Non-specific esterase activity in bovine acrosomes

Synopsis Fixed and unfixed smears of bovine spermatozoa were stained for esterase activity at pH 6.1–6.2 using 1-naphthyl acetate as substrate and hexazonium pararosaniline as coupler. Best results were obtained with unfixed smears prepared from suspensions of spermatozoa washed free of seminal plasma. The most intense staining was obtained when smears were incubated at 4°C for 40 hr with replacement of the incubating medium every 12 hr. No staining was detected in spermatozoa when smears were incubated in medium lacking substrate or when heat-inactivated smears were incubated in complete medium. Enzyme activity was restricted to the acrosomal cap; no final reaction product was detected in or on the post-nuclear cap, the mid-piece or the remainder of the flagellum. Enzyme activity was distributed quite uniformly throughout the acrosome with no indication of the existence of localized regions with high activity. The use of fixatives, especially those containing formalin, is not recommended for esterase studies of bovine spermatozoa.