RFamide Peptide Research Articles

Prolactin-releasing peptide receptor (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

The precursor (PRLH, P81277) for PrRP generates 31 and 20-amino-acid versions. QRFP43 (named after a pyroglutamylated arginine-phenylalanine-amide peptide) is a 43 amino acid peptide derived from QRFP (P83859) and is also known as P518 or 26RFa. RFRP is an RF amide-related peptide [29] derived from a FMRFamide-related peptide precursor (NPVF, Q9HCQ7), which is cleaved to generate neuropeptide SF, neuropeptide RFRP-1, neuropeptide RFRP-2 and neuropeptide RFRP-3 (neuropeptide NPVF).

Role of RFRP-3 in the Regulation of Kiss-1 Gene Expression in the AVPV Hypothalamic Cell Model mHypoA-50.

Kisspeptin, encoded by the Kiss-1 gene, plays a crucial role in reproductive function by governing the hypothalamic-pituitary-gonadal axis. The recently established Kiss-1-expressing cell model mHypoA-50 displays characteristics of neuronal cells of the anteroventral periventricular (AVPV) region of the mouse hypothalamus. Because Kiss-1 gene expression in these cells is upregulated by estradiol (E2), mHypoA-50 cells are regarded as a valuable model for the study of Kiss-1-expressing neurons in the AVPV region. These cells also express RFamide-related peptide-3 (RFRP-3), a mammalian homolog of gonadotropin inhibitory hormone. The RFRP-3 expression in mHypoA-50 cells was increased by melatonin stimulation. In addition, E2 stimulation increased RFRP-3 expression in these cells. Treatment of the mHypoA-50 cells with exogenous RFRP-3 resulted in the increase of Kiss-1 messenger RNA expression within the cells; however, RFRP-3 did not modify gonadotropin-releasing hormone or kisspeptin-induced Kiss-1 gene expression in these cells. In addition, we found that RFRP-3 stimulation increased the expression of corticotropin-releasing hormone, which may be involved in E2-induced positive feedback in mHypoA-50 cells. Our observations suggest that RFRP-3 might be involved in positive feedback regulation by directly or indirectly increasing Kiss-1 gene expression.

Evidence for the Direct Effect of the NPFF Peptide on the Expression of Feeding-Related Factors in Spotted Sea Bass (Lateolabrax maculatus).

Neuropeptide FF (NPFF) is a family member of RF-amide peptides, which are suggested to be involved in the control of vertebrate feeding behavior. However, little is known about the effect of the NPFF peptide on feeding-related processes in basal vertebrates. In this study, four full-length cDNAs, npff, npffr1, npffr2-1, and npffr2-2, were cloned from spotted sea bass and characterized. The conserved NPFF peptide is biologically active because it functionally interacts with different receptors expressed in cultured eukaryotic cells to enhance CRE promoter activity. Tissue distribution analysis showed that the highest npff mRNA expression occurred in the telencephalon, hypothalamus, medulla, gonad and muscle, but the npffrs mRNAs were mainly distributed within the central nervous system (CNS). In situ hybridization (ISH) detected npff-expressing cells in several specific regions ranging across the telencephalon and midbrain to the hypothalamus. Incubation of the spotted sea bass conserved NPFF peptide significantly increased the expression of orexin (orx) and neuropeptide Y (npy) mRNA and decreased the expression of leptin (lep), somatostatin (ss), and cholecystokinin (cck) mRNA in brain cells. Similarly, the conserved NPFF peptide also heightened the expression of gastrin (gas), ghrelin (ghrl), and motilin (mtl) mRNA and significantly reduced the expression of cck mRNA in the intestine and stomach. Taken together, these data suggest that the NPFF peptide may play a stimulating role in regulating feeding-related processes in spotted sea bass.

Multiple Modulation of Acid-Sensing Ion Channel 1a by the Alkaloid Daurisoline.

