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Expression of 11ß-Hydroxylase in Rat Leydig Cells

Center for Biomedical Research, The Population Council, and Rockefeller University (G.M.W., R.S.G., M.P.H.), New York, New York 10021; and Department of Pathology and Laboratory Medicine, The Miriam Hospital, Brown University School of Medicine (S.A.L., D.J.M.), Providence, Rhode Island 02906

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

11ß-Hydroxy (11ß-OH) derivatives of certain steroids function as inhibitors of 11ß-hydroxysteroid dehydrogenase isoform 1 (11ßHSD1), an enzyme expressed in Leydig cells that catalyzes the reversible oxidation of biologically active glucocorticoids to inactive 11-dehydro metabolites. 11ß-Hydroxylase is an adrenal enzyme responsible for glucocorticoid biosynthesis, catalyzing 11ß-hydroxylation of steroids and thus producing 11ß-OH-steroid derivatives. The aims of the present study were 1) to examine whether 11ß-hydroxylase is expressed in testis, 2) to define the biochemical characteristics of the testicular form of this enzyme, and 3) to establish whether 11ß-hydroxylated steroids inhibit Leydig cell 11ßHSD1 activities. 11ß-Hydroxylase mRNA was detected in purified rat Leydig cells by RT-PCR. Sequencing confirmed that the PCR products had 100% identity with the published rat adrenal enzyme cDNA sequence. Immunohistochemistry and Western blot analysis using a mouse monoclonal antibody confirmed the expression of 11ß-hydroxylase protein in Leydig cells. Moreover, 11ß-hydroxylase activity, synthesis of corticosterone from 11-deoxycorticosterone, was measurable in Leydig cells, and the K_m and maximum velocity values were 7.28 ± 0. 92 µM and 1.13 ± 0.04 µmol/10⁶ cell·h, respectively. When assayed in Leydig cells, several 11ß-hydroxylated steroids were efficient inhibitors of 11ßHSD1 dehydrogenase activity, whereas other 11-keto compounds were effective as inhibitors of oxidoreductase activity. These results provide the first direct evidence that rat Leydig cells express 11ß-hydroxylase, which may be involved in the regulation of glucocorticoid metabolism within the testis through local biosynthesis of endogenous inhibitors of 11ßHSD1.

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