Effect of cryoprotectant solutes on water permeability of human spermatozoa

Osmotic permeability characteristics and the effects of cryoprotectants are important determinants of recovery and function of spermatozoa after cryopreservation. The primary purpose of this study was to determine the osmotic permeability parameters of human spermatozoa in the presence of cryoprotectants. A series of experiments was done to: 1) validate the use of an electronic particle counter for determining both static and kinetic changes in sperm cell volume; 2) determine the permeability of the cells to various cryoprotectants; and 3) test the hypothesis that human sperm water permeability is affected by the presence of cryoprotectant solutes. The isosmotic volume of human sperm was 28.2 +/- 0.2 microns3 (mean +/- SEM), 29.0 +/- 0.3 microns3, and 28.2 +/- 0.4 microns3 at 22, 11, and 0 degrees C, respectively, measured at 285 mOsm/kg via an electronic particle counter. The osmotically inactive fraction of human sperm was determined from Boyle van't Hoff (BVH) plots of samples exposed to four different osmolalities (900, 600, 285, and 145 mOsm/kg). Over this range, cells behaved as linear osmometers with osmotically inactive cell percentages at 22, 11, and 0 degrees C of 50 +/- 1%, 41 +/- 2%, and 52 +/- 3%, respectively. Permeability of human sperm to water was determined from the kinetics of volume change in a hyposmotic solution (145 mOsm/kg) at the three experimental temperatures. The hydraulic conductivity (Lp) was 1.84 +/- 0.06 microns.min-1.atm-1, 1.45 +/- 0.04 microns.min-1.atm-1, and 1.14 +/- 0.07 microns.min-1.atm-1 at 22, 11, and 0 degrees C, respectively, yielding an Arrhenius activation energy (Ea) of 3.48 kcal/mol. These biophysical characteristics of human spermatozoa are consistent with findings in previous reports, validating the use of an electronic particle counter for determining osmotic permeability parameters of human sperm. This validated system was then used to investigate the permeability of human sperm to four different cryoprotectant solutes, i.e., glycerol (Gly), dimethylsulfoxide (DMSO), propylene glycol (PG), and ethylene glycol (EG), and their effects on water permeability. A preloaded, osmotically equilibrated cell suspension was returned to an isosmotic medium while cell volume was measured over time. A Kedem-Katchalsky model was used to determine the permeability of the cells to each solute and the resulting water permeability. The permeabilities of human sperm at 22 degrees C to Gly, DMSO, PG, and EG were 2.07 +/- 0.13 x 10(-3) cm/min, 0.80 +/- 0.02 x 10(-3) cm/min, 2.3 +/- 0.1 x 10(-3) cm/min, and 7.94 +/- 0.67 x 10(-3) cm/min, respectively. The resulting Lp values at 22 degrees C were reduced to 0.77 +/- 0.08 micron.min-1.atm-1, 0.84 +/- 0.07 micron.min-1.atm-1, 1.23 +/- 0.09 microns.min-1.atm-1, and 0.74 +/- 0.06 micron.min-1.atm-1, respectively. These data support the hypothesis that low-molecular-weight, nonionic cryoprotectant solutes affect (decrease) human sperm water permeability.