Is intracellular ice formation the cause of death of mouse sperm frozen at high cooling rates?

Mouse spermatozoa in 18% raffinose and 3.8% Oxyrase in 0.25 x PBS exhibit high motilities when frozen to -70 degrees C at 20-130 degrees C/min and then rapidly warmed. However, survival is <10% when they are frozen at 260 or 530 degrees C/min, presumably because, at those high rates, intracellular water cannot leave rapidly enough to prevent extensive supercooling and this supercooling leads to nucleation and freezing in situ (intracellular ice formation [IIF]). The probability of IIF as a function of cooling rate can be computed by coupled differential equations that describe the extent of the loss of cell water during freezing and from knowledge of the temperature at which the supercooled protoplasm of the cell can nucleate. Calculation of the kinetics of dehydration requires values for the hydraulic conductivity (Lp) of the cell and for its activation energy (Ea). Using literature values for these parameters in mouse sperm, we calculated curves of water volume versus temperature for four cooling rates between 250 and 2000 degrees C/min. The intracellular nucleation temperature was inferred to be -20 degrees C or above based on the greatly reduced motilities of sperm that underwent rapid cooling to a minimum temperature of between -20 and -70 degrees C. Combining that information regarding nucleation temperature with the computed dehydration curves leads to the conclusion that intracellular freezing should occur only in cells that are cooled at 2000 degrees C/min and not in cells that are cooled at 250-1000 degrees C/min. The calculated rate of 2000 degrees C/min for IIF is approximately eightfold higher than the experimentally inferred value of 260 degrees C/min. Possible reasons for the discrepancy are discussed.