An Alternative Method for the Study of Follicle-Stimulating Hormone Effects on Aromatase Activity in Sertoli Cell Cultures*

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Abstract
To validate the use of the RIA for the measurement of estrogen synthesis by Sertoli cells from immature rats, we have established an alternative method for the measurement of aromatase activity. This assay takes advantage of the stereospecific loss of hydrogen from positions C-lβ and C-2β of testosterone during the aromatization reaction to give H2O. [1β-3H]Testosterone was synthesized from [1β, 2β-3H]testosterone by drastic alkali treatment, and the release of 3H2 from this substrate by Sertoli cells in culture formed the basis of the aromatization assay. Sertoli cells were isolated from 11-day-old Wistar rats and were maintained in culture for 4 days in a chemically defined medium. Untreated cells released amounts of 3H2O which approximated those of blank values (200–300 dpm/ mg protein) when incubated with 0.4 μCi [1β-3H]testosterone for 4 h. Cells treated with 300 ng NIH-FSH-Sl3/ml for 24 h before incubation with substrate released significantly greater amounts of 3H2O (2000–3000 dpm$/mg protein) into the culture medium. The rate of production of 3H2O was linear throughout the 4-h incubation of FSH-treated Sertoli cells. Using the same culture conditions as those employed for the 3H2O assay, the synthesis of estrogen from exogenous unlabeled testosterone was measured by RIA to determine if this method under these conditions would also reflect FSH stimulation of aromatase activity. Cells stimulated with FSH for 24 h before incubation with exogenous testosterone synthesized 300 pg estrogen/ mg protein·4 h, whereas untreated cells synthesized only low levels. The number of picomoles of estrogen synthesized by Sertoli cells, as assessed by RIA, was not significantly different from the number of picomoles of testosterone metabolized, as calculated from the amount of 3H2O released. Androst-4-ene-3,6,17-trione (a potent inhibitor of aromatase activity), added immediately before the addition of testosterone, reduced the apparent effect of FSH on both products of the aromatase reaction, i.e. estrogen and 3H2O. In the presence of 19-hydroxyandrostenedione and 19-hydroxytestosterone, which are also substrates for the aromatization reaction, the effect of FSH on the release of 3H2O from [1β-3H]testosterone was no longer apparent, confirming the specificity of the 3H2O release assay for aromatization in Sertoli cells.