Ana M. Gómez

Perspectives on fish gonadotropins and their receptors.

Teleosts lack a hypophyseal portal system and hence neurohormones are carried by nerve fibers from the preoptic region to the pituitary. The various cell types in the teleost pituitary are organized in discrete domains. Fish possess two gonadotropins (GtH) similar to FSH and LH in other vertebrates; they are heterodimeric hormones that consist of a common alpha subunit non-covalently associated with a hormone-specific beta subunit. In recent years the availability of molecular cloning techniques allowed the isolation of the genes coding for the GtH subunits in 56 fish species representing at least 14 teleost orders. Advanced molecular engineering provides the technology to produce recombinant GtHs from isolated cDNAs. Various expression systems have been used for the production of recombinant proteins. Recombinant fish GtHs were produced for carp, seabream, channel and African catfish, goldfish, eel, tilapia, zebrafish, Manchurian trout and Orange-spotted grouper. The hypothalamus in fishes exerts its regulation on the release of the GtHs via several neurohormones such as GnRH, dopamine, GABA, PACAP, IGF-I, norepinephrine, NPY, kisspeptin, leptin and ghrelin. In addition, gonadal steroids and peptides exert their effects on the gonadotropins either directly or via the hypothalamus. All these are discussed in detail in this review. In mammals, the biological activities of FSH and LH are directed to different gonadal target cells through the cell-specific expression of the FSH receptor (FSHR) and LH receptor (LHR), respectively, and the interaction between each gonadotropin-receptor couple is highly selective. In contrast, the bioactivity of fish gonadotropins seems to be less specific as a result of promiscuous hormone-receptor interactions, while FSHR expression in Leydig cells explains the strong steroidogenic activity of FSH in certain fish species.

Evidence for two distinct KiSS genes in non-placental vertebrates that encode kisspeptins with different gonadotropin-releasing activities in fish and mammals.

Kisspeptins, the products of KiSS-1 gene, have recently emerged as fundamental regulators of reproductive function in different mammalian and, presumably, non-mammalian species. To date, a single form of KiSS-1 has been described in mammals, and recently, in several fish species and Xenopus. We report herein the cloning and characterization of two distinct KiSS-like genes, namely, KiSS-1 and KiSS-2, in the teleost sea bass. While KiSS-1 encodes a peptide identical to rodent kisspeptin-10, the predicted KiSS-2 decapeptide diverges at 4 amino acids (FNFNPFGLRF). Genome database searches showed that both genes are present in non-placental vertebrate genomes. Indeed, phylogenetic and genome mapping analyses suggest that KiSS-1 and KiSS-2 are paralogous genes that originated by duplication of an ancestral gene, although KiSS-2 is lost in placental mammals. KiSS-1 and KiSS-2 mRNAs are present in brain and gonads of sea bass, medaka and zebrafish. Comparative functional studies demonstrated that KiSS-2 decapeptide was significantly more potent than KiSS-1 peptide in inducing LH and FSH secretion in sea bass. In contrast, KiSS-2 decapeptide only weakly elicited LH secretion in rats, whereas KiSS-1 peptide was maximally effective. Our data are the first to provide conclusive evidence for the existence of a second KiSS gene, KiSS-2, in non-placental vertebrates, whose product is likely to play a dominant stimulatory role in the regulation of the gonadotropic axis at least in teleosts.

Comparative insights of the kisspeptin/kisspeptin receptor system: Lessons from non-mammalian vertebrates

Kisspeptins, the peptide products of the Kiss1 gene, were initially identified in mammals as ligands of the G protein-coupled receptor 54 (GPR54; also termed Kiss1R) with ability to suppress tumor metastasis. In late 2003, the indispensable role of kisspeptins in the control of reproductive function was disclosed by the seminal observations that humans and mice carrying inactivating mutations of GPR54 displayed hypogonadotropic hypogonadism. Since then, numerous experimental studies, conducted initially in several mammalian species, have substantiated the roles of kisspeptins as essential players in the physiologic regulation of key aspects of reproductive maturation and function, including the timing of puberty onset, the dynamic control of gonadotropin secretion via stimulation of GnRH neurons, the transmission of the negative and positive feedback effects of sex steroids, the metabolic regulation of fertility and the control of reproductive function by environmental (photoperiodic) cues. Notably, while studies about kisspeptins in non-mammals appeared initially to lag behind, significant efforts have been devoted recently to define the genomic organization and functional characteristics of kiss/kisspeptins and gpr54 in different non-mammalian species, including fish, reptiles and amphibians. These analyses, which will be comprehensively revised herein, have not only substantiated the conserved, essential roles of kisspeptins in the control of reproduction, but have also disclosed intriguing evolutionary aspects of kisspeptins and their receptors. Such comparative approaches will be instrumental to fuel further studies on the molecular regulation and physiological roles of kisspeptins, thus helping to unveil the complex biology of this system as indispensable regulator of the reproductive axis in a wide diversity of animal species.

