Characterization of the somatostatin system in tilapia: implications for growth and reproduction.

Somatostatin (SST) plays diverse physiological roles in vertebrates, particularly in regulating growth hormone secretion from the pituitary. While the function of SST as a neuromodulator has been studied extensively, its role in fish and mammalian reproduction remains poorly understood. To address this gap, we investigated the involvement of the somatostatin system in the regulation of growth and reproductive hormones in tilapia. RNA sequencing of mature tilapia brain tissue revealed the presence of three SST peptides: SST6, SST3, and low levels of SST1. Four different isoforms of the somatostatin receptor (SSTR) subfamily were also identified in the tilapia genome. Phylogenetic and synteny analysis identified tiSSTR2-like as the root of the tree, forming two mega clades, with SSTR1 and SSTR4 in one and SSTR2a, SSTR3a, and SSTR5b in the other. Interestingly, the tiSSTR-5 isoforms 5x1, 5x2, and 5x3 were encoded in the sstr3b gene and were an artifact of misperception in the nomenclature in the database. RNA-seq of separated pituitary cell populations showed that SSTRs were expressed in gonadotrophs, with sstr3a enriched in luteinizing hormone (LH) cells and sstr3b significantly enriched in follicle-stimulating hormone (FSH) cells. Notably, cyclosomatostatin, an SSTR antagonist, induced cAMP activity in all SSTRs, with SSTR3a displaying the highest response, whereas octreotide, an SSTR agonist, showed a binding profile like that observed in human receptors. Binding site analysis of tiSSTRs from tilapia pituitary cells revealed the presence of canonical binding sites characteristic of peptide-binding class A G-protein-coupled receptors. Based on these findings, we explored the effect of somatostatin on gonadotropin release from the pituitary in vivo. Whereas cyclosomatostatin increased LH and FSH plasma levels at 2 h post-injection, octreotide decreased FSH levels after 2 h, but the LH levels remained unaffected. Overall, our findings provide important insights into the somatostatin system and its mechanisms of action, indicating a potential role in regulating growth and reproductive hormones. Further studies of the complex interplay between SST, its receptors, and reproductive hormones may advance reproductive control and management in cultured populations.

Identifying the Interaction of the Brain and the Pituitary in Social - and Reproductive - State of Tilapia by Transcriptome Analyses.

INTRODUCTION: As in all vertebrates, reproduction in fish is regulated by gonadotrophin-releasing hormone (GnRH) control on gonadotrophic hormones (GtHs) activity. However, the neuroendocrine factors that promote GnRH and GtH activity are unknown. In Nile tilapia (Oreochromis niloticus), sexual activity and reproduction ability depend on social rank; only dominant males and females reproduce. Here, this characteristic of dominant fish allows us to compare brain and pituitary gene expression in animals that do and do not reproduce, aiming to reveal mechanisms that regulate reproduction. METHODS: An extensive transcriptome analysis was performed, combining two sets of transcriptomes: a novel whole-brain and pituitary transcriptome of established dominant and subordinate males, together with a cell-specific transcriptome of luteinizing hormone (LH) and follicle-stimulating hormone cells. Pituitary incubation assay validated the direct effect of steroid application on chosen genes and GtH secretion. RESULTS: In most dominant fish, as determined behaviorally, the gonadosomatic index was higher than in subordinate fish, and the leading upregulated pituitary genes were those coding for GtHs. In the brain, various neuropeptide genes, including isotocin, cholecystokinin, and MCH, were upregulated; these may be related to reproductive status through effects on behavior and feeding. In a STRING network analysis combining the two transcriptome sets, brain aromatase, highly expressed in LH cells, is the most central gene with the highest number of connections. In the pituitary incubation assay, testosterone and estradiol increased the secretion of LH and specific gene transcription. CONCLUSIONS: The close correlation between behavioral dominance and reproductive capacity in tilapia allows unraveling novel genes that may regulate the hypothalamic-pituitary-gonadal axis, highlighting aromatase as the main factor affecting the brain and pituitary in maintaining a sexually active organism.

