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Dianne O. Hardy, Ren-Shan Ge, James F. Catterall, Yong-tai Hou, Trevor M. Penning, Matthew P. Hardy, Identification of the Oxidative 3α-Hydroxysteroid Dehydrogenase Activity of Rat Leydig Cells as Type II Retinol Dehydrogenase, Endocrinology, Volume 141, Issue 5, May 2000, Pages 1608–1617, https://doi.org/10.1210/endo.141.5.7445
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Abstract
Dihydrotestosterone (DHT) is the most potent naturally occurring androgen, and its production in the testis may have important consequences in developmental and reproductive processes. In the rat testis, three factors can contribute to intracellular DHT levels: 1) synthesis of DHT from T by 5α-reductase, 2) conversion of DHT to 5α-androstane-3α,17β-diol (3α-DIOL) by the reductive activity of 3α-hydroxysteroid dehydrogenase (3α-HSD), and 3) conversion of 3α-DIOL by an oxidative 3α-HSD activity. While the type I 3α-HSD enzyme (3α-HSD1 or AKR1C9) is an oxidoreductase in vitro and could theoretically be responsible for factors 2 and 3, we have shown previously that rat Leydig cells have two 3α-HSD activities: a cytosolic NADP(H)- dependent activity, characteristic of 3α-HSD1, and a microsomal NAD(H)-dependent activity. The two activities were separable by both developmental and biochemical criteria, but the identity of the second enzyme was unknown. To identify the microsomal NAD(H)-dependent 3α-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 dehydrogenase type II (RoDH2) as the prevalent species in purified Leydig cells. RoDH2 cDNA was subcloned into expression vectors and transiently transfected into CHOP and COS-1 cells. Its properties were compared with transiently transfected 3α-HSD1. When measured in intact CHOP and COS-1 cells, RoDH2 cDNA produced a protein that catalyzed the conversions of 3α-DIOL to DHT and androsterone to androstanedione, but not the reverse reactions. Therefore, the 3α-HSD activity of RoDH2 was exclusively oxidative. In contrast, type I 3α-HSD cDNA produced a protein that was exclusively a 3α-HSD reductase. In cell homogenates and subcellular fractions, RoDH2 catalyzed both 3α-HSD oxidation and reduction reactions that were NAD(H) dependent, and the enzyme activities were located in the microsomes. Type I 3α-HSD also catalyzed both oxidation and reduction, but was located in the cytosol and was NADP(H) dependent. We conclude that type I 3α-HSD and RoDH2 have distinct 3α-HSD activities with opposing catalytic directions, thereby controlling the rates of DHT production by Leydig cells.
