The effect of extracellular ice and cryoprotective agents on the water permeability parameters of human sperm plasma membrane during freezing

A firm biophysical basis for the cryopreservation of human spermatozoa is l imited by a lack of knowledge regarding the water permeability characterist ics during freezing in the presence of extracellular ice and cryoprotective agents (CPA). Cryomicroscopy cannot be used to measure dehydration during freezing in human spermatozoa because of their highly non-spherical shape a nd their small dimensions which are at the limits of light microscopic reso lution. Using a new shape-independent differential scanning calorimeter (DS C) technique, volumetric shrinkage during freezing of human sperm cell susp ensions was obtained at cooling rates of 5 and 10 degrees C/min in the pres ence of extracellular ice and CPA. Using previously published data, the hum an sperm cell was modelled as a cylinder of length 40.2 mu m and a radius o f 0.42 mu m with an osmotically inactive cell volume, V-b, of 0.23V(o), whe re V-o is the isotonic cell volume. By fitting a model of water transport t o the experimentally obtained volumetric shrinkage data, the best fit membr ane permeability parameters (L-pg and E-Lp) were determined. The 'combined best fit' membrane permeability parameters at 5 and 10 degrees C/min for hu man sperm cells in modified media are: L-pg = 2.4 x 10(-14) m(3)/Ns (0.14 m u m/min-atm) and E-Lp = 357.7 kJ/mol (85.5 kcal/mol) (R-2 = 0.98), and in C PA media (with 6% glycerol and 10% egg yolk) are L-pg[cpa] = 0.67x10(-14) m (3)/Ns (0.04 mu m/min-atm) and E-Lp[cpa] = 138.9 kJ/mol (33.2 kcal/mol) (R- 2 = 0.98). These parameters are significantly different from previously pub lished parameters for human spermatozoa obtained at suprazero temperatures and at subzero temperatures in the absence of extracellular ice. The parame ters obtained in this study also suggest that damaging intracellular ice fo rmation (IIF) could occur in human sperm cells at cooling rates as low as 2 5-45 degrees C/min, depending on the concentrations of the CPA. This may he lp to explain the discrepancy between the empirically determined optimal cr yopreservation cooling rates (<100 degrees C/min) and the numerically predi cted optimal cooling rates (>7000 degrees C/min) obtained using previously published suprazero human sperm permeability parameters which do not accoun t for the presence of extracellular ice.