Publications

Full-length cDNA encoding two leptin sequences (tLepA and tLepB) and one leptin receptor sequence (tLepR) were identified in tilapia (Oreochromis niloticus). The full-length cDNA of tLepR was 3423bp, encoding a protein of 1140 amino acid (aa) which contained all functionally important domains conserved among vertebrate leptin receptors. The cDNAs of tLepA and tLepB were 486bp and 459bp in length, encoding proteins of 161 aa and 152 aa, respectively. Modeling the three-dimensional structures of tLepA and tLepB predicted strong conservation of tertiary structure with that of human leptin, comprised of four helixes. Using synteny, the tLeps were found near common genes, such as IMPDH1 and LLRC4. The cDNA for tLepA and tLepB was cloned and synthetic cDNA optimized for expression in Escherichia coli was prepared according to the cloned sequence. The tLepA- and tLepB-expressing plasmids were transformed into E. coli and expressed as recombinant proteins upon induction with nalidixic acid, found almost entirely in insoluble inclusion bodies (IBs). The proteins were solubilized, refolded and purified to homogeneity by anion-exchange chromatography. In the case of tLepA, the fraction eluted contained a mixture of monomers and dimers. The purified tLepA and tLepB monomers and tLepA dimer showed a single band of ∼15kDa on an SDS-polyacrylamide gel in the presence of reducing agent, whereas the tLepA dimer showed one band of ∼30kDa in the absence of reducing agent, indicating its formation by S-S bonds. The three tLeps were biologically active in promoting proliferation of BAF/3 cells stably transfected with the long form of human leptin receptor (hLepR), but their activity was four orders of magnitude lower than that of mammalian leptin. Furthermore, the three tLeps were biologically active in promoting STAT-LUC activation in COS7 cells transfected with the identified tLepR but not in cells transfected with hLepR. tLepA was more active than tLepB. Low or no activity likely resulted from low identity (9-22%) to mammalian leptins. In an in vivo experiment in which tilapia were fed ad libitum or fasted, there was no significant difference in the expressions of tLepA, tLepB or tLepR in the brain between the two groups examined both by real-time PCR and RNA next generation sequencing. In conclusion, in the present report we show novel, previously unknown sequences of tilapia leptin receptor and two leptins and prepare two biologically active recombinant leptin proteins.

Tilapias are emerging as one of the most important fish in worldwide aquaculture and are also widely used as model fish in the study of reproduction and behavior. During the reproductive season, male tilapia are highly territorial and form spawning pits in which the dominant males court and spawn with available females. Non-territorial males stand a much lower chance of reproducing. Using transgenic tilapia in which follicle stimulating hormone (FSH) gonadotrophs were fluorescently labeled with enhanced green fluorescent protein (EGFP), we studied the effect of social dominance on the hormonal profile and pituitary cell populations in dominant and non-dominant males. Immunofluorescence studies showed that FSH-EGFP-transgenic fish reliably express EGFP in FSH-secreting cells. EGFP expression pattern differed from that of luteinizing hormone. Dominant males had larger gonads as well as higher levels of androgens and gonadotropins in the plasma. Pituitaries of dominant males exhibited higher gonadotropin content and gene expression. Flow cytometry revealed pituitary hyperplasia as well as FSH cell hyperplasia and increased granulation. Taken together, these findings suggest that gonadotroph hyperplasia as well as increased production by individual cells underlie the increased reproductive activity of dominant tilapia males.

The gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and their receptors play critical roles in vertebrate reproduction. In order to study intra- and interspecies ligand promiscuity of gonadotropins, COS-7 cells were transiently transfected with one of the gonadotropin receptor genes, FSHR or LHR, and tested for activation by gonadotropins from representative fish orders: Aquilliformes (eel; e), Salmoniformes (trout; tr), and Perciformes (tilapia; ta), and of mammalian origin: porcine (p), bovine (b) and human (h). The study reveals complex relations between the gonadotropin hormones and their receptors. Each gonadotropin activated its own cognate receptor. However, taLHR was also activated by hCG and eLHR was activated by hFSH, hCG, and trFSH. For FSHR, the only cross-reactivity detected was for hFSHR, which was activated by pFSH and bFSH. These findings are of great interest and applicability in the context of activation of various GTHRs by their ligands and by ligands from other vertebrates. Analysis of the three-dimensional models of the structures highlights the importance of residues outside of the currently established hormone-receptor interface region. In addition, the interface residues in taFSHR and the effect of exon duplication, which causes an insert in the LRR domain, are suggested to affect the interaction and binding of taFSH.

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.

The gonadotropins (GTHs) follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are the key regulators of reproduction. We determined the competence of heterologous recombinant GTHs at eliciting steroid secretion from carp ovaries at different reproductive stages. We collected carp ovaries at: early, mid and end vitellogenesis, when most of the oocytes still contained a germinal vesicle (GV) at a central stage, and mature ovaries with a migrating GV. Plasma estradiol (E2) levels at early vitellogenesis were high and decreased thereafter. Basal secretion levels of E2 increased with oocyte diameter and GSI value, whereas 17α,20β-dihydroxy-4-pregnen-3-one (DHP) was detected only in females with mature follicles. Carp ovary fragments were exposed to recombinant fish GTHs belonging to different teleost orders: Japanese eel (Anguilla japonica, Anguilliformes), Manchurian trout (Brachymystax lenok, Salmoniformes), and Nile tilapia (Oreochromis niloticus); to mammalian GTHs (pFSH and hCG), or to carp and tilapia pituitary extract (CPE and TPE, respectively). All of the recombinant GTHs tested stimulated steroid secretion. However, the steroid secretion differed according to the type of GTH and the developmental state of the ovary. CPE increased the secretion of both E2 and DHP at almost all stages of ovarian maturity. In mature ovarian fragments, DHP secretion was higher in response to recombinant LHs (eel and tilapia) than to recombinant FSH. Early- and mid-vitellogenic ovaries showed no secretion of DHP and high secretion of E2 in response to all recombinant GTHs tested. This is in line with the hypothesis that LH regulates the final stages of maturation, when the involvement of FSH is marginal. These results may contribute to understanding the mechanisms that determine differential activation of steroid secretion and specificity in fish.

Aquaculture is one of the fastest rising sectors of world food production. Hundreds of fish species are cultured, providing an affordable, high quality food source. Two aspects of sexual development are critically important for the continued improvement of cultured fish stocks: sexual dimorphism and control of reproduction. In this paper, we review the main methods used to control sex determination in fish and their application in some of the most widely cultured species. Specifically, we review the techniques available for the production of all-male, all-female, and sterile populations. Techniques for endocrinological control of reproduction are also discussed.

The KISS1 gene encodes the kisspeptin neuropeptide, which activates the KISS1 receptor (KISS1R; G protein-coupled receptor 54; GPR54) and participates in neuroendocrine regulation of GnRH secretion. To study the physiological function(s) and evolutionary conservation of KISS1, we cloned opossum, Xenopus, and zebrafish kiss1 cDNAs. Processing zebrafish, Xenopus, or opossum KISS proteins would liberate a carboxy-terminal amidated peptide with 52, 54, or 53 amino acid residues, respectively. Phylogenetic analysis of all known vertebrate KISS1 peptides showed clear clustering of the sequences according to canonical vertebrate classes. The zebrafish kiss1 gene consists of two exons and one intron. Real-time PCR analysis of two kiss1R cloned from zebrafish brain found expression of kiss1, kiss1ra, and kiss1rb, with kiss1ra-more similar to other piscine Kiss1 receptors-highly expressed in the gonads and kiss1rb in other nonbrain tissues. In females kiss1 mRNA levels gradually increased during the first few weeks of life to peak in fish with ovaries containing mature oocytes, while in males kiss1 mRNA levels peaked after 6 wk postfertilization when the testes exhibited initial stages of spermatogenesis and decreased after puberty. Zebrafish kiss1ra and kiss1rb were expressed differentially with similar patterns in both genders. These results indicate that the Kiss1/Kiss1r system may participate in puberty initiation in fish as well. Like human KISS1R, Kiss1ra transduces its activity via the PKC pathway, whereas Kiss1rb does so via both PKC and PKA pathways. The human KISS1R was highly activated by both huKISS10amide and zfKISS10amide, whereas both zebrafish Kiss1 receptor types were less sensitive to amidation.

