrenomedullin 2 and 5 activate the calcitonin receptor-like receptor (clr) - Receptor activity-modifying protein 3 (ramp3) receptor complex in Xenopus tropicalis.

The adrenomedullin (AM) family is involved in diverse biological functions, including cardiovascular regulation and body fluid homeostasis, in multiple vertebrate lineages. The AM family consists of AM1, AM2, and AM5 in tetrapods, and the receptor for mammalian AMs has been identified as the complex of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 2 (RAMP2) or RAMP3. However, the receptors for AM in amphibians have not been identified. In this study, we identified the cDNAs encoding calcrl (clr), ramp2, and ramp3 receptor components from the western clawed frog (Xenopus tropicalis). Messenger RNAs of amphibian clr and ramp2 were highly expressed in the heart, whereas that of ramp3 was highly expressed in the whole blood. In HEK293T cells expressing clr-ramp2, cAMP response element luciferase (CRE-Luc) reporter activity was activated by am1. In HEK293T cells expressing clr-ramp3, CRE-Luc reporter activity was increased by the treatment with am2 at the lowest dose, but with am5 and am1 at higher dose. Our results provided new insights into the roles of AM family peptides through CLR-RAMP receptor complexes in the tetrapods.

The Arg-Phe-amide peptide 26RFa/glutamine RF-amide peptide and its receptor: IUPHAR Review 24.

The RFamide neuropeptide 26RFa was first isolated from the brain of the European green frog on the basis of cross-reactivity with antibodies raised against bovine neuropeptide FF (NPFF). 26RFa and its N-terminally extended form glutamine RF-amide peptide (QRFP) have been identified as cognate ligands of the former orphan receptor GPR103, now renamed glutamine RF-amide peptide receptor (QRFP receptor). The 26RFa/QRFP precursor has been characterized in various mammalian and non-mammalian species. In the brain of mammals, including humans, 26RFa/QRFP mRNA is almost exclusively expressed in hypothalamic nuclei. The 26RFa/QRFP transcript is also present in various organs especially in endocrine glands. While humans express only one QRFP receptor, two isoforms are present in rodents. The QRFP receptor genes are widely expressed in the CNS and in peripheral tissues, notably in bone, heart, kidney, pancreas and testis. Structure-activity relationship studies have led to the identification of low MW peptidergic agonists and antagonists of QRFP receptor. Concurrently, several selective non-peptidic antagonists have been designed from high-throughput screening hit optimization. Consistent with the widespread distribution of QRFP receptor mRNA and 26RFa binding sites, 26RFa/QRFP exerts a large range of biological activities, notably in the control of energy homeostasis, bone formation and nociception that are mediated by QRFP receptor or NPFF2. The present report reviews the current knowledge concerning the 26RFa/QRFP-QRFP receptor system and discusses the potential use of selective QRFP receptor ligands for therapeutic applications.

Production and characterization of neurosecretory protein GM using Escherichia coli and Chinese Hamster Ovary cells.

Neurosecretory protein GL (NPGL) and neurosecretory protein GM (NPGM) are paralogs recently discovered in birds and in mammals. The post-translational products of NPGL and of NPGM genes include a signal peptide sequence, a glycine amidation signal, and a dibasic amino acid cleavage site. This suggests that the mature forms of NPGL and of NPGM are small proteins secreted in the hypothalamus and containing an amidated C-terminus. However, endogenous NPGL and NPGM have not yet been identified. Chicken NPGL and NPGM have two highly conserved Cys residues that are likely to form a disulfide bond, while mammalian NPGM has one additional Cys residue located between the two conserved Cys residues and the correct disulfide bond pattern is unclear. In this study, we prepared rat NPGM to elucidate the structure of its mature form. We first expressed the predicted mature NPGM, containing an extra C-terminal Gly, in Escherichia coli SHuffle cells, which are engineered to promote the formation of native disulfide bridges in recombinant proteins. We observed the presence of a disulfide bond between the N-terminal Cys residue and the second Cys residue, while the C-terminal Cys residue was free. Secondly, we transfected a construct containing the entire NPGM open reading frame into Chinese Hamster Ovary cells, and observed that NPGM was cleaved immediately after the signal peptide and that it was secreted into the medium. Furthermore, the protein presented a disulfide bond at the same location observed in recombinant NPGM.

