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.     

RFRP-3对哺乳动物生殖功能和能量平衡的影响

哺乳动物的生殖功能受体内状态和外部环境综合作用的影响,这种综合作用通过作用于HPG轴的刺激因子和抑制因子之间的相对平衡来调控生殖。RFRP-3是目前下丘脑中唯一已知的HPG轴抑制因子。大量研究证实,RFRP-3能够抑制GnRH和LH的分泌,进而影响生殖功能。然而,RFRP-3对LH分泌的抑制作用是发生在垂体水平还是下丘脑水平尚不清楚。此外,RFRP-3还可能参与了MLT对哺乳动物季节性繁殖调控的信号通路,但是MLT对RFRP-3神经元的作用方式仍不清楚。此外,RFRP-3还可能在能量平衡和动物行为的调控中发挥着重要作用。文章就RFRP-3对HPG轴的调节机制以及其在能量平衡调节和行为调控中的作用进行了系统的阐述,并针对目前尚待解决的一些问题进行了探讨。     

Gonadotropin releasing hormone (GnRH) and gonadotropin inhibitory hormone (GnIH) in the songbird hippocampus: Regional and sex differences in adult zebra finches

Hypothalamic gonadotropin releasing hormone (GnRH) and gonadotropin inhibitory hormone (GnIH) are vital to reproduction in all vertebrates. These neuropeptides are also present outside of the hypothalamus, but the roles of extra-hypothalamic GnRH and GnIH remain enigmatic and widely underappreciated. We used immunohistochemistry and PCR to examine whether multiple forms of GnRH (chicken GnRH-I (GnRH1), chicken GnRH-II (GnRH2) and lamprey GnRH-III (GnRH4)) and GnIH are present in the hippocampus (Hp) of adult zebra finches (Taeniopygia guttata). Using immunohistochemistry, we provide evidence that GnRH1, GnRH2 and GnRH4 are present in hippocampal cell bodies and/or fibers and that GnIH is present in hippocampal fibers only. There are regional differences in hippocampal GnRH immunoreactivity, and these vary across the different forms of GnRH. There are also sex differences in hippocampal GnRH immunoreactivity, with generally more GnRH1 and GnRH2 in the female Hp. In addition, we used PCR to examine the presence of GnRH1 mRNA and GnIH mRNA in micropunches of Hp. PCR and subsequent product sequencing demonstrated the presence of GnRH1 mRNA and the absence of GnIH mRNA in the Hp, consistent with the pattern of immunohistochemical results. To our knowledge, this is the first study in any species to systematically examine multiple forms of GnRH in the Hp or to quantify sex or regional differences in hippocampal GnRH. Moreover, this is the first demonstration of GnIH in the avian Hp. These data shed light on an important issue: the sites of action and possible functions of GnRH and GnIH outside of the HPG axis.     

RNA interference of gonadotropin-inhibitory hormone gene induces arousal in songbirds

Gonadotropin-inhibitory hormone (GnIH) was originally identified in quail as a hypothalamic neuropeptide inhibitor of pituitary gonadotropin synthesis and release. However, GnIH neuronal fibers do not only terminate in the median eminence to control anterior pituitary function but also extend widely in the brain, suggesting it has multiple roles in the regulation of behavior. To identify the role of GnIH neurons in the regulation of behavior, we investigated the effect of RNA interference (RNAi) of the GnIH gene on the behavior of white-crowned sparrows, a highly social songbird species. Administration of small interfering RNA against GnIH precursor mRNA into the third ventricle of male and female birds reduced resting time, spontaneous production of complex vocalizations, and stimulated brief agonistic vocalizations. GnIH RNAi further enhanced song production of short duration in male birds when they were challenged by playbacks of novel male songs. These behaviors resembled those of breeding birds during territorial defense. The overall results suggest that GnIH gene silencing induces arousal. In addition, the activities of male and female birds were negatively correlated with GnIH mRNA expression in the paraventricular nucleus. Density of GnIH neuronal fibers in the ventral tegmental area was decreased by GnIH RNAi treatment in female birds, and the number of gonadotropin-releasing hormone neurons that received close appositions of GnIH neuronal fiber terminals was negatively correlated with the activity of male birds. In summary, GnIH may decrease arousal level resulting in the inhibition of specific motivated behavior such as in reproductive contexts.     