Seasonal changes in gonadal expression of gonadotropin receptors, steroidogenic acute regulatory protein and steroidogenic enzymes in the European sea bass

The endocrine regulation of gametogenesis, and particularly the roles of gonadotropins, is still poorly understood in teleost fish. This study aimed to investigate transcript levels of both gonadotropin receptors (FSHR and LHR) during an entire reproductive cycle in male and female sea bass (Dicentrarchus labrax). To have a more comprehensive understanding of how different key factors interact to control sea bass gonadal function, changes in the transcript abundance of two important steroidogenic enzymes, P450 11beta-hydroxylase (CYP11B1) and P450 aromatase (CYP19A1), and the steroidogenic acute regulatory protein (StAR), were also studied. These expression profiles were analysed in relation to changes in the plasma levels of important reproductive hormones and histological data. Expression of the FSHR was connected with early stages of gonadal development, but also with the spermiation/maturation-ovulation periods. The expression profile of the LHR seen in both sexes supports the involvement of LH in the regulation of the final stages of gamete maturation and spermiation/ovulation. In both sexes StAR expression was strongly correlated with LHR expression. In females high magnitude increments of StAR expression levels were observed during the maturation-ovulation stage. In males, gonadotropin receptors and CYP11B1 mRNA levels were found to be correlated. In females, the expression profiles of FSHR and CYP19A1 and the changes in plasma estradiol (E2) indicate that the follicular production of E2 could be under control of FSH through the regulation of aromatase expression. This study supports the idea that FSH and LH may have different roles in the control of sea bass gonadal function.

Molecular characterization of two sea bass gonadotropin receptors: cDNA cloning, expression analysis, and functional activity.

The follicle-stimulating hormone (FSH) and the luteinizing hormone (LH) play central roles in vertebrate reproduction. They act through their cognate receptors to stimulate testicular and ovarian functions. The present study reports the cloning and characterization of two sea bass (Dicentrarchus labrax) cDNAs encoding a FSH receptor (sbsFSHR) and a LH receptor (sbsLHR). The mature proteins display typical features of the glycoprotein hormone receptor family members, but the sbsFSHR also contains some remarkable differences when compared with other fish or mammalian FSHRs. Among them, a distinct extracellular N-terminal cysteine domain as regards to its length and cysteine number, and the presence of an extra leucine-rich repeat. Expression analysis revealed that the sbsFSHR is exclusively expressed in gonadal tissues, specifically in the follicular wall of previtellogenic and early-vitellogenic follicles. On the contrary, sbsLHR mRNA was found to be widely distributed in sea bass somatic tissues. When stably expressed in mammalian cell lines, sbsFSHR was specifically stimulated by bovine FSH, while sbsLHR was activated by both bovine LH and FSH. Nevertheless, specific stimulation of the sbsLHR was observed when recombinant sea bass gonadotropins were used. The isolation of a FSHR and a LHR in sea bass opens new ways to study gonadotropin action in this species.

