Tomohiro Osugi

Gonadotropin-inhibitory hormone (GnIH) and its control of central and peripheral reproductive function.

Identification of novel neurohormones that regulate the reproductive axis is essential for the progress of neuroendocrinology. The decapeptide gonadotropin-releasing hormone (GnRH) is the primary factor responsible for the hypothalamic control of gonadotropin secretion. Gonadal sex steroids and inhibin modulate gonadotropin secretion via feedback from the gonads, but a neuropeptide that directly inhibits gonadotropin secretion was unknown in vertebrates until 2000 when a hypothalamic dodecapeptide serving this function was discovered in quail. Because of its action on cultured pituitary in quail, it was named gonadotropin-inhibitory hormone (GnIH). GnIH acts on the pituitary and on GnRH neurons in the hypothalamus via a novel G protein-coupled receptor (GPR147). GPR74 may also be a possible candidate GnIH receptor. GnIH decreases gonadotropin synthesis and release, inhibiting gonadal development and maintenance. Melatonin stimulates the expression and release of GnIH via melatonin receptors expressed by GnIH neurons. GnIH actions and interactions with GnRH seem common not only to several avian species, but also to mammals. Thus, GnIH is considered to have an evolutionarily conserved role in controlling vertebrate reproduction, and GnIH homologs have also been identified in the hypothalamus of mammals. As in birds, mammalian GnIH homologs act to inhibit gonadotropin release in several species. More recent evidence in birds and mammals indicates that GnIH may operate at the level of the gonads as an autocrine/paracrine regulator of steroidogenesis and gametogenesis. Importantly, GnIH in birds and mammals appears to act at all levels of the hypothalamo-pituitary-gonadal (HPG) axis, and possibly over different time-frames (minutes-days). Thus, GnIH and its homologs appear to act as key neurohormones controlling vertebrate reproduction. The discovery of GnIH has enabled us to understand and manipulate vertebrate reproduction from an entirely new perspective.

Hypophysiotropic role of RFamide-related peptide-3 in the inhibition of LH secretion in female rats

Gonadotropin-inhibitory hormone (GnIH), a newly discovered hypothalamic RFamide peptide, inhibits reproductive activity by decreasing gonadotropin synthesis and release in birds. The gene of the mammalian RFamide-related peptides (RFRP) is orthologous to the GnIH gene. This Rfrp gene gives rise to the two biologically active peptides RFRP-1 (NPSF) and RFRP-3 (NPVF), and i.c.v. injections of RFRP-3 suppress LH secretion in several mammalian species. In this study, we show whether RFRP-3 affects LH secretion at the pituitary level and/or via the release of GnRH at the hypothalamus in mammals. To investigate the suppressive effects of RFRP-3 on the mean level of LH secretion and the frequency of pulsatile LH secretion in vivo, ovariectomized (OVX) mature rats were administered RFRP-3 using either i.c.v. or i.v. injections. Furthermore, the effect of RFRP-3 on LH secretion was also investigated using cultured female rat pituitary cells. With i.v. administrations, RFRP-3 significantly reduced plasma LH concentrations when compared with the physiological saline group. However, after i.c.v. RFRP-3 injections, neither the mean level of LH concentrations nor the frequency of the pulsatile LH secretion was affected. When using cultured pituitary cells, in the absence of GnRH, the suppressive effect of RFRP-3 on LH secretion was not clear, but when GnRH was present, RFRP-3 significantly suppressed LH secretion. These results suggest that RFRP-3 does not affect LH secretion via the release of GnRH, and that RFRP-3 directly acts upon the pituitary to suppress GnRH-stimulated LH secretion in female rats.

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.

