RFamide Peptide Research Articles

The roles of RFamide-related peptides (RFRPs), mammalian gonadotropin-inhibitory hormone (GnIH) orthologues in female reproduction.

Objective(s):To benefit from reproduction and deal with challenges in the environmental conditions, animals must adapt internal physiology to maximize the reproduction rate. Maladaptive variations in the neurochemical systems and reproductive system can lead to manifestation of several significant mammalian reprocesses, including mammalian ovarian lifespan. RFamide-related peptide (RFRP, Rfrp), mammalian orthologues of gonadotropin-inhibitory hormone (GnIH), which is a regulator to prevent the gonadotropin-releasing hormone (GnRH) neural activity, is known to be related to reproductive traits. This review aimed to summarize recent five-year observations to outline historic insights and novel perspectives into the functions of RFRPs in coding the mammalian reproductive physiology, especially highlight recent advances in the impact on RFRPs in regulating mammalian ovary lifespan.Materials and Methods:We reviewed the recent five-year important findings of RFRP system involved in mammalian ovary development. Data for this review were collected from Google Scholar and PubMed using the RFRP keyword combined with the keywords related to physiological or pathological reproductive functions.Results:Recent discoveries are focused on three major fronts in research on RFRP role in female reproduction including reproductive functions, energy balance, and stress regulation. The roles of RFRPs in various development phases of mammal reproduction including prepuberty, puberty, estrous cycle, pregnancy, milking, menopause, and/or ovarian diseases have been shown.Conclusion:Overall, these recent advances demonstrate that RFRPs serve as critical mediators in mammalian ovarian development.

Comparative and Evolutionary Aspects of Gonadotropin-Inhibitory Hormone and FMRFamide-Like Peptide Systems.

Gonadotropin-inhibitory hormone (GnIH) is a hypothalamic neuropeptide that was found in the brain of Japanese quail when investigating the existence of RFamide peptides in birds. GnIH was named because it decreased gonadotropin release from cultured anterior pituitary, which was located in the hypothalamo-hypophysial system. GnIH and GnIH precursor gene related peptides have a characteristic C-terminal LPXRFamide (X = L or Q) motif that is conserved in jawed vertebrates. Orthologous peptides to GnIH are also named RFamide related peptide or LPXRFamide peptide from their structure. A G-protein coupled receptor GPR147 is the primary receptor for GnIH. Similarity-based clustering of neuropeptide precursors in metazoan species indicates that GnIH precursor of vertebrates is evolutionarily related to FMRFamide precursor of mollusk and nematode. FMRFamide peptide is the first RFamide peptide that was identified from the ganglia of the venus clam. In order to infer the evolutionary history of the GnIH-GnIH receptor system we investigate the structural similarities between GnIH and its receptor and well-studied nematode Caenorhabditis elegans (C. elegans) FMRFamide-like peptides (FLPs) and their receptors. We also compare the functions of FLPs of nematode with GnIH of chordates. A multiple sequence alignment and phylogenetic analyses of GnIH, neuropeptide FF (NPFF), a paralogous peptide of GnIH, and FLP precursors have shown that GnIH and NPFF precursors belong to different clades and some FLP precursors have structural similarities to either precursor. The peptide coding regions of FLP precursors in the same clade align well with those of GnIH or NPFF precursors. Alignment of GnIH (LPXRFa) peptides of chordates and FLPs of C. elegans grouped the peptides into five groups according to the last C-terminal amino acid sequences, which were MRFa, LRFa, VRFa, IRFa, and PQRFa. Phylogenetic analysis of receptors suggested that GPR147 has evolutionary relationships with FLP receptors, which regulate reproduction, aggression, locomotion, and feeding. GnIH and some FLPs mediate the effect of stress on reproduction and behavior, which may also be a conserved property of these peptide systems. Future studies are needed to investigate the mechanism of how neuropeptide precursor genes are mutated to evolve new neuropeptides and their inheritance.

