Binding of Bovine Thyrotropin to Receptors in Rat Testis and Its Interaction with Gonadotropins*

Abstract

Preparations of hCH [human chorionic gonadotropin] bind to bovine thyroid membranes, as judged from their ability both to inhibit the binding of 125I-labeled bovine TSH (bTSH) and to activate adenylate cyclase. In the present studies, 125I-labeled, highly purified bTSH ([125I]bTSH) bound specifically and saturably to receptors in a particulate fraction from rat testis. At 37.degree. C, binding was rapid, reaching a maximum level in less than 15 min, but then declining markedly during the next several hours. At 22.degree. C, binding reached a steady state after 2 h and remained unchanged for another 22 h. Binding of [125I]bTSH was greatest at pH 5.5, at which pH more than 50% of [125I]bTSH was bound in the presence of 330 .mu.g/ml particulate protein, the concentration of protein that yielded maximum binding. Nevertheless, the majority of experiments were conducted at lesser protein concentrations and at physiological pH (7.45), under which conditions total binding was only 25% of that measured at pH 5.5. Scatchard plots indicated the presence of a single binding site with a dissociation constant of 5.8 .times. 10-8 M and a binding capacity of 0.22 nmol/mg protein on the basis of data obtained at 22.degree. C and pH 7.45. Both crude and highly purified preparations of hCG inhibited the binding of [125I]bTSH to testis particulate fraction; crude hCH had 46 times the activity, and purified hCG had only one-tenth the activity of bTSH itself in this respect. This was true despite the fact that with respect to the displacement of [125I]hCG, crude and purified hCG were almost equally active. Bovine LH had one-third the activity of bTSH in displacing [125I]bTSH. Human FSH [follicle stimulating hormone] inhibited [125I]bTSH binding only slightly at the highest concentration tested while glucagon, insulin, PRL [prolactin] and GH [growth hormone] were inactive. Purified bTSH inhibited the binding of [125I]hCG to testis particulate fraction but contained only about 2% of the activity of purified hCG. Lineweaver-Burk analysis suggested that inhibition of [125I]hCG binding by bTSH was competitive in nature. Purified bTSH stimulated cAMP production in Leydig cells, but with only about 0.1% of the activity of purified hCG. bTSH binds reversibly, saturably, and with relatively high affinity to receptors in rat testis that are either the same as receptors for hCG and LH or that interact therewith. bTSH, like hCG, is capable of stimulating the production of cAMP in rat Leydig cells, but is much less potent than hCG in this regard. Preparations of crude hCG contain a factor lacking hCG activity in bioassay, immunoassay, and receptor assay that is especially potent in displacing [125I]bTSH from receptors in testis, as was earlier described for bTSH receptors in bovine thyroid membranes.