Thyroid hormone induces a 52 kDa soluble protein in goat testis Leydig cell which stimulates androgen release

Incubation of goat testicular Leydig cells with 3,5,3'-triiodothyronine (T3) induces the generation of a proteinaceous factor (factors) which was located in the soluble supernatant fraction (100 000 x g supernatant, 100 k sup) of sonicated Leydig cells. Addition of this factor to Leydig cell incubation greatly stimulated androgen release. This factor(s) was purified based on its biological properties, i.e., its addition to Leydig cell incubation augmented the release of androgen. This was designated as TIP (T3-induced protein) activity. 100 k sup prepared from Leydig cells incubated in the absence (control) or presence of T3 was gel filtered through Sephadex G-100. 100 k sup from T3 incubated gave two protein peaks, P-I and P-II, control 100 k sup had similar nature of P-I, but P-II was not well marked. Incubation in the presence of [14C]leucine clearly showed TCA precipitable radioactivity only in the P-II region of T3 incubate. 5 microg of P-II protein stimulated androgen release from Leydig cells cells (1 x 10(6) cells/well) to more than 5-fold as compared to control. P-II protein was further purifies by FPLC Mono-Q column chromatography where one unadsorbed (MQ-I) and two adsorbed (MQ-II and MQ-III) protein peaks could be detected. MQ-II, which was eluted with 0.20 M NaCl gradient, demonstrated strong TIP activity (2 microg protein released 4.8-fold more androgen as compared to control). MQ-II was passed through FPLC Superose-6 column where it gave two peaks and Peak-I(SP-I) showed strong TIP activity (2 microg protein stimulated a 6-fold increase in androgen release as compared to control). Polyacrylamide gel electrophoresis (PAGE) indicated SP-I to be a homogeneous protein and SDS-PAGE demonstrated it to be a 52 kDa monomer protein. Results show that T3 induces the synthesis of a 52 kDa protein in testicular Leydig cells which in turn causes stimulation of androgen release suggesting this protein to be a novel mediator of T3 function in Leydig cells.