Role of RFRP-3 in the development of cold stress-induced polycystic ovary phenotype in rats

RFamide-related peptide (RFRP-3) is a regulator of GnRH secretion from the brain, but it can also act in human ovary to influence steroidogenesis. We aimed to study the putative local role of RFRP-3 in the ovary and its potential participation in the development of a polycystic ovary phenotype induced by chronic sympathetic stress (cold stress). We used adult Sprague–Dawley rats divided into control and stressed groups. In both groups, we studied the effect of intraovarian exposure to RFRP-3 on follicular development and plasma ovarian steroid concentrations. We also tested the effect of RFRP-3 on ovarian steroid production in vitro. Chronic in vivo intraovarian exposure to RFRP-3 decreased basal testosterone concentrations and cold stress-induced progesterone production by the ovary. In vitro, RFRP-3 decreased hCG-induced ovarian progesterone and testosterone secretion. Immunohistochemistry and mRNA expression analysis showed a decrease in Rfrp and expression of its receptor in the ovary of stressed rats, a result which is in line with the increased testosterone levels found in stressed rats. In vivo application of RFRP-3 recovered the low levels of secondary and healthy antral follicles found in stressed rats. Taken together, our data indicate a previously unknown response of hypothalamic and ovarian RFRP-3 to chronic cold stress, influencing ovarian steroidogenesis and follicular dynamics. Thus, it is likely that RFRP-3 modulation in the ovary is a key component of development of the polycystic ovary phenotype.

Central Injection of Substance P Antagonizes the RF Amide-Related Peptide-3 Impacts on Hypothalamic KISS-1 and GnRH Gene Expressions in Male Wistar Rats

Background: Gonadotropin-inhibitory hormone and its mammalian orthologues (RFRP: RF amid related peptide) are known to inhibit the secretion of gonadotropins. In addition, substance P (SP) a member of tachykinin’s family is demonstrated that can increase the firing rate of kisspeptin/neurokinin B/dynorphin (KNDy) neurons and provokes the of secretion gonadotropins. In this experimental study we investigated the effects of co-administration of RFRP-3 and SP on the expression of KISS-1 and GnRH genes in male rats. Methods: forty-two mature Wistar rats were randomly allocated in 7 groups (n=6 in each group). Animals in each group intracerebroventricularly received either saline+DMSO, 1nmol SP, 5 nmol RFRP-3, 1nmol SP plus 5 nmol RFRP-3, 1 nmol SP plus10 nmol RF9 (RFRP-3 receptor antagonist), 1 nmol SP plus 1 nmol P234 (kisspeptin receptor antagonist) plus 5 nmol RFRP-3 or 1 nmol SP plus 5 nmol CP-96,345 (SP receptor antagonist) plus 5 nmol RFRP-3 in final volume of 3 µl. After two hours following injections hypothalamic samples were collected for evaluating the expression of target genes by real-time PCR technique. Results: Injections in SP and SP plus RF9 groups increase the expression of the both GnRH and KISS-1 genes (P<0.05). Injections in RFRP-3 group and SP plus RFRP-3 plus SP antagonist group significantly decrease the expression of both GnRH and KISS-1 genes (P<0.05). Injections of SP plus RFRP-3 group and SP plus RFRP-3 plus P234 group did not significantly change the expression of GnRH and KISS-1 genes. Conclusion: The results indicate that SP antagonizes the effects of RFRP-3 on the expression of Hypothalamic KISS-1 and GnRH genes.

