Potential Role in Controlling Fish Reproduction

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 known to be involved in reproduction. Zebrafish tac3a mRNA levels puberty. We report here the identification of previously un- 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- SCIENCES

an optimum time for offspring survival. Fish show an enormous AGRICULTURAL 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-function 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 | June 26, 2012 | vol. 109 | no. 26 | 10269–10274 Downloaded by guest on October 1, 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, shows that TAC3R and TAC1R are closest to each other, suggesting that their separation was a more recent evolutionary event (Fig. 1B and Fig. S2B). Fish have one gene of Tac2r, and two genes each of Tac1r and Tac3r. Surprisingly, three Tac3r were A found in zebrafish, the only species to have a third receptor of any one type. However, we were unable to clone this gene from brain mRNA, so it was excluded from further biological analysis. The similarity and identity among the various TAC3 receptors is shown in Table S3. We found two forms of tac3 genes in zebrafish and salmon, but more evolved fish contained only one tac3 ortholog; however, all fish species exhibit two forms of NKB receptors, suggesting that the piscine NKB/NKBR can provide an excellent model for under- standing the molecular coevolution of the peptide/receptor pairs.

Gene Organization of tac3 and Chromosomal Synteny of Tac3 and Tac3 Receptor. The in silico analyses of fish genomic structure verified that the zftac3 consists of seven exons (Fig. 1C). In B mammals the tac3 gene contains seven exons, five of which are translated to form the prepro-NKB protein (11). Notably, the NKBa peptide sequence was encoded in the fifth exon [like in mammals (13, 14)], whereas NKBb spans exons 3–5 (Fig. 1C). Surprisingly, unlike in mammalian NKBs (11), the deduced amino acid sequences of both zftac3 genes encoded an additional putative TK sequence flanked by a Gly C-terminal amidation signal, and typical endoproteolytic sites at both termini, suggesting that additional TK peptides (YNDIDYDSFVGLM-NH2 and YDDIDYDSFVGLM-NH2, spliced from Tac3a and Tac3b, respectively) (Fig. 1C and Fig. S1) are produced by the same precursors. Intriguingly, we found this additional peptide in tac3 not only in zebrafish but in all other fish species identified in this study (11 species), but not in chicken, lizard, or alligator. These peptides possess an N-terminal dibasic cleavage site with potential to release the peptide, and the common NKB motif C FVGLM at their C terminal; therefore, we termed this unique peptide neurokinin F (NKF) because it has only been found in fish species to date. As Page et al. (11) anticipated, the vertebrate TAC3 gene encoded an additional TK in exon 3, in a similar position to substance P in TAC1, and endokinin A/B in TAC4. This TK (NKF) still exists in fish but was lost from other species during evolution. Interestingly, in Tac4 there is a similar loss of one active peptide in mammals (the C-terminal peptide in Tac4 as opposed to the N-terminal peptide in Tac3), whereas most fish species retain putative active peptides in both locations. Fig. 1. Unrooted phylogenetic tree of neurokinin (A) or neurokinin receptor Chromosome syntenic analysis revealed that the locus of tac3 (B) sequences generated with neighbor-joining (ClustalW 2.1) and maximum is highly conserved between teleosts (Fig. S4). zftac3a is located likelihood (Phylip 3.69, ProML) on the basis of alignments performed both by on chromosome 23 and tac3b on chromosome 6. The only tac3 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 found in medaka is located on chromosome 7 (Fig. S4B). For the sequences identified in this study are marked in bold. Gene nomenclature has zftac3 gene, the nearest neighboring gene (c1galt1a) is non- been standardized to Tac3, and species are indicated for illustration and syntenic, whereas the next nearest ones (b4galnt1a and slc6a1) comparison. Numbers at nodes indicate the bootstrap values from 1,000 rep- were found in inverse order in humans (Fig. S4A). Despite nearly licates. (Scale bar: the substitution rate per residue.) GenBank accession num- perfect preservation of synteny, we found substantial shuffling of bers are detailed in the legend to Fig. S2.(C) Gene organization of zebrafish gene order along corresponding chromosome arms between tac3a and tac3b. Each gene is consists of seven exons and encode both NKF and zebrafish and human. The neighborhoods of gene loci of tac3a NKB. SP, signal peptide; ATG and TGA, start and stop codon, respectively. were conserved in the zebrafish and medaka (Fig. S4B). We then

