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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.

Discovery of gonadotropin-inhibitory hormone (GnIH) in the Japanese quail in 2000 was the first to demonstrate the existence of a hypothalamic neuropeptide inhibiting gonadotropin release. We now know that GnIH regulates reproduction by inhibiting gonadotropin synthesis and release via action on the gonadotropin-releasing hormone (GnRH) system and the gonadotrope in various vertebrates. GnIH peptides identified in birds and mammals have a common LPXRF-amide (X = L or Q) motif at the C-terminus and inhibits pituitary gonadotropin secretion. However, the function and structure of GnIH peptides were diverse in fish. Goldfish GnIHs possessing a C-terminal LPXRF-amide motif had both stimulatory and inhibitory effects on gonadotropin synthesis or release. The C-terminal sequence of grass puffer and medaka GnIHs were MPQRF-amide. To investigate the evolutionary origin of GnIH and its ancestral structure and function, we searched for GnIH in agnathans, the most ancient lineage of vertebrates. We identified GnIH precursor gene and mature GnIH peptides with C-terminal QPQRF-amide or RPQRF-amide from the brain of sea lamprey. Lamprey GnIH fibers were in close proximity to GnRH-III neurons. Further, one of lamprey GnIHs stimulated the expression of lamprey GnRH-III peptide in the hypothalamus and gonadotropic hormone β mRNA expression in the pituitary. We further identified the ancestral form of GnIH, which had a C-terminal RPQRF-amide, and its receptors in amphioxus, the most basal chordate species. The amphioxus GnIH inhibited cAMP signaling in vitro. In sum, the original forms of GnIH may date back to the time of the emergence of early chordates. GnIH peptides may have had various C-terminal structures slightly different from LPXRF-amide in basal chordates, which had stimulatory and/or inhibitory functions on reproduction. The C-terminal LPXRF-amide structure and its inhibitory function on reproduction may be selected in later-evolved vertebrates, such as birds and mammals.

Gonadotropin-inhibitory hormone (GnIH), a 12 amino acid peptide, is expressed in the avian brain and inhibits luteinizing hormone secretion. Additionally, exogenous injection of GnIH causes increased food intake of chicks although the central mechanism mediating this response is poorly understood. Hence, the purpose of our study was to elucidate the central mechanism of the GnIH orexigenic response using 12 day post hatch layer-type chicks as models. Firstly, via mass spectrometry we deduced the chicken GnIH amino acid sequence: SIRPSAYLPLRFamide. Following this we used chicken GnIH to demonstrate that intracerebroventricular (ICV) injection of 2.6 and 7.8 nmol causes increased food intake up to 150 min following injection with no effect on water intake. The number of c-Fos immunoreactive cells was quantified in appetite-associated hypothalamic nuclei following ICV GnIH and only the lateral hypothalamic area (LHA) had an increase of c-Fos positive neurons. From whole hypothalamus samples following ICV GnIH injection abundance of several appetite-associated mRNA was quantified which demonstrated that mRNA for neuropeptide Y (NPY) was increased while mRNA for proopiomelanocortin (POMC) was decreased. This was not the case for mRNA abundance in isolated LHA where NPY and POMC were not affected but melanin-concentrating hormone (MCH) mRNA was increased. A comprehensive behavior analysis was conducted after ICV GnIH injection which demonstrated a variety of behaviors unrelated to appetite were affected. In sum, these results implicate activation of the LHA in the GnIH orexigenic response and NPY, POMC and MCH are likely also involved.

Tomohiro Osugi,1 Jun Iwamoto,2 Michio Yamazaki,1 Masayuki Takakuwa3 1Department of Rehabilitation, Takakuwa Orthopaedic Nagayama Clinic, Asahikawa, Hokkaido, 2Institute for Integrated Sports Medicine, Keio University School of Medicine, Shinjuku-ku, Tokyo, 3Department of Orthopaedic Surgery, Takakuwa Orthopaedic Nagayama Clinic, Asahikawa, Hokkaido, Japan Abstract: A randomized controlled trial was conducted to clarify the beneficial effect of whole body vibration (WBV) exercise plus squat training on body balance, muscle power, and walking ability in the elderly with knee osteoarthritis and/or spondylosis. Of 35 ambulatory patients (14 men and 21 women) who were recruited at our outpatient clinic, 28 (80.0%, 12 men and 16 women) participated in the trial. The subjects (mean age 72.4 years) were randomly divided into two groups (n=14 in each group), ie, a WBV exercise alone group and a WBV exercise plus squat training group. A 4-minute WBV exercise (frequency 20 Hz) was performed 2 days per week in both groups; squat training (20 times per minute) was added during the 4-minute WBV training session in the WBV exercise plus squat training group. The duration of the trial was 6 months. The exercise and training program was safe and well tolerated. WBV exercise alone improved indices of body balance and walking velocity from baseline values. However, WBV exercise plus squat training was more effective for improving tandem gait step number and chair-rising time compared with WBV exercise alone. These results suggest the benefit and safety of WBV exercise plus squat training for improving physical function in terms of body balance and muscle power in the elderly. Keywords: whole body vibration exercise, squat training, body balance, walking velocity, muscle power

