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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.

journal_name

Gen Comp Endocrinol

journal_title

General and comparative endocrinology

authors

Chauvigné F,Verdura S,Mazón MJ,Boj M,Zanuy S,Gómez A,Cerdà J

doi

10.1016/j.ygcen.2014.10.009

subject

Has Abstract

pub_date

2015-09-15 00:00:00

pages

75-85

eissn

0016-6480

issn

1095-6840

pii

S0016-6480(14)00410-9

journal_volume

221

pub_type

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