Kinetics and activation energy of recrystallization of intracellular ice in mouse oocytes subjected to interrupted rapid cooling

Abstract

Intracellular ice formation (IIF) is almost invariably lethal. In most cases, it results from the too rapid cooling of cells to below -40 degrees C, but in some cases it is manifested, not during cooling, but during warming when cell water that vitrified during cooling first devitrifies and then recrystallizes during warming. Recently, Mazur et al. [P. Mazur, I.L. Pinn, F.W. Kleinhans, Intracellular ice formation in mouse oocytes subjected to interrupted rapid cooling, Cryobiology 55 (2007) 158-166] dealt with one such case in mouse oocytes. It involved rapidly cooling the oocytes to -25 degrees C, holding them 10 min, rapidly cooling them to -70 degrees C, and warming them slowly until thawed. No IIF occurred during cooling but intracellular freezing, as evidenced by blackening of the cells, became detectable at -56 degrees C during warming and was complete by -46 degrees C. The present study differs in that the oocytes were warmed rapidly from -70 degrees C to temperatures between -65 and -50 degrees C and held for 3-60 min. This permitted us to determine the rate of blackening as function of temperature. That in turn allowed us to calculate the activation energy (E(a)) for the blackening process; namely, 27.5 kcal/mol. This translates to about a quadrupling of the blackening rate for every 5 degrees C rise in temperature. These data then allowed us to compute the degree of blackening as a function of temperature for oocytes warmed at rates ranging from 10 to 10,000 degrees C/min. A 10-fold increase in warming rate increased the temperature at which a given degree of blackening occurred by 8 degrees C. These findings have significant implications both for cryobiology and cryo-electron microscopy.

Fig. 1

Experimental temperature/time protocols for mouse oocytes in 1.5 M EG/PBS. The numbers 1, 2, 3, 4, 5, 6, and 7 refer to the Linkam cryostage temperature/time ramps described in Table 1. This schematic illustrates a hold for 10 min at −60°C.

Fig. 2

A sequence of photomicrographs of Class 4 mouse oocytes cooled to −70°C and then warmed to −62.5°C and held for 24 min (C–H). They were then warmed and thawed (I [taken at −46°C], J [taken at + 20°C]). The oocytes were suspended in 1.5 M EG/PBS and subjected to the ramps described in Table 1 and Fig. 1. The normal oocyte (A) is 75 μm in diameter.

Fig. 3

(A) The mean degree of blackening of mouse oocytes vs the time they were held at the indicated temperatures after warming from −70°C. Table 2 gives the numbers of ooctyes in each curve. (B) An example of three individual runs at holding temperatures of −60, −62.5, and −65°C illustrating how the slopes of the curves (darkening rates) were calculated.

Fig. 4

Arrhenius plot of the logarithm of the mean rate of darkening of mouse oocytes vs. the reciprocal of the absolute temperature of the hold. The hold temperatures from left to right were −55, −57.5, −60, −62.5, −65, and −70°C. The error bars are standard errors (standard deviations of the mean). The darkening rate for −50°C (1/T = 0.00448) has not been plotted for reasons given in the text. The log_e rate for it was 1.618.

Fig. 5

Degree of blackening (from 0 to 3) as a function of temperature for oocytes warmed at indicated rates from −70°C. The lines are values computed from the measured rates of blackening at various temperatures, the computed activation energy for blackening, and the algorithm described in the text. The closed circles are data obtained in [13] for twelve Class 4 oocytes warmed at 10°C/min. The open circles are data obtained in the present study for six Class 4 oocytes warmed at 100°C/min. The experimental oocytes were suspended in 1.5 M EG/PBS, cooled rapidly to −25°C, held 10 or 12 min, cooled rapidly to −70°C, and then warmed at 10° or 100°C/min to + 20°C. The plots were computed using a temperature increment of 0.1°C in our BASIC program (See text). These plots are indistinguishable from those using a temperature increment of 0.01°C.