Neurokinin Bs and neurokinin B receptors in zebrafish-potential role in controlling fish reproduction

Abstract

The endocrine regulation of vertebrate reproduction is achieved by the coordinated actions of several peptide neurohormones, tachykinin among them. To study the evolutionary conservation and physiological functions of neurokinin B (NKB), we identified tachykinin (tac) and tac receptor (NKBR) genes from many fish species, and cloned two cDNA forms from zebrafish. Phylogenetic analysis showed that piscine Tac3s and mammalian neurokinin genes arise from one lineage. High identity was found among different fish species in the region encoding the NKB; all shared the common C-terminal sequence. Although the piscine Tac3 gene encodes for two putative tachykinin peptides, the mammalian ortholog encodes for only one. The second fish putative peptide, referred to as neurokinin F (NKF), is unique and found to be conserved among the fish species when tested in silico. tac3a was expressed asymmetrically in the habenula of embryos, whereas in adults zebrafish tac3a-expressing neurons were localized in specific brain nuclei that are known to be involved in reproduction. Zebrafish tac3a mRNA levels gradually increased during the first few weeks of life and peaked at pubescence. Estrogen treatment of prepubertal fish elicited increases in tac3a, kiss1, kiss2, and kiss1ra expression. The synthetic zebrafish peptides (NKBa, NKBb, and NKF) activated Tac3 receptors via both PKC/Ca(2+) and PKA/cAMP signal-transduction pathways in vitro. Moreover, a single intraperitoneal injection of NKBa and NKF significantly increased leuteinizing hormone levels in mature female zebrafish. These results suggest that the NKB/NKBR system may participate in neuroendocrine control of fish reproduction.

Fig. 1.

Unrooted phylogenetic tree of neurokinin (A) or neurokinin receptor (B) sequences generated with neighbor-joining (ClustalW 2.1) and maximum likelihood (Phylip 3.69, ProML) on the basis of alignments performed both by ClustalW and Muscle (3.8.31), and visualized with FigTree 1.3.1. The Tac1 and Tac4 (A) and Tacr1 and Tacr2 (B) were grouped (full trees in Fig. S2). The sequences identified in this study are marked in bold. Gene nomenclature has been standardized to Tac3, and species are indicated for illustration and comparison. Numbers at nodes indicate the bootstrap values from 1,000 replicates. (Scale bar: the substitution rate per residue.) GenBank accession numbers are detailed in the legend to Fig. S2. (C) Gene organization of zebrafish tac3a and tac3b. Each gene is consists of seven exons and encode both NKF and NKB. SP, signal peptide; ATG and TGA, start and stop codon, respectively.

Fig. 2.

Expression of zebrafish tac3a, tac3b, tac3ra, or tac3rb mRNA in various parts of the brain (A) and changes in zebrafish tac3a at various ages toward puberty (B) as determined by real-time PCR. The relative abundance of the mRNAs was normalized to the amount of elongation factor 1 α (ef1α) by the comparative threshold cycle method; the comparative threshold reflects the relative amount of the transcript. Results are means ± SEM (n = 11–15). Means marked with different letters differ significantly (P < 0.05).

Fig. 3.

(A–O) Localization of tac3a during early stages of development, as detected by whole-mount ISH. tac3a is dominantly expressed in the right habenular nuclei, midbrain, and hindbrain. Dorsal view of larva heads, anterior is to the left (A–E). High magnification of boxed area in Upper panel. Note the unilateral expression of tac3a to right of the midline (dotted line) in the habenula (F–J), lateral view of larva heads (K–O). rHbn, right habenula; MB, midbrain; HB, hindbrain. (P–R) Localization of tac3a in adult zebrafish brain as indicated by ISH (nomenclature according to ref. 40). Tac3a mRNA-expressing cells were observed in the ventral (Hav) and medial (Ham) habenula (P) tac3a mRNA-expressing cells detected in the periventricular nucleus of posterior tuberculum (TPp), dorsal (Hd), and ventral zone (Hv) of periventricular hypothalamus (Q) tac3a mRNA-expressing cells observed in the posterior tuberal nucleus (PTN) and central zone (Hc) of periventricular hypothalamus (R). (Magnification: A–E and K–O, 40×; F–J, 120×).

Fig. 4.

Ligand selectivity of the NKB receptors. Human NKBR (A and D) zebrafish Tac3ra (B and E) or zebrafish Tac3rb (C and F), each together with SRE-Luc (A–C) or CRE-Luc (D–F). The cells were treated with various concentrations of human (hu) NKB: DMHDFFVGLM-NH₂; zebrafish (zf) NKBa: EMHDIFVGLM-NH₂; zfNKBb: STGINREAHLPFRPNMNDIFVGLL-NH₂; or zfNKF: YNDIDYDSFVGLM-NH₂. Data are expressed as the increase in luciferase activity over basal activity. Each point was determined in quadruplicate and is given as a mean ± SEM. (G) Ribbon representation of human and zebrafish NKBs structural model. The PDB ID for the human structure is 1p9f.

Fig. 5.

Exposure to estradiol (18 nM) by immersion caused a significant increase in mRNA expression of ligands (A) or receptor (B) genes, in the brain of juvenile zebrafish. sGnRHa, zfNKBa, zfNKBb, zfNKF, or senktide (see legend to Fig. 4 for details), were injected intraperitoneally to mature zebrafish and blood was collected 6 h thereafter (C). Hormone values are means ± SEM. Statistical significance vs. corresponding control values: **P < 0.01; *P < 0.05.