Effect of Cryoprotectant Solutes on Water Permeability of Human Spermatozoa1

Abstract

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.

An improved cryopreservation method for a mouse embryonic stem cell line

“…V b was determined to be 49.8% of isosmotic cell volume. This value is comparable to the V b of human spermatozoa (50% of isosmotic cell volume) [54] and human red blood cells (43% of isosmotic cell volume) [55], and is notably higher than the range of V b measured for other stem cells, including human umbilical cord blood CD34+ cells (27% and 32% of their respective isosmotic cell volumes) [13;56], and human bone marrowderived hematopoietic progenitor cells (20.5% of isosmotic cell volume) [57].…”

Aquaporins in spermatozoa and testicular germ cells: identification and potential role

“…When subjected to extreme ranges of osmolality (70-1 500 mmol kg −1 ) , they shrink or swell rapidly, behaving almost like perfect osmometers (human [3,4]; boar [5,6]; mouse [7,8]), enabling the hypo-osmotic swelling test to be used as a simple clinical test for sperm membrane integrity, which is manifested in the swollen shapes and thus reflects cell viability [9]. Nonselective water permeability through plasma membranes is very low, as water molecule is polar and diffusion of polar compounds is hindered by lipid bilayers.…”

Prevention of Lethal Osmotic Injury to Cells During Addition and Removal of Cryoprotective Agents: Theory and Technology

“…However, more recent literature suggests that aquaporins in cell membrane are highly selective, with nonionic solute transport occurring mainly through the lipid bilayer or through other channels that are distinct from the aquaporins (Gilmore et al, 1995;Preston et al, 1992). In this case, the estimation of σ as independent parameter may be inappropriate and may not be relevant from a biological stand point (Kleinhans, 1998).…”