Quantification of salmon alpha- and thyrotropin (TSH) beta-subunit messenger RNA by an RNase protection assay: regulation by thyroid hormones.

In order to study salmon thyroid-stimulating hormone (TSH), we designed a highly specific, sensitive, and rapid RNase protection assay (RPA) for quantification of steady-state levels of salmon TSH beta-subunit mRNA expression. The cDNA encoding the beta-subunit of TSH was isolated from coho salmon pituitary total RNA by reverse-transcriptase PCR, partially sequenced, and used as template for synthesizing a radioactively labeled, sequence-specific, antisense probe, and sense standard for the RPA. This assay, along with a similar RPA previously designed for coho salmon total alpha-subunit mRNA, was used to examine the effects of feeding T3 (0, 10, 100 micrograms/g) and methimazole (a thyroid inhibitor) (2.5 mg/g) on TSH subunit gene expression after 2 and 4 weeks. The low dose of T3 (10 micrograms/g) caused no change in TSH beta mRNA after 2 and 4 weeks and a transient increase in alpha mRNA after 2 weeks, followed by no significant effect after 4 weeks. The high dose of T3 (100 micrograms/g) caused a decrease in TSH beta mRNA after 4 weeks and no change in total alpha mRNA after 2 and 4 weeks. In contrast, methimazole treatment caused significant increases in both TSH beta mRNA (250%) and alpha mRNA (50%) levels after 4 weeks. These findings confirm that, as in mammals, TSH alpha- and beta-subunit expression in teleosts may be differentially regulated by negative feedback from the thyroid hormones.