Differential Regulation of Gonadotropins as Revealed by Transcriptomes of Distinct LH and FSH Cells of Fish Pituitary.

From mammals to fish, reproduction is driven by luteinizing hormone (LH) and follicle-stimulating hormone (FSH) temporally secreted from the pituitary gland. Teleost fish are an excellent model for addressing the unique regulation and function of each gonadotropin cell since, unlike mammals, they synthesize and secrete LH and FSH from distinct cells. Only very distant vertebrate classes (such as fish and birds) demonstrate the mono-hormonal strategy, suggesting a potential convergent evolution. Cell-specific transcriptome analysis of double-labeled transgenic tilapia expressing GFP and RFP in LH or FSH cells, respectively, yielded genes specifically enriched in each cell type, revealing differences in hormone regulation, receptor expression, cell signaling, and electrical properties. Each cell type expresses a unique GPCR signature that reveals the direct regulation of metabolic and homeostatic hormones. Comparing these novel transcriptomes to that of rat gonadotrophs revealed conserved genes that might specifically contribute to each gonadotropin activity in mammals, suggesting conserved mechanisms controlling the differential regulation of gonadotropins in vertebrates.

Characterization of gonadotropin receptors Fshr and Lhr in Japanese medaka, Oryzias latipes.

Reproduction in vertebrates is controlled by the brain-pituitary-gonad axis, where the two gonadotropins follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) play vital parts by activating their cognate receptors in the gonads. The main purpose of this work was to study intra- and interspecies ligand promiscuity of teleost gonadotropin receptors, since teleost receptor specificity is unclear, in contrast to mammalian receptors. Receptor activation was investigated by transfecting COS-7 cells with either Fsh receptor (mdFshr, tiFshr) or Lh receptor (mdLhr, tiLhr), and tested for activation by recombinant homologous and heterologous ligands (mdFshßα, mdLhßα, tiFshßα, tiLhßα) from two representative fish orders, Japanese medaka (Oryzias latipes, Beloniformes) and Nile tilapia (Oreochromis niloticus, Cichliformes). Results showed that each gonadotropin preferentially activates its own cognate receptor. Cross-reactivity was detected to some extent as mdFshßα was able to activate the mdLhr, and mdLhßα the mdFshr. Medaka pituitary extract (MPE) stimulated CRE-LUC activity in COS-7 cells expressing mdlhr, but could not stimulate cells expressing mdfshr. Recombinant tiLhßα, tiFshßα and tilapia pituitary extract (TPE) could activate the mdLhr, suggesting cross-species reactivity for mdLhr. Cross-species reactivity was also detected for mdFshr due to activation by tiFshßα, tiLhßα, and TPE, as well as for tiFshr and tiLhr due to stimulation by mdFshßα, mdLhßα, and MPE. Tissue distribution analysis of gene expression revealed that medaka receptors, fshr and lhr, are highly expressed in both ovary and testis. High expression levels were found for lhr also in brain, while fshr was expressed at low levels. Both fshr and lhr mRNA levels increased significantly during testis development. Amino acid sequence alignment and three-dimensional modelling of ligands and receptors highlighted conserved beta sheet domains of both Fsh and Lh between Japanese medaka and Nile tilapia. It also showed a higher structural homology and similarity of transmembrane regions of Lhr between both species, in contrast to Fshr, possibly related to the substitution of the conserved cysteine residue in the transmembrane domain 6 in medaka Fshr with glycine. Taken together, this is the first characterization of medaka Fshr and Lhr using homologous ligands, enabling to better understand teleost hormone-receptor interactions and specificities. The data suggest partial ligand promiscuity and cross-species reactivity between gonadotropins and their receptors in medaka and tilapia.

Ontogeny of the specificity of gonadotropin receptors and gene expression in carp.

The pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), are the principle endocrine drivers of reproductive processes in the gonads of jawed vertebrates. Canonically, FSH recruits and maintains selected ovarian follicles for maturation and LH induces the stages of germinal vesicle breakdown and ovulation. In mammals, LH and FSH specifically activate cognate G-protein-coupled receptors that affect the proteins involved in steroidogenesis, protein hormone synthesis, and gametogenesis. This dual-gonadotropin model also exists in some fish species, but not in all. In fact, due to their diverse number of species, extended number of ecological niches, and remarkably flexible reproductive strategies, fish are appropriate as models to understand the co-evolution of gonadotropins and their receptors. In this study, we cloned and characterized the expression profile over the final stages of ovarian maturation of carp (Cyprinus carpio) LHCGR and FSHR. Expression of both gonadotropin receptors increased in the later stage of early vitellogenesis, suggesting that both LH and FSH play a role in the development of mature follicles. We additionally tested the activation of cLHCGR and cFSHR using homologous and heterologous recombinant gonadotropins in order to gain insight into an evolutionary model of permissive gonadotropin receptor function. These data suggest that carp (Cyprinus carpio) gonad development and maturation depends on a specific gonadotropin profile that does not reflect the temporally distinct dual-gonadotropin model observed in salmonids or mammals, and that permissive gonadotropin receptor activation is a specific feature of Ostariophysi, not all teleosts.

Establishment of specific enzyme-linked immunosorbent assay (ELISA) for measuring Fsh and Lh levels in medaka (Oryzias latipes), using recombinant gonadotropins.

The paucity of information on understanding the regulatory mechanisms that are involved in the control of piscine Fsh and Lh synthesis, secretion, and function, prompted the present work. Part of the problem is related to the molecular heterogeneity and the unavailability of Fsh and Lh assays for quantifying gonadotropins, in particular assays regarding the measurement of Fsh, and such assays are available today for only a few teleost species. The present study reports the development and validation of competitive ELISAs for quantitative determination of medaka Fsh and Lh by first producing medaka recombinant (md) gonadotropins mdFshß, mdLhß, mdFshßα, and mdLhßα by Pichia pastoris, generating specific antibodies against their respective ß subunits, and their use within the development of ELISAs. The advantages of this protocol include: •The reproducibility of the ELISA demonstrated was relatively high, as shown by reasonably low intra- (Fsh 2.7%, Lh 3%) and interassay CVs (Fsh 5.3%, Lh 5.7%).•The high degree of parallelism between serial dilutions of the recombinant and native pituitary-derived Fsh and Lh, may be a sign of similar structures and immunologically similarity.•Two new competitive ELISAs for the quantification of medaka Fsh and Lh were established for the first time.

Agouti-Related Protein 2 Is a New Player in the Teleost Stress Response System.

Agouti-related protein (AgRP) is a hypothalamic regulator of food consumption in mammals. However, AgRP has also been detected in circulation, but a possible endocrine role has not been examined. Zebrafish possess two agrp genes: hypothalamically expressed agrp1, considered functionally equivalent to the single mammalian agrp, and agrp2, which is expressed in pre-optic neurons and uncharacterized pineal gland cells and whose function is not well understood. By ablation of AgRP1-expressing neurons and knockout of the agrp1 gene, we show that AgRP1 stimulates food consumption in the zebrafish larvae. Single-cell sequencing of pineal agrp2-expressing cells revealed molecular resemblance to retinal-pigment epithelium cells, and anatomic analysis shows that these cells secrete peptides, possibly into the cerebrospinal fluid. Additionally, based on AgRP2 peptide localization and gene knockout analysis, we demonstrate that pre-optic AgRP2 is a neuroendocrine regulator of the stress axis that reduces cortisol secretion. We therefore suggest that the ancestral role of AgRP was functionally partitioned in zebrafish by the two AgRPs, with AgRP1 centrally regulating food consumption and AgRP2 acting as a neuroendocrine factor regulating the stress axis.

Data on Western blot and ELISA analysis of medaka (Oryzias latipes) follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) using recombinant proteins expressed with Pichia pastoris.

