Subzero water permeability parameters of mouse spermatozoa in the presenceof extracellular ice and cryoprotective agents

Optimization of techniques for cryopreservation of mammalian sperm is limit ed by a lack of knowledge regarding water permeability characteristics duri ng freezing in the presence of extracellular ice and cryoprotective agents (CPAs). Cryomicroscopy cannot be used to measure dehydration during freezin g in mammalian sperm because they are highly nonspherical and their small d imensions are at the limits of light microscopic resolution. Using a new sh ape-independent differential scanning calorimeter (DSC) technique, volumetr ic shrinkage during freezing of ICR mouse epididymal sperm cell suspensions was obtained at cooling rates of 5 and 20 degrees C/min in the presence of extracellular ice and CPAs. Using previously published data, the mouse spe rm cell was modeled as a cylinder (122-mu m long, radius 0.46 mu m) with an osmotically inactive cell volume (V-b) of 0.61 V-o, where V-o is the isoto nic cell volume. By fitting a model of water transport to the experimentall y obtained volumetric shrinkage data, the best-fit membrane permeability pa rameters (L-pg and E-Lp) were determined. The "combined best-fit" membrane permeability parameters at 5 and 20 degrees C/min for mouse sperm cells in solution are as follows: in D-PBS: L-pg = 1.7 x 10(-15) m(3)/Ns (0.01 mu m/ min-atm) and E-Lp = 94.1 kJ/mole (22.5 kcal/mole) (R-2 = 0.94); in "low" CP A media (consisting of 1% glycerol, 16% raffinose, and 15% egg yolk in D-PB S): L-pg[cpa] 1.7 x 10(-15) m(3)/Ns (0.01 mu m/min-atm) and E-Lp[cpa] = 122 .2 kJ/mole (29.2 kcal/mole) (R-2 = 0.98); and in "high" CPA media (consisti ng of 4% glycerol, 16% raffinose, and 15% egg yolk in D-PBS): L-pg[cpa] = 0 .68 x 10(-15) m(3)/Ns (0.004 mu m/min-atm) and E-Lp[cpa] = 63.6 kJ/mole (15 .2 kcal/mole) (R-2 = 0.99). These parameters are significantly different th an previously published parameters for mammalian sperm obtained at suprazer o temperatures and at subzero temperatures in the absence of extracellular ice. The parameters obtained in this study also suggest that damaging intra cellular ice formation (IIF) could occur in mouse sperm cells at cooling ra tes as low as 25-45 degrees C/min, depending on the concentrations of the C PAs. This may help to explain the discrepancy between the empirically deter mined optimal cryopreservation cooling rates, 10-40 degrees C/min, and the numerically predicted optimal cooling rates, greater than 5000 degrees C/mi n, obtained using suprazero mouse sperm permeability parameters that do not account for the presence of extracellular ice. As an independent test of t his prediction, the percentages of viable and motile sperm cells were obtai ned after freezing at two different cooling rates ("slow" or 5 degrees C/mi n; "fast," or 20 degrees C/min) in both the low and high CPA media. The gre atest sperm motility and viability was found with the low CPA media under f ast (20 degrees C/min) cooling conditions.