Takayoshi Ubuka

Variation in Kisspeptin and RFamide-Related Peptide (RFRP) Expression and Terminal Connections to Gonadotropin-Releasing Hormone Neurons in the Brain: A Novel Medium for Seasonal Breeding in the Sheep

Reproductive activity in sheep is seasonal, being activated by short-day photoperiods and inhibited by long days. During the nonbreeding season, GnRH secretion is reduced by both steroid-independent and steroid-dependent (increased response to estradiol negative feedback) effects of photoperiod. Kisspeptin (also known as metastin) and gonadotropin-inhibitory hormone (GnIH, or RFRP) are two RFamide neuropeptides that appear critical in the regulation of the reproductive neuroendocrine axis. We hypothesized that expression of kisspeptin and/or RFRP underlies the seasonal change in GnRH secretion. We examined kisspeptin and RFRP (protein and mRNA) expression in the brains of ovariectomized (OVX) ewes treated with estradiol (OVX+E) during the nonbreeding and breeding seasons. In OVX+E ewes, greater expression of kisspeptin and Kiss1 mRNA in the arcuate nucleus and lesser expression of RFRP (protein) in the dorsomedial nucleus of the hypothalamus were concurrent with the breeding season. There was also a greater number of kisspeptin terminal contacts onto GnRH neurons and less RFRP-GnRH contacts during the breeding season (compared with the nonbreeding season) in OVX+E ewes. Comparison of OVX and OVX+E ewes in the breeding and nonbreeding season revealed a greater effect of steroid replacement on inhibition of kisspeptin protein and Kiss1 mRNA expression during the nonbreeding season. Overall, we propose that the two RFamide peptides, kisspeptin and RFRP, act in concert, with opposing effects, to regulate the activity of GnRH neurons across the seasons, leading to the annual change in fertility and the cyclical seasonal transition from nonbreeding to breeding season.

Identification of Human GnIH Homologs, RFRP-1 and RFRP-3, and the Cognate Receptor, GPR147 in the Human Hypothalamic Pituitary Axis

The existence of a hypothalamic gonadotropin-inhibiting system has been elusive. A neuropeptide named gonadotropin-inhibitory hormone (GnIH, SIKPSAYLPLRF-NH2) which directly inhibits gonadotropin synthesis and release from the pituitary was recently identified in quail hypothalamus. Here we identify GnIH homologs in the human hypothalamus and characterize their distribution and biological activity. GnIH homologs were isolated from the human hypothalamus by immunoaffinity purification, and then identified as MPHSFANLPLRF-NH2 (human RFRP-1) and VPNLPQRF-NH2 (human RFRP-3) by mass spectrometry. Immunocytochemistry revealed GnIH-immunoreactive neuronal cell bodies in the dorsomedial region of the hypothalamus with axonal projections to GnRH neurons in the preoptic area as well as to the median eminence. RT-PCR and subsequent DNA sequencing of the PCR products identified human GnIH receptor (GPR147) mRNA expression in the hypothalamus as well as in the pituitary. In situ hybridization further identified the expression of GPR147 mRNA in luteinizing hormone producing cells (gonadotropes). Human RFRP-3 has recently been shown to be a potent inhibitor of gonadotropin secretion in cultured sheep pituitary cells by inhibiting Ca2+ mobilization. It also directly modulates GnRH neuron firing. The identification of two forms of GnIH (RFRP-1 and RFRP-3) in the human hypothalamus which targets human GnRH neurons and gonadotropes and potently inhibit gonadotropin in sheep models provides a new paradigm for the regulation of hypothalamic-pituitary-gonadal axis in man and a novel means for manipulating reproductive functions.

Distribution of a novel avian gonadotropin-inhibitory hormone in the quail brain

We recently identified a novel hypothalamic neuropeptide inhibiting gonadotropin release in the quail brain and termed it gonadotropin inhibitory hormone (GnIH). In this study, we investigated the localization and distribution of GnIH in both sexes of adult quails by immunohistochemistry with a specific antiserum against GnIH and in situ hybridization. Quantitative analysis demonstrated that the concentration of GnIH in the diencephalon was greater than that in the mesencephalon without sex difference. GnIH concentrations in the cerebrum and cerebellum were below the level of detectability. Clusters of GnIH-like immunoreactive (GnlH-ir) cell bodies were localized in the paraventricular nucleus (PVN) of the hypothalamus. There was no significant difference in the number of GnlH-ir cells in the PVN between males and females. By double immunostaining with antisera reacting with GnIH or avian posterior pituitary hormones (vasotocin and mesotocin), GnIH-ir cells were found to be parvocellular neurons in the ventral portion of PVN, which showed no immunoreaction with the antisera against vasotocin and mesotocin. In situ hybridization revealed the cellular localization of GnIH mRNA in the PVN. GnIH-ir nerve fibers were however widely distributed in the diencephalic and mesencephalic regions. Dense networks of immunoreactive fibers were found in the ventral paleostriatum, septal area, preoptic area, hypothalamus, and optic tectum. The most prominent fibers were seen in the median eminence of the hypothalamus and the dorsal motor nucleus of the vagus in the medulla oblongata. Thus, GnIH may participate not only in neuroendocrine functions, but also in behavioral and autonomic mechanisms.

