Thyroid hormone induces the generation of a novel putative protein in piscine ovarian follicle that stimulates the conversion of pregnenolone to progesterone.

Abstract

Ovarian follicles were collected from the perch belonging to the vitellogenic stage and incubated in vitro for 4 h in the absence (control) or presence of triiodothyronine (T3). Addition of T3 (40 ng/ml) to the follicle incubation caused a two-fold increase of [3H] pregnenolone conversion to radiolabelled progesterone (P4) as compared to the control. The increase in P4 formation in the ovarian follicle could be blocked completely by the inhibitors of protein synthesis, actinomycin D and cycloheximide (50 micrograms/ml), suggesting a protein or peptide mediator of the T3 stimulatory effect. To search for this mediator, ovarian follicles from the control or T3 incubate were homogenized and ultracentrifuged and different fractions were added separately to fresh follicle incubations. Only the 100,000 g supernatant from T3 incubate showed a significant (p < 0.01) increase in P4 formation, while the corresponding 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 pregnenolone to P4 in the ovarian follicle, the T3-induced protein (TIP) was purified to homogeneity by employing Sephadex G-75 gel filtration, FPLC Mono-Q and FPLC Superose-6 chromatography. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of purified TIP showed it to be a 52 K monomer protein. Addition of TIP in increasing concentrations to follicle incubations caused a linear increase in P4 formation. Experiments with radiolabelled TIP ([125I] TIP) indicate its entry through the follicular cell membrane within the limited period of incubation. Results suggest that TIP activates ovarian 3 beta-hydroxysteroid dehydrogenase enzyme, thus effecting a greater conversion of pregnenolone to P4.