SALMON THYROID-STIMULATING HORMONE - ISOLATION, CHARACTERIZATION, ANDDEVELOPMENT OF A RADIOIMMUNOASSAY

Coho salmon (Oncorhynchus kisutch) thyroid-stimulating hormone (TSH) w as isolated by ethanol extraction of pituitary glands from mature coho salmon. Extraction was followed by gel-filtration chromatography on S ephadex G-100 superfine, anion-exchange chromatography on a Whatman DE -52 column, and finally by reverse-phase high-performance liquid chrom atography. Fractions were monitored for TSH content by a homologous in vivo bioassay and by immunoblots using anti-human TSH beta-subunit an tisera. In vivo treatment of coho salmon parr with coho salmon TSH cau sed a dose-dependent increase in plasma thyroxine level similar to tha t induced by bovine TSH. The N-terminal sequence (25 residues) of the salmon TSH beta subunit has 56% sequence identity to that of human TSH beta subunit and is identical to the deduced amino acid sequence of t rout TSH beta subunit. The N-terminal sequence (25 residues) of the sa lmon TSH alpha subunit is identical to gonadotropin alpha-II subunit. Molecular sizes of the alpha and beta subunits are 18,000 and 24,000 d altons, respectively, as estimated by SDS-PAGE. Antiserum generated ag ainst salmon TSH, which was preadsorbed with alpha subunit using an al pha-subunit affinity column, detected only salmon TSH beta subunit by immunoblot and specifically stained thyrotropin-producing cells of the pituitary gland. A homologous radioimmunoassay (RIA) was developed us ing purified salmon TSH standard, iodinated TSH beta subunit, and anti serum generated against salmon TSH. Cross-reactivities of GTH I, GTH I I, GTH I beta and GTH II beta subunits, alpha subunit, growth hormone, prolactin, and somatolactin were less than 1%. Displacement curves fo r serial dilutions of plasma and pituitary extracts of various salmoni d species, as well as coho salmon pituitary cell culture medium, were parallel to the coho salmon TSH standards. In contrast, plasma of hypo physectomized juvenile coho salmon and pituitary extracts of Pacific t omcod (Microgadus proximus) did not displace bound radiolabeled salmon TSH. Finally, in vivo injection of juvenile coho salmon with triiodot hyronine decreased plasma TSH levels, whereas the goitrogen, methimazo le, increased plasma TSH levels. Injection of gonadotropin-releasing h ormone agonist did not alter plasma TSH. These data suggest that the R IA is specific for TSH and confirm a negative-feedback relationship be tween the thyroid hormones and TSH. (C) 1997 Academic Press.