Conopeptides promote itch through human itch receptor hMgprX1

Members of Mas related G-protein coupled receptors (Mrgpr) are known to mediate itch. To date, several compounds have been shown to activate these receptors, including chloroquine, a common antimalarial drug, and peptides of the RF-amide family. However, specific ligands for these receptors are still lacking and there is a need for novel compounds that can be used to modulate the receptors in order to understand the cellular and molecular mechanism in which they mediate itch. Some cone snail venoms were previously shown to induce itch in mice. Here, we show that the venom of Conus textile induces itch through activation of itch-sensing sensory neurons, marked by their sensitivity to chloroquine. Two RF-amide peptides, CNF-Tx1 and CNF-Tx2, were identified in a C. textile venom gland transcriptome. These belong to the conorfamide family of peptides which includes previously described peptides from the venoms of Conus victoriae (CNF-Vc1) and Conus spurius (CNF-Sr1 and CNF-Sr2). We show that CNF-Vc1 and CNF-Sr1 activate MrgprC11 whereas CNF-Vc1 and CNF-Tx2 activate the human MrgprX1 (hMrgprX1). The peptides CNF-Tx1 and CNF-Sr2 do not activate MrgprC11 or hMrgprX1. Intradermal injection of CNF-Vc1 and CNF-Tx2 into the cheek of a transgenic mouse expressing hMrgprX1 instead of endogenous mouse Mrgprs resulted in itch-related scratching thus demonstrating the in vivo activity of these peptides. Using truncated analogues of CNF-Vc1, we identified amino acids at positions 7–14 as important for activity against hMrgprX1. The conopeptides reported here are tools that can be used to advance our understanding of the cellular and molecular mechanism of itch mediated by Mrgprs.

Immunolocalization of RFamide-related peptide 3 in a desert rodent Gerbillus tarabuli during seminiferous epithelium cycle.

Gerbillus tarabuli is a nocturnal seasonal breeder desert rodent with a main breeding season in spring and summer, and sexual quiescence in winter. This species is an interesting model for studying testis function in rodents. Therefore, the present study was performed firstly to investigate the stages of seminiferous epithelium cycle of Gerbillus tarabuli with a histological, morphometric and statistical study. And secondly to investigate the expression and possible variations in cellular distribution of RFamide-related peptide-3 (RFRP-3) - the mammalian ortholog of avian gonadotropin-inhibitory hormone (GnIH) - during seminiferous epithelium cycle using immunohistochimestry. Our results showed for the first time that the seminiferous epithelium cycle in Gerbillus tarabuli comprises 14 well-defined stages according to the tubular morphology method. The seminiferous epithelium thickness showed a significant difference during the epithelium cycle, thus it was the only morphometric classification criterion of seminiferous epithelium cycle in Gerbillus tarabuli. The immunohistochemical study reveals, for the first time, the presence of RFRP-3 in Gerbillus tarabuli testes, in both testicular compartments: the tubular and the interstitial. RFRP-3 is expressed differently according to the seminiferous epithelium cycle, RFRP-3 seemed to be more expressed at the stages V-VII and XIII. RFRP-3 was detected in Sertoli cells (≈12%), spermatocytes I (≈19%), round and elongated spermatids (≈13%), and with a more important signal in Leydig cells (26.87%±0.07). These results indicated the importance of RFRP-3 in testicular function in Gerbillus tarabuli; its expression at the interstitial and germinal levels argues in favor of an involvement in androgens synthesis and in spermatogenesis specifically in meiosis and spermiogenesis. This action seems primordial from stages V-VII and XIII. Also, the study of the seminiferous epithelium cycle will enrich the histological identity of the species.

Ontogeny of QRFP and its receptor expression in mouse testis during post-natal development

Pyroglutamylated RFamide peptide (QRFP), a neuropeptide, binds to its receptor GPR103 and influences various biological functions. In the present study, expression and localization of QRFP and GPR103 in mouse testis during post-natal development were investigated. The results showed that QRFP and GPR103 and also the isoforms of the receptor, viz. GPR103A and GPR103B were expressed in mouse testis during post-natal development. Expression of QRFP and its receptor was high during the early periods of post-natal development. Immunohistochemical study demonstrated the localization of QRFP and GPR103 in both interstitial and tubular compartments of the testis throughout post-natal development. A shift in the germ cell types expressing QRFP and its receptor along the course of testicular development and also a prominent immunoreactivity of QRFP in germ cells and of GPR103 in Sertoli cells was observed. Further, the immunoreactivity of QRFP and GPR103 appeared to be stage-specific in spermatogenetically active testis. Besides in intercellular spaces, localization of QRFP was also noticed in nuclei of germ cells. In conclusion, the present results suggest potential involvement of QRFP system in the development, proliferation and differentiation of testicular cells, possibly by regulating the energy requirement for these processes.

