Glycine调控猪卵母细胞钙稳态失衡诱导的内质网应激的机制研究

Oocyte in vitro maturation (IVM) process is highly susceptible to external stimulation due to its isolation from follicular microenvironment, which results in stress response, thus blocking the meiotic process of oocyte. Our previous studies found that glycine plays a positive role in improving mitochondrial function and alleviating endoplasmic reticulum stress (ERS), however this mechanism is still unclear. Base on the importance of calcium ion transport for homeostasis and the pivotal role of calcium channel protein IP3R1 in regulating ERS and mitochondrial function. The aim of this research was to investigate the regulation, influence and mechanism of glycine on calcium homeostasis imbalance ERS in porcine oocytes IVM process. Given the importance of the regulation of glycine on ERS, a calcium homeostasis imbalance ERS and the regulatory mechanism of glycine on ERS with calcium homeostasis imbalance was elucidated, which provided important theoretical model was established. Through siRNA interference with IP3R1 expression, cell nuclear maturation, UPR signaling pathway key factors, ER distribution, calcium level, mitochondrial function and apoptosis were detected. In addition, the key role of IP3R1 in regulation was clarified, support for understanding the regulation mechanism of mammalian oocyte development and maturation, and provided basis for optimizing oocyte culture system in vitro.

卵母细胞体外成熟（IVM）培养过程中，由于脱离了卵泡微环境，极易受到外界刺激，引起应激反应，从而阻滞卵母细胞减数分裂进程。课题组前期研究发现甘氨酸（glycine）对改善线粒体功能和缓解内质网应激（ERS）均具有积极作用，但机制尚不明确。基于钙离子的传递对内环境稳态维持的重要性及钙通道蛋白IP3R1在调节ERS和线粒体功能上的枢纽作用。本项目拟探讨猪卵母细胞IVM过程中glycine对钙稳态失衡ERS的调控作用、效果及机制，在明确glycine对ERS的调控作用基础上，建立钙稳态失衡ERS模型，通过siRNA干扰IP3R1的表达，检测细胞核质成熟、UPR信号通路关键因子、ER分布、钙离子水平、线粒体功能及细胞凋亡情况，明确IP3R1在此调控中的关键作用，阐明glycine对钙稳态失衡ERS的调控机制，为解析哺乳动物卵母细胞的成熟调控机制提供重要理论支持，为优化卵母细胞体外培养体系提供依据。

高质量的成熟卵母细胞对受精后植入前胚胎发育潜能至关重要。但是，在卵母细胞的体外培养过程中，微环境的改变会造成钙稳态失衡引起的ERS，并造成卵母细胞成熟质量下降。本研究旨在探究猪卵母细胞体外成熟过程中glycine对钙稳态失衡诱导的ERS的调控作用、效果及机制，从而提高卵母细胞体外成熟效率和质量。本项目首先研究了glycine对猪卵母细胞ER功能的调控作用，结果表明添加glycine后显著提高卵母细胞成熟质量及ER功能，显著下调CHOP、Xbp1、ATF4基因，并调节[Ca2+]i、[Ca2+]m、[Ca2+]ER水平，证实glycine对猪卵母细胞ER功能和钙稳态具有调控作用。其次探究了glycine对猪卵母细胞钙稳态失衡诱导的ERS的调控效果，以Ca2+-ATPase 抑制剂TG建立猪卵母细胞ERS模型，glycine显著提高TG诱导的卵母细胞成熟率、线粒体及ER功能，缓解TG诱导的ERS，减少细胞凋亡，提高TG处理后早期胚胎发育能力，对Ca2+水平检测发现，glycine通过恢复IP3R1、VDAC1蛋白在细胞内的分布位置，促进其形成皮质簇，同步下调IP3R1、Grp75、VDAC1基因的表达，进一步改善钙离子水平失衡的现象，证实glycine对猪卵母细胞TG诱导的ERS具有较好的调控效果。最后探讨了glycine改善ERS的可能途径，锁定钙通道蛋白IP3R1，比较了抑制IP3R1和glycine对ERS的改善效果，结果表明添加glycine或抑制IP3R1效果相似，均可通过影响IP3R1的分布与表达改善TG诱导成熟率、ER功能和Ca2+水平异常，提高PA后早期胚胎发育能力。综上所述，glycine可通过影响IP3R1的分布与表达调控钙稳态来改善猪卵母细胞体外成熟过程中由TG诱导的ERS，从而改善卵母细胞质量，提高猪卵母细胞体外成熟能力以及促进早期胚胎发育。