FSH induction of aromatase in cultured rat granulosa cells measured by a radiometric assay

摘要: Abstract The effects of FSH on the aromatase activity rat granulosa cells in culture were studied by measuring stereospecific transfer 3 H from [1,2,6,7- H]testosterone or [1β- into 2 O. use O release as a specific assay for aromatization was validated various means. days after plating, cultures released only minimal amounts during 3-h incubation, whereas which had been treated with FSH, (Bu) cAMP plus 3-isobutyl-1-methylxanthine (MIX), time plating considerable medium. cultured inhibited aromatizable androgens, 19-hydroxy testosterone and 19-hydroxyandrostenedione, indicating specificity method aromatization. In order to study mechanism enhanced O, optimal conditions cell-free sonicates determined. Optimal achieved incubating at 37°C presence 20 mM phosphate buffer (pH 7.4) containing 5 dithiothreitol, MgCl , 0.5 NADP + glucose 6-phosphate U/ml glucose-6-phosphate dehydrogenase. A concentration 0.25 μM 0.1 μCi tritium used standard assay. Under these linear 1 h up 150 μg protein having maximal activity. When stimulated purified 48 increased dose-dependent fashion. ED 50 Sairam's S-1528 C2 12 ng/ml. It concluded studies that increases estrogen levels primarily inducing an active rather than influencing secretion availability substrate cofactor reaction.