The gonadotropins follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) play essential roles in vertebrate reproduction. This article presents data on molecular weight validation of recombinant medaka (Oryzias latipes) (md) gonadotropins Fshß (mdFshß), Lhß (mdLhß), Fshßα (mdFshßα), and Lhßα (mdLhßα) generated by Pichia pastoris, as well as data on a validation of produced antibodies against Fshß and Lhß by Western blot analysis. Furthermore, the article includes data on Fsh and Lh protein levels in male medaka pituitaries using recombinant mdFshßα and mdLhßα within enzyme-linked immunosorbent assays (ELISAs), in which protein amounts were analyzed related to body weight and age of the fish. This dataset is associated with the research article entitled "Medaka Follicle-stimulating hormone (Fsh) and Luteinizing hormone (Lh): Developmental profiles of pituitary protein and gene expression" (Burow et al., in press).

Medaka follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh): Developmental profiles of pituitary protein and gene expression levels.

The two gonadotropins follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) are of particular importance within the hypothalamic-pituitary-gonadal (HPG) axis of vertebrates. In the current study, we demonstrate the production and validation of Japanese medaka (Oryzias latipes) recombinant (md) gonadotropins Fshß (mdFshß), Lhß (mdLhß), Fshßα (mdFshßα), and Lhßα (mdLhßα) by Pichia pastoris, the generation of specific rabbit antibodies against their respective ß subunits, and their use within the development and validation of competitive enzyme-linked immunosorbent assays (ELISAs) for quantification of medaka Fsh and Lh. mdFsh and mdLh were produced as single-chain polypeptides by linking the α subunit with mdFshß or mdLhß mature protein coding sequences to produce a "tethered" polypeptide with the ß-chain at the N-terminal and the α-chain at the C-terminal. The specificity of the antibodies raised against mdFshß and mdLhß was determined by immunofluorescence (IF) for Fshß and Lhß on medaka pituitary tissue, while comparison with fluorescence in situ hybridization (FISH) for fshb and lhb mRNA was used for validation. Competitive ELISAs were developed using antibodies against mdFshß or mdLhß, and the tethered proteins mdFshßα or mdLhßα for standard curves. The standard curve for the Fsh ELISA ranged from 97.6 pg/ml to 50 ng/ml, and for the Lh ELISA from 12.21 pg/ml to 6.25 ng/ml. The sensitivity of the assays for Fsh and Lh was 44.7 and 70.8 pg/ml, respectively. A profile of pituitary protein levels of medaka Fsh and Lh comparing juveniles with adults showed significant increase of protein amount from juvenile group (body length from 12 mm to 16.5 mm) to adult group (body length from 21 mm to 26.5 mm) for both hormones in male medaka. Comparing these data to a developmental profile of pituitary mRNA expression of medaka fshb and lhb, the mRNA expression of lhb also increased during male maturation and a linear regression analysis revealed a significant increase of lhb expression with increased body length that proposes a linear model. However, fshb mRNA expression did not change significantly during male development and therefore was not correlated with body length. In summary, we have developed and validated homologous ELISA assays for medaka Fsh and Lh based on proteins produced in P. pastoris, assays that will be used to study the functions and regulations of Fsh and Lh in more detail.

Characterization of carp gonadotropins: Structure, annual profile, and carp and zebrafish pituitary topographic organization.

