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 limited by a lack of knowledge regarding the water permeability characteristics 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 and their small dimensions which are at the limits of light microscopic resolution. Using a new shape-independent differential scanning calorimeter (DSC) technique, volumetric shrinkage during freezing of human sperm cell suspensions was obtained at cooling rates of 5 and 10°C/min in the presence of extracellular ice and CPA. Using previously published data, the human sperm cell was modelled as a cylinder of length 40.2 μm and a radius of 0.42 μm with an osmotically inactive cell volume, V(b), of 0.23V(o), where V(o) is the isotonic cell volume. By fitting a model of water transport to the experimentally obtained volumetric shrinkage data, the best fit membrane permeability parameters (L(pg) and E(Lp)) were determined. The 'combined best fit' membrane permeability parameters at 5 and 10°C/min for human sperm cells in modified media are: L(pg) = 2.4 x 10^-14 m³/Ns (0.14 μm/min-atm) and E(Lp) = 357.7 kJ/mol (85.5 kcal/mol) (R² = 0.98), and in CPA media (with 6% glycerol and 10% egg yolk) are L(pg)[cpa] = 0.67 x 10^-14 m³/Ns (0.04 μm/min-atm) and E(Lp)[cpa] = 138.9 kJ/mol (33.2 kcal/mol) (R² = 0.98). These parameters are significantly different from previously published parameters for human spermatozoa obtained at suprazero temperatures and at subzero temperatures in the absence of extracellular ice. The parameters obtained in this study also suggest that damaging intracellular ice formation (IIF) could occur in human sperm cells at cooling rates as low as 25-45°C/min, depending on the concentrations of the CPA. This may help to explain the discrepancy between the empirically determined optimal cryopreservation cooling rates (<100°C/min) and the numerically predicted optimal cooling rates (>7000°C/min) obtained using previously published suprazero human sperm permeability parameters which do not account for the presence of extracellular ice.