Microwave-assisted solid-phase peptide synthesis of neurosecretory protein GL composed of 80 amino acid residues.

We recently identified a novel cDNA encoding a small secretory protein of 80 amino acid residues, termed neurosecretory protein GL (NPGL), from the chicken hypothalamus. Homologs of NPGL have been reported to be present in mammals, such as human and rat. NPGL is amidated at its C-terminus, contains an intramolecular disulfide bond, and is hydrophobic in nature. In this study, we have optimized the synthesis of the entire 80-amino acid peptide sequence of rat NPGL by microwave-assisted solid-phase peptide synthesis. NPGL was obtained with a 10% yield when the coupling reactions were performed using 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxid hexafluorophosphate (HATU) at 50 °C for 5 min, and Fmoc deprotections were performed using 40% piperidine containing 0.1 M HOBt. Furthermore, the disulfide bond of NPGL was formed with 20% yield with the use of glutathione-containing redox buffer and 50% acetonitrile.

Central administration of chicken growth hormone-releasing hormone decreases food intake in chicks.

Growth hormone-releasing hormone (GHRH) is well known as a stimulator of growth hormone (GH) secretion. GHRH not only stimulates GH release but also modifies feeding behavior and energy homeostasis in rodents. In chickens (Gallus gallus domesticus), on the other hand, two types of GHRH, namely, chicken GHRH (cGHRH) and cGHRH-like peptide (cGHRH-LP), have been identified. The purpose of the present study was to investigate the effect of central injection of cGHRH and cGHRH-LP on feeding behavior in chicks. Intracerebroventricular (ICV) injection of both cGHRH and cGHRH-LP (0.04 to 1 nmol) significantly decreased food intake without any abnormal behavior in chicks. Furthermore, the feeding-inhibitory effect was not abolished by co-injection of the antagonist for pituitary adenylate cyclase-activating polypeptide (PACAP) or corticotropin-releasing hormone (CRH) receptors, suggesting that the anorexigenic effect of cGHRH and cGHRH-LP might not be related to the PACAP and CRH systems in the brain of chicks. Finally, 24-h food deprivation increased mRNA expression of cGHRH but not cGHRH-LP in the diencephalon. These results suggest that central cGHRH is related to inhibiting feeding behavior and energy homeostasis in chicks.

Identification, expression, and physiological functions of Siberian hamster gonadotropin-inhibitory hormone.

Gonadotropin-inhibitory hormone (GnIH) is a hypothalamic neuropeptide that inhibits gonadotropin secretion in birds and mammals. To further understand its physiological roles in mammalian reproduction, we identified its precursor cDNA and endogenous mature peptides in the Siberian hamster brain. The Siberian hamster GnIH precursor cDNA encoded two RFamide-related peptide (RFRP) sequences. SPAPANKVPHSAANLPLRF-NH(2) (Siberian hamster RFRP-1) and TLSRVPSLPQRF-NH(2) (Siberian hamster RFRP-3) were confirmed as mature endogenous peptides by mass spectrometry from brain samples purified by immunoaffinity chromatography. GnIH mRNA expression was higher in long days (LD) compared with short days (SD). GnIH mRNA was also highly expressed in SD plus pinealectomized animals, whereas expression was suppressed by melatonin, a nocturnal pineal hormone, administration. GnIH-immunoreactive (-ir) neurons were localized to the dorsomedial region of the hypothalamus, and GnIH-ir fibers projected to hypothalamic and limbic structures. The density of GnIH-ir perikarya and fibers were higher in LD and SD plus pinealectomized hamsters than in LD plus melatonin or SD animals. The percentage of GnRH neurons receiving close appositions from GnIH-ir fiber terminals was also higher in LD than SD, and GnIH receptor was expressed in GnRH-ir neurons. Finally, central administration of hamster RFRP-1 or RFRP-3 inhibited LH release 5 and 30 min after administration in LD. In sharp contrast, both peptides stimulated LH release 30 min after administration in SD. These results suggest that GnIH peptides fine tune LH levels via its receptor expressed in GnRH-ir neurons in an opposing fashion across the seasons in Siberian hamsters.

Identification and characterization of antimicrobial peptides from the skin of the endangered frog Odorrana ishikawae.

