EFFECT OF CRYOPROTECTANT SOLUTES ON WATER PERMEABILITY OF HUMAN SPERMATOZOA

Osmotic permeability characteristics and the effects of cryoprotectant s are important determinants of recovery and function of spermatozoa a fter cryopreservation. The primary purpose of this study was to determ ine the osmotic permeability parameters of human spermatozoa in the pr esence of cryoprotectants. A series of experiments was done to: 1) val idate the use of an electronic particle counter for determining both s tatic and kinetic changes in sperm cell volume; 2) determine the perme ability of the cells to various cryoprotectants; and 3) test the hypot hesis that human sperm water permeability is affected by the presence of cryoprotectant solutes. The isosmotic volume of human sperm was 28. 2 +/- 0.2 mu m(3) (mean +/- SEM), 29.0 +/- 0.3 mu m(3), and 28.2 +/- 0 .4 mu m(3) at 22, 11, and O degrees C, respectively, measured at 285 m Osm/kg via an electronic particle counter. The osmotically inactive fr action 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 wit h osmotically inactive cell percentages at 22, 11, and 0 degrees C of 50 +/- 1%, 41 +/- 2%, and 52 +/- 3%, respectively. Permeability of hum an sperm to water was determined from the kinetics of volume change in a hyposmotic solution [145 mOsm/kg] at the three experimental tempera tures. The hydraulic conductivity (L(p)) was 1.84 +/- 0.06 mu m . min( -1). atm(-1), 1.45 +/- 0.04 mu m . min(-1). atm(-1), and 1.14 +/- 0.07 mu m . min(-1). atm(-1) at 22, 11, and 0 degrees C, respectively, yie lding an Arrhenius activation energy (E(a)) of 3.48 kcal/mol. These bi ophysical characteristics of human spermatozoa are consistent with fin dings in previous reports, validating the use of an electronic particl e counter for determining osmotic permeability parameters of human spe rm. This validated system was then used to investigate the permeabilit y of human sperm to four different cryoprotectant solutes, i.e., glyce rol (Gly), dimethylsulfoxide (DMSO), propylene glycol (PG), and ethyle ne 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 mo del was used to determine the permeability of the cells to each solute and the resulting water permeability. The permeabilities of human spe rm 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 L(p) va lues at 22 degrees C were reduced to 0.77 +/- 0.08 mu m . min(-1). atm (-1), 0.84 +/- 0.07 mu m . min(-1). atm(-1), 1.23 +/- 0.09 mu m . min( -1). atm(-1), and 0.74 +/- 0.06 mu m . min(-1). atm(-1), respectively. These data support the hypothesis that low-molecular-weight, nonionic cryoprotectant solutes affect (decrease) human sperm water permeabili ty.