Molecular Evolution of Multiple Forms of Kisspeptins and GPR54 Receptors in Vertebrates

Kisspeptin and its receptor GPR54 play important roles in mammalian reproduction and cancer metastasis. Because the KiSS and GPR54 genes have been identified in a limited number of vertebrate species, mainly in mammals, the evolutionary history of these genes is poorly understood. In the present study, we have cloned multiple forms of kisspeptin and GPR54 cDNAs from a variety of vertebrate species. We found that fish have two forms of kisspeptin genes, KiSS-1 and KiSS-2, whereas Xenopus possesses three forms of kisspeptin genes, KiSS-1a, KiSS-1b, and KiSS-2. The nonmammalian KiSS-1 gene was found to be the ortholog of the mammalian KiSS-1 gene, whereas the KiSS-2 gene is a novel form, encoding a C-terminally amidated dodecapeptide in the Xenopus brain. This study is the first to identify a mature form of KiSS-2 product in the brain of any vertebrate. Likewise, fish possess two receptors, GPR54-1 and GPR54-2, whereas Xenopus carry three receptors, GPR54-1a, GPR54-1b, and GPR54-2. Sequence identity and genome synteny analyses indicate that Xenopus GPR54-1a is a human GPR54 ortholog, whereas Xenopus GPR54-1b is a fish GPR54-1 ortholog. Both kisspeptins and GPR54s were abundantly expressed in the Xenopus brain, notably in the hypothalamus, suggesting that these ligand-receptor pairs have neuroendocrine and neuromodulatory roles. Synthetic KiSS-1 and KiSS-2 peptides activated GPR54s expressed in CV-1 cells with different potencies, indicating differential ligand selectivity. These data shed new light on the molecular evolution of the kisspeptin-GPR54 system in vertebrates.

Discovery and Evolutionary History of Gonadotrophin―Inhibitory Hormone and Kisspeptin: New Key Neuropeptides Controlling Reproduction

Gonadotrophin‐releasing hormone (GnRH) is the primary hypothalamic factor responsible for the control of gonadotrophin secretion in vertebrates. However, within the last decade, two other hypothalamic neuropeptides have been found to play key roles in the control of reproductive functions: gonadotrophin‐inhibitory hormone (GnIH) and kisspeptin. In 2000, we discovered GnIH in the quail hypothalamus. GnIH inhibits gonadotrophin synthesis and release in birds through actions on GnRH neurones and gonadotrophs, mediated via GPR147. Subsequently, GnIH orthologues were identified in other vertebrate species from fish to humans. As in birds, mammalian and fish GnIH orthologues inhibit gonadotrophin release, indicating a conserved role for this neuropeptide in the control of the hypothalamic‐pituitary‐gonadal axis across species. Subsequent to the discovery of GnIH, kisspeptin, encoded by the KiSS‐1 gene, was discovered in mammals. By contrast to GnIH, kisspeptin has a direct stimulatory effect on GnRH neurones via GPR54. GPR54 is also expressed in pituitary cells, but whether gonadotrophs are targets for kisspeptin remains unresolved. The KiSS‐1 gene is also highly conserved and has been identified in mammals, amphibians and fish. We have recently found a second isoform of KiSS‐1, designated KiSS‐2, in several vertebrates, but not birds, rodents or primates. In this review, we highlight the discovery, mechanisms of action, and functional significance of these two chief regulators of the reproductive axis.

Evolutionary origin and divergence of PQRFamide peptides and LPXRFamide peptides in the RFamide peptide family: Insights from novel lamprey RFamide peptides

