Phase transition temperature and chilling sensitivity of bovine oocytes
Phase transition temperature and chilling sensitivity of bovine oocytes
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DOI：
10.1006/cryo.1996.0062
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发表时间：
1996-12-01
期刊：
影响因子：
2.7
通讯作者：
Crowe, JH
中科院分区：
文献类型：
--
作者：
Arav, A;Zeron, Y;Crowe, JH
实现卵母细胞冷冻保存的限制因素是在冷却期间发生的直接冷害（DCI）。DCI，或冷休克，被定义为暴露于低温（但不是冷冻）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，DCI在精子和热致相变的膜脂之间的关联被证明在本研究中，我们研究了在冷却过程中的未成熟和体外成熟的牛卵母细胞的膜脂的相变，用傅立叶变换红外光谱（FTIR）。在萌发囊泡（GV）阶段卵母细胞膜脂的相变发生在13和20 ℃之间，而在成熟卵母细胞（MII）阶段观察到一个非常广泛的相变，其中心在10 ℃左右。热致相变被证明是相关的温度下，DCI影响卵母细胞膜的完整性。当未成熟的卵母细胞被冷却到13摄氏度时，较少的卵母细胞（40%）保持其膜完整性比暴露于4摄氏度（51%）或将它们保持在38摄氏度（78%）后，（如通过二乙酸黄绿素-FDA测试所确定的）。这一发现可能表明，将未成熟的卵母细胞保持在相变温度下比暴露在较低的温度下对其膜的损害更大。相比之下，没有显着差异，膜完整性观察时，在体外成熟的卵母细胞冷却到相同的温度。随后，GV卵母细胞被冷却到4 ℃，26%的卵母细胞进行了体外成熟，19%的卵母细胞进行了体外受精。在体外成熟的卵母细胞，冷却到4摄氏度显示受精率略有下降;总体受精率为60%，多精受精率为24%，而不是76%的受精率，多精受精率为12%。多精受精的高比率表明在牛卵母细胞的冷却过程中，质膜以外的部位受到影响。将有核牛GV卵母细胞与体外成熟和去核的卵母细胞电融合，然后冷却至4 ℃。融合卵母细胞的膜完整性的评估表明，这些卵母细胞是耐冷的，这强烈表明，卵母细胞的膜组成的改变可以改变细胞的低温敏感性。这一发现导致了冷冻保存后卵母细胞存活率的提高。(C)出版社：Academic Press，Inc.
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 vitro-matured 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. (C) 1996 Academic Press, Inc.