Acid-sensing ion channels (ASICs) are proton-gated sodium-selective channels that are expressed in the peripheral and central nervous systems. ASIC1a is one of the most intensively studied isoforms due to its importance and wide representation in organisms, but it is still largely unexplored as a target for therapy. In this study, we demonstrated response of the ASIC1a to acidification in the presence of the daurisoline (DAU) ligand. DAU alone did not activate the channel, but in combination with protons, it produced the second peak component of the ASIC1a current. This second peak differs from the sustained component (which is induced by RF-amide peptides), as the second (DAU-induced) peak is completely desensitized, with the same kinetics as the main peak. The co-application of DAU and mambalgin-2 indicated that their binding sites do not overlap. Additionally, we found an asymmetry in the pH activation curve of the channel, which was well-described by a mathematical model based on the multiplied probabilities of protons binding with a pool of high-cooperative sites and a single proton binding with a non-cooperative site. In this model, DAU targeted the pool of high-cooperative sites and, when applied with protons, acted as an inhibitor of ASIC1a activation. Moreover, DAU’s occupation of the same binding site most probably reverses the channel from steady-state desensitization in the pH 6.9–7.3 range. DAU features disclose new opportunities in studies of ASIC structure and function.

Dynamic expressions of hypothalamic genes regulate seasonal breeding in a natural rodent population.

Seasonal breeding is a universal reproductive strategy in many animals. Hypothalamic genes, especially type 2 and 3 iodothyronine deiodinases (Dio2/3), RFamide-related peptide 3 (Rfrp-3), kisspeptin (Kiss-1) and gonadotropin-releasing hormone (GnRH), are involved in a photoperiodic pathway that encodes seasonal signals from day length in many vertebrate species. However, the seasonal expression patterns of these genes in wild mammals are less studied. Here, we present a four-year field investigation to reveal seasonal rhythm and age-dependent reproductive activity in male Brandt's voles (Lasiopodomys brandtii) and to detect relationships among seasonal expression profiles of hypothalamic genes, testicular activity, age and annual day length. From breeding season (April) to nonbreeding season (October), adult male voles displayed a synchronous peak in gonadal activity with annual day length around summer solstice, which was jointly caused by age structure shifts and age-dependent gonadal development patterns. Overwintered males maintained reproductive activity until late in the breeding season, whereas most newborn males terminated gonadal development completely, except for a minority of males born early in spring. Consistently, the synchronous and opposite expression profiles of Dio2/3 suggest their central function to decode photoperiodic signals and to predict the onset of the nonbreeding season. Moreover, changes in Dio2/3 signals may guide the actions of Kiss-1 and Rfrp-3 to regulate the age-dependent divergence of reproductive strategy in wild Brandt's vole. Our results provide evidence on how hypothalamic photoperiod genes regulate seasonal breeding in a natural rodent population.

The dromedary camel displays annual variation in hypothalamic kisspeptin and Arg-Phe-amide-related peptide-3 according to sex, season, and breeding activity.

The dromedary camel (Camelus dromedarius) is a desert mammal whose cycles in reproductive activity ensure that the offspring's birth and weaning coincide with periods of abundant food resources and favorable climate conditions. In this study, we assessed whether kisspeptin (Kp) and arginine-phenylalanine (RF)-amide related peptide-3 (RFRP-3), two hypothalamic peptides known to regulate the mammalian hypothalamo-pituitary gonadal axis, may be involved in the seasonal control of camel's reproduction. Using specific antibodies and riboprobes, we found that Kp neurons are present in the preoptic area (POA), suprachiasmatic (SCN), and arcuate (ARC) nuclei, and that RFRP-3 neurons are present in the paraventricular (PVN), dorsomedial (DMH), and ventromedial (VMH) hypothalamic nuclei. Kp fibers are found in various hypothalamic areas, notably the POA, SCN, PVN, DMH, VMH, supraoptic nucleus, and the ventral and dorsal premammillary nucleus. RFRP-3 fibers are found in the POA, SCN, PVN, DMH, VMH, and ARC. POA and ARC Kp neurons and DMH RFRP-3 neurons display sexual dimorphism with more neurons in female than in male. Both neuronal populations display opposed seasonal variations with more Kp neurons and less RFRP-3 neurons during the breeding (December-January) than the nonbreeding (July-August) season. This study is the first describing Kp and RFRP-3 in the camel's brain with, during the winter period lower RFRP-3 expression and higher Kp expression possibly responsible for the HPG axis activation. Altogether, our data indicate the involvement of both Kp and RFRP-3 in the seasonal control of the dromedary camel's breeding activity.