Rapid inhibition of female sexual behavior by gonadotropin-inhibitory hormone (GnIH)

Gonadotropin-releasing hormone (GnRH) is largely responsible for the initiation of sexual behaviors; one form of GnRH activates a physiological cascade causing gonadal growth and gonadal steroid feedback to the brain, and another form is thought to act as a neurotransmitter to enhance sexual receptivity. In contrast to GnRH, gonadotropin-inhibitory hormone (GnIH) inhibits gonadotropin release. The distribution of GnIH in the avian brain suggests that it has not only hypophysiotropic actions but also unknown behavioral actions. GnIH fibers are present in the median eminence (ME) and are in apparent contact with chicken GnRH (cGnRH)-I and -II neurons and fibers. In birds, cGnRH-I regulates pituitary gonadotropin release, whereas cGnRH-II enhances copulation solicitation in estradiol-primed females exposed to male song. In the present study, we determined the effects of GnIH administered centrally to female white-crowned sparrows. A physiological dose of GnIH reduced circulating LH and inhibited copulation solicitation, without affecting locomotor activity. Using rhodaminated GnIH, putative GnIH binding sites were seen in the ME close to GnRH-I fiber terminals and in the midbrain on or close to GnRH-II neurons. These data demonstrate direct effects of GnIH upon reproductive physiology and behavior, possibly via separate actions on two forms of GnRH.     

The orexigenic effect of GnIH is mediated by central opioid receptors in chicks

Gonadotropin-inhibiting hormone (GnIH) is a newly discovered hypothalamic hormone which suppresses gonadotropin synthesis and release from the anterior pituitary. Recently, we found that intracerebroventricular (ICV) injection of GnIH stimulated feeding behavior of chicks (Gallus gallus) and suggested that GnIH is one of orexigenic peptides. However, the mechanism underlying the orexigenic effect is still unknown. In the present study, we examined whether the orexigenic effect of GnIH is related to opioid and nitric oxide (NO) systems. The orexigenic effect of ICV-injected GnIH was attenuated by co-injection of beta-funaltrexamine (an opioid mu-receptor antagonist) but not ICI-174,864 and nor-binaltorphimine (antagonists of opioid delta- and kappa-receptors, respectively). The co-injection of non-selective NO synthase inhibitor did not affect GnIH-induced feeding behavior. The present study demonstrated that the GnIH-induced feeding might be mediated by opioid mu-receptor in chicks.     

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.     

Immunohistochemical localization of GnRH and RFamide-related peptide-3 in the ovaries of mice during the estrous cycle

Gonadotropin releasing hormone (GnRH) has now been suggested as an important intraovarian regulatory factor. Gonadotropin inhibitory hormone (GnIH) a hypothalamic dodecapeptide, acts opposite to GnRH. GnRH, GnIH and their receptors have been demonstrated in the gonads. In order to find out the physiological significance of these neuropeptides in the ovary, we aim to investigate changes in the abundance of GnRH I and GnIH in the ovary of mice during estrous cycle. The present study investigated the changes in GnRH I, GnRH I-receptor and RFRP-3 protein expression in the ovary of mice during estrous cycle by immunohistochemistry and immunoblot analysis. The immunoreactivity of GnRH I and its receptor and RFRP-3 were mainly localized in the granulosa cells of the healthy and antral follicles during proestrus and estrus and in the luteal cells during diestrus 1 and 2 phases. The relative abundance of immunoreactivity of GnRH I, GnRH I-receptor and RFRP-3 undergo significant variation during proestrus and thus may be responsible for selection of follicle for growth and atresia. A significant increase in the concentration of RFRP-3 during late diestrus 2 coincided with the decline in corpus luteum activity and initiation of follicular growth and selection. In general, immunolocalization of GnRH I, GnRH I-receptor and RFRP-3 were found in close vicinity suggesting functional interaction between these peptides. It is thus, hypothesized that interaction between GnRH I-RFRP-3 neuropeptides may be involved in the regulation of follicular development and atresia.     

Stimulation of sexual behavior in the male rat by galanin-like peptide

Galanin-like peptide (GALP) is a recently described neuropeptide, which shares a partial sequence identity with galanin but is derived from a separate gene. Central injections of GALP stimulate the secretion of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) and induce the expression of Fos in several brain areas known to regulate male sexual behavior in the rat. We postulated that GALP may also stimulate sexual behavior in concert with its stimulatory effect on the hypothalamic-pituitary-gonadal (HPG) axis. To test this hypothesis, we administered GALP, galanin, or the vehicle (artificial cerebrospinal fluid, aCSF) alone to sexually experienced male rats and assessed the effects of these agents on sexual behavior. We observed that compared to aCSF alone, GALP significantly increased all aspects of male-typical sexual behavior, whereas galanin inhibited all of these same behaviors. To examine whether the stimulatory effects of GALP on sexual behavior were mediated by GALP's stimulatory effects on the HPG axis, we castrated the same male rats and repeated the behavioral experiment. We found that GALP maintained its inductive action on male-typical sexual behaviors in the castrated animals, suggesting that the effects of GALP on sexual behavior are not the result of GALP's ability to stimulate testosterone secretion. These observations suggest that GALP neurons are part of the hypothalamic circuitry controlling sexual behavior in the male rat.     

