College of Veterinary Medicine, Yunnan Agricultural University, Kunming, Yunnan, People's Republic of China.
Yunnan Provincial Engineering Laboratory of Animal Genetic Resource Conservation and Germplasm Enhancement, Yunnan Animal Science and Veterinary Institute, Kunming, Yunnan, People's Republic of China.
Mol Reprod Dev. 2019 Nov;86(11):1615-1627. doi: 10.1002/mrd.23249. Epub 2019 Aug 1.
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.
在冷冻保存后，提高不成熟卵母细胞的体外成熟（IVM）条件至关重要，尤其是在从特定供体中收集到有限数量的卵母细胞的情况下。本研究的目的是确定在 IVM 期间与新鲜卵母细胞共培养是否可以提高玻璃化猪不成熟卵母细胞的质量。为了区分新鲜卵母细胞和玻璃化卵母细胞，我们使用了两种共培养系统：（a）Transwell 两室共培养；（b）在常规培养过程中使用 CellTracker™ Green CMFDA 标记和追踪新鲜卵母细胞。共培养系统显著加速了玻璃化卵母细胞的减数分裂进程，并显著提高了孤雌激活和体细胞核转移后囊胚的形成率。共培养条件改善了玻璃化卵母细胞中活性氧的产生，玻璃化卵母细胞和新鲜卵母细胞之间的细胞内谷胱甘肽水平没有显著差异。两种共培养系统都显著增加了玻璃化卵母细胞中线粒体的正常分布率，但对线粒体的荧光强度没有影响。在与新鲜卵母细胞共培养的玻璃化卵母细胞中，正常内质网（ER）分布和 ER 荧光强度的卵母细胞比例显著更高。在 IVM 20 小时后，来自玻璃化卵母细胞和共培养系统的卵丘细胞中 COX2、HAS2、PTX3 和 TNFAIP6 的 mRNA 表达仍然显著升高，并且这些基因的表达显著降低。此外，共培养方法防止了玻璃化卵母细胞中 BMP15、ZAR1、POU5F1 和 DNMT3A 的 mRNA 表达减少。总之，通过在 IVM 期间与新鲜卵母细胞共培养，玻璃化猪不成熟卵母细胞的质量和随后的胚胎发育得到了显著改善。
