Characteristics and kinetics of subzero chilling injury in Drosophila embryos.

Abstract Drosophila embryos manifest unusually high sensitivity to chilling in that they are killed with increased rapidity by exposure to temperatures between 0 and −25 °C in the absence of ice formation. Thus, 50% of 15-h eggs succumb in 35, 4, and 1 h at 0, −9, and −15 °C, respectively. The sensitivity becomes substantially greater in embryos at stages of development earlier than 12 h, especially at 3 and 6 h. The killing kinetics at given subzero temperatures between 0 and −25 °C are characterized by a shoulder followed by a more-or-less linear decrease in survival with time. The lower the temperature, the shorter the shoulder and the faster the postshoulder decline. The rate of both components follows Arrhenius kinetics, i.e., plots of log rate vs 1/absolute temperature are linear, the slopes being proportional to the activation energy. In both cases the activation energy is high and negative; namely, −46.5 kcal/mol for the shoulder length and −24.7 kcal/mol for the postshoulder inactivation. Negative activation energies are unusual, and according to absolute reaction rate theory, they exist only when the entropy of activation is negative, which suggests that the activated state is more ordered. By combining the duration of the shoulder as a function of time and temperature with the rate of postshoulder inactivation, one can compute survival as a function of temperature for embryos cooled at various rates. For those cooled at ⩽ 1 °C/min, the computed curve of survival vs temperature agrees closely with observed survivals. But for embryos cooled at ~10 °C/min, the drop in survival occurs some 7 to 10 ° above that computed. Embryos exposed to 0 °C for >5 min undergo conditioning that renders them more resistant to subsequent exposure to lower temperatures, and those cooled at 10 °C/min presumably lack sufficient time at 0 °C to undergo such conditioning; hence the discrepancy between observed and computed survivals. As a test of the possibility that chilling injury is a consequence of the loss of synchrony of coupled reactions involved in embryological development, embryos were rendered anoxic prior to chilling, a treatment that has been shown by Foe and Alberts to reversibly halt development of early stages. Although anoxia somewhat reduced chilling injury in 6-h eggs, it had no effect on 15-h eggs. The sensitivity of embryos to chilling injury below −25 °C cannot be experimentally assessed because differential thermal analysis shows that intact eggs freeze between −27 and −32 °C with lethal consequences. But death from chilling occurs so rapidly between −15 and −25 °C that the use of orthodox slow freezing procedures to remove intracellular water prior to entering the intraembryonic nucleation zone will not succeed; the embryos will succumb to chilling even before they have reached that zone. Unless ways can be found to reduce the accelerating rate of chilling injury as cooling progresses, the only solution is to “outrace” it by cooling at high rates and hope that intraembryonic ice formation can be avoided by the introduction of solutes that enhance vitrification.