Delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity in two distinct density Leydig cells from immature...

The present studies examined the responsiveness to human chorionic gonadotropin (hCG) or 8-bromoadenosine 3',5'-monophosphate (8-Br-cAMP) of delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity of cultured immature Band 2 (low density) or Band 3 (high density) Leydig cells isolated on Percoll gradients. Enzyme activity increased in relation to the dose of hCG or 8-Br-cAMP in both bands; however, activity in Band 2 cells increased about 200% above control, while activity in Band 3 cells increased only about 30-60% above control following 6 days of treatment. Maximal responses were observed 4-6 days following exposure to hCG or 8-Br-cAMP in both bands. Because elevated 5 alpha-reductase activity prevents testosterone accumulation in immature Leydig cells, Band 2 or Band 3 cells were cultured in the presence of 4-methyl-4-aza-3-oxo-pregnan-(20S)-carboxylate, a 5 alpha-reductase inhibitor, to assess the relationship between changes in delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity and testosterone formation. Although hCG or 8-Br-cAMP-stimulated delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity began to decline by day 8 or 10 of treatment, testosterone levels progressively increased for 10 days before declining in both bands. Thus, changes in enzyme activity did not strictly correlate with testosterone synthesizing capacity of cultured Leydig cells. Enzyme activity also was measured in cells cultured with the 5 alpha-reductase inhibitor to determine whether the responsiveness of delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity of Band 2 or Band 3 cells was due, in part, to low testosterone levels. delta 5-3 beta-Hydroxysteroid dehydrogenase-isomerase activity in Band 2 or Band 3 cells cultured without or with the inhibitor was similar, suggesting that testosterone does not inhibit the enzyme in immature Leydig cells. The greater responsiveness of delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity in Band 2 cells was not due to Leydig cell replication as neither total DNA or [3H]thymidine incorporation into DNA was altered by hCG or cAMP.