Potential Role in Controlling Fish Reproduction
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Neurokinin Bs and neurokinin B receptors in zebrafish- potential role in controlling fish reproduction Jakob Birana, Ori Palevitcha, Shifra Ben-Dorb, and Berta Levavi-Sivana,1 aDepartment of Animal Sciences, The Robert H. Smith Faculty of Agriculture, Food, and Environment, Hebrew University of Jerusalem, Rehovot 76100, Israel; and bDepartment of Biological Services, Weizmann Institute of Science, Rehovot 76100, Israel Edited by John E. Halver, University of Washington, Seattle, WA, and approved April 27, 2012 (received for review December 2, 2011) The endocrine regulation of vertebrate reproduction is achieved by zebrafish (Danio rerio) as a model, was to examine the in- the coordinated actions of several peptide neurohormones, tachy- volvement of NKB in fish reproduction. kinin among them. To study the evolutionary conservation and NKB is a member of the tachykinin (TK) family of peptides. physiological functions of neurokinin B (NKB), we identified tachy- TKs are characterized by a common carboxyl-terminal amino acid kinin (tac) and tac receptor (NKBR) genes from many fish species, sequence of FXGLM-NH2 (where X is a hydrophobic residue), and cloned two cDNA forms from zebrafish. Phylogenetic analysis and include substance P, neurokinin A (NKA) and NKB, as well γ showed that piscine Tac3s and mammalian neurokinin genes arise as neuropeptide K, neuropeptide- , and hemokinin-1 (7). NKB is from one lineage. High identity was found among different fish the only TK synthesized from the preprotachykinin-B gene (8), species in the region encoding the NKB; all shared the common C- which is currently designated as TAC3 in mammals, except for terminal sequence. Although the piscine Tac3 gene encodes for two rodents, where it was named Tac2. Because there are different names for the gene encoding NKB in different species (TAC3 or putative tachykinin peptides, the mammalian ortholog encodes for fi Tac2), in this article we will refer to mRNA products of this gene only one. The second sh putative peptide, referred to as neuro- as tac3 mRNA and to the peptides as NKB. The receptor that kinin F (NKF), is unique and found to be conserved among the fish tac3a binds NKB, which is termed NKBR in humans, will be termed species when tested in silico. was expressed asymmetrically in tac3r at the mRNA level and Tac3r at the protein level. fi tac3a the habenula of embryos, whereas in adults zebra sh -ex- Until now, NKB was not cloned from any fish species, nor was pressing neurons were localized in specific brain nuclei that are the NKB/NKBR system shown to be involved in reproduction or SCIENCES known to be involved in reproduction. Zebrafish tac3a mRNA levels puberty. We report here the identification of previously un- AGRICULTURAL gradually increased during the first few weeks of life and peaked at identified fish NKB/NKBR genes and their possible involvement pubescence. Estrogen treatment of prepubertal fish elicited in- in the control of reproduction. creases in tac3a, kiss1, kiss2, and kiss1ra expression. The synthetic zebrafish peptides (NKBa, NKBb, and NKF) activated Tac3 receptors Results and Discussion 2+ via both PKC/Ca and PKA/cAMP signal-transduction pathways Cloning Two Types of tac3 and tac3r and Their Phylogenetic Analysis. in vitro. Moreover, a single intraperitoneal injection of NKBa and As the first step toward examining the involvement of the NKB/ NKF significantly increased leuteinizing hormone levels in mature NKBRs (tac3r) in the control of reproduction in fish, we report female zebrafish. These results suggest that the NKB/NKBR system here the identification of the full-length tac3a and tac3b cDNA may participate in neuroendocrine control of fish reproduction. from zebrafish brain using real-time PCR with specificprimers (Table S1). Tac3a contains the decapeptide sequence EMH- gonadotropin-releasing hormone | kisspeptin | teleost | gonadotropin DIFVGLM (Fig. S1A) (accession no. JN392856), whereas tac3b contains a 24-aa peptide (STGINREAHLPFRPNMNDIFVGLL) eproduction is a highly integrated and complex function that (Fig. S1B) (accession no. JN392857), both with the TK signature motif (FXGLM-NH ) flanked by potential dibasic cleavage sites requires synchronized production of gametes by both sexes at 2 R and an adjacent glycine at the C terminus for amidation (9). Typ- an optimum time for offspring survival. Fish show an enormous ically, following prohormone convertase action, a carboxypepti- variety of reproductive strategies (1), and were recently chosen as dase removes the C-terminal dibasic residues, and a peptidylglycine models for the study of growth, metabolism, and human diseases. fi a-amidating enzyme converts the exposed glycine into a C-terminal The hypothalamic