Anti-apoptotic regulation contributes successful nuclear reprogramming using cryopreserved oocytes

The cryopreservation has a negative effect on the quality of oocytes and may be closely associated with increased levels of reactive oxygen species (ROS) and apoptotic events. The purpose of the present study was to evaluate the detrimental effects on the developmental competence of somatic cell nuclear transferred (SCNT)- mouse embryos using vitrified (cryopreserved) oocytes and to evaluate the recovery effects of melatonin on cryo-damage in cloned embryos. Development of SCNT embryos using cryopreserved oocyte cytoplasm (SCNT-CROC) was inferior to those using fresh cytoplasm (SCNT-FOC). Using RNA-sequencing analysis, we found upregulation of 8 pro-apoptotic-related genes (Cyct, Dapk2, Dffb, Gadd45g, Hint2, Mien1, P2rx7, and Pmaip) in the SCNT-CROC group. Furthermore, the addition of melatonin, an agent that reduces apoptosis and ROS production, enhanced blastocyst formation rates in the SCNT-CROP group when compared with the melatonin-untreated group. Additionally, melatonin-treatment increased the derivation efficiency of pluripotent stem cells from cloned embryos using cryopreserved oocyte. Overall design: Here we perform RNA-Seq experiment in melatonin-treated mouse 2-cell and blastocyst embryos, respectively.

冷冻保存会对卵母细胞质量产生负面影响，且可能与活性氧（ROS）水平升高及细胞凋亡事件密切相关。本研究旨在评估使用玻璃化冷冻（冷冻保存）卵母细胞对体细胞核移植（SCNT）小鼠胚胎发育能力的损害作用，并探究褪黑素对克隆胚胎冷冻损伤的修复效果。使用冷冻卵母细胞胞质构建的体细胞核移植胚胎（SCNT-CROC），其发育能力劣于使用新鲜卵母细胞胞质构建的胚胎（SCNT-FOC）。通过RNA测序（RNA-seq）分析，我们发现SCNT-CROC组中8个促凋亡相关基因（Cyct、Dapk2、Dffb、Gadd45g、Hint2、Mien1、P2rx7及Pmaip）的表达显著上调。此外，作为一种可抑制细胞凋亡与活性氧生成的物质，褪黑素的添加可提升SCNT-CROP组的囊胚形成率，相较于未添加褪黑素的对照组更为显著。同时，褪黑素处理还提高了使用冷冻卵母细胞构建的克隆胚胎来源的多能干细胞衍生效率。整体实验设计：本研究分别对经褪黑素处理的小鼠2-细胞胚胎及囊胚开展了RNA-seq实验。