Gonadotropin-inhibitory hormone (GnIH) is a newly identified hypothalamic neuropeptide that inhibits pituitary hormone secretion in vertebrates. GnIH has an LPXRFamide (X = L or Q) motif at the C-terminal in representative species of gnathostomes. On the other hand, neuropeptide FF (NPFF), a neuropeptide characterized as a pain-modulatory neuropeptide, in vertebrates has a PQRFamide motif similar to the C-terminal of GnIH, suggesting that GnIH and NPFF have diverged from a common ancestor. Because GnIH and NPFF belong to the RFamide peptide family in vertebrates, protochordate RFamide peptides may provide important insights into the evolutionary origin of GnIH and NPFF. In this study, we identified a novel gene encoding RFamide peptides and two genes of their putative receptors in the amphioxus Branchiostoma japonicum. Molecular phylogenetic analysis and synteny analysis indicated that these genes are closely related to the genes of GnIH and NPFF and their receptors of vertebrates. We further identified mature RFamide peptides and their receptors in protochordates. The identified amphioxus RFamide peptides inhibited forskolin induced cAMP signaling in the COS-7 cells with one of the identified amphioxus RFamide peptide receptors expressed. These results indicate that the identified protochordate RFamide peptide gene is a common ancestral form of GnIH and NPFF genes, suggesting that the origin of GnIH and NPFF may date back to the time of the emergence of early chordates. GnIH gene and NPFF gene may have diverged by whole-genome duplication in the course of vertebrate evolution.

The kiss1 peptide (kisspeptin), a product of the kiss1 gene, is one of the key neuropeptides regulating vertebrate reproduction. In 2009, we identified a paralogous gene of kiss1 in the brain of amphibians and named it kiss2. Currently, the presence of the kiss2 gene and the kiss2 peptide is still obscure in amniotes compared with that in other vertebrates. Therefore, we performed genome database analyses in primates and reptiles to investigate the molecular evolution of the kiss2 gene in vertebrates. Because the mature kiss2 peptide has been identified only in amphibians, we further performed immunoaffinity purification and mass spectrometry to identify the mature endogenous kiss2 peptide in the brains of salmon and turtle that possessed the kiss2 gene. Here we provide the first evidence for the presence of a kiss2-like gene in the genome database of primates including humans. Synthetic amidated human KISS2 peptide activated human GPR54 expressed in COS7 cells, but nonamidated KISS2 peptide was inactive. The endogenous amidated kiss2 peptide may not be produced in primates because of the lack of an amidation signal in the precursor polypeptide. The kiss2-like gene may be nonfunctional in crocodilians because of premature stop codons. We identified the mature amidated kiss2 peptide in turtles and fish and analyzed the localization of kiss2 peptide mRNA expression in fish. The present study suggests that the kiss2 gene may have mutated in primates and crocodilians and been lost in birds during the course of evolution. In contrast, the kiss2 gene and mature kiss2 peptide are present in turtles and fish.

RFamide-related peptide-3 (RFRP-3), a mammalian ortholog of avian gonadotropin-inhibitory hormone, has pronounced inhibitory effects on reproduction in a number of species. RFRP-3 suppresses gonadotropin release at the hypothalamic and/or pituitary levels; however, increasing evidence also suggests putative functions within the ovary. We have now demonstrated the expression of both RFRP and its receptor (GPR147) in primary cultures of human granulosa-lutein cells. Immunohistochemical analysis of normal human ovaries from premenopausal women showed that RFRPs and GPR147 were primarily localized in the granulosa cell layer of large preovulatory follicles as well as in the corpus luteum. Treatment of human granulosa-lutein cells with RFRP-3 reduced FSH-, LH- and forskolin-stimulated progesterone production and steroidogenic acute regulatory protein expression but did not affect basal or 8-bromoadenosine 3'5'-cyclic monophosphate stimulated levels. In addition, RFRP-3 inhibited gonadotropin- and forskolin-induced intracellular cAMP accumulation, and these effects were abolished by pretreatment with an inhibitor of inhibitory G(i/o) proteins (pertussis toxin). Importantly, the effects of RFRP-3 on FSH-, LH-, and forskolin-induced cAMP and progesterone accumulation were completely eliminated by cotreatment with the bifunctional GPR147/GPR74 antagonist RF9 or by pretreatment with GPR147 small interfering RNA. These results suggest that RFRP-3 is expressed in human granulosa cells in which it acts via its receptor, GPR147, to inhibit gonadotropin signaling at the level of adenylyl cyclase via activation of a pertussis toxin-sensitive Gα(i/o) protein. This leads to reduced gonadotropin-stimulated cAMP accumulation and progesterone synthesis, likely via reduced steroidogenic acute regulatory protein expression. Thus, ovarian RFRP-3/GPR147 signaling could contribute to normal ovarian function.

