THYROID-HORMONE INDUCES THE GENERATION OF A NOVEL PUTATIVE PROTEIN INPISCINE OVARIAN FOLLICLE THAT STIMULATES THE CONVERSION OF PREGNENOLONE TO PROGESTERONE

Ovarian follicles were collected from the perch belonging to the vitel logenic stage and incubated in vitro for 4 h in the absence (control) or presence of triiodothyronine (T-3) Addition of T-3 (40 ng/ml) to th e follicle incubation caused a two-fold increase of [H-3] pregnenolone conversion to radiolabelled progesterone (P-4) as compared to the con trol, The increase in P-4 formation in the ovarian follicle could be b locked completely by the inhibitors of protein synthesis, actinomycin D and cycloheximide (50 mu g/ml), suggesting a protein or peptide medi ator of the T-3 stimulatory effect, To search for this mediator, ovari an follicles from the control or T-3 incubate were homogenized and ult racentrifuged and different fractions were added separately to fresh f ollicle incubations. Only the 100 000 g supernatant from T-3 incubate showed a significant (p < 0.01) increase in P-4 formation, while the c orresponding supernatant from control follicle incubations had no such stimulatory effect. Trypsin or heat destroyed this augmentory effect. Based on its ability to stimulate the conversion of radiolabelled pre gnenolone to P-4 in the ovarian follicle, the T-3-induced protein (TIP ) was purified to homogeneity by employing Sephadex G-75 gel filtratio n, FPLC Mono-Q and FPLC Superose-6 chromatography. Sodium dodecyl sulp hate polyacrylamide gel electrophoresis of purified TIP showed it to b e a 52 K monomer protein. Addition of TTP in increasing concentrations to follicle incubations caused a linear increase in P-4 formation. Ex periments with radiolabelled TIP ([I-125] TIP) indicate its entry thro ugh the follicular cell membrane within the limited period of incubati on, Results suggest that TIP activates ovarian 3 beta-hydroxysteroid d ehydrogenase enzyme, thus effecting a greater conversion of pregnenolo ne to P-4.