Characterization of a novel fast-growing zebrafish: a new approach to growth hormone transgenesis.

The manipulation of the somatotropic axis, governing growth, has been a focus of numerous transgenic approaches aimed at developing fast-growing fish for research, medicine and aquaculture purposes. However, the excessively high growth hormone (GH) levels in these transgenic fish often result in deformities that impact both fish health and consumer acceptance. In an effort to mitigate these issues and synchronize exogenous GH expression with reproductive processes, we employed a novel transgenic construct driven by a tilapia luteinizing hormone (LH) promoter. This approach was anticipated to induce more localized and lower exogenous GH secretion. In this study, we characterized the growth and reproduction of these transgenic LHp-GH zebrafish using hormonal and physiological parameters. Our findings reveal that LHp-GH fish exhibited accelerated growth in both length and weight, along with a lower feed conversion ratio, indicating more efficient feed utilization, all while maintaining unchanged body proportions. These fish demonstrated higher expression levels of LH and GH in the pituitary and elevated IGF-1 levels in the liver compared to wild-type fish. An examination of reproductive function in LHp-GH fish unveiled lower pituitary LH and FSH contents, smaller follicle diameter in female gonads, and reduced relative fecundity. However, in transgenic males, neither the distribution of spermatogenesis stages nor sperm concentrations differed significantly between the fish lines. These results suggest that coupling exogenous GH expression with endogenous LH expression in females directs resource investment toward somatic growth at the expense of reproductive processes. Consequently, we conclude that incorporating GH under the LH promoter represents a suitable construct for the genetic engineering of commercial fish species, providing accelerated growth while preserving body proportions.

High-resolution tracking of unconfined zebrafish behavior reveals stimulatory and anxiolytic effects of psilocybin.

Serotonergic psychedelics are emerging therapeutics for psychiatric disorders, yet their underlying mechanisms of action in the brain remain largely elusive. Here, we developed a wide-field behavioral tracking system for larval zebrafish and investigated the effects of psilocybin, a psychedelic serotonin receptor agonist. Machine learning analyses of precise body kinematics identified latent behavioral states reflecting spontaneous exploration, visually-driven rapid swimming, and irregular swim patterns following stress exposure. Using this method, we found that acute psilocybin treatment has two behavioral effects: [i] facilitation of spontaneous exploration ("stimulatory") and [ii] prevention of irregular swim patterns following stress exposure ("anxiolytic"). These effects differed from the effect of acute SSRI treatment and were rather similar to the effect of ketamine treatment. Neural activity imaging in the dorsal raphe nucleus suggested that psilocybin inhibits serotonergic neurons by activating local GABAergic neurons, consistent with psychedelic-induced suppression of serotonergic neurons in mammals. These findings pave the way for using larval zebrafish to elucidate neural mechanisms underlying the behavioral effects of serotonergic psychedelics.

In silico insights into intra- and inter-species interactions of piscine gonadotropin hormones and receptor crosstalk.

In mammals, the gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are macromolecules secreted during specific reproductive phases and display strict specificity towards their cognate receptors. However, fish gonadotropins (GTH) and their receptors (GTHR) display diverse species-specific expression patterns, secretion patterns, and intra- and interspecies cross-activation. To uncover the molecular basis of this diversity, we generated and analyzed 29 in-silico models of intra- and inter-species combinations of sturgeon, carp, tilapia, and human gonadotropins with piscine receptors and analyzed the resulting receptor activation and signal transduction of these GTHR-GTH complexes in-vitro. Our results suggest that unlike humans, the surface charge on piscine FSH/LH ß-seatbelt and N107huLHCGR/K104hFSHR homologs does not necessarily determine binding specificity. Instead, sequence and structural variations allow piscine GTHs significant conformational flexibility when binding to the receptor extracellular domain, thereby enabling cross-activation. The resulting diversity may support various reproductive strategies in different environmental niches.

Transcriptomes of testis and pituitary from male Nile tilapia (O. niloticus L.) in the context of social status.

