Ana Rocha

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.

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.

Determination of Fsh Quantity and Bioactivity During Sex Differentiation and Oogenesis in European Sea Bass

Follicle-stimulating hormone (FSH) is a glycoprotein hormone that plays a key role in the regulation of gonadal functions in vertebrates. The present study reports the monitoring of pituitary and plasma Fsh levels during sex differentiation and oogenesis in European sea bass (Dicentrarchus labrax) using a homologous immunoassay and an in vitro bioassay. Both assays were used complementarily for the first time in a fish species. High levels of Fsh bioactivity in plasma were found during the initial phases of sexual differentiation. Plasma and pituitary Fsh (quantity and bioactivity) levels and biological to immunological (B:I) ratios were higher in females than in males, suggesting sexual dimorphism in the synthesis and potency of Fsh. In females, the B:I ratios in adult were lower than during sex differentiation indicating that Fsh would be less biopotent in the adult stage. Plasma Fsh bioactivity levels increased during vitellogenesis, suggesting that Fsh would be involved in the regulation of the midphases of oogenesis, whereas luteinizing hormone would be responsible for the final events.

foxl2 and foxl3 are two ancient paralogs that remain fully functional in teleosts

FOXL2 is a forkhead transcription factor involved in mammalian development and regulation of reproduction. Two foxl2 paralogs, foxl2a and foxl2b, have been described in various teleost species and were considered as fish-specific duplicates. Here, we report the isolation and characterization of foxl2a (foxl2) and foxl2b (foxl3) in European sea bass (Dicentrarchus labrax), together with the identification of these two genes in non-teleost genomes. Phylogenetic and synteny analyses indicate that these paralogs originated from an ancient genome duplication event that happened long before the teleost specific duplication. While foxl2/foxl2a has been maintained in most vertebrate lineages, foxl2b, which we propose to rename as foxl3, was repeatedly lost in tetrapods. Gonadal expression patterns of the sea bass genes point to a strong sexual dimorphism, and the mRNA levels of foxl2 in ovary and foxl3 in testis vary significantly during the reproductive cycle. When overexpressed in cultured ovarian follicular cells, foxl2 and foxl3 produced functional transcription factors able to control the expression of reproduction-related genes. Taken together, these data suggest that Foxl2 may play a conserved role in ovarian maturation, while Foxl3 could be involved in testis physiology.

New insights into the factors mediating the onset of puberty in sea bass.

In populations of 1-year-old male European sea bass (Dicentrarchus labrax), only large males are able to acquire for the first time a functional competence of their reproductive axis; in other words, to attain puberty. To examine the causes and mechanisms involved in the onset of puberty in this species, a size sorting sampling was carried out to obtain two experimental groups of small and large male fish exhibiting different growth rates. As expected, only large fish reached full spermiogenesis (stage V of testicular development) by the end of the experiment. Our study suggests that fish size is a permissive condition to ensure full effectiveness of the hormonal (Gnrh1, gonadotropins and sexual steroids) actions. Thus, though small fish had endocrine profiles similar to those of large fish, their amplitude was much lower, and was most likely the reason why functional competence of the reproductive axis was not achieved. Moreover, this work provides evidence of the involvement of kisspeptin and Gnrh1 systems in the onset of puberty in a marine teleost fish. It also indicates that very likely kisspeptin and Gnrh1 may regulate gonadotropins and sex steroids at specific stages of testicular development.

Leptin receptor gene in the European sea bass (Dicentrarchus labrax): Cloning, phylogeny, tissue distribution and neuroanatomical organization.

In this study, we report the cloning of three transcripts for leptin receptor in the European sea bass, a marine teleost of economic interest. The two shortest variants, generated by different splice sites, encode all functional extracellular and intracellular domains but missed the transmembrane domain. The resulting proteins are therefore potential soluble binding proteins for leptin. The longest transcript (3605bp), termed sblepr, includes all the essential domains for binding and transduction of the signal. Thus, it is proposed as the ortholog for the human LEPR gene, the main responsible for leptin signaling. Phylogenetic analysis shows the sblepr clustered within the teleost leptin receptor group in 100% of the bootstrap replicates. The neuroanatomical localization of sblepr expressing cells has been assessed by in situ hybridization in brains of sea bass of both sexes during their first sexual maturation. At histological level, the distribution pattern of sblepr expressing cells in the brain shows no clear differences regarding sex or reproductive season. Transcripts of the sblepr have a widespread distribution throughout the forebrain and midbrain until the caudal portion of the hypothalamus. A high hybridization signal is detected in the telencephalon, preoptic area, medial basal and caudal hypothalamus and in the pituitary gland. In a more caudal region, sblepr expressing cells are identified in the longitudinal torus. The expression pattern observed for sblepr suggests that in sea bass, leptin is very likely to be involved in the control of food intake, energy reserves and reproduction.