Hypothalamic LPXRF-amide peptides in vertebrates: identification, localization and hypophysiotropic activity

Probing undiscovered neuropeptides that play important roles in the regulation of pituitary function in vertebrates is essential for the progress of neuroendocrinology. Recently, we identified a novel hypothalamic neuropeptide with a C-terminal LPLRF-amide sequence in the quail brain. This avian neuropeptide was shown to be located in the hypothalamo-hypophysial system and to decrease gonadotropin release from cultured anterior pituitary. We, therefore, designated this novel neuropeptide as gonadotropin-inhibitory hormone (GnIH). We further identified novel hypothalamic neuropeptides closely related to GnIH in the brains of other vertebrates, such as mammals, amphibians, and fish. The identified neuropeptides possessed a LPXRF-amide (X = L or Q) motif at their C-termini. These LPXRF-amide peptides also were localized in the hypothalamus and other brainstem areas and regulated pituitary hormone release. Subsequently, cDNAs that encode LPXRF-amide peptides were characterized in vertebrate brains. In this review, we summarize the identification, localization, and hypophysiotropic activity of these newly identified hypothalamic LPXRF-amide peptides in vertebrates.     

Effect of melatonin on GnIH precursor gene expression in Nile tilapia,Oreochromis niloticus

Sexual maturation and gonadal development of fish is greatly influenced by photic information, an external environmental factor, and melatonin mediates this information to regulate gonadotropin (GTH) secretion and gonadal activation. The relationship between gonadotropin inhibiting hormone (GnIH) and melatonin in fish, however, has not been studied to date. Here, the GnIH expression pattern and daily change of melatonin levels were compared to each other in mature tilapia (body length 16.1 ± 0.2 cm, body weight 77.7 ± 3.43 g), and the effect of melatonin injection on GnIH gene expression was investigated. GnIH gene expression increased at night when the secretion of melatonin increased, whereas gene expression decreased during the day when melatonin secretion decreased. Injecting tilapia intraperitoneally with melatonin increased GnIH gene expression and decreased the expression of gonadotropin releasing hormone (GnRH) and GTH. Furthermore, the injection decreased the 11-KT concentration in male tilapia. These results indicate that melatonin is likely to suppress the hypothalamus-pituitary-gonad (HPG) axis via the action of GnIH in this species.     

Neuronal interactions between galanin-like-peptide- and orexin- or melanin-concentrating hormone-containing neurons

Galanin-like peptide (GALP) is a novel orexigenic neuropeptide that is recently isolated from the porcine hypothalamus. GALP-containing neurons predominantly locate in the hypothalamic arcuate nucleus (ARC). The expression of GALP mRNA within the ARC is increased after the administration of leptin. GALP-containing neurons express leptin receptor and contain alpha-melanocyte-stimulating hormone. We have recently reported that neuropeptide Y (NPY)- and orexin-containing axon terminals are in close apposition with GALP-containing neurons in the ARC. In addition, GALP-containing neurons express orexin-1 receptor (OX1-R). Thus, GALP may function under the influence of leptin and orexin. However, the target neurons of GALP have not yet been clarified. To clarify the neuronal interaction between GALP-containing and other feeding regulating neurons, double-immunostaining method using antibodies against GALP- and orexin- or melanin-concentrating hormone (MCH) was performed in the rat lateral hypothalamus (LH). GALP-immunoreactive fibers appeared to project to the LH around the fornix. They were also found from the rostral to the caudal part of the ARC, paraventricular nucleus (PVH), stria terminalis (BST), medial preoptic area (MPA), and lateral septal nucleus (LSV). Moreover, GALP-like immunoreactive nerve fibers were directly contacted with orexin- and melanin-concentrating hormone (MCH)-like immunoreactive neurons in the LH. Our findings strongly suggest that GALP-containing neurons interact with orexin- and/or MCH-containing neurons in the lateral hypothalamus and that it participates in the regulation of feeding behavior in harmony with other feeding-regulating neurons in the hypothalamus.     