10270 | www.pnas.org/cgi/doi/10.1073/pnas.1119165109 Biran et al. Downloaded by guest on October 1, 2021 explored the genomic locations of tac3 receptors in humans and spermatozoa (15). The expression of the tac3b, tac3ra, and tac3rb various fish species (Fig. S4 C and D). In human, TAC3R is mRNAs in the zebrafish brain was low and did not change during located on chromosome 4, whereas in zebrafish, fugu (Takifugu sexual maturation. The increase of tac3a mRNA toward puberty, rubripes), medaka (Oryzias latipes), and tetraodon (Tetraodon consistent with kisspeptin signaling initiating puberty (15, 17), nigroviridis) tac3ra are located on chromosome 1, unplaced may indicate a possible involvement of the NKB/NKBR system (UN), 1, and 18, respectively. The nearest neighboring gene in controlling puberty. (cnga2) of the zebrafish tac3ra gene is nonsyntenic with human, but syntenic with the medaka, fugu, and tetraodon. The next- Localization of Embryonic tac3a Cells by Whole-Mount in Situ Hybridi- upstream neighboring genes (bdh2, nhedc2, and cisd2) were zation. The first appearance of tac3a expression was detected at 3 found in similar locations in all analyzed species (Fig. S4C). The d postfertilization (dpf) in the right habenula nuclei and the mid- human genome lacks tac3rb, which is present in tetraodon, fugu, brain (Fig. 3 A, F,andK). The earliest stage that the signal was and medaka. The next-upstream neighboring genes in the observed in the left habenula was at 4–5 dpf, when the signal in- zebrafish (acy3.1, acy3.2, cldnd, and glb1) were found in reverse tensity in the right habenula and midbrain increased, probably order in the tetraodon (Fig. S4D). Tac3rc had no discernible reflecting increased cell numbers (Fig. 3 B, C, G, H, L,andM). In synteny to any other family members. The presence of two forms addition, there was expression in the hindbrain. Analysis of elderly of NKB [in zebrafish and salmon (salmo salar)] and two forms of larvae (7 and 9 dpf) revealed decreased tac3a signal intensity (Fig. 3 cognate receptor genes in lower-vertebrate species supports the D, E, I, J, N,andO), and at 12 dpf it was barely detected. No signal hypothesis of two rounds of genome duplication followed by was observed at any stage by use of the tac3a sense riboprobe. degeneration and complementation of the genes. We conclude During embryogenesis tac3a was dominantly expressed in the right- that the synteny is better within fish than with mammals; more- habenula nuclei, but in adults it was expressed in both habenular over, fish tac3 and tac3r have conserved synteny with the human lobes (Fig. 3P). This asymmetrical expression of tac3a in the genome, consistent with orthology. habenula is consistent with previous findings that the habenula in zebrafish displayed left-right asymmetries in gene expression (18, Tissue Distribution of tac3 and tac3 Receptors in Zebrafish. To elu- 19). Interestingly, it was shown that neurons appear sooner in the cidate the physiological roles of the NKB/NKBR signaling sys- left than in the right habenula (20). Neuronal organization asym- tem, we next examined the tissue distribution of both ligands metries in the epithalamus (i.e., habenular nuclei and pineal com- (tac3a, tac3b) and receptors (tac3ra, tac3rb) mRNAs in zebrafish plex) are well known among vertebrates (21). by means of real-time PCR analysis, according to Biran et al. (15). We dissected the zebrafish brain into three parts, of which Localization of tac3a mRNA in the Brain of Adult Zebrafish. By using the anterior part contains the telencephalon, the midbrain con- in situ hybridization (ISH) techniques to determine the localization tains the optic tectum, diencephalon, and hypothalamus and the of tac3a mRNA in the zebrafish brain, we detected tac3a mRNA- hindbrain, the medulla oblongata and cerebellum. We mostly expressing neurons in the habenula (Fig. 3P), where kisspeptin 1 detected tac3a mRNA in the midbrain and tac3b mRNA was (kiss1) was previously shown to be expressed (22). Tac3a was also found mainly in the forebrain. Both tac3a and tac3ra were detected along the periventricular hypothalamus (Fig. 3 Q and R), expressed in the pituitary (Fig. 2A), collaborating findings in in the periventricular nucleus of the posterior tuberculum (Fig. 3Q), mammals where NKB and NKBR were expressed in the median and in the posterior tuberal nucleus (Fig. 3R). These brain nuclei eminence, which is missing in fish (6). tac3rb was expressed in the were previously found to express other important neuropeptides forebrain and was highest in the ovary. Different types of tac3 that regulate reproduction (17, 22), metabolism (23), and stress and tac3r were expressed in the ovary and testis (Fig. 2A). Dif- (24). In the mammalian ARC, KISS1 neurons that coexpress NKB ferent levels of expression of tac3 and tac3r types were found in and dynorphin were hypothesized to be a central node through extrabrain tissues (Fig. S5). The expression patterns of these which potential stress, metabolic and photoperiodic signals regulate genes in the brain-pituitary-gonad axis further support the po- GnRH release (25). The nuclear lateralis tuberis is considered as the tential role of the NKB system in fish reproduction. piscine structure homologous to the mammalian ARC (23, 26). The localization of tac3a,kisspeptin2(kiss2), kiss1 receptor b (kiss1rb), Gene Expression of the NKB/NKBR System During Sexual Maturation. leptin receptor, melanin-concentrating hormone (MCH) 2 and two SCIENCES Because it is known that the mammalian NKB/NKBR system is MCH1 receptors, Urotensin I, corticotropin-releasing factor, and AGRICULTURAL involved in reproduction, and especially in puberty initiation corticotropin-releasing factor-binding protein to the ventral zone of (16), we aimed to examine whether the piscine system fulfills the periventricular hypothalamus (Fig. 3Q)(17,22–24, 26) might a similar role. We used real-time PCR to evaluate the expression suggest that not all neuropeptide pathways are as conserved as profiles of the zfNKBs and their receptor mRNAs in the brain formerly thought. This suggestion is supported by the recent findings during several development stages. Expression of tac3a mRNA that kiss2 is not expressed in the zebrafish nuclear lateralis tuberis, was low at 2–4 wk postfertilization (wpf) (Fig. 2B); it then and that kiss2 neurons of the periventricular hypothalamus do not gradually increased, peaked at 8 wpf, when the zebrafish go directly innervate the zebrafish pituitary (22). Finally, the close through puberty, and subsequently decreased by 12 wpf (Fig. similarity between the expression patterns of zebrafish kisspeptin 2B), when the gonads contained clear, well-developed oocytes or genes and zebrafish tac3a suggests a possible interaction between

