卵母细胞激活过程中钙信号和线粒体代谢相互调节机制的研究

Abnormal body metabolism such as hypertension, hyperglycemia and hyperlipidemia will cause long-term stress in the oocyte endoplasmic reticulum and mitochondria, which seriously damage the quality of oocytes, leading to more ovulation disorders and lower pregnancy rates in women. After oocyte fertilization, the Ca2+ elevation in the cytoplasm is first initiated, and then the energy supplier mitochondria transition from the resting state to the excited state. However, there are few reports on the mechanism of Ca2+ and mitochondrial interaction during fertilization. Our previous study showed that the [Ca2+]i oscillations were significantly inhibited after mitochondria were depolarized during oocyte activation. This indicates that the maintenance of the [Ca2+]i oscillations requires continuous supply of energy. In this study, by using inhibitors of various Ca2+ channels on different membranes, we will observe disturbed [Ca2+]i oscillations patterns, mitochondrial metabolism deregulations during oocyte activation process, to study the mechanism by which mitochondrial energy metabolism is initiated by [Ca2+]i oscillations. The research results of this project can not only provide a new understanding for regulation of mammalian oocyte mitochondria by Ca2+, but also provide a theoretical basis for improving the quality of clinically assisted reproductive embryos and treating infertility.

高血压、高血糖和高血脂等代谢异常会造成卵母细胞内质网和线粒体等细胞器长期处于应激状态，严重损伤卵母细胞质量，导致妇女生殖力下降。卵母细胞受精激活首先启动细胞质中Ca2+浓度升高，随后线粒体从整体静息状态向激发状态转变。但有关卵母细胞激活过程中Ca2+和线粒体相互调控的机制还鲜有报道。前期研究结果表明，线粒体去极化可以明显抑制卵母细胞激活后的[Ca2+]i振荡，说明[Ca2+]i振荡的长期维持需要能量供应。本研究拟采用不同Ca2+通道抑制剂，扰乱[Ca2+]i振荡模式，观察线粒体代谢在卵母细胞激活过程中的变化，推测[Ca2+]i振荡启动线粒体能量代谢的机制，并尝试补偿体外成熟MII卵母细胞中Ca2+积累，提升卵母细胞激活效率。本项目研究成果不仅可以为理解哺乳动物卵母细胞Ca2+变化和线粒体功能相互调控机制提供新的认识，且可为提高临床辅助生殖胚胎质量，治疗女性不孕提供理论依据。

卵母细胞激活效率低下是受精和胚胎发育失败的最大挑战。卵母细胞停滞在第二次减数分裂的中期，直到受精起始[Ca2+]i振荡，恢复并完成第二次减数分裂，形成受精卵。线粒体功能和细胞内[Ca2+]i振荡是影响卵母细胞发育潜能的两个关键因素，而Ca2+在卵母细胞激活的调节过程中起关键作用。我们旨在了解卵母细胞中线粒体功能与Ca2+信号之间的相关性。.首先，我们使用了位于膜上的各种Ca2+通道抑制剂，包括SERCAs、TRPM7、T型Ca2+通道、Orai1抑制剂，以研究它们在卵母细胞激活过程中的作用。结果表明，SERCAs、TRPM7和T型Ca2+通道对于启动和维持[Ca2+]i振荡非常重要。.线粒体代谢从静止状态到激发态的转变需要[Ca2+]i振荡。三个线粒体膜定位Ca2+通道抑制剂包括抑制剂RU360、CGP37157和Erastin使MCU、NCLX和VDAC失活。Erastin和CGP37157均抑制线粒体活性，同时减弱了[Ca2+]i/[Ca2+]m振荡，从而引起发育阻滞。因此，NCLX和VDAC是调节卵母细胞激活的两种线粒体相关Ca2+转运蛋白。.Ca2+，尤其是线粒体基质中的Ca2+（[Ca2+]m），在线粒体能量补充中起着关键作用。引入编码的线粒体基质Ca2+探针（Mt-GCaMP6s），以观察卵母细胞成熟和激活过程中[Ca2+]m的动态变化。我们发现，在卵母细胞成熟过程中，围绕细胞核的活跃线粒体显示出更高的[Ca2+]m。在卵母细胞孤雌激活过程中，Ca2+动态变化的模式在细胞质和线粒体中是同步的。卵母细胞染色体周围较高的[Ca2+]m线粒体可能在刺激线粒体能量以钙调蛋白反应性卵母细胞纺锤体形成中具有潜在作用。而在细胞质和细胞核区域中同步的Ca2+功能对于卵母细胞有序激活很重要。.综上，卵母细胞的Ca2+和线粒体代谢之间存在紧密的相互调控关系。改善Ca2+和线粒体调控途径有望提升临床卵母细胞体外激活效率，提升辅助生殖成功率。