Altering culture conditions reveals strain-related differences in activity and immunoreactive isoforms of 3β-hydroxysteroid dehydrogenase-isomerase in mouse Leydig cells

Abstract

We previously reported that 3β-hydroxysteroid dehydrogenase-isomerase (3βHSD) activity is higher in Leydig cells from C57BL 6J mice than those from C3H HeJ mice. This study examines whether the differences in 3βHSD activity in Leydig cells from the two strains of mice are due to the expression of different 3βHSD isoforms and if a specific isoform corresponds with the amount of 3βHSD activity under various culture conditions. Leydig cells were plated in Waymouth's + 15% horse serum (HS) medium. Some culture were terminated 24 h later after plating (day 1) and assayed for 3βHSD activity or immunoreactivity. The remaining cultures were maintained in HS or changed to serum-free medium. We demonstrate for the first time that two 3βHSD immunoreactive isoforms are expressed in freshly isolated Leydig cells and those cultured for 1 day. The same two 3βHSD isoforms are detectable in both strains. Thus, the previously reported strain-related differences in 3βHSD activity are not due to the expression of different isoforms. When cultured for 8 days, the higher molecular weight 3βHSD immunoreactive band is no longer detectable in Leydig cells from either strain. When maintained in HS, 3βHSD activity in C57BL 6J Leydig cells decreases significantly by day 8, while 3βHSD activity in C3H HeJ Leydig cells does not change through day 8. When Leydig cells were cultured in serum-free medium, 3βHSD activity is maintained in cultured Leydig cells from C57BL 6J and significantly increases in C3H HeJ 3βHSD by day 8. These data suggest that HS has a strain-specific inhibitory effect on 3βHSD activity, causing a significant decrease in C57BL 6J 3βHSD activity and preventing an increase in C3H HeJ . Densitometric analysis reveals a correspondence between changes in 3βHSD activity and the lower molecular weight isoform but not the higher molecular weight isoform. Treatment with cAMP induces the immunoreactive mass of the lower molecular isoform but not the higher molecular isoform of 3βHSD. Currently, it is unclear what the function of the higher molecular weight 3βHSD isoform is in mouse Leydig cells.