AB Fig. 2. Expression of zebrafish tac3a, tac3b, tac3ra,ortac3rb 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).

Biran et al. PNAS | June 26, 2012 | vol. 109 | no. 26 | 10271 Downloaded by guest on October 1, 2021 both zftac3 receptors, as well as the hNKBR posses both PKC and PKA phosphorylation sites (Fig. S3). These findings confirm that the unique zebrafish receptors relay their signal through both PKC and PKA transduction pathways.

Ligand Models. Fig. 4G is a ribbon representation of the zfNKB structural model prediction, compared with the hNKB (PDB ID 1p9f). Intriguingly, although the three zfNKBs vary in size—10, 24, and 13 aa for NKBa, NKBb, and NKFa, respectively—all of the predicted peptides yielded high-resolution, high-quality structures with typical globular folding. These structures com- prised α-helix-loop motifs (highlighted in red in Fig. 4G). All three zfNKBs model structures approximated a binding-compe- tent conformation similar to the human NKB. Our results corroborate previous ones found for mammals that the formation of a helical conformation in the mid region of each of the TKs appears to be crucial for TK-receptor activation (28). Moreover, mammalian NKB were found to form a helical structure in the presence of dodecylphosphocholine micelles (29).

In Vivo Effect of Estradiol. Our next aim was to test the involvement of the NKB system in reproduction. In fish, clear evidence exists regarding the important role estradiol plays during the period of reproduction (1). Moreover, ISH was recently used Fig. 3. (A–O) Localization of tac3a during early stages of development, as to show that estradiol increased kisspeptin cell number in the detected by whole-mount ISH. tac3a is dominantly expressed in the right ventral hypothalamus, where the zfkiss2 neurons are homologous habenular nuclei, midbrain, and hindbrain. Dorsal view of larva heads, anterior to the estrogen-sensitive kiss1 hypothalamic neurons in medaka is to the left (A–E). High magnification of boxed area in Upper panel. Note the (17, 22, 30). Similarly to mammals, zebrafish have two forms of unilateral expression of tac3a to right of the midline (dotted line) in the GnRH: GnRH2 is localized to the midbrain tegmentum, and habenula (F–J), lateral view of larva heads (K–O). rHbn, right habenula; MB, – fi GnRH3 (considered to be the hypophysiotropic form) is located midbrain; HB, hindbrain. (P R) Localization of tac3a in adult zebra sh brain as at both the olfactory bulb terminal nerve and the preoptic area indicated by ISH (nomenclature according to ref. 40). Tac3a mRNA-expressing cells were observed in the ventral (Hav) and medial (Ham) habenula (P) tac3a (31). Because in mammals kisspeptin and NKB are expressed in mRNA-expressing cells detected in the periventricular nucleus of posterior the same neurons in the hypothalamic ARC, and play a key role tuberculum (TPp), dorsal (Hd), and ventral zone (Hv) of periventricular hypo- in physiological regulation of GnRH neurons (25), we tested the thalamus (Q)tac3a mRNA-expressing cells observed in the posterior tuberal effect of estradiol on the expression of genes along the GnRH- nucleus (PTN) and central zone (Hc) of periventricular hypothalamus (R). kisspeptin system. Estradiol treatment of prepubertal zebrafish (Magnification: A–E and K–O,40×; F–J, 120×). enhanced expression of key genes involved in reproduction (gnrh3, kiss2, and kiss1) concomitantly with significantly increased tac3a (Fig. 5A). In parallel, we also found significantly increased these two systems, as previously shown in