title: Development of a flatfish-specific enzyme-linked immunosorbent assay for Fsh using a recombinant chimeric gonadotropin.
abstract: 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 β subunit (Fshβ) and the chicken glycoprotein hormone common α subunit (CGA), respectively. Depletion of the antiserum to remove anti-CGA antibodies further enriched the sole Fshβ-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 Fshβ subunit and to develop a highly sensitive ELISA for Fsh measurements in diverse flatfishes.
authors.affiliation: IRTA-Institut de Ciències del Mar, Consejo Superior de Investigaciones Científicas (CSIC), 08003 Barcelona, Spain; Department of Biology, University of Bergen, Bergen High Technology Centre, N-5020 Bergen, Norway.
authors.forename: François
authors.initials: F
authors.lastname: Chauvigné
authors.affiliation: IRTA-Institut de Ciències del Mar, Consejo Superior de Investigaciones Científicas (CSIC), 08003 Barcelona, Spain.
authors.forename: Sara
authors.initials: S
authors.lastname: Verdura
authors.affiliation: Department of Fish Physiology and Biotechnology, Instituto de Acuicultura de Torre de la Sal (IATS), CSIC, Ribera de Cabanes, 12595 Castellón, Spain.
authors.forename: María José
authors.initials: MJ
authors.lastname: Mazón
authors.affiliation: IRTA-Institut de Ciències del Mar, Consejo Superior de Investigaciones Científicas (CSIC), 08003 Barcelona, Spain.
authors.forename: Mónica
authors.initials: M
authors.lastname: Boj
authors.affiliation: Department of Fish Physiology and Biotechnology, Instituto de Acuicultura de Torre de la Sal (IATS), CSIC, Ribera de Cabanes, 12595 Castellón, Spain.
authors.forename: Silvia
authors.initials: S
authors.lastname: Zanuy
authors.affiliation: Department of Fish Physiology and Biotechnology, Instituto de Acuicultura de Torre de la Sal (IATS), CSIC, Ribera de Cabanes, 12595 Castellón, Spain.
authors.forename: Ana
authors.initials: A
authors.lastname: Gómez
authors.affiliation: IRTA-Institut de Ciències del Mar, Consejo Superior de Investigaciones Científicas (CSIC), 08003 Barcelona, Spain. Electronic address: joan.cerda@irta.cat.
authors.forename: Joan
authors.initials: J
authors.lastname: Cerdà
mesh_terms: D000818:Animals; D000906:Antibodies / Q000378:metabolism; D001667:Binding, Competitive; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005412:Flatfishes / Q000378:metabolism*; D005640:Follicle Stimulating Hormone / Q000097:blood / Q000378:metabolism*; D037201:Follicle Stimulating Hormone, beta Subunit / Q000378:metabolism; D015292:Glycoprotein Hormones, alpha Subunit / Q000378:metabolism; D006062:Gonadotropins / Q000378:metabolism*; D006801:Humans; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism*; D012015:Reference Standards; D015203:Reproducibility of Results; D012098:Reproduction; D013045:Species Specificity
chemical_list: D000906:Antibodies; D037201:Follicle Stimulating Hormone, beta Subunit; D015292:Glycoprotein Hormones, alpha Subunit; D006062:Gonadotropins; D011994:Recombinant Proteins; D005640:Follicle Stimulating Hormone
keywords: ELISA; Flatfish; Fsh; Senegalese sole; Spermatogenesis