Cytochemical demonstration of proteolytic activity of human and rat spermatozoa.

Abstract

Summary. Proteolytic activity was demonstrated around the acrosomes of intact sperm cells with a simple cytochemical method using a gelatin substrate. The proteolytic activity was localized to the acrosomal region of both rat and human spermatozoa. Not all sperm cells showed the same digestive capacity in each sample studied. Rabbit sperm acrosomes have a denuding effect on the zona pellucida, as Srivastava, Adams & Hartree (1965a, b) have demonstrated. This finding suggested that penetration of a spermatozoon through the zona pellucida is mediated, at least in part, by the presence of hydrolytic enzymes. Recently, Stambaugh & Buckley (1969) found hyaluronidase and a 'trypsin-like' enzyme in acrosomal extracts of rabbit spermatozoa; apparently the proteolytic enzyme is mainly responsible for the ability to penetrate the zona pellucida in this species. The localization of proteolytic activity in intact sperm cells was accomplished by Gaddum & Blandau (1970), using an india-ink gelatin substrate method. The technique promised to be simple enough to allow further studies and even semiquantitative estimations of protease activity; however, in trying to repeat the method, we found that it gave inconstant results and obscured a great deal of the cytological details of the cells under study, nor was it possible to obtain clear-cut 'digestion halos' around the acrosomes, as illustrated in Gaddum & Blandau's original publication. We therefore attempted to develop a method that could clearly show proteolytic activity against gelatin, without significantly obscuring cytological detail. Human ejaculates and rat epididymal spermatozoa were used. The sperma¬ tozoa were washed twice in Hanks' solution with sodium bicarbonate 0-35 g/1 at a final pH of 7-4. The gelatin solution (5%) was prepared in a Veronalacetate buffer at a different pH ranging from 7-4 to 9. Washed spermatozoa were spread evenly on a glass slide; after 2 min at room temperature, smears were immersed once in the gelatin solution kept at about 28 to 30° C; the slides were set in a vertical position for a few minutes to obtain a thin gelatin film. After blotting the bottom edge, the slides were allowed to gel in a horizontal position in a humid chamber at room temperature from 12 to 48 hr. After incubation,