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

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