Among the RFamide peptide groups, PQRFamide peptides, such as neuropeptide FF (NPFF) and neuropeptide AF (NPAF), share a common C‐terminal Pro‐Gln‐Arg‐Phe‐NH2 motif. LPXRFamide (X = L or Q) peptides, such as gonadotropin‐inhibitory hormone (GnIH), frog growth hormone‐releasing peptide (fGRP), goldfish LPXRFamide peptide and mammalian RFamide‐related peptides (RFRPs), also share a C‐terminal Leu‐Pro‐Leu/Gln‐Arg‐Phe‐NH2 motif. Such a similar C‐terminal structure suggests that these two groups may have diverged from a common ancestral gene. In this study, we sought to clarify the evolutionary origin and divergence of these two groups, by identifying novel RFamide peptides from the brain of sea lamprey, one of only two extant groups of the oldest lineage of vertebrates, Agnatha. A novel lamprey RFamide peptide was identified by immunoaffinity purification using the antiserum against LPXRFamide peptide. The lamprey RFamide peptide did not contain a C‐terminal LPXRFamide motif, but had the sequence SWGAPAEKFWMRAMPQRFamide (lamprey PQRFa). A cDNA of the precursor encoded one lamprey PQRFa and two related peptides. These related peptides, which also had the C‐terminal PQRFamide motif, were further identified as mature endogenous ligands. Phylogenetic analysis revealed that lamprey PQRFamide peptide precursor belongs to the PQRFamide peptide group. In situ hybridization demonstrated that lamprey PQRFamide peptide mRNA is expressed in the regions predicted to be involved in neuroendocrine and behavioral functions. This is the first demonstration of the presence of RFamide peptides in the agnathan brain. Lamprey PQRFamide peptides are considered to have retained the most ancestral features of PQRFamide peptides.

Identification of gonadotropin-inhibitory hormone in the zebra finch (Taeniopygia guttata): Peptide isolation, cDNA cloning and brain distribution.

Two novel RFamide peptides, kisspeptins and gonadotropin-inhibitory hormone (GnIH) are neuropeptides that appear critical in the regulation of the reproductive neuroendocrine axis. GnIH was first identified in avian brain, however, kisspeptins have not been identified in birds. To determine biochemically the presence of kisspeptins and GnIH in the zebra finch, a study was conducted to isolate these two peptides from zebra finch brain. Peptides were isolated by immunoaffinity purification and only one peptide was characterized by mass spectrometry. This peptide was confirmed to be a 12-amino acid sequence with RFamide at its C-terminus; its sequence is SIKPFSNLPLRFamide (zebra finch GnIH). By this approach, however, identification of kisspeptin from zebra finch brain was not achieved. Cloned zebra finch GnIH precursor cDNA encoded three peptides that possess characteristic LPXRFamide (X=L or Q) motifs at the C-termini. In situ hybridization and immunohistochemical analysis revealed the cellular localization of zebra finch GnIH mRNA and peptide in the paraventricular nucleus and the dorsomedial nucleus of the hypothalamus. Fluorescent immunohistochemistry with confocal microscopy indicated that GnIH-immunoreactive (ir) fibers are very close appositions with gonadotropin-releasing hormone-I (GnRH-I) cells. Furthermore GnIH-ir nerve fibers were widely distributed in the multiple brain regions including the septum, preoptic area, median eminence, optic tectum and median eminence. The prominent fibers were seen in the ventral tegmental area, midbrain central gray and dorsal motor nucleus of the vagus in the medulla. Thus, GnIH may participate in not only neuroendocrine functions but also regulation of motivation for social behavior and autonomic mechanisms.

Synchronised expressions of LPXRFamide peptide and its receptor genes: Seasonal, diurnal and circadian changes during spawning period in grass puffer

Among the RFamide peptide family, the LPXRFamide peptide (LPXRFa) group regulates the release of various pituitary hormones and, recently, LPXRFa genes were found to be regulated by photoperiod via melatonin. As a first step towards investigating the role of LPXRFa on reproductive function in grass puffer (Takifugu niphobles), which spawns in semilunar cycles, genes encoding LPXRFa and its receptor (LPXRFa‐R) were cloned, and seasonal, diurnal and circadian changes in their absolute amounts of mRNAs in the brain and pituitary were examined by quantitative real‐time polymerase chain reaction. The grass puffer LPXRFa precursor contains two putative RFamide peptides and one possible RYamide peptide. LPXRFa and LPXRFa‐R genes were extensively expressed in the diencephalon and pituitary. The expression levels of both genes were significantly elevated during the spawning periods in both sexes in the brain and pituitary, although they were low in the spawning fish just after releasing eggs and sperm. The treatment of primary pituitary cultures with goldfish LPXRFa increased the amounts of follicle‐stimulating hormone β‐ and luteinising hormone β‐subunit mRNAs. In the diencephalon, LPXRFa and LPXRFa‐R genes showed synchronised diurnal and circadian variations with one peak at zeitgeber time 3 and circadian time 15, respectively. The correlated expression patterns of LPXRFa and LPXRFa‐R genes in the diencephalon and pituitary and the possible stimulatory effects of LPXRFa on gonadotrophin subunit gene expression suggest the functional significance of the LPXRFa and LPXRFa‐R system in the regulation of lunar‐synchronised spawning of grass puffer.

