Sooam Biotech Research Foundation, Seoul, 08359, Republic of Korea; Institute for Stem Cell and Regenerative Medicine (ISCRM), College of Veterinary Medicine, Chungbuk National University, Cheongju, 28644, Republic of Korea; Laboratory of Veterinary Embryology and Biotechnology (VETEMBIO), College of Veterinary Medicine, Chungbuk National University, Cheongju, 28644, Republic of Korea.
Sooam Biotech Research Foundation, Seoul, 08359, Republic of Korea.
Theriogenology. 2020 Jan 1;141:91-97. doi: 10.1016/j.theriogenology.2019.09.009. Epub 2019 Sep 5.
Autophagy is a critical process in early mammalian embryogenesis. Mammalian target of rapamycin (mTOR) inhibitors are major regulators of autophagy. However, mTOR plays a vital role in major signaling pathways controlling cell growth and metabolism; thus, more secure autophagy activation methods should be considered. The present study investigated the effects of supplementary trehalose, a novel mTOR-independent autophagy enhancer, on oocyte maturation and embryonic development after parthenogenetic activation (PA). Trehalose treatment during in vitro maturation (IVM) did not affect the nuclear maturation rates of oocytes. Oocytes treated with 25 mM trehalose during IVM had a significantly higher (P < 0.05) blastocyst formation rate (64.2%) after PA compared to that in control oocytes (52.0%). Blastocyst quality was also improved in the trehalose-treated group. The total cell numbers for blastocyst formation and expanded blastocyst formation were significantly increased in the trehalose-treated group (52.2% and 27.7%, respectively) compared to those in the control group (36.9% and 11.0%, respectively). Trehalose treatment led to the increased expression of LC3, an autophagy marker, in metaphase II oocytes and 4-cell stage embryos. Gene expression analysis revealed that the expression of several autophagy related genes (LAMP2, pATG5, and LC3) increased, while the Bax/Bcl2 ratio and pro-apoptotic Bak transcript levels were decreased in the trehalose-treated group. In conclusion, these results indicate that treatment with trehalose during IVM improved the developmental potential of porcine embryos by down-regulation of pro-apoptotic genes and up-regulation of autophagy-related genes and marker. Trehalose may be useful for the large-scale production of high-quality porcine blastocysts in vitro.
自噬是哺乳动物胚胎发生早期的一个关键过程。哺乳动物雷帕霉素靶蛋白（mTOR）抑制剂是自噬的主要调节剂。然而，mTOR 在控制细胞生长和代谢的主要信号通路中起着至关重要的作用；因此，应该考虑更安全的自噬激活方法。本研究探讨了新型 mTOR 非依赖性自噬增强剂海藻糖对孤雌激活（PA）后卵母细胞成熟和胚胎发育的影响。在体外成熟（IVM）期间添加海藻糖处理并不影响卵母细胞核成熟率。与对照组卵母细胞（52.0%）相比，在 IVM 期间用 25mM 海藻糖处理的卵母细胞在 PA 后具有显著更高（P<0.05）的囊胚形成率（64.2%）。海藻糖处理组的囊胚质量也得到了改善。囊胚形成和扩展囊胚形成的总细胞数在海藻糖处理组中显著增加（分别为 52.2%和 27.7%），而在对照组中分别为 36.9%和 11.0%。海藻糖处理导致中期 II 期卵母细胞和 4 细胞期胚胎中自噬标志物 LC3 的表达增加。基因表达分析显示，在海藻糖处理组中，几种自噬相关基因（LAMP2、pATG5 和 LC3）的表达增加，而 Bax/Bcl2 比值和促凋亡 Bak 转录本水平降低。总之，这些结果表明，在 IVM 期间用海藻糖处理通过下调促凋亡基因和上调自噬相关基因和标志物来提高猪胚胎的发育潜力。海藻糖可能有助于在体外大规模生产高质量的猪囊胚。