African cichlids are well established models for studying social hierarchies in teleosts and elucidating the effects social dominance has on gene expression. Ascension in the social hierarchy has been found to increase plasma levels of steroid hormones, follicle stimulating hormone (Fsh) and luteinizing hormone (Lh) as well as gonadosomatic index (GSI). Furthermore, the expression of genes related to gonadotropins and steroidogenesis and signaling along the brain-pituitary-gonad axis (BPG-axis) is affected by changes of an animal's social status. In this study, we use RNA-sequencing to obtain an in-depth look at the transcriptomes of testes and pituitaries from dominant and subordinate male Nile tilapia living in long-term stable social hierarchies. This allows us to draw conclusions about factors along the brain-pituitary-gonad axis that are involved in maintaining dominance over weeks or even months. We identify a number of genes that are differentially regulated between dominant and subordinate males and show that in high-ranking fish this subset of genes is generally upregulated. Genes differentially expressed between the two social groups comprise growth factors, related binding proteins and receptors, components of Wnt-, Tgfß- and retinoic acid-signaling pathway, gonadotropin signaling and steroidogenesis pathways. The latter is backed up by elevated levels of 11-ketotestosterone, testosterone and estradiol in dominant males. Luteinizing hormone (Lh) is found in higher concentration in the plasma of long-term dominant males than in subordinate animals. Our results both strengthen the existing models and propose new candidates for functional studies to expand our understanding of social phenomena in teleost fish.

Chemogenetic Depletion of Hypophysiotropic GnRH Neurons Does Not Affect Fertility in Mature Female Zebrafish.

The hypophysiotropic gonadotropin-releasing hormone (GnRH) and its neurons are crucial for vertebrate reproduction, primarily in regulating luteinizing hormone (LH) secretion and ovulation. However, in zebrafish, which lack GnRH1, and instead possess GnRH3 as the hypophysiotropic form, GnRH3 gene knockout did not affect reproduction. However, early-stage ablation of all GnRH3 neurons causes infertility in females, implicating GnRH3 neurons, rather than GnRH3 peptides in female reproduction. To determine the role of GnRH3 neurons in the reproduction of adult females, a Tg(gnrh3:Gal4ff; UAS:nfsb-mCherry) line was generated to facilitate a chemogenetic conditional ablation of GnRH3 neurons. Following ablation, there was a reduction of preoptic area GnRH3 neurons by an average of 85.3%, which was associated with reduced pituitary projections and gnrh3 mRNA levels. However, plasma LH levels were unaffected, and the ablated females displayed normal reproductive capacity. There was no correlation between the number of remaining GnRH3 neurons and reproductive performance. Though it is possible that the few remaining GnRH3 neurons can still induce an LH surge, our findings are consistent with the idea that GnRH and its neurons are likely dispensable for LH surge in zebrafish. Altogether, our results resurrected questions regarding the functional homology of the hypophysiotropic GnRH1 and GnRH3 in controlling ovulation.

Cloning of gonadotropin Gph-alpha, FSH-beta and LH-beta subunits and seasonal profiles of steroid hormones in wild-caught Nile perch, Lates niloticus.

The Nile perch (np; Lates niloticus) is a freshwater teleost species with a potential for aquaculture in freshwater surroundings. However, wild-caught breeders have persistently failed to spawn spontaneously in captivity. Cloning of the gonadotropin subunits and analysing seasonal variation in reproductive hormone levels for a 1-year period were done to gain knowledge on the physiological basis underlying the reproductive biology of np. The ß-follicle-stimulating hormone (FSH-ß) and ß-luteinizing hormone (LH-ß) subunits and their common α-glycoprotein (Gph-α) subunit were cloned using 3' and 5' RACE-PCR. The nucleotide sequences of the npgph-α, npfsh-ß, and nplh-ß subunits were 664, 580 and 675 nucleotides in length, encoding peptides of 124, 120 and 148 amino acids, respectively. The deduced amino acid sequence of each mature subunit showed high similarity with its counterparts in other teleost. Sequence analysis showed that npFSH-ß is more similar to higher vertebrate FSH-ßs than to higher vertebrate LH-ßs. Heterologous immunoassay was calibrated to analyse pituitary LH levels. While the LH immunoassay showed parallelism of npLH with that of tilapia (ta), no parallelism for FSH was found. Levels of pituitary LH were higher in females at gonadal stages of vitellogenic oocytes, mature secondary oocytes and mature tertiary oocytes with migrating nucleus than in pre-vitellogenic oocytes and early and late perinucleolus oocytes. Using competitive steroid ELISA, variations in the levels of the steroid hormones 11-ketotestosterone (11-KT) in males and E2 in females were characterized in relation to month and reproductive index of Nile perch. Our findings show that in females, gonadosomatic index and plasma E2 were highly correlated (R2 = 0.699, n = 172) and peaked from September to November while in males, the gonadosomatic index and plasma 11-KT peaked from October to November. In female fish, both steroid hormones were detected in the plasma but greatly varied in concentrations. E2 in particular, increased with the developmental stage of the gonads. The levels of steroid hormones, E2 and 11-KT in females and males respectively increased with fish size (total lengths) and suggest that females mature at a body length of 40-59 cm than their counter part males that mature at a total length of 60-70 cm. Taken together, we describe seasonal endocrine differences in wild-caught adult Nile perch which could potentially be exploited to manipulate the reproductive axis in cultured breeders.