Gonadotropins in European sea bass: Endocrine roles and biotechnological applications.

Follicle stimulating hormone (Fsh) and luteinizing hormone (Lh) are central endocrine regulators of the gonadal function in vertebrates. They act through specific receptors located in certain cell types found in the gonads. In fish, the differential roles of these hormones are being progressively elucidated due to the development of suitable tools for their study. In European sea bass (Dicentrarchus labrax), isolation of the genes coding for the gonadotropin subunits and receptors allowed in first instance to conduct expression studies. Later, to overcome the limitation of using native hormones, recombinant dimeric gonadotropins, which show different functional characteristics depending on the cell system and DNA construct, were generated. In addition, single gonadotropin beta-subunits have been produced and used as antigens for antibody production. This approach has allowed the development of detection methods for native gonadotropins, with European sea bass being one of the few species where both gonadotropins can be detected in their native form. By administering recombinant gonadotropins to gonad tissues in vitro, we were able to study their effects on steroidogenesis and intracellular pathways. Their administration in vivo has also been tested for use in basic studies and as a biotechnological approach for hormone therapy and assisted reproduction strategies. In addition to the production of recombinant hormones, gene-based therapies using somatic gene transfer have been offered as an alternative. This approach has been tested in sea bass for gonadotropin delivery in vivo. The hormones produced by the genes injected were functional and have allowed studies on the action of gonadotropins in spermatogenesis.

Thyroid Hormones Regulate Zebrafish Melanogenesis in a Gender-Specific Manner

Zebrafish embryos are treated with anti-thyroidal compounds, such as phenylthiourea, to inhibit melanogenesis. However, the mechanism whereby the thyroidal system controls melanin synthesis has not been assessed in detail. In this work, we tested the effect of the administration of diets supplemented with T3 (500μg/g food) on the pigment pattern of adult zebrafish. Oral T3 induced a pronounced skin paling in both adult female and male zebrafish that was reversible upon cessation of treatment. The number of visible melanophores was significantly reduced in treated fish. Accordingly, treatment down-regulated expression of tyrosinase-related protein 1 in both sexes. We also found sexually dimorphic regulation of some melanogenic genes, such as Dct/Tyrp2 that was dramatically up-regulated in females after T3 treatment. Thus, we demonstrated that melanogenesis is reversibly inhibited by thyroid hormones in adult zebrafish and make the discovery of gender-specific differences in the response of melanogenic gene expression. Thus, fish gender is now shown to be an important variable that should be controlled in future studies of fish melanogenesis.

Conserved Anti-Müllerian Hormone: Anti-Müllerian Hormone Type-2 Receptor Specific Interaction and Intracellular Signaling in Teleosts

ABSTRACT In higher vertebrates, anti-Müllerian hormone (AMH) is required for Müllerian duct regression in fetal males. AMH is also produced during postnatal life in both sexes regulating steroidogenesis and early stages of folliculogenesis. Teleosts lack Müllerian ducts, but Amh has been identified in several species including European sea bass. However, information on Amh type-2 receptor (Amhr2), the specific receptor for Amh binding, is restricted to a couple of fish species. Here, we report on cloning sea bass amhr2, the production of a recombinant sea bass Amh, and the functional analysis of this ligand-receptor couple. Phylogenetic analysis revealed that sea bass amhr2 segregates with Amhr2 from other vertebrates. This piscine receptor is capable of activating Smad proteins. Antibodies raised against sea bass Amh were used to study native and recombinant Amh, revealing proteins in the range of 66–70 kDa corresponding to the full length Amh. Once proteolytically treated, recombinant sea bass Amh generates a 12 kDa C-terminal mature protein, suggesting that contrary to what has been described for other fish Amh proteins, this protein is processed in a similar way as mammalian AMH. The mature sea bass Amh is a biologically active protein able to bind sea bass Amhr2 and, surprisingly, also human AMHR2. In prepubertal sea bass testes, Amh was detected by immunohistochemistry mostly in Sertoli cells surrounding early germ-cell generations. During spermatogenesis, a weaker staining signal could be observed in Sertoli cells surrounding spermatocytes.