Soya bean rich diet is associated with adult male rat aggressive behavior: relation to RF amide-related peptide 3-aromatase-neuroestrogen pathway in the brain.

Relation between soya bean (SB) consumption and aggressive behavior has not been elucidated yet. Thus, this study was conducted to investigate the effect of large amount of SB consumption on adult male rats' aggressive behavior through investigating changes in the expression of gonadotropin-inhibitory hormone/ RF amide-related peptide 3 (GnIH/RFRP3), neuropeptide FF receptor, cytochrome P450, family 19, subfamily A, polypeptide 1 (Cyp19A1), estrogen receptors α and β and the levels of neuroestrogen, dopamine, glutamate and testosterone as well as aromatase activity in the brain. Adult male rats were divided into three equal groups: group I, control group, received standard diet; group II and group III received 25% and 50% SB of their standard diet contents, respectively, for 12weeks. The obtained results showed that feeding male rats with large amount of SB could induce aggressive behavior in a dose dependant manner possibly through inhibition of brain GnIH/RFRP-aromatase-neuroestrogen pathway. These effects may be through decreasing aromatase activity, neuroestrogen concentration, Cyp19A1 and ER β mRNA levels and increasing ER α mRNA levels and immunostaining as well as testosterone, dopamine and glutamate levels in the brain. These findings also provide further support for the inhibitory role of RFRP3 on aggressive behavior of male rats. These data may open new avenues for the potential harmful effects of consumption large amounts of SB rich food on humans.

Role of RF-amid Related Peptide-3 (RFRP-3) in Inhibitory Effect of Orexin A on Reproductive Function in the Animal Model of Male Wistar Rats.

The aim of the present study is to examine the orexin A (OXA) signaling can leave any impact on the hypothalamic-pituitary-gonadal (HPG) axis and this impact can be relayed through the pathway of RF amide-related peptide-3 (RFRP-3, the mammalian ortholog of the avian gonadotropin-inhibitory hormone)/G-protein coupled receptor (GPR)-147 (RFRP-3 receptor) as a novel target for controlling of HPG axis in the male rats. Male rats were categorized randomly into experimental groups including control vehicle, OXA, and its antagonists' group and went through to surgical cannulation into the third ventricle. After the intracerebroventricular injection of each solution, blood samples were collected for measurements of the LH and testosterone using radioimmunoassay method. Hypothalamus of the animals were isolated for analysis of the relative expression of Rfrp-3/Gpr-147 along with Gnrh gene by Real time-PCR. Also, in the different cohort of animal sexual behavior test was done. It was shown that OXA significantly decreases the mean serum level of the LH and testosterone and, at the same time, its antagonists neutralize this impact. Moreover, we demonstrated that OXA has reduced the hypothalamic gene expression of Gnrh and increased the expression of Rfrp-3 and Gpr-147 genes. While OXA antagonists neutralize this impact. The results of this study are related to the impact of orexin on the HPG axis. It is recommended that RFRP-3/GPR-147 system as the interneural pathway relay the data of orexin to the neurons of GnRH.

RF9: May it be a new therapeutic option for hypogonadotropic hypogonadism?