RFamidergic neurons in the olfactory centers of the terrestrial slug Limax

BackgroundThe terrestrial slug Limax has long been used as a model for the study of olfactory information processing and odor learning. Olfactory inputs from the olfactory epithelium are processed in the tentacular ganglion and then in the procerebrum. Glutamate and acetylcholine are the major neurotransmitters used in the procerebrum. Phe-Met-Arg-Phe-NH2 (FMRFamide) has been shown to be involved in the regulation of the network activity of the procerebrum. Although there are thought to be various RFamide family peptides other than FMRFamide that are potentially recognized by anti-FMRFamide antibody in the central nervous system of mollusks, identifying the entire repertoire of RFamide peptides in Limax has yet to be achieved.MethodsIn the present study, we made a comprehensive search for RFamide peptide-encoding genes from the transcriptome data of Limax, and identified 12 genes. The expression maps of these RFamide genes were constructed by in situ hybridization in the cerebral ganglia including the procerebrum, and in the superior/inferior tentacles.ResultsTen of 12 genes were expressed in the procerebrum, and nine of 12 genes were expressed in the tentacular ganglia. Gly-Ser-Leu-Phe-Arg-Phe-NH2 (GSLFRFamide), which is encoded by two different genes, LFRFamide1 (Leu-Phe-Arg-Phe-NH2–1) and LFRFamide2 (Leu-Phe-Arg-Phe-NH2–2), decreased the oscillatory frequency of the local field potential oscillation in the procerebrum when exogenously applied in vitro. We also found by immunohistochemistry that the neurons expressing pedal peptide send efferent projections from the procerebrum back to the tentacular ganglion.ConclusionOur findings suggest the involvement of a far wider variety of RFamide family peptides in the olfactory information processing in Limax than previously thought.

RFamide-related peptide-3 promotes alpha TC1 clone 6 cell survival likely via GPR147

Type 2 diabetes mellitus (T2DM) is closely related to islet alpha cell mass and viability. Several types of RFamide-related peptides (RFRPs) are involved in regulating proliferation and function of islet cells. However, current understanding of the role of RFamide-related peptide-3 (RFRP-3) in pancreatic alpha cells is limited. Therefore, we investigated the expression of the RFRP-3 receptor, G protein-coupled receptor 147 (GPR147), in mouse islets and alpha TC1 clone 6 cells, and evaluated the function of RFRP-3 on alpha cells. We show that GPR147 is expressed in mouse islets and alpha cell lines. In addition, RFRP-3 promotes survival of alpha cells under conditions of hyperglycemia and serum starvation. Mechanistic evidence demonstrates that RFRP-3 activated PI3K/AKT and ERK1/2 signaling cascades and treatment with an antagonist of GPR147, 1-adamantanecarbonyl-Arg-Phe-NH₂ (RF9) blocked this function. These findings indicate a novel effect of RFRP-3 in promoting alpha cell survival, likely via GPR147.

Gonadotrophin-inhibitory hormone and its mammalian orthologue RFamide-related peptide-3: Discovery and functional implications for reproduction and stress.

