BINDING OF THYROID-HORMONE TO THE GOAT TESTICULAR LEYDIG-CELL INDUCESTHE GENERATION OF A PROTEINACEOUS FACTOR WHICH STIMULATES ANDROGEN RELEASE

Leydig cells isolated from goat testis were sonicated and pure nuclear preparations obtained for I-125-3,5,3'-triiodothyronine (T-3)-binding assay. Under optimum assay conditions of pH 7.2 at 37 degrees C and 9 0 min of incubation, binding of I-125-T-3 to Leydig cell nuclei reache d saturation at 1.2 nmol/l concentration. A Scatchard analysis of T-3 binding exhibited a K-d of 0.535 x 10(-9) mol/l and a mam binding capa city of 1.25 pmol/mg DNA. Competitive inhibition studies showed T-3 bi nding to be analogue specific. The physiological relevance of T-3 bind ing to goat Leydig cell was examined by adding increasing concentratio ns of T-3 to the Leydig cell incubation (1 x 10(6) cells/incubation). T-3 (10, 25 and 50 ng/ml or 4, 10 and 20 ng/incubation) resulted a dos e dependent increase in androgen release and in all cases stimulation of androgen release was statistically significant (P<0.01) compared wi th control. Stimulation of Leydig cell, androgen release by T-3 was si gnificantly inhibited by actinomycin-D (P<0.01) and cycloheximide (P<0 .01). T-3 had additive stimulatory effects on LH-augmented androgen re lease from Leydig cells. T-3 (50 ng/ml or 20 ng/incubation) effected a more than twofold increase in Leydig cell protein synthesis compared with control and both actinomycin-D and cycloheximide (50 mu g/ml) inh ibited it completely. The data indicated that the stimulatory effect o f T-3 on androgen release is mediated via T-3-induced protein(s). Sub- cellular fractions obtained from T-3-treated Leydig cells showed an in crease in protein synthesis in mitochondrial and soluble supernatant f ractions (100 k sup) and it was only 100 k sup which stimulated androg en release from Leydig cells in separate incubations. Treatment of 100 k sup with trypsin or heat abolished its stimulatory effect. Incubati on of Leydig cells with T-3 for different times showed an increase in protein synthesis prior to the stimulation of androgen release. The re sults therefore indicated that T-3 binding to Leydig cells induced the generation of a proteinaceous factor(s) which in turn stimulated andr ogen release.