An improved assay technique for the proteolytic activity of individual human spermatozoa

An improved substrate-film technique has been developed for the assay of released proteinase from individual human spermatozoa. During the preparation of the thin gelatin membrane, it is pretreated with formaldehyde and NaOH. These agents alter the plasma membrane and the outer acrosomal membrane of the spermatozoon, facilitating the release of acrosomal enzymes. This effect is further enhanced by the addition of albumin to the incubation mixture. More than 90% of the spermatozoa in a normal ejaculate give a reaction by this method. It reaches a maximum after 4 hours of incubation and does not increase further even up to 12 hours. No difference in the reaction between washed and unwashed ejaculated spermatozoa can be found. The nature of the proteolytic activity and its possible significance in infertility are discussed.