Regulation Of Cholesterol Side-chain Cleavage Enzyme And Testosterone Production In Leydig Cells.

Abstract

Two populations of Leydig cells were isolated by either Metrizamide or Percoll gradient centrifugation of dissociated rat testicular cells. The denser Leydig cell population (II), isolated in either type of gradient, had a greater capacity for steroid hormone synthesis than the less dense population (I). In both Leydig cell populations 25-hydroxycholesterol bypassed the cAMP-dependent transport system that delivers endogenous cholesterol to the mitochondrial cholesterol side-chain cleavage enzyme (P-450(,scc)) and therefore its metabolism to testosterone provided an index of relative P-450(,scc) activity. When primary cultures of the two Leydig cell populations were incubated with 25-hydroxycholesterol they produced more testosterone than when stimulated with cAMP. Population II produced twice as much testosterone as population I Leydig cells whether incubated with 25-hydroxycholesterol or stimulated with cAMP. The above results suggest that cAMP-dependent cholesterol transport to P-450(,scc) limits maximal cAMP-stimulated testosterone production and that a difference in P-450(,scc) activity is responsible for the functional heterogeneity of rat Leydig cells. Treatment of primary cultures of rat Leydig cells with cAMP for 2 days at ambient oxygen tension (19%) caused a 60% decrease in P-450(,scc) activity. This decrease was completely prevented when oxygen tension was reduced to 1% O(,2) or when steroid synthesis was inhibited by aminoglutethimide. When the endogenous concentration of pregnenolone was increased, by inhibiting its further metabolism, P-450(,scc) activity was reduced by 80% in unstimulated cultures, and was completely eliminated in cAMP-treated cultures. These losses were prevented when cells were maintained at 1% O(,2). The amount of immunoreactive P-450(,scc) was also reduced by treatments which reduced P-450(,scc) activity. Stimulation with cAMP or incubation with testosterone lowered the microsomal C(,17-20) lyase activity in an oxygen tension-sensitive way. These results suggest that the enhanced loss of mitochondrial and microsomal cytochrome P-450 activities in cAMP-treated cultures is caused by the increased production of pregnenolone and testosterone, respectively, which act as pseudosubstrates for these enzymes and generate reactive, damaging species derived from reduced dioxygen. Steroidogenic desensitization of cAMP-treated Leydig cells was caused by a reduction in availability of endogenous cholesterol, since it was not prevented at 1% O(,2) but was overcome when exogenous 25-hydroxycholesterol was supplied to the cells.