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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 K_d of 0·535 x 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 (1x1⁰ 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

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