Arav A, Zeron Y, Leslie S B, Behboodi E, Anderson G B, Crowe J H
Animal Science, Agriculture Research Organization (ARO), Bet Dagan, Israel.
Cryobiology. 1996 Dec;33(6):589-99. doi: 10.1006/cryo.1996.0062.
A limiting factor for achieving cryopreservation of oocytes is direct chilling injury (DCI), which occurs during cooling. DCI, or cold shock, is defined as an irreversible damage expressed shortly after exposure to low, but not freezing, temperatures. The primary target of DCI is thought to be the plasma membrane. Recently, an association between DCI in sperm and the thermotropic phase transition of their membrane lipids was demonstrated. In the present study, we examined the phase transition of the membrane lipids of immature and in vitro-matured bovine oocytes during cooling, using Fourier transform infrared spectroscopy (FTIR). The phase transition of the membrane lipids of oocytes at the germinal vesicle (GV) stage occurred between 13 and 20 degrees C, while a very broad phase transition, which centered around 10 degrees C, was observed for mature oocytes (MII) stage. Thermotropic phase transitions were demonstrated to be related to the temperature at which DCI affected the integrity of the oocyte membranes. When immature oocytes were cooled to 13 degrees C, fewer oocytes (40%) retained their membrane integrity than after exposure to 4 degrees C (51%) or holding them at 38 degrees C (78%), (as determined by the Fluorescein Diacetate-FDA test). This finding might suggest that holding immature oocytes at the phase transition temperature is more damaging to their membranes than exposure to lower temperatures. By contrast, no significant differences in membrane integrity were observed when in vitro-matured oocytes were cooled to the same temperatures. Subsequently, GV oocytes were cooled to 4 degrees C, and 26% underwent maturation and 19% underwent fertilization in vitro. In vitromatured oocytes that were cooled to 4 degrees C displayed a slightly decreased rate of fertilization; the overall fertilization was 60% with 24% polyspermy, rather than the 76% fertilization rate with 12% polyspermy obtained with those not subjected to cooling. The high rate of polyspermy indicates that a site(s) other than the plasma membrane is affected during cooling of bovine oocytes. Nucleated bovine GV oocytes were electrofused with in vitro-matured and enucleated oocytes, and then cooled to 4 degrees C. Evaluation of the membrane integrity of the fused oocytes showed that these oocytes are chilling resistant, which strongly suggests that alteration of the membrane composition of an oocyte can change the cell's susceptibility to low temperatures. This finding led to an improvement in the survival of oocytes after cryopreservation.
实现卵母细胞冷冻保存的一个限制因素是直接冷损伤（DCI），它发生在冷却过程中。DCI，即冷休克，被定义为在暴露于低温但非冷冻温度后不久表现出的不可逆损伤。DCI的主要靶点被认为是质膜。最近，精子中的DCI与其膜脂的热致相变之间的关联得到了证实。在本研究中，我们使用傅里叶变换红外光谱（FTIR）研究了未成熟和体外成熟牛卵母细胞在冷却过程中膜脂的相变。生发泡（GV）期卵母细胞的膜脂相变发生在13至20摄氏度之间，而成熟卵母细胞（MII）期则观察到一个非常宽泛的相变，其中心温度约为10摄氏度。热致相变被证明与DCI影响卵母细胞膜完整性的温度有关。当未成熟卵母细胞冷却至13摄氏度时，与暴露于4摄氏度（51%）或维持在38摄氏度（78%）相比，保留其膜完整性的卵母细胞更少（40%）（通过荧光素二乙酸酯 - FDA试验确定）。这一发现可能表明，将未成熟卵母细胞维持在相变温度对其膜的损伤比暴露于更低温度更大。相比之下，当体外成熟卵母细胞冷却至相同温度时，未观察到膜完整性的显著差异。随后，GV期卵母细胞冷却至4摄氏度，26%进行了成熟，19%进行了体外受精。冷却至4摄氏度的体外成熟卵母细胞的受精率略有下降；总体受精率为60%，多精受精率为24%，而未经过冷却的卵母细胞的受精率为76%，多精受精率为12%。高多精受精率表明，在牛卵母细胞冷却过程中，质膜以外的某个部位受到了影响。将有核的牛GV期卵母细胞与体外成熟并去核的卵母细胞进行电融合，然后冷却至4摄氏度。对融合卵母细胞膜完整性的评估表明，这些卵母细胞具有抗冷性，这强烈表明卵母细胞膜组成的改变可以改变细胞对低温的敏感性。这一发现提高了冷冻保存后卵母细胞的存活率。