Two gonadotropins, follicle stimulating hormone (FSH) and luteinizing hormone (LH), are important players in the hypothalamic-pituitary-gonadal axis of vertebrates. In the present work, we describe the construction of recombinant (r) common carp (Cyprinus carpio; c) FSH (rcFSH) and LH (rcLH) using the Pichia pastoris system, the generation of specific antibodies against their respective ß subunits, and their use in the development and validation of specific ELISAs. We produced carp rLH and rFSH as single-chain polypeptides, wherein the GTH subunit α was joined with either cLHß or cFSHß mature protein-coding sequences to form a fusion gene that encodes a yoked polypeptide, in which the GTH ß-subunit forms the N-terminal part and the α-subunit forms the C-terminal part. Competitive ELISAs were developed, using primary antibodies against rcLHß or rcFSHß, respectively, and rcLHßα or rcFSHßα for the standard curves. The standard curves for cLH paralleled those of pituitary extracts of the homologous fish and also those of other cyprinids species like the black carp (Mylopharyngodon piceus), goldfish (Carassius auratus), silver carp (Hypophthalmichthys molitrix), and grass carp (Ctenopharyngodon idella). We used the specific antibodies raised against cFSH and cLH to study the specific localization of the different GTH cells in the pituitary of carp and its taxonomic relative species - the zebrafish. Both FSH and LH cells are localized in the center of the proximal pars distalis enveloping both sides of the neurohypophysis. LH cells form a continuous population throughout the PPD, while FSH cells are more loosely distributed throughout the same area and form small aggregations. Marked annual changes were encountered in gonadosomatic index (GSI), follicle diameter, mRNA levels and protein levels of FSH and LH. From September to November, all fish had low GSI, and the ovary contained previtellogenic follicles. From December, the GSI level increased and remained high until March, the follicular diameter reached its maximum in January, where the ovary contained large fully grown follicles. Thereafter, spawning occurred through March and April and ended in May, and GSI level and follicle diameter increased again; and the ovary contained mid-vitellogenic follicles. LH pituitary content and mRNA levels were low at pre- and early vitellogenesis, increasing gradually during this process to reach a peak of LH mRNA levels in mid vitellogenic ovary and a peak of LH content in fully grown ovarian follicles. However, no significant change occurred in FSH pituitary content and mRNA levels in vitellogenic fish and in fish during final maturation stages. A dramatic difference was found in the total content of each gonadotropin in the pituitary, with higher LH than FSH. Moreover, follicle diameter was positively and significantly correlated with LH pituitary content and its transcript levels - but not with the pituitary content or mRNA levels of FSH. Taken together, these results indicate that in carp, LH alone is sufficient to regulate both vitellogenesis and final oocyte maturation while FSH may have another, yet undefined role.

Biologically active recombinant carp LH as a spawning-inducing agent for carp.

Currently, spawning is induced in carp species by carp pituitary extract (CPE) and a combination of synthetic agonist of GnRH combined with a dopamine antagonist. The main goal of this study was the production of recombinant gonadotropins (GtHs) on a large scale to serve as an alternative to currently used agents. We produced carp (c) recombinant (r) Lh as a single chain in the methylotrophic yeast Pichia pastoris Lha subunit was joined with Lhb subunit with a flexible linker of three glycine-serine repeats and six Histidines to form a mature protein, the ß-subunit formed the N-terminal part and the α-subunit formed the C-terminal part. The ability of the rcLh to elicit biological response was tested by in vivo stimulation of estradiol (E2) and 17α,20ß-dihydroxy-4-pregnen-3-one (DHP) and by its in vivo potency to induce ovulation and spawning induction. rcLh tested in this work significantly enhanced both E2 and DHP secretion in a dose-dependent manner similar to the results obtained with CPE. E2 levels showed a moderate rise following the priming injection and a subsequent decrease during the rest of the trial. DHP levels were only increased after the resolving injection, approximately 5 h before spawning. At the highest dose of rcLh (350 µg/kg BW), the recombinant protein was more efficient than CPE in terms of both spawning success and fertilization rate. It is shown here that rcLh can elicit the secretion of DHP in vivo and actually trigger spawning. These novel findings introduce the potential of utilizing recombinant gonadotropins in aquaculture.

Gonadotropins in the Russian Sturgeon: Their Role in Steroid Secretion and the Effect of Hormonal Treatment on Their Secretion.

