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.
Department of Fish Physiology and Biotechnology, Institute of Aquaculture Torre de Sal, Spanish National Research Council (CSIC), Torre de Sal s/n, 12595 Ribera de Cabanes, Castellón, Spain.
Gregorio
G
Molés
Ana
A
Gómez
Manuel
M
Carrillo
Silvia
S
Zanuy
D000367:Age Factors; D000818:Animals; D000906:Antibodies / Q000276:immunology; D001492:Bass / Q000502:physiology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000592:standards*; D005060:Europe; D005260:Female; D037201:Follicle Stimulating Hormone, beta Subunit / Q000097:blood* / Q000235:genetics / Q000276:immunology; D015292:Glycoprotein Hormones, alpha Subunit / Q000097:blood* / Q000235:genetics / Q000276:immunology; D008297:Male; D017440:Photoperiod; D010957:Plasmids / Q000235:genetics; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D015203:Reproducibility of Results; D012098:Reproduction / Q000502:physiology*; D012680:Sensitivity and Specificity; D012741:Sexual Maturation / Q000502:physiology
D000906:Antibodies; D037201:Follicle Stimulating Hormone, beta Subunit; D015292:Glycoprotein Hormones, alpha Subunit; D011994:Recombinant Proteins