PREVENTION OF OSMOTIC INJURY TO HUMAN SPERMATOZOA DURING ADDITION ANDREMOVAL OF GLYCEROL

Use of a cryoprotective agent is indispensable to prevent injury to hu man spermatozoa during the cryopreservation process, However, addition of cryoprotective agents to spermatozoa before cooling and their remo val after warming may create severe osmotic stress for the cells, resu lting in injury. The objective of this study was to test the hypothesi s that the degree (or magnitude) of human sperm volume excursion can b e used as an independent indicator to evaluate and predict possible os motic injury to spermatozoa during the addition and removal of cryo-pr otective agents. Glycerol was used as a model cryoprotective agent in the present study. To test this hypothesis, first the tolerance limits of spermatozoa to swelling in hypoosmotic solutions (iso-osmotic medi um diluted with water) and to shrinkage in hyperosmotic solutions (iso -osmotic medium with sucrose) were determined. Sperm plasma membrane i ntegrity was measured by fluorescent staining, and sperm motility was assessed by computer-assisted semen analysis before, during and after the anisosmotic exposure. The results indicate firstly that motility w as much more sensitive to anisosmotic conditions than membrane integri ty, and secondly that motility was substantially more sensitive to hyp otonic than to hypertonic conditions. Based on the experimental data, osmotic injury as a function of sperm volume excursion (swelling or sh rinking) was determined. The second step, using these sperm volume exc ursion limits and previously measured glycerol and water permeability coefficients of human spermatozoa, was to predict, by computer simulat ion, the cell osmotic injury caused by different procedures for the ad dition and removal of glycerol, The predicted sperm injury was confirm ed by experiment, Based on this study, an analytical methodology has b een developed for predicting optimal protocols to reduce osmotic injur y associated with the addition and removal of hypertonic concentration s of glycerol in human spermatozoa.