Binding of thyroid hormone to the goat testicular Leydig cell induces the generation of a proteinaceous factor which stimulates androgen release

Abstract

Abstract Leydig cells isolated from goat testis were sonicated and pure nuclear preparations obtained for 125I-3,5,3′-triiodothyronine (T3)-binding assay. Under optimum assay conditions of pH 7·2 at 37 °C and 90 min of incubation, binding of 125I-T3 to Leydig cell nuclei reached saturation at 1·2 nmol/l concentration. A Scatchard analysis of T3 binding exhibited a Kd of 0·535 × 10−9 mol/l and a maximum binding capacity of 1·25 pmol/mg DNA. Competitive inhibition studies showed T3 binding to be analogue specific. The physiological relevance of T3 binding to goat Leydig cell was examined by adding increasing concentrations of T3 to the Leydig cell incubation (1×10 cells/incubation). T3 (10, 25 and 50 ng/ml or 4, 10 and 20 ng/incubation) resulted a dose dependent increase in androgen release and in all cases stimulation of androgen release was statistically significant (P<0·01) compared with control. Stimulation of Leydig cell androgen release by T3 was significantly inhibited by actinomycin-D (P<0·01) and cycloheximide (P<0·01). T3 had additive stimulatory effects on LH-augmented androgen release from Leydig cells. T3 (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 μg/ml) inhibited it completely. The data indicated that the stimulatory effect of T3 on androgen release is mediated via T3-induced protein(s). Sub-cellular fractions obtained from T3-treated Leydig cells showed an increase in protein synthesis in mitochondrial and soluble supernatant fractions (100 k sup) and it was only 100 k sup which stimulated androgen release from Leydig cells in separate incubations. Treatment of 100 k sup with trypsin or heat abolished its stimulatory effect. Incubation of Leydig cells with T3 for different times showed an increase in protein synthesis prior to the stimulation of androgen release. The results therefore indicated that T3 binding to Leydig cells induced the generation of a proteinaceous factor(s) which in turn stimulated androgen release. Journal of Endocrinology (1994) 143, 549–556