Salmon Thyroid-Stimulating Hormone: Isolation, Characterization, and Development of a Radioimmunoassay

Abstract

Coho salmon (Oncorhynchus kisutch) thyroid-stimulating hormone (TSH) was isolated by ethanol extraction of pituitary glands from mature coho salmon. Extraction was followed by gel-filtration chromatography on Sephadex G-100 superfine, anion-exchange chromatography on a Whatman DE-52 column, and finally by reverse-phase high-performance liquid chromatography. Fractions were monitored for TSH content by a homologousin vivobioassay and by immunoblots using anti-human TSH β-subunit antisera.In vivotreatment of coho salmon parr with coho salmon TSH caused a dose-dependent increase in plasma thyroxine level similar to that induced by bovine TSH. The N-terminal sequence (25 residues) of the salmon TSH β subunit has 56% sequence identity to that of human TSH β subunit and is identical to the deduced amino acid sequence of trout TSH β subunit. The N-terminal sequence (25 residues) of the salmon TSH α subunit is identical to gonadotropin α-II subunit. Molecular sizes of the α and β subunits are 18,000 and 24,000 daltons, respectively, as estimated by SDS–PAGE. Antiserum generated against salmon TSH, which was preadsorbed with α subunit using an α-subunit affinity column, detected only salmon TSH β subunit by immunoblot and specifically stained thyrotropin-producing cells of the pituitary gland. A homologous radioimmunoassay (RIA) was developed using purified salmon TSH standard, iodinated TSH β subunit, and antiserum generated against salmon TSH. Cross-reactivities of GTH I, GTH II, GTH I β and GTH II β subunits, α subunit, growth hormone, prolactin, and somatolactin were less than 1%. Displacement curves for serial dilutions of plasma and pituitary extracts of various salmonid species, as well as coho salmon pituitary cell culture medium, were parallel to the coho salmon TSH standards. In contrast, plasma of hypophysectomized juvenile coho salmon and pituitary extracts of Pacific tomcod (Microgadus proximus) did not displace bound radiolabeled salmon TSH. Finally,in vivoinjection of juvenile coho salmon with triiodothyronine decreased plasma TSH levels, whereas the goitrogen, methimazole, increased plasma TSH levels. Injection of gonadotropin-releasing hormone agonist did not alter plasma TSH. These data suggest that the RIA is specific for TSH and confirm a negative-feedback relationship between the thyroid hormones and TSH.