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.

Vasoactive Intestinal Peptide Indirectly Elicits Pituitary LH Secretion Independent of GnRH in Female Zebrafish.

Vasoactive intestinal peptide (Vip) regulates luteinizing hormone (LH) release through the direct regulation of gonadotropin-releasing hormone (GnRH) neurons at the level of the brain in female rodents. However, little is known regarding the roles of Vip in teleost reproduction. Although GnRH is critical for fertility through the regulation of LH secretion in vertebrates, the exact role of the hypophysiotropic GnRH (GnRH3) in zebrafish is unclear since GnRH3 null fish are reproductively fertile. This phenomenon raises the possibility of a redundant regulatory pathway(s) for LH secretion in zebrafish. Here, we demonstrate that VipA (homologues of mammalian Vip) both inhibits and induces LH secretion in zebrafish. Despite the observation that VipA axons may reach the pituitary proximal pars distalis including LH cells, pituitary incubation with VipA in vitro, and intraperitoneal injection of VipA, did not induce LH secretion and lhß mRNA expression in sexually mature females, respectively. On the other hand, intracerebroventricular administration of VipA augmented plasma LH levels in both wild-type and gnrh3-/- females at 1 hour posttreatment, with no observed changes in pituitary GnRH2 and GnRH3 contents and gnrh3 mRNA levels in the brains. While VipA's manner of inhibition of LH secretion has yet to be explored, the stimulation seems to occur via a different pathway than GnRH3, dopamine, and 17ß-estradiol in regulating LH secretion. The results indicate that VipA induces LH release possibly by acting with or through a non-GnRH factor(s), providing proof for the existence of functional redundancy of LH release in sexually mature female zebrafish.

Characteristics of Neurokinin-3 Receptor and Its Binding Sites by Mutational Analysis.

NKB (Neurokinin B) is already known to play a crucial role in fish reproduction, but little is known about the structure and function of NKB receptors. Based on an in silico model of the tilapia NKB receptor Tachykinin 3 receptor a (tiTac3Ra) found in the current study, we determined the key residues involved in binding to tilapia NKB and its functional homologue NKF (Neurokinin F). Despite studies in humans suggesting the crucial role of F2516.44 and M2897.43 in NKB binding, no direct peptide interaction was observed in tilapia homologs. In-silico, Ala mutations on residues F2516.44 and M2897.43 did not influence binding affinity, but significantly affected the stability of tiTac3Ra. Moreover, in vitro studies indicated them to be critical to tiNKB/tiNKF-induced receptor activity. The binding of NKB antagonists to tiTac3Ra both in-vitro and in vivo inhibits FSH (follicle stimulating hormone) and LH (luteinizing hormone) release and sperm production in mature tilapia males. Non-peptide NKB antagonist SB-222200 had a strong inhibitory effect on the Tac3Ra activation. SB-222200 also decreased LH plasma levels; two hours post intraperitoneal injection, changed sperm volume and the ratios of the different stages along the spermatogenesis in tilapia testes.

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.

Gnrh2 maintains reproduction in fasting zebrafish through dynamic neuronal projection changes and regulation of gonadotropin synthesis, oogenesis, and reproductive behaviors.

