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Abstract

Identification of the oxidative 3α-hydroxysteroid dehydrogenase activity of rat Leydig cells as Type II retinol dehydrogenase 
Hardy,Dianne O.; Ge,Renshan; Catterall,James F.; Hou,Yong-tai; Penning,Trevor M.; Hardy,Matthew P.
Endocrinology 141(5): 1608-1617
Publication date: 2000


Dihydrotestosterone (DHT) is the most potent naturally occurring androgen,and its production in the testis may have important consequencesin developmental and reproductive processes. In the rat testis,three factors can contribute to intracellular DHT levels: 1) synthesisof DHT from T by 5-reductase, 2) conversion of DHT to 5-androstane-3,17ß-diol(3-DIOL) by the reductive activity of 3-hydroxysteroid dehydrogenase(3-HSD), and 3) conversion of 3-DIOL by an oxidative 3-HSD activity.While the type I 3-HSD enzyme (3-HSD1 or AKR1C9) is an oxidoreductasein vitro and could theoretically be responsible for factors2 and 3, we have shown previously that rat Leydig cells havetwo 3-HSD activities: a cytosolic NADP(H)- dependent activity,characteristic of 3-HSD1, and a microsomal NAD(H)-dependentactivity. The two activities were separable by both developmentaland biochemical criteria, but the identity of the second enzymewas unknown. To identify the microsomal NAD(H)-dependent 3-HSDin rat Leydig cells, degenerate primers were used to amplifya number of short-chain alcohol dehydrogenases. Sequence analysisof cloned PCR products identified retinol dehydrogenase typeII (RoDH2) as the prevalent species in purified Leydig cells.RoDH2 cDNA was subcloned into expression vectors and transientlytransfected into CHOP and COS-1 cells. Its properties were comparedwith transiently transfected 3-HSD1. When measured in intactCHOP and COS-1 cells, RoDH2 cDNA produced a protein that catalyzedthe conversions of 3-DIOL to DHT and androsterone to androstanedione,but not the reverse reactions. Therefore, the 3-HSD activityof RoDH2 was exclusively oxidative. In contrast, type I 3-HSDcDNA produced a protein that was exclusively a 3-HSD reductase.In cell homogenates and subcellular fractions, RoDH2 catalyzedboth 3-HSD oxidation and reduction reactions that were NAD(H)dependent, and the enzyme activities were located in the microsomes.Type I 3-HSD also catalyzed both oxidation and reduction, butwas located in the cytosol and was NADP(H) dependent. We concludethat type I 3-HSD and RoDH2 have distinct 3-HSD activities withopposing catalytic directions, thereby controlling the ratesof DHT production by Leydig cells.