The KISS1 gene encodes the kisspeptin neuropeptide, which activates the KISS1 receptor (KISS1R; G protein-coupled receptor 54; GPR54) and participates in neuroendocrine regulation of GnRH secretion. To study the physiological function(s) and evolutionary conservation of KISS1, we cloned opossum, Xenopus, and zebrafish kiss1 cDNAs. Processing zebrafish, Xenopus, or opossum KISS proteins would liberate a carboxy-terminal amidated peptide with 52, 54, or 53 amino acid residues, respectively. Phylogenetic analysis of all known vertebrate KISS1 peptides showed clear clustering of the sequences according to canonical vertebrate classes. The zebrafish kiss1 gene consists of two exons and one intron. Real-time PCR analysis of two kiss1R cloned from zebrafish brain found expression of kiss1, kiss1ra, and kiss1rb, with kiss1ra-more similar to other piscine Kiss1 receptors-highly expressed in the gonads and kiss1rb in other nonbrain tissues. In females kiss1 mRNA levels gradually increased during the first few weeks of life to peak in fish with ovaries containing mature oocytes, while in males kiss1 mRNA levels peaked after 6 wk postfertilization when the testes exhibited initial stages of spermatogenesis and decreased after puberty. Zebrafish kiss1ra and kiss1rb were expressed differentially with similar patterns in both genders. These results indicate that the Kiss1/Kiss1r system may participate in puberty initiation in fish as well. Like human KISS1R, Kiss1ra transduces its activity via the PKC pathway, whereas Kiss1rb does so via both PKC and PKA pathways. The human KISS1R was highly activated by both huKISS10amide and zfKISS10amide, whereas both zebrafish Kiss1 receptor types were less sensitive to amidation.

The endocrine regulation of vertebrate reproduction is achieved by the coordinated actions of several peptide neurohormones, tachykinin among them. To study the evolutionary conservation and physiological functions of neurokinin B (NKB), we identified tachykinin (tac) and tac receptor (NKBR) genes from many fish species, and cloned two cDNA forms from zebrafish. Phylogenetic analysis showed that piscine Tac3s and mammalian neurokinin genes arise from one lineage. High identity was found among different fish species in the region encoding the NKB; all shared the common Cterminal sequence. Although the piscine Tac3 gene encodes for two putative tachykinin peptides, the mammalian ortholog encodes for only one. The second fish putative peptide, referred to as neurokinin F (NKF), is unique and found to be conserved among the fish species when tested in silico. tac3a was expressed asymmetrically in the habenula of embryos, whereas in adults zebrafish tac3a-expressing neurons were localized in specific brain nuclei that are known to be involved in reproduction. Zebrafish tac3a mRNA levels gradually increased during the first few weeks of life and peaked at pubescence. Estrogen treatment of prepubertal fish elicited increases in tac3a, kiss1, kiss2, and kiss1ra expression. The synthetic zebrafish peptides (NKBa, NKBb, and NKF) activated Tac3 receptors via both PKC/Ca 2+ and PKA/cAMP signal-transduction pathways in vitro. Moreover, a single intraperitoneal injection of NKBa and NKF significantly increased leuteinizing hormone levels in mature female zebrafish. These results suggest that the NKB/NKBR system may participate in neuroendocrine control of fish reproduction.gonadotropin-releasing hormone | kisspeptin | teleost | gonadotropin R eproduction is a highly integrated and complex function that requires synchronized production of gametes by both sexes at an optimum time for offspring survival. Fish show an enormous variety of reproductive strategies (1), and were recently chosen as models for the study of growth, metabolism, and human diseases. The hypothalamic regulation of gonadotropin secretion in fish is different from that of mammals, from both endocrinal and anatomical aspects. In teleosts, the pituitary is innervated directly by neurons projecting to the vicinity of the pituitary gonadotrophs (2). Among the neuropeptides released by these nerve endings are gonadotrophin-releasing hormones (GnRHs) and dopamine, which act as stimulatory and inhibitory factors on the release of luteinizing hormone (LH) and follicle-stimulating hormone (3). However, new actors have recently entered the field of reproductive physiology: kisspeptins, neurokinin, and dynorphin have all been implicated in controlling GnRH (4).Topaloglu et al. (5) found that humans bearing loss-of-function mutations of the genes encoding either neurokinin B (NKB) or its cognate receptor, neurokinin receptor 3 (NKBR, Tac3r) displayed hypogonadotropic hypogonadism; this seminal report implicated NKB signaling as an essential factor in the onset of puberty...