Restricted food intake, either from lack of food sources or endogenous fasting, during reproductive periods is a widespread phenomenon across the animal kingdom. Considering previous studies show the canonical upstream regulator of reproduction in vertebrates, the hypothalamic Gonadotropin-releasing hormone (Gnrh), is inhibited in some fasting animals, we sought to understand the neuroendocrine control of reproduction in fasted states. Here, we explore the roles of the midbrain neuropeptide, Gnrh2, in inducing reproduction via its pituitary prevalence, gonadotropin synthesis, gametogenesis, and reproductive outputs in the zebrafish model undergoing different feeding regimes. We discovered a fasting-induced four-fold increase in length and abundance of Gnrh2 neuronal projections to the pituitary and in close proximity to gonadotropes, whereas the hypothalamic Gnrh3 neurons are reduced by six-fold in length. Subsequently, we analyzed the functional roles of Gnrh2 by comparing reproductive parameters of a Gnrh2-depleted model, gnrh2-/-, to wild-type zebrafish undergoing different feeding conditions. We found that Gnrh2 depletion in fasted states compromises spawning success, with associated decreases in gonadotropin production, oogenesis, fecundity, and male courting behavior. Gnrh2 neurons do not compensate in other circumstances by which Gnrh3 is depleted, such as in gnrh3-/- zebrafish, implying that Gnrh2 acts to induce reproduction specifically in fasted zebrafish.

Molecular characterization of kisspeptin receptors and gene expression analysis during oogenesis in the Russian sturgeon (Acipenser gueldenstaedtii).

Sturgeons belong to a subclass of fishes that derived from ray-finned fish ancestors preceding the emergence of teleosts. The Russian sturgeon (Acipenser gueldenstaedtii) is a late-maturing fish with the females reaching puberty under aquaculture conditions at 6-10 years of age. Since kisspeptin has been shown to be a key hormone involved in regulation of major reproductive processes of many vertebrate species, this study was conducted to better understand the kisspeptin receptor (KissR) in sturgeon. In this study we have cloned Russian sturgeon KissR1 from brain mRNA and observed the ontogeny of rsKissR1 mRNA expression in ovarian follicles. Multiple sequence alignment of KissR1, KissR4, and their orthologs revealed that the Russian sturgeon (rs) KissR1 sequence shares 64%-77% identity with elephant shark, coelacanth, and gar and 44-58% identity with tetrapod and teleost KissR1 sequences, while KissR4 seemed to share <65% identity to eel KissR2 and ~57% identity to Perciformes and Cypriniformes. Further rsKissR4 showed <97% identity to reed fish KissR4, <63% with Squamata (Reptiles) and gar KissR4. A phylogenetic analysis revealed that rsKissR1 is more closely related to coelacanth and gar KissR1 than teleost, while rsKissR4 was part of the KissR4 clade and shared higher similarity with Actinopterygiian sequences. We have further predicted homology models for both rsKiss receptors and performed in-silico analyses of their binding to a kiss-10 peptide. Both sturgeon and zebrafish Kiss1 and Kiss2 activated rsKissR1 via both PKC/Ca2+ and PKA/cAMP signal-transduction pathways, while rsKissR2 was found to be less effective and was not activated by stKiss peptides. Ovarian rsKissR transcript levels for 10 fishes were determined by real-time PCR and significantly increased concomitantly with oogenesis, where the highest level of expression was evident in black follicles. These data suggest that extra-neuronal expression of the kisspeptin receptor may be involved in sturgeon reproduction in a manner dependent on reproductive development.

An ex vivo Approach to Study Hormonal Control of Spermatogenesis in the Teleost Oreochromis niloticus.

As the male reproductive organ, the main task of the testis is the production of fertile, haploid spermatozoa. This process, named spermatogenesis, starts with spermatogonial stem cells, which undergo a species-specific number of mitotic divisions until starting meiosis and further morphological maturation. The pituitary gonadotropins, luteinizing hormone, and follicle stimulating hormone, are indispensable for vertebrate spermatogenesis, but we are still far from fully understanding the complex regulatory networks involved in this process. Therefore, we developed an ex vivo testis cultivation system which allows evaluating the occurring changes in histology and gene expression. The experimental circulatory flow-through setup described in this work provides the possibility to study the function of the male tilapia gonads on a cellular and transcriptional level for at least 7 days. After 1 week of culture, tilapia testis slices kept their structure and all stages of spermatogenesis could be detected histologically. Without pituitary extract (tilPE) however, fibrotic structures appeared, whereas addition of tilPE preserved spermatogenic cysts and somatic interstitium completely. We could show that tilPE has a stimulatory effect on spermatogonia proliferation in our culture system. In the presence of tilPE or hCG, the gene expression of steroidogenesis related genes (cyp11b2 and stAR2) were notably increased. Other testicular genes like piwil1, amh, or dmrt1 were not expressed differentially in the presence or absence of gonadotropins or gonadotropin containing tilPE. We established a suitable system for studying tilapia spermatogenesis ex vivo with promise for future applications.

