Opposing changes in 3 alpha-hydroxysteroid dehydrogenase oxidative and reductive activities in rat Leydig cells during pubertal development

The enzyme Sar-hydroxysteroid dehydrogenase (3 alpha-HSD) has an important role in androgen metabolism, catalyzing the interconversion of dihydrotesto sterone (DHT) and 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-DIOL). T he net direction of this interconversion will affect the amount of biologic ally active ligand available for androgen receptor binding. We hypothesize that in Leydig cells, differential expression of 3 alpha-HSD enzymes favori ng one of the two directions is a mechanism by which DHT levels are control led. In order to characterize 3a-HSD in rat Leydig cells, the following pro perties were analyzed: rates of oxidation (3 alpha-DIOL to DHT) and reducti on (DHT to 3 alpha-DIOL) and preference for the cofactors NADP(H) and NAD(H ) (i.e., the oxidized and reduced forms of both pyridine nucleotides) in Le ydig cells isolated on Days 21, 35, and 90 postpartum. Levels of 3 alpha-HS D protein were measured by immunoblotting using an antibody directed agains t the liver type of the enzyme. Levels of 3a-HSD protein and rates of reduc tion were highest on Day 21 and lowest on Day 90. The opposite was true for the rate of 3 alpha-HSD oxidation, which was barely detectable on Day 21 a nd highest on Day 90 (59.08 +/- 6.35 pmol/min per 10(6) cells, mean +/- SE) . Therefore, the level of 3 alpha-HSD protein detectable by liver enzyme wa s consistent with reduction but not with oxidation. There was a clear parti tioning of NADP(H)-dependent activity into the cytosolic fraction of Leydig cells, whereas on Days 35 anti 90, Leydig cells also contained a microsoma l NAD(H)-activated 3 alpha-HSD. We conclude that 1) the cytosolic 3 alpha-H SD in Leydig cells on Day 21 behaves as a unidirectional NADPH-dependent re ductase; 2) by Day 35, a microsomal NAD(H)-dependent enzyme activity is pre sent and may account for pre-dominance of 3 alpha-HSD oxidation over reduct ion and the resultant high capacity of Leydig cells on Day 90 to synthesize DHT from 3 alpha-DIOL.