BINDING OF THYROID-HORMONE TO FRESH-WATER PERCH LEYDIG-CELL NUCLEI-RICH PREPARATION AND ITS FUNCTIONAL RELEVANCE

Leydig cells were isolated from the testis of a freshwater perch, Anab as testudineus, belonging to the prespawning stage of reproductive cyc le. Cells were sonicated and a pure nuclei preparation was obtained fo r binding assay. Under optimum assay conditions of pH 7.4 at 30-degree s-C and 90 min incubation binding of [I-125] 3,5,3'-triiodothyronine ( T3) to Leydig cell nuclei was saturable at 1.7 nmol/l concentration. A Scatchard analysis of T3-binding exhibited a Kd of 26.8 x 10(-9) mol/ l and a maximum binding capacity (B(max)) as 1.66 pmol/mg DNA. Competi tive inhibition studies demonstrated that binding of T3 to Leydig cell nuclei is analogue specific. Biological relevance of T3 binding to pe rch Leydig cell was evaluated by adding varied concentrations of T3 to Leydig cell incubation (1 X 10(6) cells/incubation). Twenty five ng t o 100 ng of T3 resulted in a dose dependent increase in androgen relea se while 200 ng of T3 had no additional effect over 100 ng under prese nt incubation system. Stimulation of androgen release by T3 was signif icantly inhibited (p<0.01) by cycloheximide. T3 (100 ng/ml) increased protein synthesis in Leydig cell (70% as compared to control) which wa s significantly inhibited (p<0.01) by cycloheximide. Results indicate that binding of T3 to Leydig cell of perch testis triggers the synthes is of a protein (or proteins) which in turn stimulates androgen releas e.