At the turn of the millennium, a neuropeptide with pronounced inhibitory actions on avian pituitary gonadotrophin secretion was identified and named gonadotrophin-inhibitory hormone (GnIH). Across bird species, GnIH acts at the level of the pituitary and the gonadotrophin-releasing hormone (GnRH) neuronal system to inhibit reproduction. Subsequent to this initial discovery, orthologues of GnIH have been identified and characterised across a broad range of species. In many vertebrates, the actions of GnIH and its orthologues serve functional roles analogous to those seen in birds. In other cases, GnIH and its orthologues exhibit more diverse actions dependent on sex, species, season and reproductive condition. The present review highlights the discovery and functional implications of GnIH across species, focusing on research domains in which the significance of this neuropeptide has been explored most.

Action of neurotensin, corticotropin-releasing hormone, and RFamide-related peptide-3 in E2-induced negative feedback control: studies using a mouse arcuate nucleus hypothalamic cell model.

The recently established immortalized hypothalamic cell model mHypoA-55 possesses characteristics similar to those of Kiss-1 neurons in the arcuate nucleus (ARC) region of the hypothalamus. Here, we show that Kiss-1 gene expression in these cells was downregulated by 17β-estradiol (E2) under certain conditions. Both neurotensin (NT) and corticotropin-releasing hormone (CRH) were expressed in these cells and upregulated by E2. Stimulation of mHypoA-55 cells with NT and CRH significantly decreased Kiss-1 mRNA expression. A mammalian gonadotropin-inhibitory hormone homolog, RFamide-related peptide-3 (RFRP-3), was also found to be expressed in mHypoA-55 cells, and RFRP-3 expression in these cells was increased by exogenous melatonin stimulation. E2 stimulation also upregulated RFRP-3 expression in these cells. Stimulation of mHypoA-55 cells with RFRP-3 significantly increased the expression of NT and CRH. Furthermore, melatonin stimulation resulted in the increase of both NT and CRH mRNA expression in mHypoA-55 cells. On the other hand, in experiments using mHypoA-50 cells, which were originally derived from hypothalamic neurons in the anteroventral periventricular nucleus, Kiss-1 gene expression was upregulated by both NT and CRH, although E2 increased both NT and CRH expression, similarly to the mHypoA-55 cells. Our observations using the hypothalamic ARC cell model mHypoA-55 suggest that NT and CRH have inhibitory effects on Kiss-1 gene expression under the influence of E2 in association with RFRP-3 expression. Thus, these neuropeptides might be involved in E2-induced negative feedback mechanisms.

Effect of RFRP-3, the mammalian ortholog of GnIH, on the epididymis of male rats

RFamide-related peptide-3 (RFRP-3) and its receptor (GPR147) play an important role in reproduction regulation in mammals. To understand the role of RFRP-3 in male reproductive function of epididymis, we first investigated the expression changes in RFRP-3 and its receptor at different stages of development, that is, postnatal day 20 (P20), 40 (P40), 60 (P60) and 80 (P80). Our results showed that fluctuations in the expression of GnIH and GPR147 during postnatal development occurred, and the highest epididymal GnIH and GPR147 expression were both detected in P60. Subsequently, we further investigated the effect of RFRP-3 on the histology, apoptosis and autophagy of the epididymis in vivo. For in vivo study, male rats were treated intratesticularly with different doses of RFRP-3 (control, 0.1 μg, 1 μg, and 10 μg per day) for 7 days. Our results show that RFRP-3 caused dose-dependent histological changes in the epididymal duct, such as a decline in the number of spermatozoa and an increase in degenerated and vacuolated epididymal epithelial cells. Rats treated intratesticularly with RFRP-3 also showed dose-dependent effects on caspase-3 activation and the expression of apoptotic markers (whole caspase-3, cleaved caspase-3 and Bcl-2). However, the expression of autophagy markers (Beclin-1 and Atg5) exhibited a bidirectional, dose-dependent effect. It is concluded that RFRP-3 plays a regulatory role in male rat reproduction, possibly because RFRP-3 mediates the apoptosis and autophagy of the epididymis.