Hypogonadotropic hypogonadism (secondary hypogonadism), congenital or acquired, is a form of hypogonadism that is due to problems with either the hypothalamus or pituitary gland affecting gonadotropin levels. Pulsatile secretion of gonadotropin-releasing hormone (GnRH) by hypothalamus is a primer step to initiate the release of pituitary gonadotropins. Kisspeptin and gonadotropin-inhibitory hormone (GnIH) are accepted as two major players in the activation and inhibition of GnRH regarding the neuroendocrine functioning of the hypothalamic pituitary gonadal axis. Kisspeptin is known as the most potent activator of GnRH. Regarding the inhibition of GnRH, RF-amide-related peptide-3 (RFRP-3) is accepted as the mammalian orthologue of GnIH in avian species. RF9 (1-adamantane carbonyl-Arg-Phe-NH2) is an antagonist of RFRP-3/GnIH receptor (neuropeptide FF receptor 1 (NPFFR1; also termed as GPR147). In recent years, several studies have indicated that RF9 activates GnRH neurons and gonadotropins in a kisspeptin receptor (Kiss1r, formerly known as GPR54) dependent manner. These results suggest that RF9 may have a bimodal function as both an RFRP-3 antagonist and a kisspeptin agonist or it may be a kiss1r agonist rather than an RFRP-3/GnIH receptor antagonist. These interactions are possible because Kisspeptin and GnIH are members of the RF-amide family, and both possibilities are not far from explaining the potent gonadotropin stimulating effects of RF9. Therefore, we hypothesize that RF9 may be a new therapeutic option for the hypogonadotropic hypogonadism due to its potent GnRH stimulating effects. A constant or repeated administration of RF9 provides a sustained increase in plasma gonadotrophin levels. However, applications in the same way with GnRH analogues and kisspeptin may result in desensitization of the gonadotropic axis. The reasons reported above contribute to our hypothesis that RF9 may be a good option in the GnRH stimulating as a kisspeptin agonist. We suggest that further studies are needed to elucidate the potential effects of RF9 in the treatment of the hypogonadotropic hypogonadism.

An integrative view of mammalian seasonal neuroendocrinology.

Seasonal neuroendocrine cycles that govern annual changes in reproductive activity, energy metabolism and hair growth are almost ubiquitous in mammals that have evolved at temperate and polar latitudes. Changes in nocturnal melatonin secretion regulating gene expression in the pars tuberalis (PT) of the pituitary stalk are a critical common feature in seasonal mammals. The PT sends signal(s) to the pars distalis of the pituitary to regulate prolactin secretion and thus the annual moult cycle. The PT also signals in a retrograde manner via thyroid-stimulating hormone to tanycytes, which line the ventral wall of the third ventricle in the hypothalamus. Tanycytes show seasonal plasticity in gene expression and play a pivotal role in regulating local thyroid hormone (TH) availability. Within the mediobasal hypothalamus, the cellular and molecular targets of TH remain elusive. However, two populations of hypothalamic neurones, which produce the RF-amide neuropeptides kisspeptin and RFRP3 (RF-amide related peptide 3), are plausible relays between TH and the gonadotrophin-releasing hormone-pituitary-gonadal axis. By contrast, the ways by which TH also impinges on hypothalamic systems regulating energy intake and expenditure remain unknown. Here, we review the neuroendocrine underpinnings of seasonality and identify several areas that warrant further research.

Whole-exome sequencing in multiplex preeclampsia families identifies novel candidate susceptibility genes.

Preeclampsia is a common and serious heritable disorder of human pregnancy. Although there have been notable successes in identification of maternal susceptibility genes a large proportion of the heritability of preeclampsia remains unaccounted for. It is has been postulated that rare variation may account for some of this missing heritability. In this study, we performed whole-exome sequencing (WES) in multiplex families to identify rare exonic risk variants. We conducted WES in 244 individuals from 34 Australian/New Zealand multiplex preeclampsia families. Variants were tested for association with preeclampsia using a threshold model and logistic regression. We found significant association for two moderately rare missense variants, rs145743393 (Padj = 0.0032, minor allele frequency = 0.016) in the chromosome 1 open reading frame 35 (C1orf35) gene, and rs34270076 (Padj = 0.0128, minor allele frequency = 0.024) in the pyroglutamylated RFamide peptide receptor (QRFPR) gene. To replicate these associations we performed imputation in our Australian genome wide association scan for preeclampsia and found no significant exonic variants in either C1orf35 or QRFPR. However, 11 variants demonstrating nominal significance (P < 0.05) in the genomic region between QRFPR and annexin A5 (ANXA5) were identified. We further leveraged publicly available genome-wide available summary data from the UK Biobank to investigate association of these two variants with the underlying clinical phenotypes of preeclampsia and detected nominal association of the QRFPR variant (rs34270076, P = 0.03) with protein levels in females. The study represents the first to use WES in multiplex families for preeclampsia and identifies two novel genes (QRFPR and C1orf35) not previously associated with preeclampsia and find nominal association of rs34270076 with protein levels, a key clinical feature of preeclampsia. We find further support for ANXA5 previously associated with pregnancy complications, including preeclampsia.

