Noiles E E, Thompson K A, Storey B T
Center for Research on Reproduction and Women's Health, University of Pennsylvania Medical Center, Philadelphia 19104, USA.
Cryobiology. 1997 Aug;35(1):79-92. doi: 10.1006/cryo.1997.2033.
Two parameters fundamental to cell cryobiology are the water permeability (hydraulic conductivity), Lp, and its activation energy, EA. The Lp can be calculated from two experimental determinations: the critical osmolality, Osmcrit, at which 50% of the cells lyse, and the time, tcrit, to 50% lysis in a highly hyposmotic medium, based on the assumption that the cells swell to lysis with minimal resistance to swelling. We have reported [Cryobiology 32, 220-238 (1995)] that mouse sperm in hyposmotic medium show minimal swelling and so fail to meet this assumption. The concept that resistance to swelling was due to anchoring of the plasma membrane through cytoskeletal interaction was examined by treating mouse sperm with 5 microM cytochalasin D to depolymerize the cytoskeletal filamentous actin (f-actin), whose presence was established by staining with fluorescently labeled phalloidin. Diminution of fluorescence due to loss of f-actin induced by cytochalasin D was shown by flow cytometry. Mouse sperm treated with cytochalasin D showed tail curling in hyposmotic medium, similar to that observed with bovine and human sperm, indicating that the standard swelling model was applicable to these cells. Two sets of Lp values were calculated from tcrit: one using individual means of Osmcrit and one using the mean of means of Osmcrit between 37 and 4 degrees C, as these individual means were not significantly different. Values (micron.min-1.atm-1), respectively, were 9.95, 7.15 (37 degrees C); 1.51, 0.91 (22 degrees C); 0.54, 0.78 (12 degrees C); 0.47, 0.50 (4 degrees C); 0.33 (0 degree C); and 0.36 (-3 degrees C). Arrhenius plots gave EA = 13.7 and 11.7 kcal/mol, respectively. Values of t1/2 were calculated from the first-order rate constants characterizing the kinetics of cell lysis at the higher four temperatures; Lp values calculated from these, and the two sets of Osmcrit values described were 5.70, 4.09 (37 degrees C); 1.18, 0.71 (22 degrees C); 0.62, 0.90 (12 degrees C); and 0.34, 0.37 (4 degrees C). Arrhenius plots gave EA = 14.2 and 11.0 kcal/mol, respectively. We propose that these EA values are characteristic of the plasma membrane relatively unperturbed by cytoskeletal interactions. In untreated sperm, decrease of Lp with decreasing temperature and presence of cryoprotectant and the cytoskeletal interactions all act to hamper the sperm cells' ability to respond to osmotic stress encountered during freezing and thawing, such that these cells are especially sensitive to cryodamage.
细胞低温生物学的两个基本参数是水渗透性（水力传导率）Lp及其活化能EA。Lp可以通过两项实验测定来计算：临界渗透压Osmcrit，即50%的细胞发生裂解时的渗透压；以及在高度低渗介质中达到50%裂解所需的时间tcrit，前提是假设细胞以最小的膨胀阻力膨胀至裂解。我们曾报道过[《低温生物学》32卷，220 - 238页（1995年）]，处于低渗介质中的小鼠精子膨胀极小，因此不符合这一假设。通过用5微摩尔的细胞松弛素D处理小鼠精子以使细胞骨架丝状肌动蛋白（f - 肌动蛋白）解聚，来检验膨胀阻力是由于质膜通过细胞骨架相互作用而锚定的这一概念，f - 肌动蛋白的存在通过用荧光标记的鬼笔环肽染色得以证实。通过流式细胞术显示了由于细胞松弛素D诱导f - 肌动蛋白丢失导致的荧光减弱。用细胞松弛素D处理的小鼠精子在低渗介质中表现出尾部卷曲，类似于在牛和人类精子中观察到的情况，这表明标准膨胀模型适用于这些细胞。根据tcrit计算了两组Lp值：一组使用Osmcrit的个体均值，另一组使用37℃和4℃之间Osmcrit均值的平均值，因为这些个体均值没有显著差异。相应的值（微米·分钟⁻¹·大气压⁻¹）分别为：9.95，7.15（37℃）；1.51，0.91（22℃）；0.54，0.78（12℃）；0.47，0.50（4℃）；0.33（0℃）；以及0.36（-3℃）。阿累尼乌斯图给出的EA分别为13.7和11.7千卡/摩尔。根据表征较高四个温度下细胞裂解动力学的一级速率常数计算了t1/2的值；根据这些计算出的Lp值以及所描述的两组Osmcrit值分别为：5.70，4.09（37℃）；1.18，0.71（22℃）；0.62，0.90（12℃）；以及0.34，0.37（4℃）。阿累尼乌斯图给出的EA分别为14.2和