Ontogeny of Gonadotropin-Inhibitory Hormone (GnIH) in the cichlid fish Cichlasoma dimerus.

RFamide peptides are expressed in the early stages of development in most vertebrates. Gonadotropin-inhibitory hormone (GnIH) belongs to the RFamide family, and its role in reproduction has been widely studied in adult vertebrates, ranging from fish to mammals. As only three reports evaluated GnIH during development, the aim of this study was to characterise the ontogeny of GnIH in a fish model, Cichlasoma dimerus. We detected the presence of two GnIH-immunoreactive (GnIH-ir) cell clusters with spatial and temporal differences. One cluster was observed by 3 days post-hatching (dph) in the nucleus olfacto-retinalis (NOR) and the other in the nucleus posterioris periventricularis by 14 dph. The number of GnIH-ir neurons increased in both nuclei, whereas their size increased only in the NOR from hatchling to juvenile stages. These changes occurred from the moment larvae started feeding exogenously and during development and differentiation of gonadal primordia. We showed by double-label immunofluorescence that only GnIH-ir neurons in the NOR co-expressed GnRH3 associated peptide. In addition, GnIH-ir fibre density increased in all brain regions from 5 dph. GnIH-ir fibres were also detected in the retina, optic tract and optic tectum, suggesting that GnIH acts as a neuromodulator of photoreception and the integration of different sensory modalities. Also, there were GnIH-ir fibres in the pituitary from 14 dph, which were in close association with somatotropes. Moreover, GnIH-ir fibres were observed in the saccus vasculosus from 30 dph, suggesting a potential role of GnIH in the modulation of its function. Finally, we found that gnih was expressed from 1 dph, and that the pattern of variation of its transcript levels was in accordance with that of cell number. Present results are the starting point for the study of new GnIH roles during development. This article is protected by copyright. All rights reserved.

Regulation of LH secretion by RFRP-3 - From the hypothalamus to the pituitary.

RFamide-related peptides (RFRPs) have long been identified as inhibitors of the hypothalamus-pituitary-gonad axis in mammals. However, less progress has been made in the detailed roles of RFRPs in the control of LH secretion. Recent studies have suggested that RFRP-3 neurons in the hypothalamus can regulate the secretion of LH at different levels, including kisspeptin neurons, GnRH neurons, and the pituitary. Additionally, conflicting results regarding the effects of RFRP-3 on these levels exist. In this review, we collect the latest evidence related to the effects of RFRP-3 neurons in regulating LH secretion by acting on kisspeptin neurons, GnRH neurons, and the pituitary and discuss the potential role of the timely reduction of RFRP-3 signaling in the modulation of the preovulatory LH surge.

Reproductive status-dependent kisspeptin and RFamide-related peptide (Rfrp) gene expression in female Damaraland mole-rats.

Damaraland mole rats (Fukomys damarensis) are cooperatively breeding, subterranean mammals that exhibit a high reproductive skew. Reproduction is monopolised by the dominant female of the group, whereas subordinates are physiologically suppressed to the extent that they are anovulatory. In these latter animals, it is assumed that normal gonadotropin-releasing hormone secretion from the hypothalamus is disrupted. The RFamide peptides kisspeptin (Kiss1) and RFamide-related peptide-3 (RFRP-3) are considered as potent regulators of gonadotropin release. To assess whether these neuropeptides are involved in the mechanism of reproductive suppression, we investigated the distribution and gene expression of Kiss1 and Rfrp by means of in situ hybridisation in wild-caught female Damaraland mole-rats with different reproductive status. In both reproductive phenotypes, substantial Kiss1 expression was found in the arcuate nucleus and only few Kiss1-expressing cells were detected in the anteroventral periventricular nucleus (AVPV), potentially as a result of low circulating oestradiol concentrations in breeding and nonbreeding females. Rfrp gene expression occurred in the dorsomedial nucleus, the paraventricular nucleus and the periventricular nucleus. While in female breeders and nonbreeders, plasma oestradiol levels were low and not significantly different, quantification of the hybridisation signal for both genes revealed significant differences in relation to reproductive status. Reproductively active females had more Kiss1-expressing cells and a higher number of silver grains per cell in the arcuate nucleus compared to nonreproductive females. This difference was most pronounced in the caudal part of the nucleus. No such differences were found in the AVPV. Furthermore, breeding status was associated with a reduced number of Rfrp-expressing cells in the anterior hypothalamus. This reproductive status-dependent expression pattern of Kiss1 and Rfrp suggests that both neuropeptides play a role in the regulation of reproduction in Damaraland mole-rats. Enhanced long-term negative feedback effects of oestradiol could be responsible for the lower Kiss1 expression in the arcuate nucleus of reproductively suppressed females.