The endangered anuran species, Odorrana ishikawae, is endemic to only two small Japanese Islands, Amami and Okinawa. To assess the innate immune system in this frog, we investigated antimicrobial peptides in the skin using artificially bred animals. Nine novel antimicrobial peptides containing the C-terminal cyclic heptapeptide domain were isolated on the basis of antimicrobial activity against Escherichia coli. The peptides were members of the esculentin-1 (two peptides), esculentin-2 (one peptide), palustrin-2 (one peptide), brevinin-2 (three peptides) and nigrocin-2 (two peptides) antimicrobial peptide families. They were named esculentin-1ISa, esculentin-1ISb, esculentin-2ISa, palustrin-2ISa, brevinin-2ISa, brevinin-2ISb, brevinin-2ISc, nigrocin-2ISa and nigrocin-2ISb. Peptide primary structures suggest a close relationship with the Asian odorous frogs, Odorrana grahami and Odorrana hosii. These antimicrobial peptides possessed a broad-spectrum of growth inhibition against five microorganisms (E. coli, Staphylococcus aureus, methicillin-resistant S. aureus, Bacillus subtilis and Candida albicans). Nine different cDNAs encoding the precursor proteins were also cloned and showed that the precursor proteins exhibited a signal peptide, an N-terminal acidic spacer domain, a Lys-Arg processing site and an antimicrobial peptide at the C-terminus.

Biosynthesis, mode of action, and functional significance of neurosteroids in the purkinje cell.

The brain has traditionally been considered to be a target site of peripheral steroid hormones. In addition to this classical concept, we now know that the brain has the capacity to synthesize steroids de novo from cholesterol, the so-called "neurosteroids." In the middle 1990s, the Purkinje cell, an important cerebellar neuron, was identified as a major site for neurosteroid formation in the brain of mammals and other vertebrates. This discovery has provided the opportunity to understand neuronal neurosteroidogenesis in the brain. In addition, biological actions of neurosteroids are becoming clear by the studies using the Purkinje cell, an excellent cellular model, which is known to play an important role in memory and learning processes. Based on the studies on mammals over the past decade, it is considered that the Purkinje cell actively synthesizes progesterone and estradiol from cholesterol during neonatal life, when cerebellar neuronal circuit formation occurs. Both progesterone and estradiol promote dendritic growth, spinogenesis, and synaptogenesis via each cognate nuclear receptor in the developing Purkinje cell. Such neurosteroid actions mediated by neurotrophic factors may contribute to the formation of cerebellar neuronal circuit during neonatal life. 3α,5α-Tetrahydroprogesterone (allopregnanolone), a progesterone metabolite, is also synthesized in the cerebellum and considered to act as a survival factor of Purkinje cells in the neonate. This review summarizes the current knowledge regarding the biosynthesis, mode of action, and functional significance of neurosteroids in the Purkinje cell during development in terms of synaptic formation of cerebellar neuronal networks.

Unique form and osmoregulatory function of a neurohypophysial hormone in a urochordate.

The cyclic nonapeptides, oxytocin and vasopressin, are neurohypophysial hormones that regulate many significant physiological processes related especially to reproduction and osmoregulation. In this study, we characterized an oxytocin-related peptide cDNA from a urochordate, Styela plicata, thought to be a sister group to vertebrates. Sequence analysis of the deduced precursor polypeptide revealed that the precursor is composed of three segments: a signal peptide, an oxytocin-like sequence flanked by a Gly C-terminal amidation signal and a Lys-Arg dibasic processing site, and a neurophysin domain, similar to other oxytocin/vasopressin family precursors. However, unlike other members of this family, the tunicate oxytocin-like peptide (CYISDCPNSRFWST-NH2) is a tetradecapeptide. We termed this peptide Styela oxytocin-related peptide (SOP). Furthermore, analyses of mass spectrometry, in situ hybridization, and immunohistochemistry demonstrated production of mature SOP in the cerebral ganglion. To elucidate the physiological action of SOP, we kept the tunicate for 2 d under the three different concentrations of seawater, 60, 100, and 130%, and measured the expression levels of SOP mRNA in the cerebral ganglion. The greatest expression of SOP mRNA was observed in the 60% seawater. In 60% seawater, but not in 100 or 130%, the tunicate mostly closed the atrial and branchial siphons. Therefore, we investigated the contractile effects of SOP on the siphons in vitro. SOP caused contractions in both siphons in a dose-dependent manner. Taken together, these results suggest that SOP acts to prevent the influx of a low concentration of seawater into the body and thus play an important role in osmoregulation.