Central administration of substance P inhibits feeding behavior in chicks

The purpose of the present study was to determine whether central administration of substance P (SP), a tachykinin neuropeptide, influenced feeding behavior in layer chicks (Gallus gallus). Intracerebroventricular (ICV) injections of 5 nmol SP decreased food intake in 5- and 6-day-old chicks under both ad libitum and 3-h fasting conditions. There are 3 major subtypes of tachykinin receptors, namely, neurokinin 1, 2 and 3 receptors. Injection of neurokinin A and neurokinin B, which are respectively endogenous agonists for neurokinin 2 and 3 receptors, did not suppress feeding behavior in chicks, suggesting that the anorexigenic effect of SP might be mediated by the neurokinin 1 receptor rather than neurokinin 2 and 3 receptors. Chicks that received 5 nmol SP did not change their locomotion, standing, sitting or drinking time, suggesting that its anorexigenic action might not be due to SP-induced hyperactivity or sedation. ICV injection of SP increased water intake, also indicating that SP likely did not affect feeding behavior through malaise. In addition, the anorexigenic effect of SP might not be related to corticotrophin-releasing hormone (CRH) because plasma corticosterone concentration was not affected by ICV injection of SP and co-administration of the CRH receptor antagonist astressin did not affect the anorexigenic effect of SP. The present study suggests that central SP acts as an anorexigenic neuropeptide in chicks.     

Identification and functional analysis of a novel ligand for G protein-coupled receptor, Neuromedin S

We identified a novel 36-amino acid neuropeptide in rat brain as an endogenous ligand for the G protein-coupled receptors FM-3/GPR66 and FM-4/TGR-1, which were identified to date as the neuromedin U (NMU) receptors, and designated this peptide neuromedin S (NMS) because it was specifically expressed in the suprachiasmatic nucleus (SCN) of the hypothalamus. NMS shared a C-terminal core structure with NMU. NMS mRNA was highly expressed in the central nervous system, spleen and testis. In rat brain, NMS expression was restricted to the ventrolateral portion of the SCN and has a diurnal peak under light/dark cycling, but remains stable under constant darkness. Intracerebroventricular (ICV) administration of NMS in rats induced nonphotic type phase shifts in the circadian rhythm of locomotor activity. ICV injection of NMS also decreased 12-h food intake during the dark period in rats. This anorexigenic effect was more potent than that observed with the same dose of NMU. ICV administration of NMS increased proopiomelanocortin (POMC) mRNA expression in the arcuate nucleus (Arc) and corticotropin-releasing hormone mRNA in the paraventricular nucleus, and induced c-Fos expression in the POMC neurons in the Arc. These findings suggest that NMS is implicated in the regulation of circadian rhythm and feeding behavior.     

Relationship between melanin-concentrating hormone- and neuropeptide Y-containing neurons in the goldfish hypothalamus

Intracerebroventricular (ICV) administration of melanin-concentrating hormone (MCH) inhibits food intake in goldfish, unlike the orexigenic action in rodents, via the melanocortin system with suppression of neuropeptide Y (NPY) mRNA expression. We therefore investigated the neuronal relationship between MCH- and NPY-containing neurons in the goldfish brain, using a double-immunofluorescence method and confocal laser scanning microscopy. MCH- and NPY-like immunoreactivities were distributed throughout the brain. In particular, MCH-containing nerve fibers or endings lay in close apposition to NPY-containing neurons in a specific region of the hypothalamus, the nucleus posterioris periventricularis (NPPv). These observations suggest that MCH-containing neurons provide direct input to NPY-containing neurons in the NPPv of goldfish, and that MCH plays a crucial role in the regulation of feeding behavior as an anorexigenic neuropeptide, inhibiting the orexigenic activity of NPY.     

Lipopolysaccharide injection induces rapid decrease of hypothalamic GnRH mRNA and peptide, but does not affect GnIH in zebra finches