SAT-423 The Involvement of GnRH Neurons in Mediating the Effects of RFRP-3 on the Mouse Reproductive Axis

In 2000, gonadotrophin-inhibitory hormone was discovered in birds and shown to inhibit gonadotrophin secretion. The mammalian ortholog was concurrently discovered in humans and rats and termed RFamide-related peptide-3 (RFRP-3). Recent results have shown that the effects of centrally-administered RFRP-3 on gonadotrophin secretion are sex- and cycle stage-dependent in mice (Ancel et al., 2017). Indeed, intracerebroventricular injections of RFRP-3 stimulated LH secretion in males, and inhibited LH secretion in females at the time of the preovulatory LH surge. In addition, the stimulatory effect observed in males was shown to be mediated in part by GPR54, the receptor for Kisspeptins, suggesting the involvement of other pathways. In order to further our understanding of the ways in which RFRP neurons modulate the reproductive axis, we have developed a novel transgenic mouse line. Using a Cre-loxP conditional transgenic method, we knocked the receptor for RFRP-3 (GPR147) out of GnRH neurons and analysed puberty onset in these mice. While the absence of GPR147 on GnRH neurons advanced puberty onset in male mice, it resulted in a delay in female puberty, once again indicating a sex-specific role of the RFRP system in the regulation of the reproductive function. Current work is aimed at dissecting the potential involvement of GnRH neurons in the pathways mediating RFRP-3 effects on LH secretion in mice carrying a deletion of GPR147 in GnRH neurons. Both male and female mice received intracerebroventricular injections of RFRP-3 and LH levels were subsequently assayed in tail-tip blood samples. This work is still ongoing. Taken together, these new tools provide us with the possibility to advance our understanding of the structure and the functions of the RFRP neuronal system in mice.

SAT-415 Action of Neurotensin, CRH, And RFRP-3 in E2-Induced Negative Feedback Control: Studies Using a Mouse Arc 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.

Kisspeptin and RFRP3 modulate body mass in Phodopus sungorus via two different neuroendocrine pathways.

Many animals exhibit remarkable metabolic and reproductive adaptations to seasonal changes in their environment. When day length shortens, Djungarian hamsters (Phodopus sungorus) reduce their body weight and inhibit their reproductive activity, whereas the opposite occurs in springtime. These physiological adaptations are considered to depend on photoperiodic changes in hypothalamic genes encoding the peptides kisspeptin (Kp) and RFamide-related peptide 3 (RFRP3) for the control of reproduction, as well as pro-opiomelanocortin and somatostatin for metabolic regulation. The present study investigates the effect of Kp and RFRP3 on long-term body weight regulation, aiming to establish whether metabolic and reproductive hypothalamic networks may interact during adaptation to seasonal physiology. We found that chronic central administration of both Kp and RFRP3 in short photoperiod-adapted male Djungarian hamsters increased body weight, although via different pathways. The effect of Kp was dependent on testicular activity because castration prevented the body weight increase and was associated with an increase in pro-opiomelanocortin and neuropeptide Y expression. On the other hand, the orexigenic effect of RFRP3 was associated with an increase in circulating insulin and leptin levels, although it had no effect on any of the hypothalamic metabolic genes investigated, and did not change circulating levels of sex steroids. Notably, neither Kp, nor RFRP3 altered female hamster metabolic parameters. Thus, using a rodent model exhibiting seasonal changes in reproduction and metabolism, the present study demonstrates that, in addition to its role in the central control of reproduction, Kp also participates in body weight control in a sex-dependent manner via an anabolic action of testosterone. Conversely, RFRP3 affects body weight control in males mostly by acting on adiposity, with no overt effect on the reproductive system in both sexes.

