Abstract
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.
Highlights
Reproduction is an essential process that is regulated by numerous factors along the brain/hypothalamus-pituitary-gonad (HPG) axis
Since we showed an effect of Neurokinin B (NKB) analog on gonadotrophin-releasing hormones (GnRHs) synthesis in vivo, we tested whether or not this is through a direct action of huNKB Senktide tiNKB tiNKB analog tiNKF Neurokinin F (NKF) analog cAMP responsive element (CRE)-Luc was used as a reporter of protein kinase A (PKA) activation; Serum responsive element (SRE)-Luc was used as a reporter of PKC activation
The tac3 genes and their receptors were first identified in zebrafish, and it was predicted by cleavage analysis that preprotachykinin 3 encodes two mature peptides produced from each tac3 gene in many fish species [20, 34, 41]
Summary
Reproduction is an essential process that is regulated by numerous factors along the brain/hypothalamus-pituitary-gonad (HPG) axis. During the past decade the neuropeptides kisspeptin (encoded by Kiss1) and neurokinin B (NKB, encoded by tac3) were identified as important regulators of GnRH synthesis and release in vertebrates [reviewed by Topaloglu and Kotan [5], Pinilla et al [6], Parhar et al [7]]. Fish kiss was first reported in zebrafish (Danio rerio) [10, 11] but in some teleost two genes were found encoding kisspeptin-like structure [12], some fish species, such as tilapia [13], red seabream (Pagrus major) [14] and more, express only the kiss type gene. While kisspeptin is a key regulator in GnRH release in mammals [16], its role in teleosts remains controversial, especially since knockout of the two kisspeptins and two KissRs genes in zebrafish and medaka (Oryzias latipes) had no obvious effect on their reproductive capability [17, 18]. Teleost have one or two tac genes that encodes two mature peptides, NKB and NKB-related peptide
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