Neurokinin B (NKB) was recently identified as a key regulator of reproduction in mammals and fish. Fish were found to possess a specific novel neurokinin termed NKF. To study the role of NKB/NKF in the regulation of fish reproduction and to investigate the role of NKB/NKF and their receptors in the piscine pituitary, we have identified the NKB/tachikinin 3 receptor (tac3r) system in tilapia. Bioinformatics and phylogenetic analyses have demonstrated that the tilapia holds 1 putative tac3 gene and 2 NKB receptor genes (tac3ra and tac3rb) that clustered with other piscine Tac3 and NKB receptor lineages. Furthermore, we found that in African cichlids, NKB peptides differ from other vertebrate NKBs in their C-terminal sequence, possessing isoleucine instead of valine as the X in the NKB FXGLM-NH2-terminal consensus sequence. Signal transduction analysis demonstrated that tilapia NKB (tiNKB), tiNKF, and human NKB activated both CRE-luc and SRE-luc transcriptional activity of both tilapia and human NKB receptors. Two hours after ip injection of tiNKB, the plasma levels of both FSH and LH were increased, whereas tiNKF was more effective in increasing LH levels. However, tiNKB was more effective than tiNKF in increasing both FSH and LH from tilapia pituitary dispersed cells. Using in situ hybridization and fluorescent immunohistochemistry, we have shown that LH cells possess tac3, tac3ra, and tac3rb mRNAs, whereas FSH cells possess mainly tac3rb and tac3ra and tac3 to a much lesser extent. These results suggest that the members of the NKB/tac3r system may serve as paracrine/autocrine regulators of gonadotropin release in fish pituitary.
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.
Neurokinin B (NKB) and its cognate receptor (NK3R) are emerging as important components of the neuroendocrine regulation of reproduction. Unlike mammalian tac3, which encodes only one mature peptide (namely NKB), two mature peptides are predicted for each tac3 gene in fish and frogs. Therefore, it was designated as Neurokinin F (NKF). Hormone analogs with high and long-lasting biological activity are important tools for physiological and biological research; however, the availability of piscine-specific analogs is very limited. Therefore, we have developed specific NKB and NKF analogs based on the structure of the mammalian NKB analog-senktide. These analogs, specifically designed for longer half-lives by methylation of proteolysis sites, exhibited activity equal to those of the native NKB and NKF in short-term signal-transduction assays of tilapia NKB receptors. However, the analogs were found to be able to significantly increase the release of luteinizing hormone (LH), follicle stimulating hormone (FSH) and growth hormone (GH) in tilapia, as fast as 1 h after intraperitoneal (IP) injection. The impact of the analogs on LH and FSH secretion lasted longer compared to the effect of native peptides and salmon GnRH analog (sGnRHa). In addition, we harvested pituitaries 24 h post injection and measured LH, FSH and GH mRNA synthesis. Both analogs elevated mRNA levels of LH and GH, but only NKB analog increased FSH mRNA levels in the pituitary and all GnRH forms in the brain. NKB receptors were co-localized with all three types the GnRH neurons in tilapia brain in situ. We previously showed a direct effect of NKB at the pituitary level, and these new results suggest that the stronger impact of the NKB analog on GTH release is also due to an indirect effect through the activation of GnRH neurons. These results suggest that novel synthetic NKB analogs may serve as a tool for both research and agricultural purposes. Finally, the biological activity and regulatory role of NKB in tilapia brain and pituitary suggest that the NKB/NKBR system in fish is an important reproductive regulator in a similar way to the kisspeptin system in mammals.