Testicular Sterols

Abstract Crude homogenates of rat testicular tissue (centrifuged at 10,000 x g for 20 min) do not convert mevalonic acid into squalene and sterols. Enzymes in the supernatant fraction from high speed centrifugation (105,000 x g) of testicular homogenate from rats or guinea pigs catalyze the formation of prenol pyrophosphates from mevalonate. Two cell-free preparations of rat testicular tissue have been obtained that catalyze the incorporation of mevalonate into squalene and lanosterol. In one system, prenol pyrophosphate is formed by incubation of the soluble enzymes with mevalonic acid, fluoride, and appropriate cofactors. After heat inactivation of the soluble enzymes, squalene is formed during subsequent anaerobic incubation with testicular microsomes, and squalene and lanosterol are formed during subsequent aerobic incubation with a crude homogenate of testicular tissue. In the second system, microsomes dialyzed against 5 mm Mn++ and soluble enzymes dialyzed against 30 mm F- are incubated together with mevalonic acid, cofactors, an adenosine triphosphate-generating system, and a reduced triphosphopyridine nucleotide-generating system. The major product formed from mevalonate was squalene. Crude homogenates of rat testicular tissue contain many endogenous factors that affect the rate of incorporation of mevalonate into prenol pyrophosphates and nonsaponifiable lipids. Microsomes contain a very active adenosine triphosphatase that is inhibited by dialysis at 4° in the absence of Mn++ or by dialysis at 19° with Mn++. Microsomes also contain a prenol pyrophosphate pyrophosphohydrolase that is partially inhibited by 5 mm Mn++. A labile phosphatase has been isolated from the soluble fraction. The soluble phosphatase catalyzes the hydrolysis of inorganic phosphate from mevalonate 5-phosphate. The phosphatase is inhibited completely by dialysis against 30 mm F-. Successful incorporation of mevalonate into prenol pyrophosphates and squalene by simultaneous incubation of microsomes and soluble enzymes is ascribed either to suitable inactivation of the competing enzymatic processes or to compensation by the addition of cofactor-generating systems. Comparison of similar synthetic and competitive activities from rat liver and rat or guinea pig testis indicates that the ratio of synthetic activities to competitive processes is much more favorable for sterol synthesis by liver enzymes than by testicular enzymes.