Follicle-Stimulating Hormone and Luteinizing Hormone Mediate the Androgenic Pathway in Leydig Cells of an Evolutionary Advanced Teleost

ABSTRACT The endocrine pathways controlling vertebrate spermatogenesis are well established in mammals where the pituitary gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) exclusively activate the FSH receptor (FSHR) in Sertoli cells and the LH/choriogonadotropin receptor (LHCGR) in Leydig cells, respectively. In some teleosts, however, it has been shown that Lh can cross-activate the Fshra ortholog, and that Leydig cells coexpress the Lhcgrba and Fshra paralogs, thus mediating the androgenic function of Fsh in the testis. Here, we investigated whether these proposed mechanisms are conserved in an evolutionary advanced pleuronectiform teleost, the Senegalese sole (Solea senegalensis). Transactivation assays using sole Fshra- and Lhcgrba-expressing cells and homologous single-chain recombinant gonadotropins (rFsh and rLh) showed that rFsh exclusively activated Fshra, whereas rLh stimulated both Lhcgrba and Fshra. The latter cross-activation of Fshra by rLh occurred with an EC50 4-fold higher than for rFsh. Both recombinant gonadotropins elicited a significant androgen release response in vitro and in vivo, which was blocked by protein kinase A (PKA) and 3beta-hydroxysteroid dehydrogenase inhibitors, suggesting that activation of steroidogenesis through the cAMP/PKA pathway is the major route for both Lh- and Fsh-stimulated androgen secretion. Combined in situ hybridization and immunocytochemistry using cell-specific molecular markers and antibodies specifically raised against sole Fshra and Lhcgrba demonstrated that both receptors are expressed in Leydig cells, whereas Sertoli cells only express Fshra. These data suggest that Fsh-mediated androgen production through the activation of cognate receptors in Leydig cells is a conserved pathway in Senegalese sole.

Purification and characterization of follicle-stimulating hormone from pituitary glands of sea bass (Dicentrarchus labrax).

Follicle-stimulating hormone (FSH) was purified from pituitaries of sea bass (Dicentrarchus labrax), and its biochemical and biological properties were studied. Sea bass FSH (sbsFSH) was purified by ethanol extraction-precipitation (40-85%), followed by anion-exchange chromatography on a LKB Ultropac TSK-DEAE column using a linear gradient of ammonium bicarbonate (50-1000 mM) and reverse phase chromatography on a RESOURCE 15RPC column with a linear gradient of acetonitrile (0-50%), using a FPLC system. The molecular mass of the purified sbsFSH, estimated by mass spectrometry, was of 28.5 kDa for the dimer, 12.6 kDa for the glycoprotein alpha (GPalpha) and 13.6 kDa for FSHbeta subunits. After separation by SDS-PAGE under reducing condition, the intact sbsFSH was dissociated in the respective subunits (GPalpha and FSHbeta). Subunit identity was confirmed by immunological detection and N-terminal amino acid sequencing. Deglycosylation treatment with N-glycosidase F, decreased the molecular mass of both subunits. Intact sbsFSH activated the sea bass FSH receptor stably expressed in the cell line HEK 293, in a dose dependent manner. Purified sbsFSH showed gonadotropic activity, by stimulating the release of estradiol-17beta (E2) from sea bass ovary and testosterone (T) and 11-ketotestosterone (11KT) from testicular tissue cultured in vitro, in a dose and time dependent manner. These results showed that the purified sbsFSH is a heterodimeric hormone, composed of two distinct glycoprotein subunits (GPalpha and FSHbeta), and has biological activity judged by its ability to stimulate its receptor in a specific manner and to promote steroid release from gonadal tissue fragments.

Expression of kisspeptins and kiss receptors suggests a large range of functions for kisspeptin systems in the brain of the european sea bass

This study, conducted in the brain of a perciform fish, the European sea bass, aimed at raising antibodies against the precursor of the kisspeptins in order to map the kiss systems and to correlate the expression of kisspeptins, kiss1 and kiss2, with that of kisspeptin receptors (kiss-R1 and kiss-R2). Specific antibodies could be raised against the preprokiss2, but not the preoprokiss1. The data indicate that kiss2 neurons are mainly located in the hypothalamus and project widely to the subpallium and pallium, the preoptic region, the thalamus, the pretectal area, the optic tectum, the torus semicircularis, the mediobasal medial and caudal hypothalamus, and the neurohypophysis. These results were compared to the expression of kiss-R1 and kiss-R2 messengers, indicating a very good correlation between the wide distribution of Kiss2-positive fibers and that of kiss-R2 expressing cells. The expression of kiss-R1 messengers was more limited to the habenula, the ventral telencephalon and the proximal pars distalis of the pituitary. Attempts to characterize the phenotype of the numerous cells expressing kiss-R2 showed that neurons expressing tyrosine hydroxylase, neuropeptide Y and neuronal nitric oxide synthase are targets for kisspeptins, while GnRH1 neurons did not appear to express kiss-R1 or kiss-R2 messengers. In addition, a striking result was that all somatostatin-positive neurons expressed-kissR2. These data show that kisspeptins are likely to regulate a wide range of neuronal systems in the brain of teleosts.