Gonadotrophin Inhibitory Hormone Depresses Gonadotrophin α and Follicle-Stimulating Hormone β Subunit Expression in the Pituitary of the Domestic Chicken

Studies performed in vitro suggest that a novel 12 amino acid RF amide peptide, isolated from the quail hypothalamus, is a gonadotrophin inhibitory hormone (GnIH). The aim of the present study was to investigate this hypothesis in the domestic chicken. Injections of GnIH into nest‐deprived incubating hens failed to depress the concentration of plasma luteinizing hormone (LH). Addition of GnIH to short‐term (120u2003min) cultures of diced pituitary glands from adult cockerels depressed follicle‐stimulating hormone (FSH) and LH release and depressed common α and FSHβ gonadotrophin subunit mRNAs, with no effect on LHβ subunit mRNA. Hypothalamic GnIH mRNA was higher in incubating (out‐of‐lay) than in laying hens, but there was no significant difference in the amount of hypothalamic GnIH mRNA in out‐of‐lay and laying broiler breeder hens at the end of a laying year. It is concluded that avian GnIH may play a role in controlling gonadotrophin synthesis and associated constitutive release in the domestic chicken.

Effects of social cues on GnRH-I, GnRH-II, and reproductive physiology in female house sparrows (Passer domesticus)

In all vertebrates, at least two forms of gonadotropin-releasing hormone (GnRH) are present: GnRH-I and GnRH-II. GnRH-I directly influences the reproductive axis whereas the function of GnRH-II is less clear. The present experimental objectives were to determine the effect(s) of male social cues on the peripheral and neural responses of female house sparrows (Passer domesticus). We hypothesized that male breeding status would significantly influence the amount of immunoreactive GnRH-II in female house sparrow brains. In order to test this hypothesis, females were caged with a breeding male, a non-breeding male, or caged alone. The presence of breeding males did not significantly influence ovary development, luteinizing hormone, or estradiol levels, but male presence increased female body mass, and male presence and condition interacted to influence ovarian follicle size. Using immunocytochemistry, GnRH-I and GnRH-II immunoreactivity was measured in order to evaluate the neuroendocrine response to breeding status in males. When females were housed with breeding males, there were stable numbers of immunoreactive GnRH-I and -II cells but significantly lower amounts of immunoreactive GnRH-I fibre staining within the preoptic area compared to females housed with non-breeding males. Moreover, immunoreactive GnRH-II fibres in the preoptic area, ventromedial nucleus, and medial septum were significantly greater in females housed alone in chamber with non-breeding males. The data demonstrate that the GnRH system in songbirds is modulated by social context. These finding provide novel insight into the mechanisms involved with regulating avian reproductive physiology.

Molecular, cellular, morphological, physiological and behavioral aspects of gonadotropin-inhibitory hormone.

Gonadotropin-inhibitory hormone (GnIH) is a hypothalamic neuropeptide that was isolated from the brains of Japanese quail in 2000, which inhibited luteinizing hormone release from the anterior pituitary gland. Here, we summarize the following fifteen years of researches that investigated on the mechanism of GnIH actions at molecular, cellular, morphological, physiological, and behavioral levels. The unique molecular structure of GnIH peptide is in its LPXRFamide (X=L or Q) motif at its C-terminal. The primary receptor for GnIH is GPR147. The cell signaling pathway triggered by GnIH is initiated by inhibiting adenylate cyclase and decreasing cAMP production in the target cell. GnIH neurons regulate not only gonadotropin synthesis and release in the pituitary, but also regulate various neurons in the brain, such as GnRH1, GnRH2, dopamine, POMC, NPY, orexin, MCH, CRH, oxytocin, and kisspeptin neurons. GnIH and GPR147 are also expressed in gonads and they may regulate steroidogenesis and germ cell maturation in an autocrine/paracrine manner. GnIH regulates reproductive development and activity. In female mammals, GnIH may regulate estrous or menstrual cycle. GnIH is also involved in the regulation of seasonal reproduction, but GnIH may finely tune reproductive activities in the breeding seasons. It is involved in stress responses not only in the brain but also in gonads. GnIH may inhibit male socio-sexual behavior by stimulating the activity of cytochrome P450 aromatase in the brain and stimulates feeding behavior by modulating the activities of hypothalamic and central amygdala neurons.

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.