RFRP-3, the mammalian ortholog of GnIH, induces cell cycle arrest at G2/M in porcine ovarian granulosa cells

RFamide-related peptide-3 (RFRP-3), the mammalian ortholog of gonadotropin-inhibitory hormone (GnIH), has been proposed as a key inhibitory regulator of mammal reproduction. Our previous studies have demonstrated that RFRP-3 inhibited the expression of proliferation-related proteins in porcine granulose cells (GCs), but the inhibitory mechanism causing this has not been discovered. Here, we aim to elucidate the underlying mechanism and determine the cell cycle regulatory sites of action of RFRP-3 on porcine GC proliferation. To this end, the viability of porcine GCs was initially estimated by cell counting kit-8 (CCK-8). We confirmed that different doses of RFRP-3 decreased the cellular viability, suggesting that RFRP-3 could inhibit the proliferation of GCs. Subsequently, we evaluated the direct effects of RFRP-3 on the expression of cell cycle regulators. Compared to the control treated cells, 10−6 and 10−8 M of RFRP-3 effectively reduced the transcription of Cyclin B1 and CDK1 mRNAs. However, treatment with RFRP-3 did not alter Cyclin A2, Cyclin D1, CDK2, or CDK4 mRNA levels. These results suggest that RFRP-3 might be inducing G2/M-phase arrest in porcine GCs. Finally, to further determine the molecular mechanism underlying RFRP-3-mediated G2/M cell cycle arrest, we observed the levels of G2/M cell cycle regulatory factors in RFRP-3-treated porcine GCs. The results showed that RFRP-3 treatment significantly increased the expression of Myt1, p-Wee1 and p-Cdc2, whereas the level of Cyclin B1 significantly decreased in porcine GCs treated with 10−6 M of RFRP-3. Taken together, our data suggest that RFRP-3 regulates the phosphorylation or expression of G2/M cell cycle regulatory factors to induce G2/M-phase arrest via inhibition Cyclin B-CDK1 complex activation in porcine GCs, which might provide an unfavorable condition for porcine GC proliferation.

Dual Actions of Mammalian and Piscine Gonadotropin-Inhibitory Hormones, RFamide-Related Peptides and LPXRFamide Peptides, in the Hypothalamic-Pituitary-Gonadal Axis.