Warm needle moxibustion, mild moxibustion and electroacupuncture interventions have an anti-aging effect possibly by regulating hypothalamus-pituitary-testis axis in aged male rats

To observe the effect of warm needle moxibustion (WNM), mild moxibustion and electroacupuncture (EA) of "Shenshu" (BL23) and "Guanyuan" (CV4) on serum gonadal hormone levels and related gene expression in the hypothalamus and testis in aged rats, so as to explore their underlying mechanisms in anti-aging. Forty male SD male rats were randomly divided into aged control, WNM, mild moxibustion, EA and medication groups (n ＝ 8 in each group), and other 8 male young adult SD rats were used as the youth control group. WNM (acupuncture plus moxibustion), mild moxibustion or EA (2 Hz /100 Hz, 1 mA) was separately applied to CV4 and bilateral BL23 for 15 min, once daily, 5 days a week for 8 weeks. Rats of the medication group received subcutaneous injection of testosterone propionate injection (7 mg/kg), once every 3 days, and those of the aged and youth groups received the same dose of 0.9% NaCl solution for 8 weeks. The levels of serum testoste-rone (T) and gonadotropin-releasing hormone (GnRH) were measured by ELISA. The expression of Kisspeptin (KISS-1), G protein-coupled receptors 54 (GPR54), RFamide-related peptide-3 (RFRP-3) and GPR147 mRNAs in the hypothalamus and GnRH-R, GPR54 and GPR147 mRNAs in the testis tissues were detected by quantitative real-time PCR. The levels of serum T and GnRH, and the expressions of KISS-1, GPR54, RFRP-3 and GPR147 mRNAs in the hypothalamus and GnRH-R and GPR54 mRNAs in the testis in the aged rats were significantly lower than those in the youth rats (P<0.05), while the expression level of GPR147 mRNA of the testis tissue was obviously higher in the aged group than that in the youth group (P<0.05). Follo-wing the treatment, the lowered serum T and GnRH contents, the down-regulated expression levels of hypothalamic KISS-1, GPR54, RFRP-3 and GPR147 mRNAs and testicle GnRH-R and GPR54 mRNAs, as well as the up-regulated testicle GPR147 mRNA expression in the aged rats were reversed in the WNM, mild moxibustion and EA groups (not the medication group except serum T) in comparison with the aged control group (P<0.05). The therapeutic effects of WNM, mild moxibustion and EA were significantly superior to those of medication in up-regulating serum GnRH content and expression levels of KISS-1, GPR54, RFRP-3 and GPR147 mRNAs in the hypothalamus and GnRH-R, GPR54 and GPR147 mRNAs in the testis (P<0.05). No significant differences were found among the WNM, mild moxibustion and EA groups in serum T and GnRH contents, and the expression levels of KISS-1, GPR54, RFRP-3 and GPR147 mRNAs in the hypothalamus and GnRH-R and GPR54 and GPR147 mRNAs in the testis (P>0.05)．. The WNM, mild moxibustion and EA therapies have an anti-aging effect by increasing serum T and GnRH levels in aged rats, which is possibly related to their effects in up-regulating the expression of hypothalamic KISS-1, GPR54, RFRP-3 and GPR147 mRNAs and testicle GnRH-R and GPR54 mRNAs, as well as down-regulating testicle GPR147 mRNA expression. The therapeutic effect of WNM, mild moxibustion and EA is evidently superior to that of medication.