Development of a homologous enzyme-linked immunosorbent assay for European sea bass FSH. Reproductive cycle plasma levels in both sexes and in yearling precocious and non-precocious males.

Since the late 1980s, gonadotropins have been isolated and characterized in several fish species, but specific immunoassays for the follicle-stimulating hormone (FSH) have only been developed for a few. The present study reports the development and use of a specific and homologous competitive ELISA for measuring FSH in European sea bass (Dicentrarchus labrax) using a recombinant FSH and its specific antiserum. Recombinant European sea bass FSHβ and FSH heterodimer were produced in the methylotrophic yeast Pichia pastoris and a baculovirus expression system, respectively. Specific polyclonal antibodies, generated by rabbit immunization against recombinant FSHβ, were used at a final dilution of 1:8000. Recombinant FSH heterodimer was used to generate a standard curve and for coating of microplates (166 μg/ml). The sensitivity of the assay was 0.5 ng/ml [B(0)-2SD], and the intra- and inter-assay coefficients of variation were 2.12% (n=10) and 5.44% (n=16) (B(i)/B(0) ∼45%), respectively. A high degree of parallelism was observed between the standard curve and serially diluted plasma and pituitary samples of European sea bass. The ELISA developed was used to study the plasma FSH profiles of mature males and females during the reproductive cycle, and those of immature juvenile males under different light regimes. The analysis showed that FSH increased significantly during the intermediate stages of spermatogenesis and during vitellogenesis. Analyses in immature juvenile males showed that the continuous light photoperiod significantly reduced plasma FSH levels, and consequently, testicular growth and precocious puberty. In conclusion, the immunoassay developed has proven to be sensitive, specific and accurate for measuring European sea bass FSH, and it represents a valuable tool for future studies on the reproductive endocrinology of this species.

Functional and Evolutionary Analysis of Flatfish Gonadotropin Receptors Reveals Cladal- and Lineage-Level Divergence of the Teleost Glycoprotein Receptor Family

Pituitary gonadotropins, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) act via their cognate glycoprotein hormone receptors (GpHRs), FSH receptor (FSHR), and LH/choriogonadotropin receptor (LHCGR) to regulate gonad physiology. Here, we show that the flatfish Senegalese sole (Solea senegalensis) expresses functional isoforms of fshr and lhcgr, but the genomic origin, ligand activation, and tissue distribution of the receptor transcripts are more complex than expected. By integrating the molecular phylogeny of GpHRs with the syntenic loci of vertebrate orthologs, and by subsequently characterizing the physical maps with the phylogeny of flanking genes, we found that vertebrate GpHRs have undergone a divergent evolution. In Teleostei, fshr genes have a common descent and can be classified as fshra, whereas lhcgrb genes exist as alternatively coded genes even in closely related species. Structural analyses of the receptors revealed that Fshra has an elongated ligand-binding domain, containing an extra leucine-rich repeat that specifically arose in the Acanthomorpha because of exon duplication. Ectopic expression in Xenopus laevis oocytes demonstrated that sole Fshra responded to piscine Fsh and Lh, whereas Lhcgrba was preferentially activated by its cognate hormone. The expression pattern of sole fshra and lhcgrba in gonads during the reproductive cycle was consistent with earlier observations wherein Fshra regulates ovarian growth and spermatogenesis and Lhcgrb triggers gamete maturation, respectively. However, contrary to observations in other teleosts, fshra was localized exclusively in Sertoli cells of the testis, whereas lhcgrba was expressed in Leydig cells as well as in spermatids. These results demonstrate the presence of alternatively coded lhcgr isoforms (lhcgrba and lhcgrbb) in teleosts and suggest a role of the lhcgrba receptor in the differentiation of spermatids into spermatozoa in Senegalese sole.