Molecular characterization of two Russian sturgeon gonadotropin receptors: Cloning, expression analysis, and functional activity.

Sturgeons are being used in aquaculture because wild populations are now endangered due to overfishing for caviar. A challenge in working with sturgeon as an aquacultured species is its long and slow reproductive development. Reproduction is a hormonally regulated process that involves hierarchical signaling between the brain, pituitary gland, and gonads. In an effort to better understand the hormonal regulation of sturgeon reproduction, we have cloned the Russian sturgeon (st), Acipenser gueldenstaedtii, luteinizing hormone receptor (stLHR) and follicle stimulating hormone receptor (stFSHR) and measured their expression from previtellogenic to mature ovarian follicles. Sturgeon LHR and FSHR expression was elevated in early-vitellogenic and mature follicles compared with pre-vitellogenic and mid-vitellogenic follicles, and only LHR expression increased during late-vitellogenesis. Recombinant sturgeon FSH and LH both activated sturgeon LHR and FSHR in a cAMP reporter assay. Further molecular characterization of these receptors was accomplished by in silico modeling and cAMP reporter assays using heterologous recombinant gonadotropins from human and piscine species. There was no apparent trend in heterologous LH and/or FSH activation of the sturgeon LHR or FSHR. These data suggest that permissive activation of LHR and FSHR are a consequence of some yet undetermined biological characteristic(s) of different piscine species.

Ectopic over expression of kiss1 may compensate for the loss of kiss2.

Kisspeptin (KISS) is a neuropeptide which plays a central role in the regulation of the hypothalamic-pituitary-gonadal axis, and is essential for sexual maturation and fertility in mammals. Unlike mammals, which possess only one KISS gene, two paralogous genes, kiss1 and kiss2, have been identified in zebrafish and other non-mammalian vertebrates. Previous studies suggest that Kiss2, but not Kiss1, is the reproduction relevant form amongst the two. To better understand the role of each of these isoforms in reproduction, a loss of function approach was applied. Two genetic manipulation techniques-clustered regularly interspaced short palindromic repeats (CRISPR) and transcription activator-like effector nucleases (TALEN)-were used to generate kiss1 and kiss2 knockout (KO) zebrafish lines, respectively. Examination of these KO lines showed that reproductive capability was not impaired, confirming earlier observations. Further analysis revealed that KO of kiss2 caused a significant increase in expression levels of kiss1, kiss2r and tac3a, while KO of kiss1 had no effect on the expression of any of the examined genes. In situ hybridization analysis revealed that kiss1 mRNA is expressed only in the habenula in wild type brains, while in kiss2 KO fish, kiss1 mRNA-expressing cells were identified also in the ventral telencephalon, the ventral part of the entopeduncular nucleus, and the dorsal and ventral hypothalamus. Interestingly, these regions are known to express kiss2r, and the ventral hypothalamus normally expresses kiss2. These results suggest that a compensatory mechanism, involving ectopic kiss1 expression, takes place in the kiss2 KO fish, which may substitute for Kiss2 activity.

Chronic Social Defeat Stress Up-Regulates Spexin in the Brain of Nile Tilapia (Oreochromis niloticus).