De novo transcriptome assembly of the cubomedusa Tripedalia cystophora, including the analysis of a set of genes involved in peptidergic neurotransmission

BackgroundThe phyla Cnidaria, Placozoa, Ctenophora, and Porifera emerged before the split of proto- and deuterostome animals, about 600 million years ago. These early metazoans are interesting, because they can give us important information on the evolution of various tissues and organs, such as eyes and the nervous system. Generally, cnidarians have simple nervous systems, which use neuropeptides for their neurotransmission, but some cnidarian medusae belonging to the class Cubozoa (box jellyfishes) have advanced image-forming eyes, probably associated with a complex innervation. Here, we describe a new transcriptome database from the cubomedusa Tripedalia cystophora.ResultsBased on the combined use of the Illumina and PacBio sequencing technologies, we produced a highly contiguous transcriptome database from T. cystophora. We then developed a software program to discover neuropeptide preprohormones in this database. This script enabled us to annotate seven novel T. cystophora neuropeptide preprohormone cDNAs: One coding for 19 copies of a peptide with the structure pQWLRGRFamide; one coding for six copies of a different RFamide peptide; one coding for six copies of pQPPGVWamide; one coding for eight different neuropeptide copies with the C-terminal LWamide sequence; one coding for thirteen copies of a peptide with the RPRAamide C-terminus; one coding for four copies of a peptide with the C-terminal GRYamide sequence; and one coding for seven copies of a cyclic peptide, of which the most frequent one has the sequence CTGQMCWFRamide. We could also identify orthologs of these seven preprohormones in the cubozoans Alatina alata, Carybdea xaymacana, Chironex fleckeri, and Chiropsalmus quadrumanus. Furthermore, using TBLASTN screening, we could annotate four bursicon-like glycoprotein hormone subunits, five opsins, and 52 other family-A G protein-coupled receptors (GPCRs), which also included two leucine-rich repeats containing G protein-coupled receptors (LGRs) in T. cystophora. The two LGRs are potential receptors for the glycoprotein hormones, while the other GPCRs are candidate receptors for the above-mentioned neuropeptides.ConclusionsBy combining Illumina and PacBio sequencing technologies, we have produced a new high-quality de novo transcriptome assembly from T. cystophora that should be a valuable resource for identifying the neuronal components that are involved in vision and other behaviors in cubomedusae.

Isolation and identification of endogenous RFamide-related peptides 1 and 3 in the mouse hypothalamus.

Although the RFamide-related peptide (RFRP) preproprotein sequence is known in mice, until now, the molecular structure of the mature, functional peptides processed from the target precursor molecule has not been determined. In the present study, we purified endogenous RFRP1 and RFRP3 peptides from mouse hypothalamic tissue extracts using an immunoaffinity column conjugated with specific antibodies against the mouse C-terminus of RFRP-1 and RFRP-3. Employing liquid chromatography coupled with mass spectrometry, we demonstrated that RFRP1 consists of 15 amino acid residues and RFRP3 consists of 10 amino acid residues (ANKVPHSAANLPLRF-NH2 and SHFPSLPQRF-NH2, respectively). To investigate the distribution of RFRPs in the mouse central nervous system, we performed immunohistochemical staining of the brain sections collected from wild-type and Rfrp knockout animals. These data, together with gene expression in multiple tissues, provide strong confidence that RFRP-immunoreactive neuronal cells are localised in the dorsomedial hypothalamic nucleus (DMH) and between the DMH and the ventromedial hypothalamic nuclei. The identification of RFRP1 and RFRP3 peptides and immunohistochemical visualisation of targeting RFRPs neurones in the mice brain provide the basis for further investigations of the functional biology of RFRPs.

Lipopolysaccharide reduces gonadotrophin-releasing hormone (GnRH) gene expression: role of RFamide-related peptide-3 and kisspeptin.