In a study towards elucidating the role of aromatases during puberty in female grey mullet, the cDNAs of the brain (muCyp19b) and ovarian (muCyp19a) aromatase were isolated by RT-PCR and their relative expression levels were determined by quantitative real-time RT-PCR. The muCyp19a ORF of 1515bp encoded 505 predicted amino acid residues, while that of muCyp19b was 1485 bp and encoded 495 predicted amino acid residues. The expression level of muCyp19b significantly increased in the brain as puberty advanced; however, its expression level in the pituitary increased only slightly with pubertal development. In the ovary, the muCyp19a expression level markedly increased as puberty progressed. The promoter regions of the two genes were also isolated and their functionality evaluated in vitro using luciferase as the reporter gene. The muCyp19a promoter sequence (650 bp) contained a consensus TATA box and putative transcription factor binding sites, including two half EREs, an SF-1, an AhR/Arnt, a PR and two GATA-3 s. The muCyp19b promoter sequence (2500 bp) showed consensus TATA and CCAAT boxes and putative transcription binding sites, namely: a PR, an ERE, a half ERE, a SP-1, two GATA-binding factor, one half GATA-1, two C/EBPs, a GRE, a NFkappaB, three STATs, a PPAR/RXR, an Ahr/Arnt and a CRE. Basal activity of serially deleted promoter constructs transiently transfected into COS-7, alphaT3 and TE671 cells demonstrated the enhancing and silencing roles of the putative transcription factor binding sites. Quinpirole, a dopamine agonist, significantly reduced the promoter activity of muCyp19b in TE671. The results suggest tissue-specific regulation of the muCyp19 genes and a putative alternative promoter for muCyp19b.

We recently produced Oreochromis niloticus recombinant LH and FSH as single-chain polypeptides in the methylotrophic yeast Pichia pastoris. Glycoprotein subunit alpha was joined with tilapia (t) LHbeta or tFSHbeta mature protein-coding sequences to form a fusion gene that encodes a ;;tethered" polypeptide, in which the gonadotropin beta-subunit forms the N-terminal part and the alpha-subunit forms the C-terminal part. Recombinant (r) gonadotropins were used to develop specific and homologous competitive ELISAs for measurements of FSH and LH in the plasma and pituitary of tilapia, using primary antibodies against rtLHbeta or rtFSHbeta, respectively, and rtLHbetaalpha or rtFSHbetaalpha for the standard curves. The wells were coated with either rtLHbeta (2ng/ml) or rtFSHbeta (0.5ng/well), and the final concentrations of the antisera were 1:5000 (for tLH) or 1:50,000 (for tFSH). The sensitivity of the assay was 15.84pg/ml for tLH and 0.24pg/ml for tFSH measurements in the plasma, whereas for the measurements in the pituitary, the sensitivity was 2.43ng/ml and 1.52ng/ml for tLH and tFSH, respectively. The standard curves for tFSH and tLH paralleled those of serially diluted pituitary extracts of other cichlids, as well as of serially diluted pituitary extract of seabream, European seabass and hybrid bass. We examined plasma tFSH and tLH levels in the course of one reproductive cycle, between two successive spawnings, in three individual tilapia females. Plasma levels of both FSH and LH increased during the second day after the eggs had been removed, probably related to the vitellogenic phase. LH levels increased toward spawning, which occurred on the 11th day. FSH levels also increased on day of cycle, probably due to recruitment of a new generation of follicles for the successive spawning. The development of specific ELISAs using recombinant gonadotropins is expected to advance the study of the distinct functions of each of these important hormones.

