Sources of cholesterol for testosterone biosynthesis in murine Leydig cells

The sources of cholesterol for testosterone production were investigated in freshly isolated murine Leydig cells. In vitro stimulation with human CG (hCG) (0.2 IU/ml) caused a 75-fold increase in testosterone production. Leydig cells contained approximately equal amounts of free and esterified cholesterol (7.8 vs. 8.7 micrograms/mg protein). The total cholesterol content of cells stimulated for 4 h with hCG was significantly decreased compared with unstimulated cells (8.4 vs. 17.6 micrograms/mg protein); both free and esterified cholesterol decreased by about 50%. In unstimulated Leydig cells incubated with [14C]acetate for 12 h, the majority of incorporated [14C] was found in free and esterified cholesterol, whereas, in the hCG-stimulated cells, 80% of incorporated 14C was in testosterone. The activity of hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase after 4 h in hCG-stimulated cells was 20% higher than in unstimulated cells (115.5 vs. 84.4 pmol/mg protein.min). However, by 6 h, HMG-CoA reductase activity doubled in the hCG-stimulated compared with unstimulated cells. By 12 h, HMG-CoA reductase activity in hCG-stimulated cells was 4 times the preincubation level and 8 times the 12-h level in unstimulated cells. HMG-CoA reductase activity induced by hCG was blocked by aminoglutethimide, an inhibitor of the cholesterol side-chain cleavage enzyme. Lovastatin, a potent inhibitor of HMG-CoA reductase, had no effect on unstimulated or hCG-stimulated testosterone production during a 12-h incubation. Murine high density lipoproteins (mHDL) increased HMG-CoA reductase activity in both unstimulated (29%) and hCG-stimulated (20%) cells. During a 6 h incubation, mHDL increased hCG-stimulated testosterone production by 20%, but had no effect on unstimulated testosterone production. These results suggest that murine Leydig cells store enough cholesterol and cholesteryl esters to support testosterone production for at least 12 h in vitro. Although mHDL does not have a major stimulatory effect on testosterone biosynthesis, it may be involved in the regulation of de novo cholesterol synthesis.