125I-3,5,3′-tri-iodothyronine (T3) binds specifically to a pure nuclear preparation from rat granulosa cells. A Scatchard analysis of T3 binding showed a K d of 0·65 × 10−9 mol/l and a Bmax of 1·57 pmol/mg DNA. The biological relevance of T3 binding to granulosa cells was evaluated by adding T3 (20 ng/incubation) to granulosa cells (1 × 106 cells/incubation) which greatly stimulated progesterone release. T3-stimulated progesterone release was significantly inhibited by actinomycin-D (P<0·01) and cycloheximide (P<0·01). T3 caused about a twofold increase in granulosa cell protein synthesis as compared with the control which was inhibited by actinomycin-D and cycloheximide. The addition of T3 to granulosa cell incubations also resulted in a more than 2·5-fold increase in mRNA. The results indicated that T3 stimulation of progesterone release is mediated via T3-induced protein(s) or TIP. TIP was located in the soluble supernatant fraction (100 000 g supernatant; 100 k sup) from T3-incubated cells but could not be detected in the 100 k sup from the control cells or LH-incubated cells. TIP was purified based on its biological activity, i.e. its addition to granulosa cell incubations stimulated progesterone release into the medium. The 100 k sup from T3-incubated granulosa cells was subjected to Sephadex G-75 gel filtration, FPLC Mono Q and FPLC Superose 6 chromatography which resulted a 273-fold purification over the starting material and a clearly homogeneous protein was obtained. SDS-PAGE of purified TIP showed it to be a 53 kDa monomer protein. Experiments conducted with radiolabelled TIP suggested internalization of TIP into the granulosa cell. The results therefore showed that T3 induces the synthesis of mRNA and proteins in rat granulosa cells and that one of the proteins is TIP which, in turn, stimulates progesterone release from the cell, suggesting thereby that this putative protein is a novel mediator of T3 function in the granulosa cell.
Journal of Endocrinology (1996) 150, 309–318