Andrology: Prevention of osmotic injury to human spermatozoa during addition and removal of glycerol |
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| Authors: | Gao DY; Liu J; Liu C; McGann LE; Watson PF; Kleinhans FW; Mazur P; Critser ES; Critser JK |
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| Institution: | 1Cryobiology Research Institute, Methodist Hospital of Indiana Indianapolis, IN 46202
2Department of Physics, Indiana University - Purdue University at Indianapolis IN 46202, USA
3Department of Pathology, University of Alberta Edmonton, Alberta, Canada T6G 2R8
4Department of Veterinary Basic Sciences, The Royal Veterinary College London, NW 1 OTU, UK
5Biology Division, Oak Ridge National Laboratory Oak Ridge, TN 37831
6Departments of Physiology and Biophysics and Obstetrics and Gynecology, Indiana University School of Medicine Indianapolis, IN 46202, USA |
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| Abstract: | Use of a cryoprotective agent is indispensable to prevent injuryto human spermatozoa during the cryopreservation process. However,addition of cryoprotective agents to spermatozoa before coolingand their removal after warming may create severe osmotic stressfor the cells, resulting in injury. The objective of this studywas to test the hypothesis that the degree (or magnitude) ofhuman sperm volume excursion can be used as an independent indicatorto evaluate and predict possible osmotic injury to spermatozoaduring the addition and removal of cryoprotective agents. Glycerolwas used as a model cryoprotective agent in the present study.To test this hypothesis, first the tolerance limits of spermatozoato swelling in hypoosmotic solutions (iso-osmotic medium dilutedwith water) and to shrinkage in hyperosmotic solutions (iso-osmoticmedium with sucrose) were determined. Sperm plasma membraneintegrity was measured by fluorescent staining, and sperm motilitywas assessed by computer-assisted semen analysis before, duringand after the anisosmotic exposure. The results indicate firstlythat motility was much more sensitive to anisosmotic conditionsthan membrane integrity, and secondly that motility was substantiallymore sensitive to hypotonic than to hypertonic conditions. Basedon the experimental data, osmotic injury as a function of spermvolume excursion (swelling or shrinking) was determined. Thesecond step, using these sperm volume excursion limits and previouslymeasured glycerol and water permeability coefficients of humanspermatozoa, was to predict, by computer simulation, the cellosmotic injury caused by different procedures for the additionand removal of glycerol. The predicted sperm injury was confirmedby experiment. Based on this study, an analytical methodologyhas been developed for predicting optimal protocols to reduceosmotic injury associated with the addition and removal of hypertonicconcentrations of glycerol in human spermatozoa. |
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| Keywords: | glycerol/human spermatozoa/osmotic injury |
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