The 3 beta-hydroxysteroid dehydrogenase activity of cultured porcine Leydig cells in primary culture.
Previous studies have shown that there is a spontaneous progressive increase in human chorionic gonadotropin (HCG)-stimulated testosterone (T) production by immature porcine Leydig cells during primary culture for 3 days, whereas basal T production is unaltered. Preliminary data indicated that during this time, there was an increase in the activity of the 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD). The purpose of the present study was to further characterize this increase. 3 beta-HSD was assayed by measuring the conversion of tritiated dehydroepiandrosterone (DHEA) to androstenedione in the presence of NAD+. A ninefold increase in activity occurred between days 1 and 2 and only twofold between days 2 and 3. A 5- to 20-fold increase of the apparent maximum velocity of the enzyme was observed after 3 days of culture while the apparent Km remained unchanged. Cycloheximide treatment (150 ng/mL) completely abolished this spontaneous increase in enzyme activity. In parallel experiments, the stimulatory effect of HCG on T and DHEA production was inhibited by cycloheximide treatment by 90% and 60%, respectively, whereas basal T production was not affected. These results suggest that the spontaneous increase of 3 beta-HSD activity in primary cultures of porcine Leydig cells is secondary to de novo protein synthesis and that this phenomenon may be partially responsible for the increased responsiveness of these cells to HCG during the culture period.
D015096:3-Hydroxysteroid Dehydrogenases / Q000378:metabolism*; D000735:Androstenedione / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D007700:Kinetics; D007985:Leydig Cells / Q000201:enzymology*; D008099:Liver / Q000201:enzymology*; D008297:Male; D011375:Progesterone Reductase / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D013552:Swine; D013739:Testosterone / Q000378:metabolism
D011506:Proteins; D013739:Testosterone; D000735:Androstenedione; D003513:Cycloheximide; D015096:3-Hydroxysteroid Dehydrogenases; D011375:Progesterone Reductase