Mullen S F, Agca Y, Broermann D C, Jenkins C L, Johnson C A, Critser J K
Comparative Medicine Center and Department of Veterinary Pathobiology, University of Missouri at Columbia, Columbia MO 65211, USA.
Hum Reprod. 2004 May;19(5):1148-54. doi: 10.1093/humrep/deh201. Epub 2004 Apr 7.
Knowing osmotic tolerance limits is important in the design of optimal cryopreservation procedures for cells.
Mature human oocytes were exposed to anisosmotic sucrose solutions at concentrations of 35, 75, 150, 600, 1200, or 2400 (+/-5) milliosmolal (mOsm) at 37 degrees C. A control treatment at 290 mOsm was also utilized. Oocytes were randomly allocated to each experimental treatment. After the treatment, the oocytes were cultured for 1 h, then fixed in cold methanol. Immunocytochemical staining and fluorescence microscopy were used to assess the morphology of the metaphase II (MII) spindle. Logistic regression was used to determine if media osmolality had a significant effect on spindle structure.
Osmolality was a significant predictor of spindle morphology. Hyposmotic effects at 35, 75, and 150 mOsm resulted in 100, 67, and 56% of oocytes having abnormal spindles, respectively. Hyperosmotic effects at 600, 1200, and 2400 mOsm resulted in 44, 44, and 100% of the spindles with abnormal structure, respectively.
Anisosmotic conditions lead to disruption of the MII spindle in human oocytes. Applying this fundamental knowledge to human oocyte cryopreservation should result in increased numbers of cells maintaining viability.
了解渗透耐受极限对于设计细胞的最佳冷冻保存程序至关重要。
将成熟人类卵母细胞在37℃下暴露于浓度为35、75、150、600、1200或2400（±5）毫渗摩尔（mOsm）的非等渗蔗糖溶液中。还采用了290 mOsm的对照处理。卵母细胞被随机分配到每个实验处理组。处理后，将卵母细胞培养1小时，然后用冷甲醇固定。采用免疫细胞化学染色和荧光显微镜评估中期II（MII）纺锤体的形态。使用逻辑回归确定培养基渗透压是否对纺锤体结构有显著影响。
渗透压是纺锤体形态的显著预测指标。35、75和150 mOsm的低渗效应分别导致100%、67%和56%的卵母细胞纺锤体异常。600、1200和2400 mOsm的高渗效应分别导致44%、44%和100%的纺锤体结构异常。
非等渗条件会导致人类卵母细胞的MII纺锤体破坏。将这一基础知识应用于人类卵母细胞冷冻保存应能增加保持活力的细胞数量。
