Acrosomal enzymes and ultrastructure of unfrozen and cryotreated human spermatozoa

Pooled semen judged to be normal in all parameters was divided into a number of aliquots which were either 1) kept untreated; 2) mixed with glycerol (10% v/v); 3) washed by centrifugation and resuspended to the original volume with buffer; or 4) washed and resuspended in buffer with glycerol (10% v/v). The progressive motility, viability, ultrastructure, and acrosomal enzyme activity (8 different hydrolases) were studied before and after cryotreatment. The described washing procedure effectively removed seminal plasma, and did not alter sperm motility, sperm viability, sperm ultrastructure, or the acrosomal enzymes studied. Glycerol (10%, v/v) had a deleterious effect on most parameters evaluated before cryotreatment. Cryotreatment severely altered the motility and viability of the spermatozoa and their acrosomal morphology but did not cause significant decreases in most of the acrosomal hydrolases measured. However, acrosin/proacrosin levels decreased by 50-80% and were correlated to the acrosomal damage. A simple assay for the measurement of acrosin/proacrosin enzyme levels in whole sperm is presented which could be used as a monitor for acrosomal integrity. No significant differences were seen between the samples cryotreated in the absence or presence of seminal plasma.