Two types of gonadotropin-releasing hormone (GnRH) receptors were found in the pituitary of tilapia (t), named GnRHR type 3 (tGnRHR3) and GnRHR type 1, according to phylogenetic analysis. tGnRHR3 is highly expressed in the posterior part of the pituitary which contains LH and FSH cells. We characterized tGnRHR3 in terms of both LH release rate and receptor internalization rate in response to continuous exposure to GnRH. Constant exposure of tilapia pituitary fragments to salmon GnRH analog (sGnRHa) resulted in an increased secretion rate for 3h, followed by a gradual decline, taking 17-19h, to the basal secretion rate. A chimera between tGnRHR3 and green fluorescent protein (GFP) was created and used to observe the changes in receptor distribution and translocation, activated by agonist with time. The results suggested that the receptor is initially localized at the plasma membrane and upon activation by a homologous ligand (e.g. sGnRHa) undergoes relatively rapid endocytosis. In summary, the present work demonstrates that tGnRHR3 has already undergone endocytosis after 30min, while desensitization of LH release occurs only after 17-19h. It is concluded that for tGnRHR3, internalization of the receptor is not exclusively responsible for the desensitization of LH release.

In fish, FSH is generally important for early gonadal development and vitellogenesis. As in mammals, FSH is a heterodimer composed of an alpha subunit that is noncovalently associated with the hormone-specific beta subunit. The objective of the present study was to express glycosylated, properly folded, and biologically active tilapia FSH (tFSH) using the Pichia pastoris expression system. Using this material, we aimed to develop a specific ELISA and to enable the study of FSH response to GnRH. The methylotrophic yeast P. pastoris was used to coexpress recombinant genes formed by fusion of mating factor alpha leader and tilapia fshb and cga coding sequences. Western blot analysis of tilapia pituitary FSH, resolved by SDS-PAGE, yielded a band of 15 kDa, while recombinant tFSH beta (rtFSH beta) and rtFSH beta alpha had molecular masses of 17-18 kDa and 26-30 kDa, respectively. Recombinant tFSH beta alpha was found to bear only N-linked carbohydrates. Recombinant tFSH beta alpha significantly enhanced 11-ketotestosterone (11-KT) and estradiol secretion from tilapia testes and ovaries, respectively, in a dose-dependent manner (similar to tilapia pituitary extract, affinity-purified pituitary FSH, and porcine FSH). Using antibodies raised against rtFSH beta, FSH-containing cells were localized adjacent to hypothalamic nerve fibers ramifying in the proximal pars distalis (PPD), while LH cells were localized in a more peripheral region of the PPD. Moreover, FSH is under the control of hypothalamic decapeptide GnRH, an effect that was abolished through the use of specific bioneutralizing antisera, anti-rtFSH beta. It also reduced basal secretion of 11-KT.