Expression, localization and possible actions of 25-Dx, a membraneassociated putative progesterone-binding protein, in the developing Purkinje cell of the cerebellum: a new insight into the biosynthesis, metabolism and multiple actions of progesterone as a neurosteroid.

Neurosteroids are now known to be steroids that are synthesized de novo from cholesterol in the central and peripheral nervous systems of vertebrates through mechanisms at least partly independent of peripheral steroidogenic glands, such as the adrenal and gonads. A series of our studies have demonstrated that the rat Purkinje cell, a cerebellar neuron, actively produces progesterone de novo from cholesterol only during neonatal life and progesterone promotes dendritic growth, spinogenesis and synaptogenesis via its nuclear receptor in this neuron. Thus the Purkinje cell serves as an excellent cellular model for understanding the formation of cerebellar neuronal circuit in relation to genomic neurosteroid actions. Recently, we have further found that Purkinje cells express the putative membrane progesterone receptor, 25-Dx in rats. By immunocytochemistry, the expression of 25-Dx was localized in the Purkinje cell and external granule cell layer. RT-PCR and Western immunoblot analyses revealed the expressions of 25-Dx and its mRNA in the rat cerebellum, which increased during neonatal life. Therefore, progesterone would promote dendritic growth, spinogenesis and synaptogenesis via 25-Dx as well as its nuclear receptor in the Purkinje cell in the neonate. Because the subcellular localization of 25-Dx was associated with membrane structures of the endoplasmic reticulum and Golgi, 25-Dx may also play a role in the regulation of neurosteroidogenesis in the developing Purkinje cell. Here we summarize the advances made in our understanding of the expression, localization and its possible actions of 25-Dx in the developing Purkinje cell.

Mode of action and functional significance of avian gonadotropin-inhibitory hormone (GnIH): a review.

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). However, a hypothalamic neuropeptide acting at the level of the pituitary to negatively regulate gonadotropin secretion has, until recently, remained unknown in any vertebrate. In 2000, we discovered a novel hypothalamic neuropeptide inhibiting gonadotropin release at the level of the pituitary 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 further identified the receptor for GnIH and characterized its expression and binding activity in quail. The identified GnIH receptor possessed seven transmembrane domains and specifically bound to GnIH in a concentration-dependent manner. The expression of GnIH receptor was found in the pituitary and several brain regions including the hypothalamus. These results suggest 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. To understand the functional significance of GnIH in avian reproduction, we also investigated the mechanism that regulates GnIH expression. Interestingly, melatonin induced dose-dependently GnIH expression and melatonin receptor (Mel(1c)) was expressed in GnIH neurons. Thus melatonin appears to act directly on GnIH neurons via its receptor to induce GnIH expression. Based on these studies, GnIH is likely an important neuropeptide for the regulation of avian reproduction.

Neurosteroid biosynthesis in the quail brain: a review.

The brain traditionally has been considered to be a target site of peripheral steroid hormones. In contrast to this classical concept, new findings over the past decade have shown that the brain itself also has the capability of forming steroids de novo, the so-called "neurosteroids". De novo neurosteroidogenesis in the brain from cholesterol is a conserved property of vertebrates. Our studies using the quail, as an excellent animal model, have demonstrated that the avian brain possesses cytochrome P450 side-chain cleavage enzyme (P450scc), 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4)-isomerase (3beta-HSD), cytochrome P450 17alpha-hydroxylase/c17,20-lyase (P450(17alpha,lyase)), 17beta-HSD, etc., and produces pregnenolone, progesterone, 3beta, 5beta-tetrahydroprogesterone, androstenedione, testosterone and estradiol from cholesterol. However, the biosynthetic pathway of neurosteroids in the avian brain from cholesterol may be still incomplete, because we recently found that the quail brain actively produces 7alpha-hydroxypregnenolone, a previously undescribed avian neurosteroid. This paper summarize the advances made in our understanding of biosynthesis of neurosteroids in the avian brain.

Structures and diverse functions of frog growth hormone-releasing peptide (fGRP) and its related peptides (fGRP-RPs): a review.

