MODIFICATION OF THE GELATIN SUBSTRATE PROCEDURE FOR DEMONSTRATION OF ACROSOMAL PROTEOLYTIC ACTIVITY

Abstract

In situ proteolytic activity in the heads of sperm of seven mammalian species has been demonstrated using autoradiographic film as a gelatin substrate. The film is first exposed and processed and then coated with the sperm sample. Proteolytic activity is monitored by following the appearance of "halos," which are areas of gelatin digestion and are found to surround the heads of reacting sperm. The proteolytic factor appears to be released from the acrosome and may be the protease with trypsin-like activity that has been found associated with the acrosome in biochemical studies. l-Chloro-3-tosylamido-7-amino-2-heptanone and diisopropyl fluorophosphate, both of which inhibit trypsin activity, apparently interact with a substance released from the acrosome but do not interfere with the formation of halos. Sperm treated with trichloroacetic acid, urea or formalin do not form halos.