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).

Within the RFamide peptide family, PQRFamide peptides that include neuropeptide FF and AF possess a C-terminal Pro–Gln–Arg–Phe–NH 2 motif. We previously identified PQRFamide peptides, lamprey PQRFa, PQRFa-related peptide (RP)-1 and -RP-2 by immunoaffinity purification in the brain of lamprey, one of the most ancient vertebrate species [13] . Lamprey PQRFamide peptide precursor mRNA was expressed in regions predicted to be involved in neuroendocrine regulation in the hypothalamus. However, the putative function(s) of lamprey PQRFamide peptides (PQRFa, PQRFa-RP-1 and PQRFa-RP-2) were not examined nor was the distribution of PQRFamide peptides examined in other tissues besides the brain. The objective of this study was to determine tissue distribution of lamprey PQRFamide peptide precursor mRNA, and to examine the effects of PQRFamide peptides on brain gonadotropin-releasing hormone (GnRH)-I, -II, and -III protein concentrations, and pituitary gonadotropin (GTH)-β mRNA expression in adult lampreys. Lamprey PQRFamide peptide precursor mRNA was expressed in the eye and the brain. Lamprey PQRFa at 100 μg/kg increased brain concentrations of lamprey GnRH-II compared with controls. PQRFa, PQRFa-RP-1 and PQRFa-RP-2 did not significantly change brain protein concentrations of either lamprey GnRH-I, -III, or lamprey GTH-β mRNA expression in the pituitary. These data suggest that one of the PQRFamide peptides may act as a neuroregulator of at least the lamprey GnRH-II system in adult female lamprey.

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.

The optic gland, which is analogous to the anterior pituitary in the context of gonadal maturation, is found on the upper posterior edge of the optic tract of the octopus Octopus vulgaris. In mature octopus, the optic glands enlarge and secrete a gonadotrophic hormone. A peptide with structural features similar to that of vertebrate gonadotrophin-releasing hormone (GnRH) was isolated from the brain of octopus and was named oct-GnRH. Oct-GnRH showed luteinising hormone-releasing activity in the anterior pituitary cells of the Japanese quail Coturnix coturnix. Oct-GnRH immunoreactive signals were observed in the glandular cells of the mature optic gland. Oct-GnRH stimulated the synthesis and release of sex steroids from the ovary and testis, and elicited contractions of the oviduct. Oct-GnRH receptor was expressed in the gonads and accessory organs, such as the oviduct and oviducal gland. These results suggest that oct-GnRH induces the gonadal maturation and oviposition by regulating sex steroidogenesis and a series of egg-laying behaviours via the oct-GnRH receptor. The distribution and expression of oct-GnRH in the central and peripheral nervous systems suggest that oct-GnRH acts as a multifunctional modulatory factor in feeding, memory processing, sensory, movement and autonomic functions.

In vertebrates, the neuropeptide control of gonadotrophin secretion is primarily through the stimulatory action of the hypothalamic decapeptide, gonadotrophin-releasing hormone (GnRH). Gonadal sex steroids and inhibin inhibit gonadotrophin secretion via feedback from the gonads, but a hypothalamic neuropeptide inhibiting gonadotrophin secretion was, until recently, unknown in vertebrates. In 2000, we discovered a novel hypothalamic dodecapeptide that directly inhibits gonadotrophin release in quail and termed it gonadotrophin-inhibitory hormone (GnIH). GnIH acts on the pituitary and GnRH neurones in the hypothalamus via a novel G-protein-coupled receptor for GnIH to inhibit gonadal development and maintenance by decreasing gonadotrophin release and synthesis. The pineal hormone melatonin is a key factor controlling GnIH neural function. GnIH occurs in the hypothalamus of several avian species and is considered to be a new key neurohormone inhibiting avian reproduction. Thus, the discovery of GnIH provides novel directions to investigate neuropeptide regulation of reproduction. This review summarises the discovery, progress and prospects of GnIH, a new key neurohormone controlling reproduction.

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.

