Development of a homologous radioimmunoassay for Mediterranean yellowtail (Seriola dumerilii, Risso 1810) LH

Purified Mediterranean (M.) yellowtail luteinizing hormone-like gonadotropin (MyLH) and its beta-subunit (MyLHbeta) served to develop a radioimmunoassay (RIA) for MyLH. The rabbit antisera against MyLH and MyLHbeta used for this purpose were tested on pituitary sections by immunocytochemistry. Anti-MyLHbeta specifically detected a single type of cells, which were located at the periphery of the proximal pars distalis (PPD) and surrounding the pars intermedia (PI). Anti-MyLH, however, also recognized two other cell types, thyrotropin beta-immunoreactive (ir) cells and putative follicle-stimulating hormone-like (MyFSH)-producing cells. Labeling of the two latter cell types was prevented by preabsorption of anti-MyLH with M. yellowtail pituitary glycoprotein a-subunit. The standard curve for the RIA was generated using purified MyLH, 12 I-labeled MyLHbeta and anti-MyLHbeta at a dilution of 1:70,000, which resulted in the binding of 30% of the tracer added. The standard curve ranged from 0.25 to 50 ng/ml. The midrange of the assay (ED50) was obtained with 5.48-7.87 ng LH/ml. The variation between assays was less than 15%. An average cross-reactivity of FSH in the LH RIA of 8.4% was found. Serial dilutions of M. yellowtail pituitary extracts displaced radiolabelied MyLHbeta parallel to the MyLH standard. Application of the LH RIA to blood samples and pituitary cell culture medium provided physiological validation of the assay. Significant increases in LH levels were recorded after salmon GnRH treatments in vivo and in vitro. Serum LH levels from wild fish sampled at the spawning season were significantly higher than those from captive fish sampled in the same period. (C) 2002 Elsevier Science B.V. All rights reserved.