A new Arg-Phe-NH(2) (RFamide) peptide has been discovered in the amphibian hypothalamus. The cell bodies and terminals containing this peptide were localized in the suprachiasmatic nucleus and median eminence, respectively. This peptide was further revealed to have a considerable growth hormone (GH)-releasing activity in vitro and in vivo and hence designated as frog GH-releasing peptide (fGRP). Molecular cloning of cDNA encoding the fGRP precursor polypeptide revealed that it encodes fGRP and its putative gene-related peptides (fGRP-RP-1, -RP-2, and -RP-3). Subsequently, we identified these putative fGRP-RPs as mature peptides and analyzed their hypophysiotropic activities. Only fGRP-RP-2 stimulated the release of GH and prolactin (PRL) in vitro and in vivo. Thus, in addition to fGRP, fGRP-RP-2 acts as a hypothalamic factor on the frog pituitary to stimulate the release of GH and PRL.

Interactions of gonadotropin-releasing hormone (GnRH) and gonadotropin-inhibitory hormone (GnIH) in birds and mammals.

Gonadotropin-releasing hormone (GnRH) regulates secretion of both of the gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone. Thus, it is a key hormone for vertebrate reproduction. GnRH was considered to be unusual among hypothalamic neuropeptides in that it appeared to have no direct antagonist, although some neurochemicals and peripheral hormones (opiates, GABA, gonadal steroids, inhibin) can modulate gonadotropin release to a degree. Five years ago, a vertebrate hypothalamic neuropeptide that inhibited pituitary gonadotropin release in a dose-dependent manner was discovered in quail by Tsutsui et al. (2000. Biochem Biophys Res Commun 275:661-667). We now know that this inhibitory peptide, named gonadotropin-inhibitory hormone, or GnIH, is a regulator of gonadotropin release in vitro and in vivo. Its discovery has opened the door to an entirely new line of research within the realm of reproductive biology. In our collaborative studies, we have begun to elucidate the manner in which GnIH interacts with GnRH to time release of gonadotropins and thus time reproductive activity in birds and mammals. This paper reviews the distribution of GnIH in songbirds relative to GnRHs, and our findings on its modes of action in vitro and in vivo, based on laboratory and field studies. These data are simultaneously compared with our findings in mammals, highlighting how the use of different model species within different vertebrate classes can be a useful approach to identify the conserved actions of this novel neuropeptide, along with its potential importance to vertebrate reproduction.

Neural interaction of gonadotropin-regulating hormone immunoreactive neurons and the suprachiasmatic nucleus with the paraventricular organ in the Japanese grass lizard (Takydromus tachydromoides).

Our previous study demonstrated that the paraventricular organ (PVO) in the hypothalamus of the Japanese grass lizard (Takydromus tachydromoides) showed immunoreactivity against the light signal-transducing G-protein, transducin. This finding suggested that the PVO was a candidate for the deep-brain photoreceptor in this species. To understand functions of the PVO, we investigated distributions of transducin, serotonin, gonadotropin-releasing hormone (GnRH), and gonadotropin-inhibitory hormone (GnIH) in the lizard's brain. We immunohistochemically confirmed co-localization of transducin and serotonin in PVO neurons that showed structural characteristics of cerebrospinal fluid (CSF)-contacting neurons. GnRH-immunoreactive (ir) cells were localized in the posterior commissure and lateral hypothalamic area. Some of the serotonin-ir fibers extending from the PVO to the lateral hypothalamic area contacted the GnRH-ir cell bodies. GnIH-ir cells were localized in the nucleus accumbens, paraventricular nucleus, and upper medulla, and GnIH-ir fibers from the paraventricular nucleus contacted the lateral processes of serotonin-ir neurons in the PVO. In addition, we found that serotonin-ir fibers from the PVO extended to the suprachiasmatic nucleus (SCN), and the retrograde transport method confirmed the PVO projections to the SCN. These findings suggest that the PVO, by means of innervation mediated by serotonin, plays an important role in the regulation of pituitary function and the biological clock in the Japanese grass lizard.

Identification and characterization of a gonadotropin-inhibitory system in the brains of mammals.