Lipopolysaccharide (LPS) is frequently used experimentally to mimic acute infection. Through activation of the host's immune response, an LPS injection has profound effects on the adrenocortical response to stress and on behaviors including reduction in activity, water and food intake, and libido. These behavioral changes occurring during infection are collectively called "sickness behavior." It is thought that adoption of sickness behavior reallocates energy from other fitness-enhancing activities, such as reproduction, for use in the immune response. Although the behavioral effects of LPS treatment are well-known, less information is available regarding the effects of LPS on the brain in terms of controlling reproductive behavior, specifically concerning a newly discovered neuropeptide, gonadotropin-inhibitory hormone (GnIH). This study investigated the effects of an LPS injection on the behavior and the hypothalamic neuropeptides controlling reproduction [GnIH and gonadotropin-releasing hormone (GnRH)] of zebra finches (Taeniopygia guttata). Overall, there was a decrease in activity in birds injected with LPS. The number of GnRH-immunoreactive neurons was significantly reduced in birds injected with LPS when compared to controls, while the number of GnIH-releasing neurons remained unchanged. At the level of gene expression, a similar pattern was found: there was reduced expression of GnRH mRNA in LPS-injected animals, whereas GnIH expression remained unchanged. Plasma testosterone did not change significantly in LPS-injected animals, nor did plasma corticosterone. Taken together, these results indicate a rapid (within 3h) inhibition of the reproductive axis during an immune challenge mimicking an infection, specifically acting on the GnRH system. The present study expands our knowledge on the interaction between the immune system and the reproductive system.     

Regulation of food intake in the goldfish by interaction between ghrelin and orexin

Intracerebroventricular (ICV) administration of ghrelin, orexin and neuropeptide Y (NPY) stimulates food intake in goldfish. Orexin and NPY interact with each other in the regulation of feeding, while ghrelin-induced feeding has also shown to be mediated by NPY in the goldfish model. To investigate the interaction between ghrelin and orexin, we examined the effects of a selective orexin receptor-1 antagonist, SB334867, and a growth hormone secretagogue-receptor antagonist, [D-Lys(3)]-GHRP-6, on ghrelin- and orexin-A-induced feeding. Ghrelin-induced food intake was completely inhibited for 1h following ICV preinjection of SB334867, while [D-Lys(3)]-GHRP-6 attenuated orexin-A stimulated feeding. Furthermore, ICV administration of ghrelin or orexin-A at a dose sufficient to stimulate food intake increased the expression of each other's mRNA in the diencephalon. These results indicate that, in goldfish, ghrelin and orexin-A have interacting orexigenic effects in the central nervous system. This is the first report that orexin-A-induced feeding is mediated by the ghrelin signaling in any animal model.     

Evolutionary Origin of GnIH and NPFF in Chordates: Insights from Novel Amphioxus RFamide Peptides

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.     

Effect of RFRP-3 on reproduction is sex- and developmental status-dependent in the striped hamster (Cricetulus barabensis)

RFamide-related peptides (RFRPs) are orthologous to gonadotropin-inhibitory hormone (GnIH) inhibiting gonadotropin release. There are only two RFRP sequences (RFRP-1 and RFRP-3) encoded in rodents. RFRP-3, which was considered as a hypothetical inhibitor on GnRH, shows a stimulatory effect on the male Syrian and male Siberian hamster in short days. As a dominant rodent pest in northern China farmland, the striped hamster (Cricetulus barabensis) has higher reproductive activities and could act as a model to study the mechanism of reproduction. However, the effect of RFRP-3 on the reproductive activity for the striped hamster is less understood. In the study, we cloned 643 bp RFRP cDNA from the striped hamster hypothalamus, which contained an ORF of 570 bp encoding two RFamide-related peptide (RFRP) sequences: SPAPANKVPHSAANLPLRF-NH₂ (C. barabensis RFRP-1) and TLSRVPSLPQRF-NH₂ (C. barabensis RFRP-3). We also investigated the expression variation of RFRP mRNA and GnRH mRNA in the hypothalamus from hamsters with different developmental statuses (7-week-, 13-week- and 1.5-year-olds) using FQ-PCR, in which the 13-week-old female individuals were in estrous. The striped hamsters that are 7 weeks and 1.5 years old are non-breeding individuals, and those that are 13-week hamsters have breeding phenomena. The highest hypothalamus RFRP mRNA level was found in breeding males as compared to non-breeding males. Conversely, the lowest RFRP mRNA level in the hypothalamus was observed in breeding females, with no significant level when the breeding females were compared to the 7-week-old individuals. Additionally, the investigation of GnRH expression level showed a declining expression trend across the developmental stages (7-week-, 13-week- and 1.5-year-olds) in both sexes. Significant negative and positive relationships were detected in the 13-week estrous female (r = − 0.997, P = 0.035) and the 13-week male (r = 0.998, P = 0.029) striped hamsters respectively, which suggest that RFRP-3 has inhibitory and stimulatory effects on female and male adults respectively. Our results suggest that the effects of RFRP-3 on reproduction are sex- and developmental status-dependent in the striped hamster.