Identification, Localization, and Function of a Novel Avian Hypothalamic Neuropeptide, 26RFa, and Its Cognate Receptor, G Protein-Coupled Receptor-103

Several neuropeptides with the C-terminal RFamide sequence have been identified in the hypothalamus of a variety of vertebrates. Among the RFamide peptide groups, however, only LPXRFamide peptides, including gonadotropin-inhibitory hormone, have been characterized in the avian brain. In the present study, we sought for the presence of other RFamide peptides in the avian hypothalamus. We identified a cDNA encoding an RFamide peptide orthologous to 26RFa (also referred to as QRFP) in the hypothalamus of the Japanese quail. The deduced quail 26RFa precursor consisted of 120-amino-acid residues, encoding one RFamide peptide with 27 amino acids. This RFamide peptide was flanked at the N terminus by a dibasic amino acid cleavage site and at the C terminus by a glycine amidation signal. Quantitative RT-PCR analysis demonstrated specific expression of quail 26RFa mRNA in the diencephalon including the hypothalamus. Furthermore, mass spectrometry analysis revealed the presence of a peptide exhibiting the mass of mature 26RFa, indicating that the peptide is actually produced from the precursor in the diencephalon. 26RFa-producing cell bodies were localized in the anterior hypothalamic nucleus in the brain. Synthetic 26RFa increased intracellular Ca(2+) concentration in HEK293T cells transfected with the chicken G protein-coupled receptor GPR103. Intracerebroventricular injection of 26RFa in broiler chicks stimulated feeding behavior. These data provide the first evidence for the occurrence of the peptide 26RFa in the avian hypothalamus and indicate that this peptide exerts orexigenic activity.

Evolutionary origin of the structure and function of gonadotropin- inhibitory hormone: Insights from lampreys

Gonadotropin (GTH)-inhibitory hormone (GnIH) is a novel hypothalamic neuropeptide that inhibits GTH secretion in mammals and birds by acting on gonadotropes and GnRH neurons within the hypothalamic-pituitary-gonadal axis. GnIH and its orthologs that have an LPXRFamide (X = L or Q) motif at the C terminus (LPXRFamide peptides) have been identified in representative species of gnathostomes. However, the identity of an LPXRFamide peptide had yet to be identified in agnathans, the most ancient lineage of vertebrates, leaving open the question of the evolutionary origin of GnIH and its ancestral function(s). In this study, we identified an LPXRFamide peptide gene encoding three peptides (LPXRFa-1a, LPXRFa-1b, and LPXRFa-2) from the brain of sea lamprey by synteny analysis and cDNA cloning, and the mature peptides by immunoaffinity purification and mass spectrometry. The expression of lamprey LPXRFamide peptide precursor mRNA was localized in the brain and gonad by RT-PCR and in the hypothalamus by in situ hybridization. Immunohistochemistry showed appositions of lamprey LPXRFamide peptide immunoreactive fibers in close proximity to GnRH-III neurons, suggesting that lamprey LPXRFamide peptides act on GnRH-III neurons. In addition, lamprey LPXRFa-2 stimulated the expression of lamprey GnRH-III protein in the hypothalamus and GTHβ mRNA expression in the pituitary. Synteny and phylogenetic analyses suggest that the LPXRFamide peptide gene diverged from a common ancestral gene likely through gene duplication in the basal vertebrates. These results suggest that one ancestral function of LPXRFamide peptides may be stimulatory compared with the inhibitory function seen in later-evolved vertebrates (birds and mammals).