regulation of gonadotropin secretion in sh is amide (10). At the protein level, the resulting zfNKBa (Tac3a) different from that of mammals, from both endocrinal and ana- hormone precursor displayed around 25% identity with human or tomical aspects. In teleosts, the pituitary is innervated directly by mouse TAC3, 55% with putative salmon Tac3a, and 52% with neurons projecting to the vicinity of the pituitary gonadotrophs medaka Tac3 identified in this study (accession nos. BK008102 and (2). Among the neuropeptides released by these nerve endings are BK008114, respectively). zfNKBb (Tac3b) showed only around gonadotrophin-releasing hormones (GnRHs) and dopamine, 18% identity with human and mouse TAC3, 40% and 36% identity which act as stimulatory and inhibitory factors on the release of with salmon and medaka Tac3b, respectively. The zebrafish tac3s luteinizing hormone (LH) and follicle-stimulating hormone (3). shared only a 36% identity (Fig. S1 and Table S2). However, new actors have recently entered the field of re- In our search for the identification of Tac3 sequences con- productive physiology: kisspeptins, neurokinin, and dynorphin taining the NKB peptide sequence in fish mRNAs and ESTs have all been implicated in controlling GnRH (4). known to date, we encountered 30 previously unidentified Topaloglu et al. (5) found that humans bearing loss-of-func- tion mutations of the genes encoding either neurokinin B (NKB) or its cognate receptor, neurokinin receptor 3 (NKBR, Tac3r) Author contributions: J.B. and B.L.-S. designed research; J.B. and O.P. performed research; displayed hypogonadotropic hypogonadism; this seminal report J.B., S.B.-D., and B.L.-S. analyzed data; and J.B., O.P., and B.L.-S. wrote the paper. implicated NKB signaling as an essential factor in the onset of The authors declare no conflict of interest. puberty and control of gonadotropin secretion in mammals. This article is a PNAS Direct Submission. Recent studies provided evidence that, in mammals, a group of Data deposition: The sequences reported in this paper has been deposited in the GenBank neurons in the hypothalamic arcuate nucleus (ARC) are steroid- database [accession nos. JN392856 (zftac3a), JN392857 (zftac3b), JF317292 (zftac3ra), responsive and coexpress NKB, kisspeptin, dynorphin, NKBR JF317293 (zftac3rb), and BK008087–BK008126]. and estrogen receptor α(6). Compelling evidence indicates that 1To whom correspondence should be addressed. E-mail: [email protected]. these neurons function in the hypothalamic circuitry regulating This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. GnRH secretion. The main objective of the present study, using 1073/pnas.1119165109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1119165109 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 piscine Tacs. We generated a phylogenetic tree of all available phosphorylation by protein kinase C, protein kinase A, casein vertebrate neurokinin genes (Fig. 1A and Fig. S2A); it showed kinase II, tyrosine kinase, and N-myristoylation (Fig. S3). The N that the vertebrate neurokinin genes fall into several distinct and C termini are, as in other G protein-coupled receptors, the lineage groups. The identified Tac3 precursors from fish were most divergent regions. In our bioinformatic search for NKB clustered with all other previously cloned or predicted Tac3 receptors we found four additional TK receptor genes in zebra- sequences from mammals, frogs, and alligators (Fig. 1A); a sec- fish. To enable assignment of the genes to the three TK receptor ond lineage included Tac1 from both mammals and fish that subfamilies already defined in mammals (12), we identified family were identified in the present study, and the third lineage in- members in additional species, particularly in other fish. The cluded mammalian Tac4 and a unique piscine group, now named phylogenetic tree containing vertebrate TK receptors form three Tac4 (Fig. S2A). No precursors containing the exact NKB se- clearly separable groups that corresponded to TAC3R, TAC1R, quence were found in invertebrate species (11). and TAC2R (Fig. S2B). Putative orthologs of NK receptor fi We cloned the full-length tac3ra and tac3rb cDNA from members were also identi ed in several nonvertebrate species, zebrafish brain by PCR with specific primers (Table S1). The Caenorhabditis elegans, ciona, and octopus; these served as out- predicted tac3ra and tac3rb N termini have features consistent group sequences in determining the root of these three groups, with a signal peptide, as defined by SignalP program analysis which was located between TAC2R and the others, indicating (Fig. S1). Sequence analysis of the two types of zebrafish that these groups split early in the family evolution. The tree receptors identified distinct potential sites for N-glycosylation,