受精钙振荡调控端粒影响胚胎染色体稳定性的机制研究

Early embryo aneuploidy was associated with telomere shortening. After fertilization, the telomeres become shorter and it may become more serious short in some of blastomere，however its mechanism is not clear. We have to set up sensitive embryos of mice to simulate human embryos with telomeres using sperm of knockout mice. We found phenomenon similar to human that the telomeres get shorter after fertilization and are not neat among blastomeres, and assisted oocyte activation intensify the telomeres shorter and make embryo with short telomeres developmental disorders. So we suspect, activated calcium oscillation in fertilization promoted the Ca2 + related molecular pathways, such as S100A8 inhibition of telomerase activity and/or reactive oxygen species injuries to telomeres, and lead telomere inhibitors to uneven during splitting, thus cause telomeres get shorter and some are too short, so lead to chromosome instability. Therefore this research will continue to use sensitive embryo of mice, with groups of fertilization calcium oscillation strength, observe the relationship between the Ca2+ and telomerase activity or reactive oxygen species，find their distribution in the blastomere, and observed the impact of these molecular concentration on the telomeres, chromosome behavior while telomere being too short; Observe the positive and negative effects of molecular interference in S100A8/9 and reactive oxygen species; All the above results then are verified by human eggs. This study will explain the mechanism of calcium oscillation in fertilization controlling telomere, answer the molecular pathways why telomere shortening after fertilization and cause chromosome instability in some blastomeres, found the embryonic aneuploidy high-risk factors and coping strategies, so to improve the efficiency of assisted reproduction.

人早期胚胎非整倍体高发与端粒过短有关。受精后端粒变短并在部分卵裂球变短严重，其机制不清楚。我们前期用基因敲除小鼠精子建立了模拟人短端粒的小鼠胚胎模型，在小鼠发现了类似人的受精后端粒变短及卵裂球间不齐，而辅助激活后端粒更短并使短端粒胚发育障碍。故我们猜测，受精激活的钙振荡促进了Ca2+相关分子途径，如S100A8抑制端粒酶活性和/或氧自由基对端粒损伤，且分裂时导致端粒抑制成分不均，造成端粒变短且部分过短，引起染色体不稳。为此本研究将继续用小鼠胚胎模型，以受精钙振荡强弱分组，观测Ca2+、端粒酶活性、氧自由基间关系及在卵裂球的分布，观测这些分子浓度对端粒的影响，端粒过短时染色体行为；观测分子干预S100A8/9及氧自由基下的正反效果；并在人卵验证上述结果。这将阐述受精钙振荡调控端粒的机制，解答受精后端粒变短并引发部分卵裂球染色体不稳的分子途径，发现人胚非整倍体高发因素及应对策略，提高助孕效率。

本课题探究受精卵过程中尤其是辅助激活等操作后钙振荡强度的变化对受精卵子端粒长度和线粒体代谢的改变，以及对其染色体稳定性和胚胎发育潜能的进一步影响。研究构建了基于 CRISPR 技术的活细胞端粒标记技术平台，获得了能诱导着床前胚胎非整倍体细胞的小鼠端粒长度；明确在一定端粒长度范围内，辅助激活可能导致端粒的进一步缩短，并导致非整倍体的比例显著增加。进一步的机制研究中，我们发现，钙离子升高导致S100A8/A9介导的端粒酶活性下降这一途径并不能在受精和胚胎早期发育阶段影响小鼠端粒的修复；但是发现钙离子升高导致ROS增加途径具有明显作用，即辅助激活中钙离子升高频率和幅度增高，引发线粒体代谢异常，导致ROS增加，在这一阶段端粒的变短主要可能由钙过载引起的ROS所介导，并进一步导致胚胎发育表观遗传异常。为了进一步验证ROS途径，我们还通过对容易导致染色体异常的核质置换技术进行相应优化，避免钙离子与ROS因素，发现DNA完整性、染色体稳定性提高，获得了良好的胚胎发育和健康活产小鼠。.总之，本项目发现了异常激活下钙离子升高，通过ROS途径对DNA完整性有显著影响，这可能导致本来较短端粒进一步受损，线粒体代谢紊乱，引发染色体稳定性异常；而控制好这些因素可以改善DNA完整性与染色体稳定性；我们还进一步通过相关研究从多个方面探讨钙离子变化对卵子发生、成熟、受精及胚胎发育的影响，为明确辅助激活（AOA）的临床使用范围和操作规范提供依据。