Koshimoto Chihiro, Mazur Peter
Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville, TN 37392-2575, USA.
Cryobiology. 2002 Aug;45(1):49-59. doi: 10.1016/s0011-2240(02)00105-0.
We have recently reported that the survival of mouse spermatozoa is decreased when they are warmed at a suboptimal rate after being frozen at an optimal rate. We proposed that this drop in survival is caused by physical damage derived from the recrystallization of extracellular ice during slow warming. The first purpose of the present study was to determine the temperatures over which the decline in survival occurs during slow warming and the kinetics of the decline at fixed subzero temperatures. The second purpose was to examine the effects of antifreeze proteins (AFP) on the survival of slowly warmed mouse spermatozoa, the rationale being that AFP have the property of inhibiting ice recrystallization. With respect to the first point, a substantial loss in motility occurred when slow warming was continued to higher than -50 degrees C and the survival of the sperm decreased with an increase in the temperature at which slow warming was terminated. In contrast, the motility of sperm that were warmed rapidly to these temperatures remained high initially but dropped with increased holding time. At -30 degrees C, most of the drop occurred in 5 min. These results are consistent with the hypothesis that damage develops as a consequence of the recrystallization of the external ice. AFP ought to inhibit such recrystallization, but we found that the addition of AFP-I, AFP-III, and an antifreeze glycoprotein at concentrations of 1-100 microg/ml did not protect the frozen-thawed cells; rather it led to a decrease in survival that was proportional to the concentration. There was no decrease in survival from exposure to the AFP in the absence of freezing. AFP are known to produce changes in the structure and habit of ice crystals, and some have reported deleterious consequences associated with those structural changes. We suggest that such changes may be the basis of the adverse effects of AFP on the survival of the sperm, especially since mouse sperm are exquisitely sensitive to a variety of mechanical stresses.
我们最近报道，小鼠精子在以最佳速率冷冻后若以次优速率升温，其存活率会降低。我们提出，这种存活率下降是由慢速升温过程中细胞外冰重结晶导致的物理损伤引起的。本研究的首要目的是确定慢速升温过程中存活率下降所发生的温度范围以及在固定零下温度下下降的动力学。第二个目的是研究抗冻蛋白（AFP）对慢速升温的小鼠精子存活率的影响，其基本原理是AFP具有抑制冰重结晶的特性。关于第一点，当慢速升温持续到高于 -50℃时，精子活力出现大幅下降，并且精子存活率随着慢速升温终止温度的升高而降低。相比之下，快速升温至这些温度的精子最初活力仍然很高，但随着保持时间的增加而下降。在 -30℃时，大部分活力下降发生在5分钟内。这些结果与外部冰重结晶导致损伤的假设一致。AFP应该抑制这种重结晶，但我们发现添加浓度为1 - 100μg/ml的AFP-I、AFP-III和一种抗冻糖蛋白并不能保护冻融细胞；相反，它导致存活率下降，且下降程度与浓度成正比。在没有冷冻的情况下，暴露于AFP不会导致存活率下降。已知AFP会使冰晶的结构和习性发生变化，并且一些研究报道了与这些结构变化相关的有害后果。我们认为，这种变化可能是AFP对精子存活率产生不利影响的基础，特别是因为小鼠精子对各种机械应力极为敏感。