In the reproduction process of male and female fish, pituitary derived gonadotropins (GTHs) play a key role. To be able to specifically investigate certain functions of Luteinizing (LH) and Follicle stimulating hormone (FSH) in Russian sturgeon (Acipenser gueldenstaedtii; st), we produced recombinant variants of the hormones using the yeast Pichia pastoris as a protein production system. We accomplished to create in vitro biologically active heterodimeric glycoproteins consisting of two associated α- and ß-subunits in sufficient quantities. Three dimensional modelling of both GTHs was conducted in order to study the differences between the two GTHs. Antibodies were produced against the unique ß-subunit of each of the GTHs, in order to be used for immunohistochemical analysis and to develop an ELISA for blood and pituitary hormone quantification. This detection technique revealed the specific localization of the LH and FSH cells in the sturgeon pituitary and pointed out that both cell types are present in substantially higher numbers in mature males and females, compared to immature fish. With the newly attained option to prevent cross-contamination when investigating on the effects of GTH administration, we compared the steroidogeneic response (estradiol and 11-Keto testosterone (11-KT) in female and males, respectively) of recombinant stLH, stFSH, and carp pituitary extract in male and female sturgeon gonads at different developmental stages. Finally, we injected commercially available gonadotropin releasing hormones analog (GnRH) to mature females, and found a moderate effect on the development of ovarian follicles. Application of only testosterone (T) resulted in a significant increase in circulating levels of 11-KT whereas the combination of GnRH + T did not affect steroid levels at all. The response pattern for estradiol demonstrated a similar situation. FSH levels showed significant increases when GnRH + T was administered, while no changes were present in LH levels.

Stellate Cell Networks in the Teleost Pituitary.

The folliculostellate cells of the mammalian pituitary are non-endocrine cells that are implicated in long-distance communication and paracrine signaling, but to date, these cells have yet to be characterized in teleosts. We found that the stellate cells of the teleost pituitary share many common attributes with mammalian folliculostellate cells. By labeling of stellate cells in live preparations of tilapia pituitaries we investigated their distribution, association with other endocrine cells and their anatomical and functional coupling. In the pars intermedia, stellate cells were arranged around neuronal bundles and their processes extended into the pars distalis. Within the pars distalis, stellate cells formed close associations with FSH cells and, to a lesser degree, with GH and LH cells, suggesting differential paracrine regulation of the two gonadotrope populations. The production of follistatin by stellate cells further corroborates the notion of a paracrine role on FSH release. We also found stellate cells to form gap junctions that enabled dye transfer to neighboring stellate cells, implicating that these cells form a large-scale network that connects distant parts of the pituitary. Our findings represent the first wide-scale study of stellate cells in teleosts and provide valuable information regarding their functional roles in pituitary function.

Discovery of a novel insulin-like peptide and insulin binding proteins in the Eastern rock lobster Sagmariasus verreauxi.

This study reports, for the first time in any of the commercially important decapod species, the identification of an insulin-like peptide (ILP), distinct from the androgenic gland hormone. Bioinformatics analysis of the de novo assembled spiny lobster, (Sagmariasus verreauxi) transcriptome, allowed identification of Sv-ILP1 as well as eight binding proteins. Binding proteins were termed as Sv-IGFBP, due to homology with the vertebrate insulin-like growth-factor binding protein and Sv-SIBD1-7, single insulin-binding domain protein (SIBD), similar to those identified in other invertebrate species. Sv-ILP1 was found to be expressed in the eyestalk, gonads and antennal gland of both sexes and to a lesser extent in male muscle, androgenic gland and hepatopancreas. The expression profiles of each binding protein were found to vary across tissues, with Sv-SIBD5, 6 and 7 showing higher expression in the gonad, demonstrated by PCR and digital gene expression. Further spatial investigations, using in-situ hybridisation, found Sv-ILP1 to be expressed in the neurosecretory cells of the thoracic ganglia, in keeping with the tissue expression of Drosophila ILP7 (DILP7). This correlative tissue expression, considered with the phylogenetic clustering of Sv-ILP1 and DILP7, suggests Sv-ILP1 to be a DILP7 orthologue. The broad expression of Sv-ILP1 strongly suggests that ILPs have a role beyond that of masculinisation in decapods. The function of these novel peptides may have application in enhancing aquaculture practices in the commercially important decapod species.

Production, gene structure and characterization of two orthologs of leptin and a leptin receptor in tilapia.

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.