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.

Development of a flatfish-specific enzyme-linked immunosorbent assay for Fsh using a recombinant chimeric gonadotropin.

In flatfishes with asynchronous and semicystic spermatogenesis, such as the Senegalese sole (Solea senegalensis), the specific roles of the pituitary gonadotropins during germ cell development, particularly of the follicle-stimulating hormone (Fsh), are still largely unknown in part due to the lack of homologous immunoassays for this hormone. In this study, an enzyme-linked immunosorbent assay (ELISA) for Senegalese sole Fsh was developed by generating a rabbit antiserum against a recombinant chimeric single-chain Fsh molecule (rFsh-C) produced by the yeast Pichia pastoris. The rFsh-C N- and C-termini were formed by the mature sole Fsh ß subunit (Fshß) and the chicken glycoprotein hormone common α subunit (CGA), respectively. Depletion of the antiserum to remove anti-CGA antibodies further enriched the sole Fshß-specific antibodies, which were used to develop the ELISA using the rFsh-C for the standard curve. The sensitivity of the assay was 10 and 50 pg/ml for Fsh measurement in plasma and pituitary, respectively, and the cross-reactivity with a homologous recombinant single-chain luteinizing hormone was 1%. The standard curve for rFsh-C paralleled those of serially diluted plasma and pituitary extracts of other flatfishes, such as the Atlantic halibut, common sole and turbot. In Senegalese sole males, the highest plasma Fsh levels were found during early spermatogenesis but declined during enhanced spermiation, as found in teleosts with cystic spermatogenesis. In pubertal males, however, the circulating Fsh levels were as high as in adult spermiating fish, but interestingly the Fsh receptor in the developing testis containing only spermatogonia was expressed in Leydig cells but not in the primordial Sertoli cells. These results indicate that a recombinant chimeric Fsh can be used to generate specific antibodies against the Fshß subunit and to develop a highly sensitive ELISA for Fsh measurements in diverse flatfishes.

Administration of follicle-stimulating hormone in vivo triggers testicular recrudescence of juvenile European sea bass (Dicentrarchus labrax).

Follicle-stimulating hormone (Fsh) is thought to act early in the process of spermatogenesis; however, its action in fish has not yet been clearly established. In the present work, we analyzed the effects of recombinant Fsh in sea bass (Dicentrarchus labrax) spermatogenesis according to two different approaches: direct injection of recombinant single-chain Fsh hormone (scFSH) and injection of scFSH coding sequence. Both approaches were efficient in increasing plasma Fsh at 7 and 15 days, respectively, after injection. The Fsh increment caused a significant increase in plasma 11-ketotestosterone levels and induced dramatic changes at the testicular level. Fsh-treated groups showed an increase in germ cell proliferation at Day 7, and cysts of spermatocytes and spermatids were observed at the end of the experiment. After treatment with Fsh, a suppression in amh transcripts and an increase of lhr transcripts were detected at Day 7 and Day 15, respectively, and an increment in fshr expression became evident at Day 23. These results show that Fsh initiates germ cell proliferation, triggering spermatogenesis in sea bass via androgen production and regulation of spermatogenesis-related genes.

Isolation and characterization of Ff1 and Gsdf family genes in European sea bass and identification of early gonadal markers of precocious puberty in males.

Puberty represents the transition from an immature to a mature reproductive stage. The mechanisms underlying the onset of normal or precocious puberty have not yet been elucidated. With the goal of gaining an understanding of early events that occur in the testes of precocious animals during this process, a hemigonadectomy was performed on male juvenile sea bass and expression levels of candidate mRNAs were determined through quantitative real-time RT-PCR. For this purpose, the gonadal soma-derived factors gsdf1 and gsdf2, the nuclear receptor 5 subfamily members nr5a1a (ff1b), nr5a1b (ff1d), nr5a2 (ff1a) and nr5a5 (ff1c) and the proliferating cell nuclear antigen or pcna, genes with a putative role in the beginning of spermatogenesis, were isolated and cloned. Hemigonadectomy proved to be a suitable strategy for the study of gonadal stages prior to the appearance of histological differences between precocious and non-precocious fish, as it allowed the subsequent classification of these gonads. The upregulation of the gene encoding the steroidogenic acute regulatory protein (Star) in precocious testes indicates that sex steroids could play a role in the onset of spermatogenesis in sea bass. In contrast, the downregulation observed in ff1b expression indicates that this initial surge in star expression is not the result of Ff1b transactivation, suggesting an alternative pathway for this transcriptional activation. Finally, a decrease in gsdf1 expression in precocious animals suggests that this gene may play a role in the onset of puberty, while its correlation with ff1b expression points to gsdf1 as a putative target for Ff1b-mediated transactivation.

Luteinizing hormone plasmid therapy results in long-lasting high circulating Lh and increased sperm production in European sea bass (Dicentrarchus labrax).

The present work aimed at evaluating the potential of intramuscular injection of a hormone-coding gene as an approach for gene therapy in fish. A plasmid containing luteinizing hormone (Lh) in a single-chain (sc) form, pCMV-scLh, was chosen as the coding gene, and sea bass was chosen as the target species. In vivo injection of pCMV-scLh in muscle of juvenile sea bass rendered plasma Lh levels higher than 50 ng/ml in 40% of the injected fish, while these Lh levels were only detected in 4% of controls. Injections performed on spermiating broodstock demonstrated that this strategy produced an active Lh able to increase sperm production without affecting its quality, in terms of density. Compared with the injection of a recombinant single-chain Lh, plasmid injection provoked longer-lasting and higher plasma Lh levels. These results show that sea bass skeletal muscle is able to uptake plasmid DNA and to secrete the encoded protein to the bloodstream. Therefore, we propose somatic gene transfer as a realistic approach for hormone therapy of dysfunctions due to low hormone levels in fish or just to synchronize spawning.