25-Hydroxycholesterol-Supported and 8-Bromo-Adenosine 3′,5′ -Monophosphate- Stimulated Testosterone Production by Primary Cultures of Two Populations of Rat Leydig Cells*

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Abstract
The metabolism of 25-hydroxycholesterol in primary cultures of both populations [I and II] of Leydig cells was examined. 25-Hydroxycholesterol bypasses the cAMP-dependent transport mechanism to the mitochondrial cytochrome P-450 side-chain cleavage enzyme (P-450scc) required by cholesterol and thus provides an index of the relative activity of P-450scc. Incubation of Leydig cells with increasing concentrations of 25-hydroxycholesterol resulted in a concentration-dependent increase in the amount of testosterone produced, with maximal amounts in both populations being reached at 25-hydroxycholesterol concentrations of .gtoreq. 5 .mu.M. Population II produced more than twice as much testosterone as population I Leydig cells, whether incubated with 25-hydroxycholesterol or with 8-bromo-cAMP. Each population of Leydig cells produced 2.5-fold greater amounts of testosterone when incubated with 25-hydroxycholesterol than when incubated with 8-bromo-cAMP. In both populations of Leydig cells, cAMP-stimulated testosterone production was not different in cells cultured for 24 h from that in freshly isolated Leydig cells. Cholesterol transport to P-450scc limits maximal testosterone production, and the difference in hCG [human chorionic gonadotropin]- or cAMP-stimulated testosterone production between the 2 populations of Leydig cells is primarily due to differences in P-450scc activity between the 2 populations and is not a result of population I consisting mostly of damaged Leydig cells.

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