Evidence for the existence of a functional Kiss1/Kiss1 receptor pathway in fish
Introduction
Puberty in vertebrates is defined as the period during which a sexually immature animal acquires the capacity to reproduce. The initiation of puberty in teleosts occurs following gonadal sex differentiation [38] and is characterized by the onset of spermatogenesis in males [35] and vitellogenic ovarian development in females [33].
In teleosts, as in other vertebrates, the brain–pituitary–gonad (BPG) axis controls sexual development and reproduction. Gonadotropin-releasing hormone (GnRH) produced in the brain governs the release of the gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), from the pituitary, which then regulate gonadal development and function [27]. The cascades of hormones and growth factors that regulate gonad growth and gamete maturation in fish have been well characterized in many fish species, but the triggers and mechanisms that initiate puberty are not known for any fish species [41].
Recently, exciting developments have been made in the study of puberty in mammals, where kisspeptins, peptide products of the Kiss1 gene, and their receptor (Kiss1r; previously designated GPR54) have been shown to be key factors regulating the onset of puberty [8], [34], [36], [39]. In mammals, Kiss1r is mainly expressed in the placenta and specific areas of the adult brain (including the hypothalamus and pituitary), as well as a variety of other tissues including the pancreas, thymus, spleen and testes [8], [25]. Mammalian Kiss1 is mainly expressed in the placenta, with considerably lower levels of expression in the brain, testes, liver, pancreas and small intestine [8]. In situ hybridization studies in rats and mice have demonstrated that Kiss1r transcripts are co-localized with GnRH neurons in the hypothalamus [17], [23]. In mammals, mutations in either the Kiss1r or the Kiss1 gene have been shown to result in hypogonadotropic hypogonadism, a syndrome that is characterized by complete or partial failure of pubertal development due to impaired secretion of LH and FSH [10], [11], [14], [37]. Kiss1- or Kiss1r-deficient mice also had developmental and histopathological abnormalities in the genitalia in both males and females (small testes in males and a delay in vaginal opening in females) [10], [11], [14], [37]. These mice had normal levels of GnRH in the hypothalamus, their gonads remained sensitive to exogenous gonadotropins, and the pituitary gonadotropes remained responsive to stimulation by GnRH, indicating that the Kiss1/Kiss1r pathway influences the processing or release of GnRH [10], [37]. Supporting the role of the Kiss1/Kiss1r pathway in GnRH release, in hypogonadal, Kiss1- or Kiss1r-deficient mice, GnRH neurons showed normal projections to the median eminence and were anatomically normal [10], [23]. Administration of kisspeptins in immature mammals has been shown to induce the precocious activation of the gonadotropic axis [29]. These effects were not observed after administration of kisspeptins to Kiss1r-deficient mice [23], or in adult male rats that were pre-treated with a GnRH antagonist (acycline [36]). In contrast, administration of kisspeptins to Kiss1-deficient mice resulted in the release of LH [10]. Together, these results demonstrate that kisspeptins act directly and uniquely by means of Kiss1r-GnRH signaling.
Recently, the Kiss1 receptor has been characterized in fish, including in tilapia (Oreochromis niloticus [32]), grey mullet (Mugil cephalus [30]), cobia (Rachycentron canadum [24]) and fathead minnow (Pimephales promelas [13]). Gene expression studies in fish have shown that the levels of Kiss1r mRNA increase dramatically during the onset of puberty, suggesting that the Kiss1/Kiss1r pathway plays a role in the initiation of puberty in fish as it does in mammals. Kiss1, however, has not yet been isolated and characterized in non-mammalian species. In the present study, we sought for evidence for the presence of the Kiss1/Kiss1r pathway in fish, using a combination of bioinformatics (comparative genomics) and empirical studies. Putative sequences for kiss1 and kiss1r were identified in the genomes of five teleost species and this information was used to clone cDNAs for these genes from zebrafish. The expression of kiss1 and its receptor, kiss1r, was determined in adult zebrafish across a range of tissues using RT-PCR. The structure of zebrafish Kiss1 was investigated for the presence of a putative signal peptide, cleavage and amidation sites and compared to its mammalian orthologues in order to uncover evidence for functional similarity. The region containing the kiss1 locus in the zebrafish genome was also investigated for conservancy across vertebrate genomes.
Section snippets
Animals and tissue sample collection
Zebrafish (Danio rerio) were obtained from breeding stocks at the University of Exeter. Male and female (n = 3 for each sex) adult fish were sacrificed humanely using an overdose of benzocaine (0.5 g/L; ethyl-p-aminobenzoate; Fisher, UK) according to UK Home Office guidelines. Brain, eye, heart, liver, intestine, testis, ovary, and adipose tissues were isolated, snap-frozen in liquid nitrogen and stored at −80 °C until use for RNA extraction.
Comparative genomics
The sequence of human kisspeptin-10 (YNWNSFGLRF-NH2) was
Characterization of zebrafish kiss1r
A putative sequence for zebrafish kiss1r was found in the NCBI EST database and a partial cDNA sequence of kiss1r (809 bp) was subsequently isolated from zebrafish brains using RT-PCR. During the course of this study, a predicted putative full-length sequence for zebrafish kiss1r was also found in the genome (chromosome 2; ENSDART00000041353). Comparisons of this predicted gene with our cDNA sequence for kiss1r obtained by PCR showed that they were identical (Fig. 1A). Zebrafish Kiss1r showed
Discussion
Mammalian Kiss1 is processed into a number of kisspeptins, including kisspeptin-54, -14, -13, and -10 (C-terminal cleavage fragments of Kiss1), all of which have been shown to bind and activate the Kiss1 receptor in a similar manner [19], [25], [31]. Kisspeptin-10, the shortest kisspeptin and part of the C-terminal end of all other kisspeptins found, is required for biological activation of the Kiss1 receptor [19], [25], [31]. Overall, the sequences obtained for Kiss1 for the fish species
Acknowledgments
We would like to thank Dr. Eduarda Santos for helpful comments on an earlier version of this manuscript. R.v.A. was funded by the Natural Environment Research Council (NERC) and the Environment Agency, UK (Grant ref. NE/C002369/1). A.L. was funded by NERC (Grant refs. NE/D002818/1 and NE/D002508/1) to CRT.
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