Identification of the oxidative 3 alpha-hydroxysteroid dehydrogenase activity of rat Leydig cells as type II retinol dehydrogenase

Dihydrotestosterone (DHT) is the most potent naturally occurring androgen, and its production in the testis may have important consequences in develop mental and reproductive processes. In the rat testis, three factors can con tribute to intracellular DHT levels: 1) synthesis of DHT from T by 5 alpha- reductase, 2) conversion of DHT to 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-DIOL) by the reductive activity of 3 alpha-hydroxysteroid dehydrog enase (3 alpha-HSD), and 3) conversion of 3 alpha-DIOL by an oxidative 3 al pha-HSD activity. While the type I 3 alpha-HSD enzyme (3 alpha-HSD 1 or AKR 1C9) is an oxidoreductase in vitro and could theoretically be responsible f or factors 2 and 3, we have shown previously that rat Leydig cells have two Sor-HSD activities: a cytosolic NADP(H)- dependent activity, characteristi c of 3 alpha-HSD1, and a microsomal NAD(H)-dependent activity. The two acti vities were separable by both developmental and biochemical criteria, but t he identity of the second enzyme was unknown. To identify the microsomal NA D(H)-dependent 3 alpha-HSD in rat Leydig cells, degenerate primers were use d to amplify a number of short-chain alcohol dehydrogenases. Sequence analy sis of cloned PCR products identified retinol dehydrogenase type II(RoDH(2) ) as the prevalent species in purified Leydig cells. RoDH(2) cDNA was subcl oned into expression vectors and transiently transfected into CHOP and COS- 1 cells. Its properties were compared with transiently transfected 3 alpha- HSD1, When measured in intact CHOP and COS-1 cells, RoDH2 cDNA produced a p rotein that catalyzed the conversions of 3 alpha-DIOL to DHT and androstero ne to androstanedione, but not the reverse reactions. Therefore, the 3 alph a-HSD activity of RoDH2 was exclusively oxidative. In contrast, type I 3 al pha-HSD cDNA produced a protein that was exclusively a 3 alpha-HSD reductas e. In cell homogenates and subcellular fractions, RoDH2 catalyzed both 3 al pha-HSD oxidation and reduction reactions that were NAD(H) dependent, and t he enzyme activities were located in the microsomes. Type I 3 alpha-HSD als o catalyzed both oxidation and reduction, but was located in the cytosol an d was NADP(H) dependent. We conclude that type I 3 alpha-HSD and RoDH2 have distinct 3 alpha-HSD activities with opposing catalytic directions, thereb y controlling the rates of DHT production by Leydig cells.