Gonadotropin-inhibitory hormone (GnIH) is a hypothalamic neuropeptide that decreases gonadotropin synthesis and release by directly acting on the gonadotrope or by decreasing the activity of gonadotropin-releasing hormone (GnRH) neurons. GnIH is also called RFamide-related peptide in mammals or LPXRFamide peptide in fishes due to its characteristic C-terminal structure. The primary receptor for GnIH is GPR147 that inhibits cAMP production in target cells. Although most of the studies in mammals, birds, and fish have shown the inhibitory action of GnIH in the hypothalamic–pituitary–gonadal (HPG) axis, several in vivo studies in mammals and many in vivo and in vitro studies in fish have shown its stimulatory action. In mouse, although the firing rate of the majority of GnRH neurons is decreased, a small population of GnRH neurons is stimulated by GnIH. In hamsters, GnIH inhibits luteinizing hormone (LH) release in the breeding season when their endogenous LH level is high but stimulates LH release in non-breeding season when their LH level is basal. Besides different effects of GnIH on the HPG axis depending on the reproductive stages in fish, higher concentration or longer duration of GnIH administration can stimulate their HPG axis. These results suggest that GnIH action in the HPG axis is modulated by sex-steroid concentration, the action of neuroestrogen synthesized by the activity of aromatase stimulated by GnIH, estrogen membrane receptor, heteromerization and internalization of GnIH, GnRH, and estrogen membrane receptors. The inhibitory and stimulatory action of GnIH in the HPG axis may have a physiological role to maintain reproductive homeostasis according to developmental and reproductive stages.

The roles of kisspeptin and gonadotropin inhibitory hormone in stress-induced reproductive disorders.

Several kinds of stress suppress the hypothalamic-pituitary-gonadal (HPG) axis and reproductive behavior in humans and animals. These changes can eventually cause diseases and disorders, such as amenorrhea and infertility. In previous studies, it has been shown that stress-related factors, e.g., corticotropin-releasing hormone, cortisol, and pro-inflammatory cytokines, promote the stress-induced suppression of the HPG axis. However, these mechanisms are not sufficient to explain how stress suppresses HPG axis activity, and it has been suggested that some other factors might also be involved. In the early 21st century, novel neuroendocrine peptides, kisspeptin and gonadotropin inhibitory hormone (GnIH)/RFamide-related peptide 3 (RFRP-3), which directly regulate GnRH/gonadotropin synthesis and secretion, were newly discovered. Growing evidence indicates that kisspeptin and GnIH/RFRP-3 play pivotal roles in the stress-induced disruption of the HPG axis and reproductive behavior in addition to their physiological functions. This review summarizes what is currently known about the roles of kisspeptin and GnIH/RFRP-3 in stress-induced reproductive disorders.

Decreased Expression of Arginine-Phenylalanine-Amide-Related Peptide-3 Gene in Dorsomedial Hypothalamic Nucleus of Constant Light Exposure Model of Polycystic Ovarian Syndrome.

BackgroundAn abnormality in pulse amplitude and frequency of gonadotropin releasing hormone (GnRH) secretion is the most characteristics of polycystic ovarian syndrome (PCOS). On the other hand, arginine-phenylalanine-amide (RFamide)-related peptide-3 (RFRP3) inhibits the secretion of GnRH in mammalian hypothalamus. The current study performed in order to investigate the expression of RFRP3 mRNA in the dorsomedial hypothalamic nucleus (DMH) after the induction of PCOS in a rat model of constant light exposure, and the possible role of parity on occurrence of PCOS.Materials and MethodsIn the experimental study, female nulliparous (n=12) and primiparous (n=12) rats were randomly subdivided into control and PCOS subgroups (n=6). PCOS were induced by 90 days exposure to constant light. After 90 days, blood, brain, and ovaries were sampled. Serum levels of follicle stimulating hormone (FSH), luteinizing hormone (LH), and testosterone were evaluated. In addition, six adult female ovariectomized rats as a control of real-time polymerase chain reaction (PCR) tests were prepared and in the DMH of all rats, the relative mRNA expression of RFRP3 was assessed.ResultsHistological evaluation of ovaries represented the polycystic features. In addition, serum concentrations of testosterone in the PCOS subgroups were more than the controls (P<0.05). Furthermore, the relative expression of RFRP3 mRNA in PCOS subgroups was lower than the controls (P<0.05).ConclusionConstant light model of the PCOS-induced rats decreased the gene expression of RFRP3 in the DMH that suggests the decrease of RFRP3 may reduce its inhibitory effect on GnRH during the PCOS pathogenesis. This effect was stronger in the nulliparous rats than the primiparous.

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.