Acrosin, Proacrosin, and Acrosin Inhibitor of Human Spermatozoa: Extraction, Quantitation, and Stability

Ten different methods were evaluated to determine the treatment for obtaining maximal amounts of acrosin, proacrosin, and acrosin inhibitor (the acrosin system) from human spermatozoa by a single technique. Optimal results were obtained by extraction of the gametes with 10% glycerol at pH 2.8 for at least 12 hours. A single treatment by this technique provided approximately 95% of all available acrosin. The use of benzamidlne during extraction was essential for maximal recovery of proacrosin, i.e., to prevent zymogen activation. Benzamidlne did not affect the total amount of acrosin recovered. The levels of acrosin Inhibitor obtained did not vary with the method of treatment but were greatly influenced by the molecular pore size of the membrane used for dialysis of the extracts. An assay method for the simple determination of the components of the acrosin system is presented. Using the optimal extraction method and described assay technique, 42 pooled semen samples, each consisting of four to 14 ejaculates, were analyzed for the acrosin system. The total acrosin averaged 106 mlU/10 7 spermatozoa (S.D. = ±22 mlU/10 7 sperm). Almost all the acrosin was in the zymogen (proacrosin) form (93% ± 2%). After proacrosin activation, enough inhibitor was present to inhibit 94% ±5% of the acrosin. Acrosin and proacrosin were stable in spermatozoa kept in seminal plasma at room temperature for at least 6 hours. After solubilization, the enzymes were stable for at least one week when stored at pH 3.0,4 C, if the proacrosin was not activated to acrosin. After activation, the extracts were much less stable. Maximal conversion of proacrosin to acrosin occurred in 10 minutes when the extracts were incubated at pH 8.0 at room temperature. The presence of calcium ions severely retarded activation. The described extraction and assay techniques provide a standard method by which the acrosin system of spermatozoa can be measured, and may now be applied to the spermatozoa of infertile men to determine whether any abnormalities exist In the acrosin system of these gametes.