Quality of vitrified porcine immature oocytes is improved by coculture with fresh oocytes during in vitro maturation
Quality of vitrified porcine immature oocytes is improved by coculture with fresh oocytes during in vitro maturation
复制标题
玻璃化猪未成熟卵母细胞在体外成熟过程中与新鲜卵母细胞共培养可提高其质量。
DOI：
10.1002/mrd.23249
复制
发表时间：
2019-11-01
影响因子：
2.5
通讯作者：
Wu, Guo-Quan
中科院分区：
文献类型：
--
作者：
Jia, Bao-Yu;Xiang, De-Cai;Wu, Guo-Quan
提高冷冻保存后未成熟卵母细胞的体外成熟（IVM）条件至关重要，特别是如果从特定供体收集的卵母细胞数量有限。本研究的目的是确定在体外成熟过程中，与新鲜卵母细胞共培养是否能提高玻璃化冷冻猪未成熟卵母细胞的质量。为了区分新鲜卵母细胞与玻璃化冷冻卵母细胞，我们使用了两种类型的共培养系统：（a）transwell双室共培养;（B）在常规培养期间用CellTracker™绿色CMFDA标记和追踪新鲜卵母细胞。共培养系统显着加速玻璃化冷冻卵母细胞减数分裂进程，显着增加孤雌激活和体细胞核移植后的囊胚形成率。在玻璃化冷冻卵母细胞的活性氧的产生改善的共培养条件下，与新鲜和玻璃化冷冻卵母细胞的细胞内谷胱甘肽水平没有显着差异。两种共培养体系均能显著提高玻璃化冷冻卵母细胞线粒体的正常分布率，但对线粒体荧光强度无影响。玻璃化冷冻卵母细胞与新鲜卵母细胞共培养后，内质网（ER）分布正常的卵母细胞比例和ER荧光强度均显著增高。IVM 20小时后，COX 2、HAS 2、PTX 3和TNFAIP 6的mRNA表达在来自玻璃化冷冻卵母细胞的卵丘细胞中仍然显著较高，并且共培养系统显著降低了这些基因的表达。此外，共培养方法防止了玻璃化冷冻卵母细胞中BMP 15、ZAR 1、POU 5 F1和DNMT 3A的mRNA表达的降低。总之，新鲜猪卵母细胞体外成熟过程中的共培养可显著提高猪未成熟卵母细胞玻璃化冷冻后的卵母细胞质量和后续胚胎发育。
It is essential to enhance the in vitro maturation (IVM) condition for immature oocytes after cryopreservation, particularly if limited numbers of oocytes collected from specific donors. The objective of this study was to determine if quality of vitrified porcine immature oocytes was enhanced by coculturing with fresh oocytes during IVM. To distinguish fresh versus vitrified oocytes, we used two types of coculture systems: (a) transwell two‐chamber coculture; (b) labeling and tracing fresh oocytes with CellTracker™ Green CMFDA during conventional culture. Coculture systems significantly accelerated meiotic progression of vitrified oocytes and significantly increased blastocyst formation rates following parthenogenetic activation and somatic cell nuclear transfer. Reactive oxygen species generation in vitrified oocytes was ameliorated by the coculture conditions, with no significant difference between fresh and vitrified oocytes for intracellular glutathione level. Both coculture systems significantly increased rate of normal mitochondrial distribution in vitrified oocytes, but did not affect fluorescence intensity of mitochondria. The percentage of oocytes with normal endoplasmic reticulum (ER) distribution and ER fluorescence intensity were significantly higher in vitrified oocytes cocultured with fresh oocytes. After 20 hr of IVM, mRNA expression of COX2, HAS2, PTX3, and TNFAIP6 remained significantly higher in cumulus cells derived from vitrified oocytes and coculture systems significantly decreased the expression of these genes. Additionally, coculture methods prevented the reduction of mRNA expression for BMP15, ZAR1, POU5F1, and DNMT3A in vitrified oocytes. In conclusion, oocyte quality and subsequent embryo development of vitrified porcine immature oocytes were significantly improved by fresh oocyte coculture during IVM.