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.

Production of recombinant channel catfish (Ictalurus punctatus) FSH and LH in S2 Drosophila cell line and an indication of their different actions.

Due to the lack of purified, native gonadotropins (GtH) for almost all species of fish, we designed a system for the production of recombinant bioactive luteinizing hormone (LH) and follicle stimulating hormone (FSH) using the channel catfish (Ictalurus punctatus) as a model animal. The strategy was to produce the three subunits composing FSH and LH, i.e. the common alpha-subunit (alpha-glycoprotein hormone (alpha-GP)), beta-FSH, and beta-LH subunit, individually in stable recombinant insect cells (S2) with C-terminal His-tag. This expression system was also used to co-express the alpha-subunit without the His-tag with each of the His-tagged beta-subunits. The recombinant S2 cells were capable of secreting FSH and LH heterodimers and alpha-GP in abundance; however, expression of the individual beta-subunits was much less successful. The recombinant GtHs were partially purified from the cell medium by immobilized metal affinity chromatography to ~15% purity with a yield of 7 and 4 mg per liter of medium for FSH and LH respectively. These recombinant GtHs activated their receptors in vitro, enhanced estrogen secretion, up-regulated several steroidogenic enzyme genes in channel catfish ovarian follicles, and increased androgen secretion from African catfish testis. Interestingly, the FSH and LH dose-response curves for each of these biological activities clearly demonstrate differences in their cellular action and physiological roles. This expression system may be an important development for the production of species-specific GtHs so that FSH- and LH-specific mechanisms of actions within the reproductive endocrine processes can finally be examined with homologous, albeit recombinant, hormones.

Molecular cloning of follicle-stimulating hormone (FSH)-beta subunit cDNA from duck pituitary.

We have cloned FSH-beta cDNA from duck pituitary gland by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE) methods. The cloned duck FSH-beta cDNA contains 1909-bp nucleotides including 396-bp of open-reading frame and 1491-bp of 3'-untranslational region. The open-reading frame encodes a 131-amino acid protein with a putative 20-amino acid signal peptide and a putative 111-amino acid mature protein. The deduced amino acid sequence shows a remarkable similarity (94-98%) to those of other avian FSH-beta subunits; while it exhibits lower similarities with those of turtles (82-84%), mammals (63-71%), and amphibians (53-57%). The structural model analysis of duck FSH suggests that the cysteine-knot and beta-strands for maintaining the specific structural frame, and the "seat-belt" loop for specific binding to FSH receptor have been conserved in tetrapodian FSH-betas.

Isolation and characterization of the follicle-stimulating hormone beta subunit gene and 5' flanking region of the Chinook salmon.

In this study we have isolated the follicle-stimulating hormone beta subunit gene from the Chinook salmon (csFSHbeta). This gene encodes for a protein that is highly similar to those isolated from other salmonids and shares all of the structural constraints seen in mammalian gonadotropins, including twelve conserved cysteines and a putative N-linked glycosylation site. The organization of the gene follows the conserved pattern regarding the numbers and positions of the introns, although the csFSHbeta gene contains a particularly large 6.2-kb first intron due to the inclusion of several transposon-like elements. Isolation of 1.2 kb of the 5' flanking region of the csFSHbeta gene and subsequent analysis in silico have revealed a number of putative elements which appear highly conserved in teleost FSHbeta gene promoters and are thus likely involved in basal and hormone-induced transcriptional regulation. The functionality of this 1.2-kb fragment in driving expression of a reporter gene and its response to GnRH was shown in gonadotropes, while the overexpression of AP-1 factors, Sf-1, estrogen receptor or Smad1 revealed that the promoter is responsive to these transcription factors. Our current study has opened the way for future analysis to verify the role of these factors in mediating hormonally induced transcription of this gene.