Cytochemical Demonstration of Acrosomal Proteinase in Mammalian and Avian Spermatozoa by a Silver Proteinate Method

A silver proteinate method developed by Takamatsu et al. (1963) was employed for visual demonstration of the active sites of proteinase in the spermatozoa of nine mammalian species (golden hamster, mouse, deer mouse, rat, guinea pig, rabbit, dog, bull, and human) and one avian species (cock). Proteinase activity was demonstrated only in the acrosomal caps of all mammalian species studied. Strong proteinase activity was observed in the acrosomes of cock spermatozoa. In the golden hamster, activity was demonstrated in the acrosomes of not only mature spermatozoa, but also of immature spermatozoa as well as early spermatids. The optimum activity of the acrosomal proteinase in all of these species was generally in the acidic range. Studies using golden hamster and rabbit spermatozoa showed that proteinase activity was not inhibited by soybean and lima bean trypsin inhibitors, but it was drastically inhibited by tetrabutylammonium iodide, and completely inhibited by HgCl2, KCN, KMnO4, heparin sodium salt, phosphorylated hesperidin and a hydroquinone sulphonic acid-formaldehyde polymer. It was inferred that the hamster and rabbit sperm acrosomal proteinase demonstrable by this technique represents a proteinase conjugated with hyaluronidase. The function of such a proteinase was judged to be depolymerization of the matrix of the cumulus oophorus in close association with the action of hyaluronidase.