Development of a specific enzyme-linked immunosorbent assay (ELISA) for determining FSH levels in sea bass (Dicentrarchus labrax), using recombinant gonadotropins

Resumen del trabajo presentado en el 25th Conference of European Comparative Endocrinologists, celebrada en Pécs (Hungría), del 31 de agosto al 4 de septiembre de 2010The follicle-stimulating hormone (FSH) is a key gonadotropin for control of reproduction in vertebrates. FSH is a heterodimeric glycoprotein secreted by the pituitary gland, formed by the non-covalent association of two subunits (alpha-beta) where beta-subunit confers hormone specificity. Since late 1980¿s, gonadotropins have been isolated and characterized in several fish species but the development of specific immunoassays for FSH has been accomplished only for a few ones. The present study reports the production of recombinant sea bass FSH-beta in the methylotrophic yeast Pichia pastoris and the development of a specific and homologous competitive ELISA for FSH determination in plasma and pituitary. The coding sequence of the mature sea bass FSH-beta was inserted in-frame into the pPIC9K expression vector, which was used to transform the host strain GS115 by electroporation. After large-scale production and purification of recombinant FSH-beta, this was used to generate polyclonal primary antibodies by rabbit immunization. Sea bass FSH dimer produced in our laboratory in a baculovirus expression system was used both as coating and standard curve. Wells were coated with FSH (0.5 mg/well), and the final concentration of the antisera was 1:8000. Validation of this assay showed a high degree of parallelism between the standard curve and serially diluted plasma and pituitary samples of sea bass but not with others perciforms. The sensitivity of the assay was 1.59 ng/ml [Bo-2SD] and the intra- and inter-assays coefficients of variation of 2.12% (n=10) and 5.44% (n=16) (Bi/Bo 45%), respectively. Plasma FSH levels of males and females sea bass were measured during one reproductive cycle using this ELISA. In females, plasma FSH levels increased during vitellogenesis and decreased in the maturation-ovulation stage, whereas in males increased during late-spermatogenesis (stage IV). In conclusion, a sensitive and accurate immunoassay for analysis of FSH in biological samples of sea bass has been developed. This assay represents a valuable tool to investigate the role of this hormone in the reproductive endocrinology of this species.Research supported by MICINN (AGL2005-0079, AGL200604672 and CSD2007-00002 / Aquagenomics