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.

Reproduction in vertebrates is controlled by the brain-pituitary-gonad axis, where the two gonadotropins follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) play vital parts by activating their cognate receptors in the gonads. The main purpose of this work was to study intra- and interspecies ligand promiscuity of teleost gonadotropin receptors, since teleost receptor specificity is unclear, in contrast to mammalian receptors. Receptor activation was investigated by transfecting COS-7 cells with either Fsh receptor (mdFshr, tiFshr) or Lh receptor (mdLhr, tiLhr), and tested for activation by recombinant homologous and heterologous ligands (mdFshβα, mdLhβα, tiFshβα, tiLhβα) from two representative fish orders, Japanese medaka (Oryzias latipes, Beloniformes) and Nile tilapia (Oreochromis niloticus, Cichliformes). Results showed that each gonadotropin preferentially activates its own cognate receptor. Cross-reactivity was detected to some extent as mdFshβα was able to activate the mdLhr, and mdLhβα the mdFshr. Medaka pituitary extract (MPE) stimulated CRE-LUC activity in COS-7 cells expressing mdlhr, but could not stimulate cells expressing mdfshr. Recombinant tiLhβα, tiFshβα and tilapia pituitary extract (TPE) could activate the mdLhr, suggesting cross-species reactivity for mdLhr. Cross-species reactivity was also detected for mdFshr due to activation by tiFshβα, tiLhβα, and TPE, as well as for tiFshr and tiLhr due to stimulation by mdFshβα, mdLhβα, and MPE. Tissue distribution analysis of gene expression revealed that medaka receptors, fshr and lhr, are highly expressed in both ovary and testis. High expression levels were found for lhr also in brain, while fshr was expressed at low levels. Both fshr and lhr mRNA levels increased significantly during testis development. Amino acid sequence alignment and three-dimensional modelling of ligands and receptors highlighted conserved beta sheet domains of both Fsh and Lh between Japanese medaka and Nile tilapia. It also showed a higher structural homology and similarity of transmembrane regions of Lhr between both species, in contrast to Fshr, possibly related to the substitution of the conserved cysteine residue in the transmembrane domain 6 in medaka Fshr with glycine. Taken together, this is the first characterization of medaka Fshr and Lhr using homologous ligands, enabling to better understand teleost hormone-receptor interactions and specificities. The data suggest partial ligand promiscuity and cross-species reactivity between gonadotropins and their receptors in medaka and tilapia.

In brief: The hypothalamus–pituitary–gonad axis integrates environmental and internal signals to tune reproductive functions. We hypothesized that in fish, Gnrh predominantly stimulates luteinizing hormone (LH) secretion, while Cck selectively regulates follicle-stimulating hormone (FSH), thereby uncovering a novel role for Cck as a metabolic gatekeeper that links reproductive activity with nutritional status. In fish, the hypothalamus–pituitary–gonad axis governs reproduction in response to environmental and internal signals. FSH regulates gonadal growth, while LH controls maturation. Gonadotropin-releasing hormone (Gnrh) plays a key role in this process, while the role of the satiety hormone cholecystokinin (Cck) is under investigation. We hypothesized that Gnrh and Cck differentially regulate tilapia LH and FSH, reflecting distinct reproductive and metabolic roles. Gnrh receptor (r) 1 was localized to FSH cells and detected only in juvenile fish, while GnRrhr3 was found in LH cells across both juvenile and mature stages. GnRH stimulation significantly increased LH release, while FSH levels showed only a moderate rise. Structural analysis revealed that Gnrhr3 exhibits more stable and rapid ligand binding and activates protein kinase A and protein kinase C (PKC) pathways, whereas Gnrhr1 signals exclusively through the PKC/Ca ²⁺ pathway. Elevated FSH levels and gnrhr 1 expression were observed during early vitellogenesis, coinciding with increased cck-rba expression in FSH cells. Cck-positive neurons originating in the brain were observed to terminate in the adenohypophysis, specifically near FSH-secreting cells. In addition, c ck-rba was exclusively expressed in FSH cells, and tilapia Cck injection significantly elevated plasma FSH levels while reducing food intake, highlighting the role of Cck in linking reproduction to nutritional status. Together, these findings indicate that Gnrh predominantly regulates LH secretion via Gnrhr3, while Cck, through Cck-rba, specifically controls FSH, linking reproduction to nutritional status. Cck acts as a metabolic gatekeeper, aligning gonadal development with energy availability and ensuring reproduction when energy is sufficient.

NKB (Neurokinin B) is already known to play a crucial role in fish reproduction, but little is known about the structure and function of NKB receptors. Based on an in silico model of the tilapia NKB receptor Tachykinin 3 receptor a (tiTac3Ra) found in the current study, we determined the key residues involved in binding to tilapia NKB and its functional homologue NKF (Neurokinin F). Despite studies in humans suggesting the crucial role of F251^6.44 and M289^7.43 in NKB binding, no direct peptide interaction was observed in tilapia homologs. In-silico, Ala mutations on residues F251^6.44 and M289^7.43 did not influence binding affinity, but significantly affected the stability of tiTac3Ra. Moreover, in vitro studies indicated them to be critical to tiNKB/tiNKF-induced receptor activity. The binding of NKB antagonists to tiTac3Ra both in-vitro and in vivo inhibits FSH (follicle stimulating hormone) and LH (luteinizing hormone) release and sperm production in mature tilapia males. Non-peptide NKB antagonist SB-222200 had a strong inhibitory effect on the Tac3Ra activation. SB-222200 also decreased LH plasma levels; two hours post intraperitoneal injection, changed sperm volume and the ratios of the different stages along the spermatogenesis in tilapia testes.

The gonadotropins follicle stimulating hormone (FSH) and luteinizing hormone (LH) are key regulators of sexual development and the reproductive cycle in vertebrates. Unlike most G protein-coupled receptors (GPCR), the FSHR and LHR have large extracellular domains containing multiple leucine-rich repeats, which leads to an elaborate mechanism of receptor activation via orthosteric sites that is difficult to manipulate synthetically. To bypass the orthosteric mechanism, in this study using carp as a model organism we identified allosteric sites capable of receptor activation on the transmembrane domain, which are spatially separated from the orthosteric sites. We have further generated pharmacophore hypothesis based on the structural motifs and exposed residues of these cavities. Using available online small compound libraries consisting of >70000 small molecules, we have thereon used receptor cavity-based hypothesis and other screening stages to identify potential modulators of the allosteric binding site on the carp FSHR and LHR in-silico . We then examined by in vitro transactivation assay the effect of four candidate compounds on FSHR and LHR, as compared to the activity of native ligands. Our results reveal both specific and dual effective allosteric modulators for FSHR and LHR, demonstrating the potential of our approach for efficient pharmacophore-based screening.