mammals; however, this expression of tac3ra, tac3rb, and kiss1ra (Fig. 5B). Both Tac3 needs further investigation. receptors bound the zfNKBs (Fig. 4 A–F), and Kiss1ra was shown previously to bind Kiss2, considered to be the more important Pharmacological Analysis and Signal Transduction Pathways of zfTac3 form, with higher affinity than Kiss1 (32). In mammals there is Receptors. We used functional expression analysis with COS-7 strong evidence of sexual dimorphism of NKB neurons: larger cells to evaluate the response, binding selectivity, and signal numbers of NKB neurons have been identified in ARC of ewes transduction pathways of the unique TK receptors to their ago- than of rams (33). The transcription of NKB could be directly nists. We previously showed the specificity of the reporter serum altered by estrogen receptors, as sequences corresponding to the responsive element (SRE)-Luc and cAMP responsive element estrogen responsive element and imperfect palindromic estrogen (CRE)-Luc, to activation of PKC/Ca2+ and PKA/cAMP signal responsive element have been reported upstream of the TAC3 transduction pathways, respectively (15). Graded concentrations gene transcriptional start site (8). In fish estradiol is involved in of the zfTKs (NKBa, NKBb, and NKF), and of hNKB and its both early oogenesis and the beginning of the first wave of vi- agonist senktide were applied to COS-7 cells that expressed tellogenesis that precede puberty (34), collaborating our finding hNKBR, zfTac3ra, or zfTac3rb. The EC50 values of TKs for each that tac3a expression peaked in prepubertal fish (Fig. 2B). receptor are summarized (Table S4). Both human and piscine Moreover, increased levels of estradiol are a required charac- TKs induced concentration-dependent increases in both SRE- teristic of both follicular growth and final oocyte maturation in Luc and CRE-Luc activity (Fig. 4 A–F). For hNKBR, in both fish (3, 35), pointing toward the involvement of the piscine NKB signal transduction systems, zfNKBa and NKF showed high po- system in control of reproduction, probably in concert with tency, very similar to human NKB, but zfNKBb peptide exhibited kisspeptin and GnRH. relatively low potency (Fig. 4 A–D). For both zfTac3 receptors, zfNKBa and NKF (both derived from tac3a) were similarly In Vivo Effect of NKBs. We next tested the in vivo biological highly potent in both signal transduction pathways (Fig. 4). function of zebrafish NKB peptides. Single intraperitoneal in- These results confirmed that both zfNKBa and NKF were en- jection of zfNKBa or zfNKF elicited significant LH secretion in dogenous ligands of Tac3 receptors, and it is noteworthy that this sexually mature female zebrafish (Fig. 5C). The magnitude of the report of activation of TK receptors by a second peptide derived induced LH discharge was comparable with that observed in from the NKB gene (zfNKF) is unique. However, NKBb was less response to GnRH. LH response to the hNKBR agonist, senk- effective than the other forms in eliciting luciferase activity by tide, or zfNKBb was less pronounced (Fig. 5C), in a similar way both signal transduction pathways. It was previously shown un- to the order of potency obtained in the transactivation assay (Fig. equivocally that hNKBR potentially can couple directly to both 4). Our findings corroborate previous findings that activation of phospholipase C and adenylate cyclase, and stimulate both NKB receptors evoked potent LH-secretory responses in phosphoinositides hydrolysis and cAMP formation (27). Indeed rodents, sheep, and monkey (4, 36–38).

