Gomes Claudia Messias, Silva Cristine Ane Silva E, Acevedo Nicole, Baracat Edmund, Serafini Paulo, Smith Gary D
Department of Obstetrics and Gynecology, University of Sao Paulo, Sao Paulo, Brazil.
Fertil Steril. 2008 Oct;90(4 Suppl):1396-404. doi: 10.1016/j.fertnstert.2007.08.025. Epub 2008 Mar 24.
To evaluate influences of vitrification and warming of metaphase II (MII) mouse oocytes on survival, spindle dynamics, spindle morphology, and chromatin alignment on metaphase plates.
Experimental animal study.
University animal laboratory.
ANIMAL(S): Eight-week-old B6D2F1 mice.
INTERVENTION(S): Denuded MII oocytes were used fresh (control), exposed to vitrification/warming solutions (Sol Expos), or vitrified and warmed (Vitr).
MAIN OUTCOME MEASURE(S): Oocyte recovery and survival after warming and the influence of solution exposure and cryopreservation on spindle dynamics and chromatin alignment.
RESULT(S): Cryopreservation of two or 10 oocytes per straw resulted in recovery (100% +/- 0% and 95% +/- 4%, respectively; mean +/- SE) and survival (95% +/- 2% and 98% +/- 2%, respectively). Immediately after warming (Vitr), significantly fewer oocytes assessed with immunocytochemistry contained spindles, compared with control and Sol Expos. When oocytes were placed into a 37 degrees C environment for 2 hours after exposure or warming, the ability to recognize spindles by immunocytochemistry was not significantly different between groups. Using live-cell time-lapse imaging with LC-Polscope, similar time-dependent spindle formation dynamics were observed. At 2 hours after collection or treatment, spindle morphology and length were not significantly different between the groups, nor was the incidence of aberrant alignment of chromatin on metaphase plates.
CONCLUSION(S): Immediately after warming of vitrified MII oocytes, beta-tubulin is depolymerized and chromatin remains condensed on the metaphase plate. Within a 2-hour period, beta-tubulin repolymerizes, forming morphologically normal metaphase spindles with properly aligned chromatin.
评估中期II（MII）期小鼠卵母细胞玻璃化和复温对其存活、纺锤体动力学、纺锤体形态以及中期板上染色质排列的影响。
实验动物研究。
大学动物实验室。
8周龄的B6D2F1小鼠。
去除卵丘细胞的MII期卵母细胞分为新鲜使用（对照组）、暴露于玻璃化/复温溶液（溶液暴露组）、玻璃化并复温（玻璃化组）。
复温后的卵母细胞回收率和存活率，以及溶液暴露和冷冻保存对纺锤体动力学和染色质排列的影响。
每管冷冻保存2个或10个卵母细胞后，回收率分别为100%±0%和95%±4%（平均值±标准误），存活率分别为95%±2%和98%±2%。与对照组和溶液暴露组相比，玻璃化组复温后立即通过免疫细胞化学评估发现含有纺锤体的卵母细胞明显减少。当卵母细胞在暴露或复温后置于37℃环境中2小时，各组间通过免疫细胞化学识别纺锤体的能力无显著差异。使用带有LC-Polscope的活细胞延时成像观察到，纺锤体形成动力学在时间上相似。在收集或处理后2小时，各组间纺锤体形态和长度无显著差异，中期板上染色质异常排列的发生率也无显著差异。
玻璃化的MII期卵母细胞复温后立即出现β微管蛋白解聚，染色质在中期板上仍保持凝聚状态。在2小时内，β微管蛋白重新聚合，形成形态正常且染色质排列正确的中期纺锤体。
