Identification of the Oxidative 3 -Hydroxysteroid Dehydrogenase Activity of Rat Leydig Cells as Type II Retinol Dehydrogenase

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Abstract
Dihydrotestosterone (DHT) is the most potent naturally occurring androgen, and its production in the testis may have important con- sequences in developmental and reproductive processes. In the rat testis, three factors can contribute to intracellular DHT levels: 1) synthesis of DHT from T by 5a-reductase, 2) conversion of DHT to 5a-androstane-3a,17b-diol (3a-DIOL) by the reductive activity of 3a- hydroxysteroid dehydrogenase (3a-HSD), and 3) conversion of 3a- DIOL by an oxidative 3a-HSD activity. While the type I 3a-HSD enzyme (3a-HSD1 or AKR1C9) is an oxidoreductase in vitro and could theoretically be responsible for factors 2 and 3, we have shown pre- viously that rat Leydig cells have two 3a-HSD activities: a cytosolic NADP(H)- dependent activity, characteristic of 3a-HSD1, and a mi- crosomal NAD(H)-dependent activity. The two activities were sepa- rable by both developmental and biochemical criteria, but the identity of the second enzyme was unknown. To identify the microsomal NAD(H)-dependent 3a-HSD in rat Leydig cells, degenerate primers were used to amplify a number of short-chain alcohol dehydrogenases. Sequence analysis of cloned PCR products identified retinol dehy- drogenase type II (RoDH2) as the prevalent species in purified Leydig cells. RoDH2 cDNA was subcloned into expression vectors and tran- siently transfected into CHOP and COS-1 cells. Its properties were compared with transiently transfected 3a-HSD1. When measured in intact CHOP and COS-1 cells, RoDH2 cDNA produced a protein that catalyzed the conversions of 3a-DIOL to DHT and androsterone to androstanedione, but not the reverse reactions. Therefore, the 3a- HSD activity of RoDH2 was exclusively oxidative. In contrast, type I 3a-HSD cDNA produced a protein that was exclusively a 3a-HSD reductase. In cell homogenates and subcellular fractions, RoDH2 cat- alyzed both 3a-HSD oxidation and reduction reactions that were NAD(H) dependent, and the enzyme activities were located in the microsomes. Type I 3a-HSD also catalyzed both oxidation and reduc- tion, but was located in the cytosol and was NADP(H) dependent. We conclude that type I 3a-HSD and RoDH2 have distinct 3a-HSD ac- tivities with opposing catalytic directions, thereby controlling the rates of DHT production by Leydig cells. (Endocrinology 141: 1608 - 1617, 2000)

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