10272 | www.pnas.org/cgi/doi/10.1073/pnas.1119165109 Biran et al. Downloaded by guest on October 1, 2021 AB C G hNKBR zfTac3ra zfTac3rb

4 3 4 zfNKBa 3 zfNKBb 3 2 zfNKF 2 2 hNKB 1 1 Senktide (fold induction) 1 SRE-Luc activity

0 0 0 .0-11 -10 -9 -8 -7 -6 -5 .0-11 -10 -9 -8 -7 -6 -5 .0-11 -10 -9 -8 -7 -6 -5 DE12 F 10 10 8 zfNKBa 8 8 6 zfNKBb 6 6 zfNKF 4 4 4 hNKB

(fold induction) 2 CRE-Luc activity 2 2 Senktide

0 0 0 .0-11 -10 -9 -8 -7 -6 -5 .0-11 -10 -9 -8 -7 -6 -5 .0-11 -10 -9 -8 -7 -6 -5 peptide (logM) peptide (logM) peptide (logM)

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.

In conclusion, we provided detailed information on the orga- Isolation of Zebrafish tac3 Ligands and Receptors. We designed specific pri- nization of the NKB systems in teleosts, including the splice tac3 mers for cloning the putative zfTac3 ligands and receptors (Table S1). The fi fi (NKF). We also show that the zftac3a, unlike zftac3b, expression fragments were PCR-ampli ed from an adult zebra sh brain cDNA library and were cloned into pCRII-TOPO vector (Invitrogen). increased toward puberty, increased in response to estradiol treatment, caused increase in LH secretion, and was abundantly Tissue Distribution and Expression Profiles. Tissue distributions of zftac3a, expressed in the brain, notably in the hypothalamus. Tac3 zftac3b, zftac3ra, and zftac3rb were determined by real-time PCR as previously receptors were expressed in the pituitary and gonads, suggesting described (15). Tissue samples were collected from sexually mature post- that these ligand-receptor pairs are likely involved in the control vitellogenic female and milt-producing male zebrafish, total RNA extraction, of reproductive functions, during puberty or during diverse steps and cDNA samples were prepared as previously described (15). To study gene of reproduction. expression of the NKB/NKBR system at different ages, 15 fish were randomly sampled at ages 2, 4, 6, 8, and 12 wpf. The brain was removed, and the pubertal Materials and Methods stage classification was determined as described previously (15). Elongation factor 1α (39) was used as a reference gene. The primer sequences, R2 values, Animals. Wild-type zebrafish were purchased from a commercial supplier (A & and slopes of the real-time PCR analyses, calculated by linear regression, are H). All experimental procedures were approved by the Hebrew University

