This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hardy, D. O. Articles by Hardy, M. P. Search for Related Content PubMed PubMed Citation Articles by Hardy, D. O. Articles by Hardy, M. P.

Identification of the Oxidative 3-Hydroxysteroid Dehydrogenase Activity of Rat Leydig Cells as Type II Retinol Dehydrogenase¹

Population Council and The Rockefeller University (D.O.H., R.-S.G., J.F.C., M.P.H.), New York, New York 10021; and the Department of Pharmacology (Y-t.H., T.M.P.), University of Pennsylvania, Philadelphia, Pennsylvania 19104

Address all correspondence and requests for reprints to: Dianne O. Hardy, Population Council, 1230 York Avenue, New York, New York 10021. E-mail: d-hardy{at}popcbr.rockefeller.edu

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 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 oxidoreductase in vitro and could theoretically be responsible for factors 2 and 3, we have shown previously that rat Leydig cells have two 3-HSD activities: a cytosolic NADP(H)- dependent activity, characteristic of 3-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 3-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 3-HSD1. When measured in intact CHOP and COS-1 cells, RoDH2 cDNA produced a protein that catalyzed the conversions of 3-DIOL to DHT and androsterone to androstanedione, but not the reverse reactions. Therefore, the 3-HSD activity of RoDH2 was exclusively oxidative. In contrast, type I 3-HSD cDNA produced a protein that was exclusively a 3-HSD reductase. In cell homogenates and subcellular fractions, RoDH2 catalyzed both 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, but was located in the cytosol and was NADP(H) dependent. We conclude that type I 3-HSD and RoDH2 have distinct 3-HSD activities with opposing catalytic directions, thereby controlling the rates of DHT production by Leydig cells.

This article has been cited by other articles:

				T. L. Rizner, H. K. Lin, D. M. Peehl, S. Steckelbroeck, D. R. Bauman, and T. M. Penning Human Type 3 3{alpha}-Hydroxysteroid Dehydrogenase (Aldo-Keto Reductase 1C2) and Androgen Metabolism in Prostate Cells Endocrinology, July 1, 2003; 144(7): 2922 - 2932. [Abstract] [Full Text] [PDF]

				R. Ivell, M. Balvers, R. J. K. Anand, H.-J. Paust, C. McKinnell, and R. Sharpe Differentiation-Dependent Expression of 17{beta}-Hydroxysteroid Dehydrogenase, Type 10, in the Rodent Testis: Effect of Aging in Leydig Cells Endocrinology, July 1, 2003; 144(7): 3130 - 3137. [Abstract] [Full Text] [PDF]

				L. D. Griffin and S. H. Mellon Biosynthesis of the Neurosteroid 3{alpha}-Hydroxy-4-pregnen-20-one (3{alpha}HP), a Specific Inhibitor of FSH Release Endocrinology, November 1, 2001; 142(11): 4617 - 4622. [Abstract] [Full Text] [PDF]

				H. M. Theobald, B. L. Roman, T.-M. Lin, S. Ohtani, S.-W. Chen, and R. E. Peterson 2,3,7,8-Tetrachlorodibenzo-p-dioxin Inhibits Luminal Cell Differentiation and Androgen Responsiveness of the Ventral Prostate without Inhibiting Prostatic 5{alpha}-Dihydrotestosterone Formation or Testicular Androgen Production in Rat Offspring Toxicol. Sci., December 1, 2000; 58(2): 324 - 338. [Abstract] [Full Text] [PDF]