Cholesterol side-chain cleavage activity in rat fetal gonads: A limiting step for ovarian steroidogenesis

Abstract

The aim of this study was to examine the first step in steroidogenesis in male and female gonads of fetal rats. Pregnenolone production was measured by radioimmunoassay in organ culture, conversion of [ 3H]cholesterol to [ 3H]pregnenolone was evaluated in isolated mitochondria and cytochrome P-450 scc was revealed by immunoblotting and immunocytochemical techniques. Our results clearly showed that in fetal testes (1) pregnenolone was produced in media where testes were cultured in the presence of trilostane and spironolactone, indicating an important metabolism of pregnenolone, (2) [ 3H]cholesterol was converted into [ 3H]pregnenolone in mitochondria, (3) cytochrome P-450 scc was revealed in immunoblots with a molecular weight of 50,000, (4) cytochrome P-450 scc was localized in Leydig cells from 15.5-day-old fetal testes onwards. With respect to fetal ovaries, we were unable to detect any scc activity, except after treatment with dibutyryl cyclic AMP. A lag period of 18 h was necessary to induce pregnenolone synthesis. However, the immunoperoxidase staining did not localize ovarian positive cells. Cytochrome P-450 scc could be revealed in postnatal ovaries by immunoblotting and some interstitial positive cells were observed with immunostaining; the reaction was enhanced in luteinizing hormone-pretreated ovaries. These data indicate that (a) the cholesterol scc activity is present in fetal testes, (b) the conversion of cholesterol to pregnenolone is a limiting step for steroidogenesis in fetal ovaries. The inductive effect of the nucleotide on the enzyme suggests that the absence of gonadotrophic receptors in fetal female gonads could explain the lack of steroidogenesis before birth.