猪GV期卵母细胞冷冻保存的内质网应激损伤及干预研究

The cryoinjuries have limited the potential applications of oocytes cryopreservation in practice; however, its mechanisms and corresponding resolving measures are still not fully understood. Endoplasmic reticulum stress (ERS) is provoked by the damage of endoplasmic reticulum, and excessive ERS results in cell injuries or death. We previously found that the protein expressions of GRP78/Bip and CHOP increased in cryopreserved oocytes, which indicated the cryopreservation caused severe ERS in the oocytes. For the purpose of further revealing injury mechanisms of ERS in cryopreserved oocytes, porcine oocytes at the GV stage are vitrified in this study. We observe the expression levels of ERS-related proteins in oocytes and cumulus cells. Based on this, the vitrified porcine GV oocytes are treated with ERS inhibitor during the process of maturation, the endoplasmic reticulum functions, oxidative stress, apoptosis and developmental competence of oocytes and the DNA fragmentation and expansion-related genes of cumulus cells are analyzed. The results obtained will redound to demonstrate the mechanisms of ERS mediated-injuries in porcine GV oocytes following vitrification and seek to improve the potential of the cells. This study is not only helpful to understand the injury mechanisms of vitrified oocytes, but also enrich the intension of ERS knowledge.

超低温冷冻保存致猪卵母细胞损伤是限制该技术应用和推广的重要原因，其冷冻损伤机制及相应解决措施尚未彻底阐明。内质网作为细胞内重要细胞器，受损后会触发内质网应激（ERS），若ERS过度则可成为细胞损伤的重要机制。申请人前期研究发现，冷冻导致卵母细胞中ERS标志蛋白GRP78/Bip和CHOP表达升高，表明细胞内发生了严重的ERS。为进一步揭示冷冻卵母细胞的ERS损伤机制，本项目拟对猪GV期卵母细胞玻璃化冷冻保存，分别检测卵母细胞和卵丘细胞中ERS相关蛋白表达；在此基础上，通过体外成熟阶段给予ERS抑制剂干预，观察卵母细胞的内质网功能、氧化应激、细胞凋亡、发育潜力的变化情况，并分析卵丘细胞的DNA片段化和扩展相关基因表达，旨在探明ERS在猪GV期卵母细胞冷冻损伤中的作用，寻求改善冷冻卵母细胞质量的措施。研究结果不仅能丰富卵母细胞的冷冻损伤机制，也可以拓宽ERS的知识内涵。

目前，卵母细胞的内质网冷冻损伤还缺乏深入研究。本项目以猪GV期卵母细胞为对象，对其进行玻璃化冷冻及体外成熟培养，然后研究内质网损伤情况，同时期望通过内质网应激（ERS）抑制剂改善卵母细胞质量。研究发现，冷冻卵母细胞的内质网空间分布异常、活力降低、内质网钙含量下降；部分ERS相关基因mRNA表达水平异常、ERS标志性蛋白XBP1表达升高、ERS凋亡途径caspase-12活性上升。卵母细胞在玻璃化冷冻及体外成熟过程中采用ERS抑制剂干预，可缓解上述内质网损伤情况，同时也能够减轻氧化应激、增强线粒体活性、降低Bax mRNA表达水平、提高孤雌激活囊胚发育率。研究结果说明，玻璃化冷冻导致卵母细胞内质网损伤，这可能是冷冻卵母细胞质量下降的原因之一。在本项目的部分资助下，课题组也优化了猪GV期卵母细胞玻璃化冷冻保存技术体系。截止目前，已发表SCI收录论文3篇、北大核心期刊收录论文3篇，培养硕士研究生1名。