Spexin (SPX), a neuropeptide evolutionarily conserved from fish to mammals, is widely distributed in the brain and peripheral tissues and associated with various physiological functions. Recently SPX has been suggested to be involved in neurological mechanism of stress. The current study investigates the involvement of SPX in chronic social defeat stress, using male teleost, the Nile tilapia (Oreochromis niloticus) as an animal model due to its distinct social hierarchy of dominant and subordinate relationship. The tilapia genome has SPX1a and SPX1b but has no SPX2. In the Nile tilapia, we localized SPX1a and SPX1b in the brain using in-situ hybridization. Next, using qPCR we examined gene expression of SPX1a and SPX1b in chronically stress (socially defeated) fish. SPX1a expressing cells were localized in the semicircular torus of the midbrain region and SPX1b expressing cells in the telencephalon. Chronically stress fish showed elevated plasma cortisol levels; with an upregulation of SPX1a and SPX1b gene expression in the brain compared to non-stress (control) fish. Since social defeat is a source of stress, the upregulated SPX mRNA levels during social defeat suggests SPX as a potentially inhibitory neuropeptide capable of causing detrimental changes in behaviour and physiology.

Spexin and a Novel Cichlid-Specific Spexin Paralog Both Inhibit FSH and LH Through a Specific Galanin Receptor (Galr2b) in Tilapia.

Spexin (SPX) is a 14 amino acid peptide hormone that has pleiotropic functions across vertebrates, one of which is involvement in the brain-pituitary-gonad axis of fish. SPX(1) has been identified in each class of vertebrates, and a second SPX (named SPX2) has been found in some non-mammalian species. We have cloned two spexin paralogs, designated as Spx1a and Spx1b, from Nile tilapia (Oreochromis niloticus) that have varying tissue distribution patterns. Spx1b is a novel peptide only identified in cichlid fish, and is more closely related to Spx1 than Spx2 homologs as supported by phylogenetic, synteny, and functional analyses. Kisspeptin, Spx, and galanin (Gal) peptides and their corresponding kiss receptors and Gal receptors (Galrs), respectively, are evolutionarily related. Cloning of six tilapia Galrs (Galr1a, Galr1b, Galr2a, Galr2b, Galr type 1, and Galr type 2) and subsequent in vitro second-messenger reporter assays for Gαs, Gαq, and Gαi suggests that Gal and Spx activate Galr1a/Galr2a and Galr2b, respectively. A decrease in plasma follicle stimulating hormone and luteinizing hormone concentrations was observed with injections of Spx1a or Spx1b in vivo. Additionally, application of Spx1a and Spx1b to pituitary slices decreased the firing rate of LH cells, suggesting that the peptides can act directly at the level of the pituitary. These data collectively suggest an inhibitory mechanism of action against the secretion of gonadotropins for a traditional and a novel spexin paralog in cichlid species.

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.

Synteny and phylogenetic analysis of paralogous thyrostimulin beta subunits (GpB5) in vertebrates.

At some point early in the vertebrate lineage, two whole genome duplication events (1R, 2R) took place that allowed for the diversification and sub-/neo-functionalization of the glycoprotein hormones (GpHs). All jawed vertebrates possess the GpHs luteinizing hormone (LH), follicle stimulating hormone (FSH), and thyroid stimulating hormone (TSH), each of which are heterodimers with a common alpha subunit and unique beta subunits. In 2002, a novel glycoprotein hormone named thyrostimulin was described to have unique GpA2 and GpB5 subunits that were homologous to the vertebrate alpha and beta subunits. The presence of GpA2 and GpB5 in representative protostomes and deuterostomes indicates their ancestry in the GpH family. There are several reports of GpH subunit evolution, but none have included GpA2 and GpB5 for species in each major vertebrate class. Thus, we addressed the ancestry of two paralogous GpB5 subunits (GpB5a and GpB5b) that were previously only recognized in two teleost species. Our search for orthologous GpB5a and GpB5b sequences in representative vertebrates and phylogenetic analysis, in addition to the currently published evolutionary scenarios of the GpH family, supports that GpB5a and GpB5b are paralogs that arose from the first vertebrate whole genome duplication event (1R). Syntenic analysis supports lineage specific losses of GpB5a in chondrichthyes, basal actinopterygians, and tetrapods, and retention in coelacanth and teleosts. Additionally, we were unable to identify GpA2 transcripts from tilapia mRNA, suggesting that this species does not produce heterodimeric thyrostimulin. While the conserved or even species-specific functional role of thyrostimulin or its individual subunits are still unknown in vertebrates, the analyses presented here provide context for future studies on the functional divergence of the GpH family.