presented in Table S1. SCIENCES Administrative Panel for Laboratory Animal Care. AGRICULTURAL ISH Analysis of Embryos and Adults. ISH was conducted on embryos and adults Data Mining, Phylogenetic Analysis, and Chromosomal Synteny. The putative according to Mitani et al. (30) and Palevitch et al. (40) respectively, as de- ﬁ Tac3 gene sequences were isolated from zebra sh by using a stepwise tailed in SI Materials and Methods. evolutionary strategy, with the mouse Tac2 protein (NP_033338.2) as ﬁrst-

step input, and extension to other genomes and ESTs, as described in the SI Peptide Synthesis. Zebraﬁsh NKBa (EMHDIFVGLM-NH2), NKBb (STGIN- Materials and Methods. Details of sequences, phylogenetic analyses and REAHLPFRPNMNDIFVGLLEMHDIFVGLM-NH2), and NKF (YNDIDYDSFVG- synteny are presented in SI Materials and Methods. LM-NH2) were synthesized by the automated solid-phase method by

AB C

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.

Biran et al. PNAS | June 26, 2012 | vol. 109 | no. 26 | 10273 Downloaded by guest on October 1, 2021 applying Fmoc active-ester chemistry, purified by HPLC to >95% purity Each group of 10 fish was kept in a 3-L aquarium with water maintained at (GeneMed), and the carboxyl terminus of each peptide was amidated. 28 °C and replaced daily. After 3 d, fish were anesthetized and brains were removed. RNA extraction, reverse transcription of RNA and real-time PCR Receptor Transactivation Assay and Protein Structure Modeling. To study the were carried out as previously described (15). signaling pathways of the recently identified zfNKBs, the entire coding Adult female zebrafish were injected intraperitoneally with 20 pmol/g 6 9 regions of zftac3ra and zftac3rb were inserted into pcDNA3.1 (Invitrogen). body weight of either saline, salmon GnRH analog [(D-Ala ,Pro -Net)- The cDNA clone for hNKBR was obtained from the Missouri S&T cDNA Re- mammalian GnRH], zfNKBa, zfNKBb, zfNKF, or senktide (n =8fish per source Center (www.cdna.org), and the luciferase assay was conducted (15). group). Six hours postinjection the fish were bled (44). Blood was Protein structures of zebrafish NKBa, NKBb, or NKF were predicted on the centrifuged at 970 × g 30 min and the plasma was separated and stored at I-Tasser server (41, 42). −20 °C. Plasma from two animals were pooled (within each treatment) and analyzed for LH using ELISA for other Cyprinidae, carp, according to In Vivo Experiments. Two-month-old prepubertal zebrafish were exposed to Aizen et al. (35, 45). E2 (Sigma) at a concentration of 5 μg/L (18 nM), or to vehicle (ethanol 100% fi μ to a nal concentration of 33 L/L), by immersion for 3 d. This relatively low ACKNOWLEDGMENTS. This research was funded by United States-Israel concentration was used because the natural E2 concentration in the plasma Binational Science Foundation Grant 2005096, and by Binational Agricul- of adult vitellogenic female zebrafish was determined as 3–4 ng/mL (43). tural Research and Development Fund MD-8719-08.