Successful reproduction requires maintenance of the reproductive axis within fine operating limits through negative feedback actions of sex steroids. Despite the importance of this homeostatic process, our understanding of the neural loci, pathways, and neurochemicals responsible remain incomplete. Here, we reveal a neuropeptidergic pathway that directly links gonadal steroid actions to regulation of the reproductive system. An RFamide (Arg-Phe-NH2) peptide that inhibits gonadotropin release from quail pituitary was recently identified and named gonadotropin-inhibitory hormone (GnIH). Birds are known to have specialized adaptations associated with gonadotropin-releasing hormone (GnRH) regulation to optimize reproduction (e.g., encephalic photoreceptors), and the existence of a hypothalamic peptide inhibiting gonadotropins may or may not be another such specialization. To determine whether GnIH serves as a signaling pathway for sex steroid regulation of the reproductive axis, we used immunohistochemistry and in situ hybridization to characterize the distribution and functional role of this peptide in hamsters, rats, and mice. GnIH-immunoreactive (GnIH-ir) cell bodies are clustered in the mediobasal hypothalamus with pronounced projections and terminals throughout the CNS. In vivo GnIH administration rapidly inhibits luteinizing hormone secretion. Additionally, GnIH-ir neurons form close appositions with GnRH cells, suggesting a direct means of GnRH modulation. Finally, GnIH-ir cells express estrogen receptor-alpha and exhibit robust immediate early gene expression after gonadal hormone stimulation. Taken together, the distribution of GnIH efferents to neural sites regulating reproductive behavior and neuroendocrine secretions, expression of steroid receptors in GnIH-ir nuclei, and GnIH inhibition of luteinizing hormone secretion indicate the discovery of a system regulating the mammalian reproductive axis.

Gonadotropin-inhibitory hormone inhibits gonadal development and maintenance by decreasing gonadotropin synthesis and release in male quail.

Until recently, any neuropeptide that directly inhibits gonadotropin secretion had not been identified. We recently identified a novel hypothalamic dodecapeptide that directly inhibits gonadotropin release in quail and termed it gonadotropin-inhibitory hormone (GnIH). The action of GnIH on the inhibition of gonadotropin release is mediated by a novel G protein-coupled receptor in the quail pituitary. This new gonadotropin inhibitory system is considered to be a widespread property of birds and provides us with an unprecedented opportunity to study the regulation of avian reproduction from an entirely novel standpoint. To understand the physiological role(s) of GnIH in avian reproduction, we investigated GnIH actions on gonadal development and maintenance in male quail. Continuous administration of GnIH to mature birds via osmotic pumps for 2 wk decreased the expressions of gonadotropin common alpha and LHbeta subunit mRNAs in a dose-dependent manner. Plasma LH and testosterone concentrations were also decreased dose dependently. Furthermore, administration of GnIH to mature birds induced testicular apoptosis and decreased spermatogenic activity in the testis. In immature birds, daily administration of GnIH for 2 wk suppressed normal testicular growth and rise in plasma testosterone concentrations. An inhibition of juvenile molt also occurred after GnIH administration. These results indicate that GnIH inhibits gonadal development and maintenance through the decrease in gonadotropin synthesis and release. GnIH may explain the phenomenon of photoperiod-induced gonadal regression before an observable decline in hypothalamic GnRH in quail. To our knowledge, GnIH is the first identified hypothalamic neuropeptide inhibiting reproductive function in any vertebrate class.

Gonadotropin-inhibiting hormone stimulates feeding behavior in chicks.

Neuropeptides containing a C-terminal Arg-Phe-NH2 motif (RFamide peptides) are suggested to be involved in the control of feeding behavior in both invertebrates and vertebrates. Gonadotropin-inhibitory hormone (GnIH) is the first identified avian RFamide peptide that inhibits gonadotropin release from the pituitary. The GnIH precursor encodes one GnIH and its related peptides (GnIH-RP-1 and -RP-2) that shared the same C-terminal motif, Leu-Pro-Xaa-Arg-Phe-NH2 (Xaa = Leu or Gln) (LPXRFamide). GnIH neurons are localized in the paraventricular nucleus, with their fibers visible in multiple brain locations including the median eminence and brainstem. In this study, we therefore investigated the action of GnIH and its related peptides on feeding behavior. Intracerebroventricular (ICV) injection of GnIH, GnIH-RP-1 and GnIH-RP-2 significantly stimulated food intake in chicks. The chicken pentapeptide LPLRFamide, a degraded C-terminus of GnIH and GnIH-RP-1, did not stimulate feeding thereby demonstrating the importance of the N-terminus of GnIH and its related peptides for the orexigenic effect. Anti-GnIH antiserum suppressed appetite induced by fasting, but did not modify feeding under ad libitum conditions. The present study suggests that GnIH and its related peptides act as endogenous orexigenic factors in the brain of chicks.