玻璃化冷冻对牛卵母细胞钙库的影响机制及其调控

The vitrification of mamalian oocytes contributes greatly to the rapid development of embryo biotechnologies. Many experiments have shown that vitrification leads to the abnormal increase of calcium ion (Ca2+) concentration in vitrified oocytes, resulting in the pre-release of cortical granule, hardening of zona pellucida (ZP) and decreasing the fertilizing rate, which greatly limits the wide application of vitrified oocytes. However, the mechanism via which vitrification increases the Ca2+ concentration in oocytes still remains unclear, and much less about the method to positively regulate Ca2+concentration in vitrified oocytes. Therefore, the present study is designed to utilize bovine oocytes vitrified with Ca2+-free vitrification solution as the model to investigate the effect of vitrification on the main Ca2+ pool in oocytes, including endoplasmic reticulum and mitochodria. The regulation chemicals of intracellular Ca2+ (BAPTA-AM；ruthenium red, RR) are also used to positively regulate the Ca2+ concentration in vitrified bovine oocytes. The results of the present study will contribute to illustrating the mechanism via which vitrification increases the Ca2+ concentration in oocytes and providing the efficient approach to positively regulate the Ca2+ concentration in vitrified oocytes, which are supposed to increase the efficiency of in vitro fertilization and the developmental potential of vitrified oocytes.

卵母细胞的玻璃化冷冻保存对于促进胚胎生物技术的快速发展，具有重要的理论意义和应用价值。但是，玻璃化冷冻会引起卵母细胞中Ca2+浓度异常升高，导致皮质颗粒提前释放，透明带硬化，受精率降低，这已经成为影响冷冻卵母细胞应用潜力的重要因素。目前，玻璃化冷冻引起卵母细胞中Ca2+浓度升高的机制还不十分清楚，更缺乏能够正向调控冷冻卵母细胞中Ca2+浓度的有效措施。针对这一问题，本课题采用无Ca2+玻璃化冷冻液对牛MII期卵母细胞进行冷冻保存，从胞内钙库（内质网、线粒体）角度深入研究了玻璃化冷冻对卵母细胞中Ca2+浓度的影响机制，并采用Ca2+调控物质（BAPTA-AM、钌红（RR））正向调控冷冻牛卵母细胞中的Ca2+浓度，进而提高其受精和发育能力，这对于促进玻璃化冷冻卵母细胞的广泛应用具有十分重要的意义。

玻璃化冷冻能引起卵母细胞胞质Ca2+（[Ca2+]i）浓度异常升高，进而导致透明带硬化、受精率降低，这严重影响了玻璃化冷冻卵母细胞应用范围。然而，其相关机制还不十分清楚，更缺乏有效调控措施。针对该问题，本课题进行了深入研究，结果表明：（1）玻璃化冷冻促使ER Ca2 +释放到胞质中去，提高[Ca2+]i和mCa2 +水平，引起CG提前释放和线粒体功能损伤，降低冷冻卵母细胞精子结合、体外受精和发育能力。二甲基亚砜，是诱导ER Ca2 +释放到细胞质的重要因素。玻璃化冷冻会造成内质网和IP3R1分布异常，以及钙震荡次数明显减少，发生时间延迟，间隔时间、平均最高振幅均显著升高；（2）BAPTA处理能显著降低冷冻牛卵母细胞[Ca2+]i水平，能显著提高冷冻牛卵母细胞CGs环形分布和IVF时正常受精的比例，提高卵裂率；BAPTA处理能够提高冷冻卵母细胞中钙振荡发生次数，并且使钙振荡发生时间显著提前，而间隔时间、振幅显著缩短，对IP3R1和内质网无显著影响。（3）RR处理显著抑制[Ca2 +]i进入线粒体，维持mCa2 +水平和线粒体功能正常，RR处理对冷冻卵母细胞钙振荡事件、IP3R1和内质网分布、IVF卵裂率无显著影响。BAPT和RR联合处理，可显著提高IVF后卵裂率和囊胚率；（4）褪黑素能够显著性降低玻璃化冷冻牛卵母细胞中mCa2+，抑制线粒体介导的凋亡通路，进而提高冷冻卵母细胞的发育能力；（5）玻璃化冷冻会引起牛卵母细胞钙调控基因CALM和SSRG的下调。另外，在本课题的部分资助下课题组发现了玻璃化冷冻影响小鼠囊胚表观遗传特性的潜在通路。这些成果分别发表在《J Pineal Res》、 《Sci Rep》、《PLoS One》、《Reprod Domest Anim》上，对于提高冷冻卵母细胞体外受精效率、进而提高其应用范围，都有重要的理论意义和实用价值。