Gonadotropin-inhibitory hormone (GnIH) is a hypothalamic dodecapeptide (SIKPSAYLPLRF-NH2) that directly inhibits gonadotropin synthesis and release from quail pituitary. The action of GnIH is mediated by a novel G-protein coupled receptor. This gonadotropin-inhibitory system may be widespread in vertebrates, at least birds and mammals. In these higher vertebrates, histological evidence suggests contact of GnIH immunoreactive axon terminals with GnRH neurons, thus indicating direct regulation of GnRH neuronal activity by GnIH. In this study we investigated the interaction of GnIH and GnRH-I and -II neurons in European starling (Sturnus vulgaris) brain. Cloned starling GnIH precursor cDNA encoded three peptides that possess characteristic LPXRF-amide (X = L or Q) motifs at the C termini. Starling GnIH was further identified as SIKPFANLPLRF-NH2 by mass spectrometry combined with immunoaffinity purification. GnIH neurons, identified by in situ hybridization and immunocytochemistry (ICC), were clustered in the hypothalamic paraventricular nucleus. GnIH immunoreactive fiber terminals were present in the external layer of the median eminence in addition to the preoptic area and midbrain, where GnRH-I and GnRH-II neuronal cell bodies exist, respectively. GnIH axon terminals on GnRH-I and -II neurons were shown by GnIH and GnRH double-label ICC. Furthermore, the expression of starling GnIH receptor mRNA was identified in both GnRH-I and GnRH-II neurons by in situ hybridization combined with GnRH ICC. The cellular localization of GnIH receptor has not previously been identified in any vertebrate brain. Thus, GnIH may regulate reproduction of vertebrates by directly modulating GnRH-I and GnRH-II neuronal activity, in addition to influencing the pituitary gland.

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.

The neuropeptide control of gonadotropin secretion is primarily through the stimulatory action of the hypothalamic decapeptide, GnRH. We recently identified a novel hypothalamic dodecapeptide with a C-terminal LeuPro-Leu-Arg-Phe-NH2 sequence in the domestic bird, Japanese quail (Coturnix japonica). This novel peptide inhibited gonadotropin release in vitro from the quail anterior pituitary; thus it was named gonadotropin-inhibitory hormone (GnIH). GnIH may be an important factor regulating reproductive activity not only in domesticated birds but also in wild, seasonally breeding birds. Thus, we tested synthetic quail GnIH in seasonally breeding wild bird species. In an in vivo experiment, chicken gonadotropin-releasing hormone-I (cGnRH-I) alone or a cGnRH-I/quail GnIH cocktail was injected i.v. into non-breeding song sparrows (Melospiza melodia). Quail GnIH rapidly (within 2 min) attenuated the GnRH-induced rise in plasma LH. Furthermore, we tested the effects of quail GnIH in castrated, photostimulated Gambel's white-crowned sparrows (Zonotrichia leucophrys gambelii), using quail GnIH or saline for injection. Again, quail GnIH rapidly reduced plasma LH (within 3 min) compared with controls. To characterize fully the action of GnIH in wild birds, the identification of their endogenous GnIH is essential. Therefore, in the present study a cDNA encoding GnIH in the brain of Gambel's white-crowned sparrow was cloned by a combination of 3' and 5' rapid amplification of cDNA ends and compared with the quail GnIH cDNA previously identified. The deduced sparrow GnIH precursor consisted of 173 amino acid residues, encoding one sparrow GnIH and two sparrow GnIH-related peptides (sparrow GnIH-RP-1 and GnIH-RP-2) that included Leu-Pro-Xaa-Arg-Phe-NH2 (Xaa=Leu or Gln) at their C-termini. All these peptide sequences were flanked by a glycine C-terminal amidation signal and a single basic amino acid on each end as an endoproteolytic site. Although the homology of sparrow and quail GnIH precursors was approximately 66%, the C-terminal structures of GnIH, GnIH-RP-1 and GnIH-RP-2 were all identical in two species. In situ hybridization revealed the cellular localization of sparrow GnIH mRNA in the paraventricular nucleus (PVN) of the hypothalamus. Immunohistochemical analysis also showed that sparrow GnIH-like immunoreactive cell bodies and terminals were localized in the PVN and median eminence respectively. Thus, only the sparrow PVN expresses GnIH, which appears to be a hypothalamic inhibitory factor for LH release, as evident from our field injections of GnIH into free-living breeding white-crowned sparrows. Sparrow GnIH rapidly (within 2 min) reduced plasma LH when injected into free-living Gambel's white-crowned sparrows on their breeding grounds in northern Alaska. Taken together, our results indicate that, despite amino acid sequence differences, quail GnIH and sparrow GnIH have similar inhibitory effects on the reproductive axis in wild sparrow species. Thus, GnIH appears to be a modulator of gonadotropin release.