Reproductive Neuroendocrine Pathways of Social Behavior

Social behaviors are key components of reproduction, because they are essential for successful fertilization. Social behaviors, such as courtship, mating, and aggression, are strongly associated with sex steroids, such as testosterone, estradiol, and progesterone. Secretion of sex steroids from the gonads is regulated by the hypothalamus–pituitary–gonadal (HPG) axis in vertebrates. Gonadotropin-releasing hormone (GnRH) is a pivotal hypothalamic neuropeptide that stimulates gonadotropin release from the pituitary. In recent years, the role of neuropeptides containing the C-terminal Arg–Phe–NH2 (RFamide peptides) has been emphasized in vertebrate reproduction. In particular, two key RFamide peptides, kisspeptin and gonadotropin-inhibitory hormone (GnIH), emerged as critical accelerator and suppressor of gonadotropin secretion. Kisspeptin stimulates GnRH release by directly acting on GnRH neurons, whereas GnIH inhibits gonadotropin release by inhibiting kisspeptin, GnRH neurons, or pituitary gonadotropes. These neuropeptides can regulate social behavior by regulating the HPG axis. However, distribution of neuronal fibers of GnRH, kisspeptin, and GnIH neurons is not limited within the hypothalamus, and the existence of extrahypothalamic neuronal fibers suggests direct control of social behavior within the brain. It has traditionally been shown that central administration of GnRH can stimulate female sexual behavior in rats. Recently, it was shown that Kiss1, one of the paralogs of kisspeptin peptide family, regulates fear responses in zebrafish and GnIH inhibits sociosexual behavior in birds. Here, we highlight recent findings regarding the role of GnRH, kisspeptin, and GnIH in the regulation of social behaviors in fish, birds, and mammals and discuss their importance in future biological and biomedical research.

Inhibitory action of gonadotropin-inhibitory hormone on the signaling pathways induced by kisspeptin and vasoactive intestinal polypeptide in GnRH neuronal cell line, GT1-7

Gonadotropin‐inhibitory hormone (GnIH) acts as a negative regulator of reproduction by acting on gonadotropes and gonadotropin‐releasing hormone (GnRH) neurons. Despite its functional significance, the molecular mechanism of GnIH action in the target cells has not been fully elucidated. To expand our previous study on GnIH actions in gonadotropes, we investigated the potential signal transduction pathway that conveys the inhibitory action of GnIH in GnRH neurons by using the GnRH neuronal cell line, GT1–7. We examined whether GnIH inhibits the action of kisspeptin and vasoactive intestinal polypeptide (VIP), positive regulators of GnRH neurons. Although GnIH significantly suppressed the stimulatory effect of kisspeptin on GnRH release in hypothalamic culture, GnIH had no inhibitory effect on kisspeptin stimulation of serum response element and nuclear factor of activated T‐cell response element activities and ERK phosphorylation, indicating that GnIH may not directly inhibit kisspeptin signaling in GnRH neurons. On the contrary, GnIH effectively eliminated the stimulatory effect of VIP on p38 and ERK phosphorylation, c‐Fos mRNA expression, and GnRH release. The use of pharmacological modulators strongly demonstrated the specific inhibitory action of GnIH on the adenylate cyclase/cAMP/protein kinase A pathway, suggesting a common inhibitory mechanism of GnIH action in GnRH neurons and gonadotropes.—Son, Y. L., Ubuka, T., Soga, T., Yamamoto, K., Bentley, G. E., Tsutsui, K. Inhibitory action of gonadotropin‐inhibitory hormone on the signaling pathways induced by kisspeptin and vasoactive intestinal polypeptide in GnRH neuronal cell line, GT1–7. FASEB J. 30, 2198–2210 (2016). www.fasebj.org

A Journey through the Gonadotropin-Inhibitory Hormone System of Fish

Gonadotropin-inhibitory hormone (GnIH) is a hypothalamic neuropeptide that belongs to the RFamide peptide family and was first identified in the quail brain. From the discovery of avian GnIH, orthologous GnIH peptides have been reported in a variety of vertebrates, including mammals, amphibians, teleosts and agnathans, but also in protochordates. It has been clearly established that GnIH suppresses reproduction in avian and mammalian species through its inhibitory actions on brain GnRH and pituitary gonadotropins. In addition, GnIH also appears to be involved in the regulation of feeding, growth, stress response, heart function and social behavior. These actions are mediated via G protein-coupled GnIH receptors (GnIH-Rs), of which two different subtypes, GPR147 and GPR74, have been described to date. With around 30,000 species, fish represent more than one-half of the total number of recognized living vertebrate species. In addition to this impressive biological diversity, fish are relevant because they include model species with scientific and clinical interest as well as many exploited species with economic importance. In spite of this, the study of GnIH and its physiological effects on reproduction and other physiological processes has only been approached in a few fish species, and results obtained are in some cases conflicting. In this review, we summarize the information available in the literature on GnIH sequences identified in fish, the distribution of GnIH and GnIH-Rs in central and peripheral tissues, the physiological actions of GnIH on the reproductive brain-pituitary-gonadal axis, as well as other reported effects of this neuropeptide, and existing knowledge on the regulatory mechanisms of GnIH in fish.