Studies on Hypothalamus-Pituitary-Thyroid Control System of Bullfrog

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To clarify the control mechanisms of metamorphosis on Amphibian it is important to isolate and characterize the thyroid stimulating hormone (TSH) and TSH releasing hormone (TRH).We have isolated the TSH from the pituitary glands of bullfrogs, Rana catesbeiana, using various chromatographic techniques. The bioactivity of this preparation, as measured in vitro by the release of T_4 into culture medium from the thyroid gland taken from hypophysectomized prometamorphic bullfrog tadpole, was about 2 times higher than that of bovine TSH. The molecular weight and multiple isoelectric points. of this TSH were about 32,000 and 4.5-5.0, respectively. The amino acid sequence of the alpha-subunit of TSH is identical to those of bullfrog LH and FSH, having about 60% identity to that of mammalian one. However, the amino acid sequence of beta-subunit of this TSH has no homology with mammalian TSH-beta hitherto known, while beta-subunit of bullfrog LH and FSH have resemblance to those of mammalian LH and FSH, 40 and 60 % identities, respectively.A double antibody radioimmunoassay (RIA) system for bullfrog TSH-beta was established using polyclonal antibody after raised against beta-subunit of this TSH. Intact LH, FSH and their subunits from bullfrog did not react in this system. Serum TSH-beta in larval bullfrogs of various developmental stages were measured using this RIA system. Serum TSH-beta levels were relatively low during prometamorphosis. The levels rose markedly at the onset of climax and declined at the end of climax, as in the case of T_4.Synthetic TRH (5-20ug/100g body weight) were injected into the cannulated vena abdominalis of adult bullfrogs. However, no significant elevation of plasma TSH was observed during the experimental period up to 8 hours. These probably suggest the existence of another type TRH in bullfrog.