RFamide-related peptide (RFRP)-3 reduces luteinising hormone (LH) secretion in rodents. Stress has been shown to upregulate the expression of the RFRP gene (Rfrp) with a concomitant reduction in LH secretion, but an effect on expression of the gonadotrophin-releasing hormone (GnRH) gene (Gnrh1) has not been shown. We hypothesised that lipopolysaccharide (LPS)-induced stress affects expression of Rfrp, the gene for kisspeptin (Kiss1) and/or Gnrh1, leading to suppression of LH levels in rats. Intracerebroventricular injections of RFRP-3 (0.1, 1, 5 nmol) or i.v. LPS (15μgkg-1) reduced LH levels. Doses of 1 and 5 nmol RFRP-3 were then administered to analyse gene expression by in situ hybridisation. RFRP-3 (5 nmol) had no effect on Gnrh1 or Kiss1 expression. LPS stress reduced GnRH and Kiss1 expression, without affecting Rfrp1 expression. These data indicate that LPS stress directly or indirectly reduces Gnrh1 expression, but this is unlikely to be due to a change in Rfrp1 expression.

Neuropeptide FF modulates neuroendocrine and energy homeostasis through hypothalamic signaling.

Neuropeptide FF (NPFF) is known as a morphine-modulating peptide and was first isolated in 1985. It has been characterized as an RF-amide peptide. The traditional role of NPFF is mediation of the pain response, and it displays both anti-opioid and pro-opioid actions through central nervous system. In the recent decade, additional evidence has revealed some untraditional features of NPFF, such as regulation of the neuroendocrine system, energy homeostasis, anti-inflammation, pain transmission, and peripheral modulation of adipose tissue macrophages. Neuropeptide FF receptor 2 (NPFFR2) is a physiological receptor of NPFF, and the actions of NPFF may occur through downstream NPFFR2 signaling. NPFF and NPFFR2 increase the neuronal activity in various areas of the hypothalamus to modulate the hypothalamic-pituitary-adrenal axis, the autonomic nervous system, food intake, and energy balance. These underlying cellular mechanisms have been explored in the past few years. Here, we review the impact of NPFF and related RF-amide peptides on hypothalamic function. The interaction of NPFF with NPFFR2 in the hypothalamus is emphasized, and NPFF-NPFFR2 system may represent an important therapeutic target in hypothalamic-related disorders in the future.

Immunohistochemical detection of prolactin-releasing peptide2 in the brain of the inshore hagfish Eptatretus burgeri

Prolactin-releasing peptide2 (PrRP2) belongs to the RFamide peptide group and is a paralog of prolactin-releasing peptide (PrRP). Recent studies demonstrated that PrRP2, but not PrRP, regulates prolactin release in teleosts. The evolutionary origin of PrRP and PrRP2 dates back to at least early vertebrates because homologs of PrRP/PrRP2 were identified in lampreys, one of the earliest branch of vertebrates class Agnatha. However, PrRP/PrRP2 remains to be identified in hagfish, another representative species of class Agnatha. Here, we examined the distribution of PrRP2 in the brain and pituitary of the inshore hagfish Eptatretus burgeri to obtain further understanding of the neuroendocrine system of PrRP2. PrRP2-immunoreactive (ir) cell bodies were detected in the infundibular nucleus of hypothalamus (HYinf). PrRP2-ir fibers were restricted around PrRP2-ir cell bodies and were not detected in the dorsal wall of the neurohypophysis compared to the abundant PrRP2-ir fiber distribution in the brain and innervation to the pituitary in other vertebrates. To examine possible reciprocal connections of PrRP2 and other neuropeptides, we further conducted dual-label immunohistochemistry of PrRP2 and the PQRFamide (PQRFa) peptide or corticotropin-releasing hormone (CRH). Reciprocal connections are suggested between PrRP2 and PQRFa neurons as well as between PrRP2 and CRH neurons. The present study demonstrates, for the first time, that PrRP2 is expressed in the brain of inshore hagfish. The restricted distribution of PrRP2-ir fibers in the HYinf suggests that PrRP2 does not directly regulate the pituitary gland, but regulates the function of the HYinf where PQRFa and CRH are expressed.