Sturgeons are known throughout the world as the source of black caviar. Their declining populations in their native habitats, mainly the Caspian Sea, due to over-fishing for meat and caviar production, destruction of their spawning grounds and water pollution, have led to their introduction into aquaculture in areas with suitable conditions, including Israel. Recently, we noticed an unusual phenomenon in these normally gonochoristic species. Several 5-year-old female sturgeons were found to have one or more testicular sections in each of their two gonads, forming an intersexual gender. Further examination of other fish from the same age group revealed 14% fish with intersex gonads among a population of 5000 fish that had been pre-selected as females. This phenomenon has not been found however in other age groups of Russian sturgeons, cultured at the same facility. Sturgeons are a generally gonochoristic species, and hermaphroditism is only very infrequently observed under natural or normal breeding conditions. Moreover, these rare cases have all been from polluted habitats. The present work is the first description of fish containing intersex gonads in Russian sturgeon (Acipenser gueldenstaedtii). We describe the phenomenon anatomically and histologically, and examine plasma steroid levels and pituitary gonadotropin gene expression by comparing fish with intersex gonads with normal females and males of the same age group. Intersex gonads were typical female ovaries with one or more white testicular components embedded in each. The testis components were not uniform in size or location among the two gonads of each fish or among different fish, and they showed marked differences in distribution. The ovarian component of the intersex gonad was at the pre-vitellogenic stage as in normal females, and the testis component contained spermatids and mature spermatozoa as in normal males of the same age. However, in terms of estradiol and 11-ketotestosterone plasma levels, as well as of pituitary gonadotropin (betaLH and betaFSH) gene-expression levels, the fish with intersex gonads were more similar to the normal males than to the normal females, even though the testis part of the intersex gonad was smaller than the ovarian part. To examine the possibility that the fish containing intersex gonads were hybrids, phylogenetic trees were constructed from the consensus sequences of Cytochrome b and control region (D-loop) genes. Results indicated no differences between the fish with intersex gonads and normal males or females of the same age group. However, statistically significant differences were found between different age groups of Russian sturgeon, as well as of white sturgeons (A. transmontanus), grown under the same culture conditions.

The Russian sturgeon, Acipenser gueldenstaedtii, is a late-maturing Acipenseriformes. To elucidate the role of FSH and LH in its reproduction, we cloned its glycoprotein alpha-subunit (GPalpha) and gonadotropin beta-subunits (FSHbeta and LHbeta) using 5' and 3' RACE-PCR. The nucleotide sequences of the Russian sturgeon (st) GPalpha, FSHbeta, and LHbeta are 345, 384, and 411 bp long, encoding peptides of 91, 115, and 114 amino acids, respectively. The deduced amino acid sequence of each mature subunit showed high similarity with those of other teleosts. Sequence analysis showed that stFSHbeta is more similar to higher vertebrate FSHbetas (35-37%) than to higher vertebrate LHbetas (26-30%). The next objective of this work was to compare the development of sturgeon gonads at the very first stages of their growth with the expression of their gonadotropins. Sturgeons at ages 1, 2, 3 or 4 years were sacrificed. The expression of their gonadotropin beta-subunits was determined using quantitative real-time PCR, and their gonads were examined histologically, followed by a determination of the plasma levels of estradiol in females and 11-ketotestosterone (11-KT) in males. The expression levels of stFSHbeta subunit was found to be higher in fish at 3 and 4 years of age than in 1-year olds. mRNA levels of stLHbeta were higher than those of stFSHbeta in both genders. Moreover mRNA levels of stFSHbeta detected in females were significantly higher than those found in males. Even at age 4 years, all female Russian sturgeons tested contained gonads at the pre-vitellogenic stage, with small oocytes and very low levels of estradiol in the plasma. However, among the males, at ages 3 and 4 years, we found testes that contained spermatids and spermatozoa. Those males were found to have significantly high GSI (gonadosomatic index; gonadal weight as a percentage of BW) levels, stLHbeta expression and 11-KT levels.

In fish, luteinizing hormone (LH) stimulates processes leading to final oocyte maturation and ovulation in females, and spermiation in males. The hormone is a heterodimeric glycoprotein composed of two non-covalently associated subunits. In this study, we describe the expression of tilapia LH (tLH) as a biologically active, single-chain polypeptide using the methylotrophic yeast Pichia pastoris. The tLHbeta and alpha mature protein-coding sequences were joined to form a fusion gene that encodes a "tethered" polypeptide in which the tLHbeta-chain forms the N-terminal part and the alpha-chain forms the C-terminal part. A "linker" sequence of six amino acids (three Gly-Ser pairs) was placed between the beta- and alpha-chains to assist in the chimerization of the subunits, and a six-His tail was placed at the end of the beta-subunit, to enable purification of the recombinant protein. Western blot analysis of the pituitary LH resolved by SDS-PAGE yielded a band of 35 kDa, while the recombinant tLHbetaalpha had a molecular mass of 45 kDa, and was found to possess only N-linked carbohydrates. Recombinant tLHbetaalpha stimulated the release of 11-ketotestosterone from mature testes, whereas its release from immature testes was less pronounced.