1. Yaron Z, Levavi-Sivan B (2006) Fish reproduction. Physiology of Fishes, eds Evans DH, 26. Liu Q, et al. (2010) Expression of leptin receptor gene in developing and adult ze- Claiborne JB (CRC, New York), 3rd Ed, pp 345–388. brafish. Gen Comp Endocrinol 166:346–355. 2. Ball JN (1981) Hypothalamic control of the pars distalis in fishes, amphibians, and 27. Nakajima Y, Tsuchida K, Negishi M, Ito S, Nakanishi S (1992) Direct linkage of three reptiles. Gen Comp Endocrinol 44:135–170. tachykinin receptors to stimulation of both phosphatidylinositol hydrolysis and cyclic 3. Levavi-Sivan B, Bogerd J, Mañanós EL, Gómez A, Lareyre JJ (2010) Perspectives on fish AMP cascades in transfected Chinese hamster ovary cells. J Biol Chem 267: gonadotropins and their receptors. Gen Comp Endocrinol 165:412–437. 2437–2442. 4. Navarro VM, et al. (2011) Interactions between kisspeptin and neurokinin B in the 28. Almeida TA, et al. (2004) Tachykinins and tachykinin receptors: Structure and activity control of GnRH secretion in the female rat. Am J Physiol Endocrinol Metab 300: relationships. Curr Med Chem 11:2045–2081. E202–E210. 29. Mantha AK, Chandrashekar IR, Baquer NZ, Cowsik SM (2004) Three dimensional 5. Topaloglu AK, et al. (2009) TAC3 and TACR3 mutations in familial hypogonadotropic structure of mammalian tachykinin peptide neurokinin B bound to lipid micelles. hypogonadism reveal a key role for Neurokinin B in the central control of re- J Biomol Struct Dyn 22:137–148. production. Nat Genet 41:354–358. 30. Mitani Y, Kanda S, Akazome Y, Zempo B, Oka Y (2010) Hypothalamic Kiss1 but not 6. Rance NE, Krajewski SJ, Smith MA, Cholanian M, Dacks PA (2010) Neurokinin B and Kiss2 neurons are involved in estrogen feedback in medaka (Oryzias latipes). Endo- – the hypothalamic regulation of reproduction. Brain Res 1364:116 128. crinology 151:1751–1759. – 7. Page NM (2004) Hemokinins and endokinins. Cell Mol Life Sci 61:1652 1663. 31. Steven C, et al. (2003) Molecular characterization of the GnRH system in zebrafish 8. Page NM, Woods RJ, Lowry PJ (2001) A regulatory role for neurokinin B in placental (Danio rerio): Cloning of chicken GnRH-II, adult brain expression patterns and pitui- – physiology and pre-eclampsia. Regul Pept 98:97 104. tary content of salmon GnRH and chicken GnRH-II. Gen Comp Endocrinol 133:27–37. 9. Seidah NG, Prat A (2002) Precursor convertases in the secretory pathway, cytosol and 32. Lee YR, et al. (2009) Molecular evolution of multiple forms of kisspeptins and GPR54 – extracellular milieu. Essays Biochem 38:79 94. receptors in vertebrates. Endocrinology 150:2837–2846. 10. Eipper BA, Stoffers DA, Mains RE (1992) The biosynthesis of neuropeptides: Peptide 33. Cheng G, Coolen LM, Padmanabhan V, Goodman RL, Lehman MN (2010) The kiss- – alpha-amidation. Annu Rev Neurosci 15:57 85. peptin/neurokinin B/dynorphin (KNDy) cell population of the arcuate nucleus: Sex 11. Page NM, Morrish DW, Weston-Bell NJ (2009) Differential mRNA splicing and pre- differences and effects of prenatal testosterone in sheep. Endocrinology 151: cursor processing of neurokinin B in neuroendocrine tissues. Peptides 30:1508–1513. 301–311. 12. Maggi CA (1995) The mammalian tachykinin receptors. Gen Pharmacol 26:911–944. 34. Lubzens E, Young G, Bobe J, Cerdà J (2010) Oogenesis in teleosts: How eggs are 13. Bonner TI, Affolter H-U, Young AC, Young WS, 3rd (1987) A cDNA encoding the formed. Gen Comp Endocrinol 165:367–389. precursor of the rat neuropeptide, neurokinin B. Brain Res 388:243–249. 35. Aizen J, Kasuto H, Levavi-Sivan B (2007) Development of specific enzyme-linked im- 14. Kotani H, Hoshimaru M, Nawa H, Nakanishi S (1986) Structure and gene organization munosorbent assay for determining LH and FSH levels in tilapia, using recombinant of bovine neuromedin K precursor. Proc Natl Acad Sci USA 83:7074–7078. gonadotropins. Gen Comp Endocrinol 153:323–332. 