Is Intracellular Ice Formation the Cause of Death of Mouse Sperm Frozen at High Cooling Rates?1

Abstract

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.

Production of F1Offspring with Vitrified Sperm from a Live-Bearing Fish, the Green SwordtailXiphophorus hellerii

“…While some studies suggest that at cooling rates as high as 3000°C/min there is no formation of intracellular ice, 61,62 other studies suggest that intracellular ice starts forming at a cooling rate of 2000°C/min but not in cells that are cooled at 250°C-1000°C/min. 63 The intracellular nucleation temperature inferred by DSC for X. hellerii was -30°C. 58 In a similar live-bearing fish (Poecilia reticulata) the nucleation temperature was calculated to be in the range of -25°C to -32°C at cooling rates from 5°C/min to 100°C/ min.…”

Kinetic Vitrification of Spermatozoa of Vertebrates: What Can We Learn from Nature?

“…Table 1 represents just two examples when the Isachenkos published their paper, and other cryobiologists, who came to the same conclusions, namely: -There is no proof of the absence of vitrification inside the sperm even at quite slow cooling [Morris, 2006]; the role of intracellular ice in the death of fast cooling mouse sperm is also questioned in [Mazur & Koshimoto, 2002]. -Some cells can be vitrified in "diluted" solutions at relatively slow rate of cooling but very fast warming is essential for kinetic VF [Mazur & Seki, 2011] Thus, both cryobiologists have reported similar findings as the Isachenkos observations, but they unfortunately fell short of mentioning Isachenkos in their own publications and presentations (e.g., in Cryo-2010 in Bristol), which might have made looking their observations (that were solid, of course) for an unfamiliar reader as "pioneering" or even as "a new paradigm for cryopreservation by vitrifcation" [Mazur & Seki, 2011] ].…”

“…2) acting as an osmotic buffer that prevents excessive shrinkage and other "solute effects" [Lovelock, 1953;Mazur, 1970Mazur, , 1984Mazur & Koshimoto, 2002]. The effect of protective action of the CPA is much more pronounced for larger bone marrow cells while small erythrocytes perfectly survive the absence of CPA if cooled fast enough.…”

Numerical Prediction of the Intracellular ICE Formation Zone during Cryosurgery on a Nodular Basal Cell Carcinoma Using Liquid Nitrogen Spray

“…The present study defines the lowest CR that can trigger IIF as the critical cooling rate (CCR). For the time being, it is reasonable to assume a CCR of 17°C s -1 [192] for BCC and dermal cells. Using information on the temperature distribution and the local CR at t = 3 s, one can determine where IIF occurs.…”