Kisspeptin is an important regulator of reproduction in many vertebrates. The involvement of the two kisspeptins, Kiss1 and Kiss2, and their receptors, Gpr54-1 and Gpr54-2, in controlling reproduction was studied in the brains of the modern teleosts, striped and hybrid basses. In situ hybridization and laser capture microdissection followed by quantitative RT (QRT)-PCR detected coexpression of kiss1 and kiss2 in the hypothalamic nucleus of the lateral recess. Neurons expressing gpr54-1 and gpr54-2 were detected in several brain regions. In the preoptic area, gpr54-2 was colocalized in GnRH1 neurons while gpr54-1 was expressed in cells attached to GnRH1 fibers, indicating two different modes of GnRH1 regulation. The expression of all four genes was measured in the brains of males and females at different life stages using QRT-PCR. The levels of kiss1 and gpr54-1 mRNA, the latter being expressed in minute levels, were consistently lower than those of kiss2 and gpr54-2. While neither gene's expression increased at prepuberty, all were dramatically elevated in mature females. The levels of kiss2 mRNA increased also in mature males. Kiss1 peptide was less potent than Kiss2 in elevating plasma luteinizing hormone levels and in up-regulating gnrh1 and gpr54-2 expression in prepubertal hybrid bass in vivo. In contrast, during recrudescence, Kiss1 was more potent than Kiss2 in inducing luteinizing hormone release, and Kiss2 down-regulated gnrh1 and gpr54-2 expression. This is the first report in fish to demonstrate the alternating actions and the importance of both neuropeptides for reproduction. The organization of the kisspeptin system suggests a transitional evolutionary state between early to late evolving vertebrates.

LPXRFamide (LPXRFa) peptides have been characterized for their ability to inhibit gonadotropin (GTH) release in birds and stimulate growth hormone (GH) release in frogs. However, their involvement in regulating the reproductive hypothalamo-pituitary-gonadal axis in mammals and fish is inconclusive. To study the role of LPXRFa peptides in the regulation of GTH secretion, we cloned tilapia LPXRFa and LPXRF receptor (LPXRF-R). Processing of the tilapia preproLPXRFa liberated three mature LPXRFa peptides that varied in size and post-translational modifications. Phylogenetic analysis of LPXRFa and the closely related RFamide peptide PQRFa showed clear clustering of each peptide sequence with its orthologs from various vertebrates. Signal-transduction analysis of the tilapia LPXRF-R in COS-7 cells showed clear stimulation of CRE-dependent luciferase activity, whereas the human NPFFR1 showed suppression of forskolin-induced CRE-dependent activity in this system. Administration of the tilapia pyroglutaminated LPXRFa-2 peptide to primary cell culture of tilapia pituitaries, or to reproductive female tilapia by ip injection, positively regulated both LH and FSH release in vivo and in vitro. Using double-labeled fluorescent in-situ hybridization and immunofluorescence, βLH cells were found to co-express both tilapia lpxrf and tilapia lpxrf-r mRNA, whereas some of the βFSH cells coexpressed only lpxrf-r mRNA. No coexpression of tilapia lpxrf-r was identified in GH-positive cells. These findings suggest that the LPXRFa system is a potent positive regulator of the reproductive neuroendocrine axis of tilapia.

The hypothalamus is a brain region which regulates homeostasis by mediating endocrine, autonomic and behavioral functions. It is comprised of several nuclei containing distinct neuronal populations producing neuropeptides and neurotransmitters that regulate fundamental body functions including temperature and metabolic rate, thirst and hunger, sexual behavior and reproduction, circadian rhythm, and emotional responses. The identity, number and connectivity of these neuronal populations are established during the organism’s development and are of crucial importance for normal hypothalamic function. Studies have suggested that developmental abnormalities in specific hypothalamic circuits can lead to obesity, sleep disorders, anxiety, depression and autism. At the molecular level, the development of the hypothalamus is regulated by transcription factors (TF), secreted growth factors, neuropeptides and their receptors. Recent studies in zebrafish and mouse have demonstrated that some of these molecules maintain their expression in the adult brain and subsequently play a role in the physiological functions that are regulated by hypothalamic neurons. Here, we summarize the involvement of some of the key developmental factors in hypothalamic development and function by focusing on the mouse and zebrafish genetic model organisms.