15. Biran J, Ben-Dor S, Levavi-Sivan B (2008) Molecular identification and functional 36. Ramaswamy S, et al. (2010) Neurokinin B stimulates GnRH release in the male monkey characterization of the kisspeptin/kisspeptin receptor system in lower vertebrates. (Macaca mulatta) and is colocalized with kisspeptin in the arcuate nucleus. Endocri- Biol Reprod 79:776–786. nology 151:4494–4503. 16. Navarro VM, et al. (2012) Role of neurokinin B in the control of female puberty and its 37. Billings HJ, et al. (2010) Neurokinin B acts via the neurokinin-3 receptor in the ret- modulation by metabolic status. J Neurosci 32:2388–2397. rochiasmatic area to stimulate luteinizing hormone secretion in sheep. Endocrinology 17. Kitahashi T, Ogawa S, Parhar IS (2009) Cloning and expression of kiss2 in the zebrafish – and medaka. Endocrinology 150:821–831. 151:3836 3846. 38. García-Galiano D, et al. (2012) Kisspeptin signaling is indispensable for neurokinin B, 18. Aizawa K, et al. (2007) Responses of embryonic germ cells of the radiation-sensitive Medaka mutant to gamma-irradiation. J Radiat Res (Tokyo) 48:121–128. but not glutamate, stimulation of gonadotropin secretion in mice. Endocrinology – 19. Bianco IH, Carl M, Russell C, Clarke JD, Wilson SW (2008) Brain asymmetry is encoded 153:316 328. fi at the level of axon terminal morphology. Neural Dev 3:9. 39. Tang R, Dodd A, Lai D, McNabb WC, Love DR (2007) Validation of zebra sh (Danio 20. Roussigné M, Bianco IH, Wilson SW, Blader P (2009) Nodal signalling imposes left- rerio) reference genes for quantitative real-time RT-PCR normalization. Acta Biochim – right asymmetry upon neurogenesis in the habenular nuclei. Development 136: Biophys Sin (Shanghai) 39:384 390. fi 1549–1557. 40. Palevitch O, et al. (2007) Ontogeny of the GnRH systems in zebra sh brain: In situ 21. Concha ML, Wilson SW (2001) Asymmetry in the epithalamus of vertebrates. J Anat hybridization and promoter-reporter expression analyses in intact animals. Cell Tissue 199:63–84. Res 327:313–322. 22. Servili A, et al. (2011) Organization of two independent kisspeptin systems derived 41. Zhang Y (2008) I-TASSER server for protein 3D structure prediction. BMC Bio- from evolutionary-ancient kiss genes in the brain of zebrafish. Endocrinology 152: informatics 9:40. 1527–1540. 42. Roy A, Kucukural A, Zhang Y (2010) I-TASSER: A unified platform for automated 23. Berman JR, Skariah G, Maro GS, Mignot E, Mourrain P (2009) Characterization of two protein structure and function prediction. Nat Protoc 5:725–738. melanin-concentrating hormone genes in zebrafish reveals evolutionary and physi- 43. Heiden TK, Carvan MJ, 3rd, Hutz RJ (2006) Inhibition of follicular development, vi- ological links with the mammalian MCH system. J Comp Neurol 517:695–710. tellogenesis, and serum 17beta-estradiol concentrations in zebrafish following 24. Alderman SL, Bernier NJ (2007) Localization of corticotropin-releasing factor, ur- chronic, sublethal dietary exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol Sci otensin I, and CRF-binding protein gene expression in the brain of the zebrafish, 90:490–499. Danio rerio. J Comp Neurol 502:783–793. 44. Detrich HW, Westerfield M, Zon LI (2011) The Zebrafish: Cellular and Developmental 25. Lehman MN, Coolen LM, Goodman RL (2010) Minireview: Kisspeptin/neurokinin B/ Biology (Elsevier, Amsterdam), Vol 101, p 92. dynorphin (KNDy) cells of the arcuate nucleus: A central node in the control of go- 45. Aizen J, et al. (2012) Steroidogenic response of carp ovaries to piscine FSH and LH de- nadotropin-releasing hormone secretion. Endocrinology 151:3479–3489. pends on the reproductive phase. Gen Comp Endocrinol 178:28–36.

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