Neurohormones similar to those of mammals are carried in fish by hypothalamic nerve fibers to regulate directly follicle-stimulating hormone (FSH) and luteinizing hormone (LH). Gonadotropin-releasing hormone (GnRH) stimulates the secretion of FSH and LH and the expression of the glycoprotein hormone alpha (GPalpha), FSHbeta, and LHbeta, as well as their secretion. Its signal transduction leading to LH release is similar to that in mammals although the involvement of cyclic AMP-protein kinase A (cAMP-PKA) cannot be ruled out. Dopamine (DA) acting through DA D2 type receptors may inhibit LH release, but not that of FSH, at sites distal to activation of protein kinase C (PKC) and PKA. GnRH increases the steady-state levels of GPalpha, LHbeta, and FSHbeta mRNAs. Pituitary adenylate cyclase-activating polypeptide (PACAP) 38 and neuropeptide Y (NPY) potentiate GnRH effect on gonadotropic cells, and also act directly on the pituitary cells. Whereas PACAP increases all three subunit mRNAs, NPY has no effect on that of FSHbeta. The effect of these peptides on the expression of the gonadotropin subunit genes is transduced differentially; GnRH regulates GPalpha and LHbeta via PKC-ERK and PKA-ERK cascades, while affecting the FSHbeta transcript through a PKA-dependent but ERK-independent cascade. The signals of both NPY and PACAP are transduced via PKC and PKA, each converging at the ERK level. NPY regulates only GPalpha- and LHbeta-subunit genes whereas PACAP regulates the FSHbeta subunit as well. Like those of the mammalian counterparts, the coho salmon LHbeta gene promoter is driven by a strong proximal tripartite element to which three different transcription factors bind. These include Sf-1 and Pitx-1 as in mammals, but the function of the Egr-1 appears to have been replaced by the estrogen receptor (ER). The GnRH responsive region in tilapia FSHbeta 5' flanking region spans the canonical AP1 and CRE motifs implicating both elements in conferring GnRH responsiveness. Generally, high levels of gonadal steroids are associated with high LHbeta transcript levels whereas those of FSHbeta are reduced when pituitary cells are exposed to high steroid levels. Gonadal or hypophyseal activin also participate in the regulation of FSHbeta and LHbeta mRNA levels. However, gonadal effects are dependent on the gender and stage of maturity of the fish.

1. The D2-type dopamine receptors are thought to inhibit adenylyl cyclase (AC), via coupling to pertussis toxin (PTX)-sensitive G proteins of the Gi family. We examined whether and to what extent the various D2 receptors (D2S, D2L, D3S, D3L, and D4) couple to the PTX-insensitive G protein Gz, to produce inhibition of AC activity. 2. COS-7 cells were transiently transfected with the individual murine dopamine receptors alone, as well as together with the alpha subunit of Gz. PTX treatment was employed to inactivate endogenous alpha i, and coupling to Gi and Gz was estimated by measuring the inhibition of cAMP accumulation induced by quinpirole, in forskolin-stimulated cells. 3. D2S or D2L receptors can couple to the same extent to Gi and to Gz. The D4 dopamine receptor couples preferably to Gz, resulting in about 60% quinpirole-induced inhibition of cAMP accumulation. The D3S and D3L receptor isoforms couple slightly to Gz and result in 15 and 30% inhibition of cAMP accumulation, respectively. 4. We have demonstrated for the first time that the two D3 receptor isoforms, and not any of the other D2 receptor subtypes, also couple to Gs in both COS-7 and CHO transfected cells, in the presence of PTX. 5. Thus, the differential coupling of the D2 dopamine receptor subtypes to various G proteins may add another aspect to the diversity of dopamine receptor function.