Choi Hyerin, Lee Joohyeong, Yoon Junchul David, Hwang Seon-Ung, Cai Lian, Kim Mirae, Kim Gahye, Oh Dongjin, Kim Eunhye, Hyun Sang-Hwan
Laboratory of Veterinary Embryology and Biotechnology (VETEMBIO), College of Veterinary Medicine, Chungbuk National University, 1 Chungdae-ro, Seowon-gu, Cheongju 28644, South Korea; Institute of Stem Cell & Regenerative Medicine, Chungbuk National University, Cheongju, Republic of Korea.
Laboratory of Veterinary Embryology and Biotechnology (VETEMBIO), College of Veterinary Medicine, Chungbuk National University, 1 Chungdae-ro, Seowon-gu, Cheongju 28644, South Korea; Institute of Stem Cell & Regenerative Medicine, Chungbuk National University, Cheongju, Republic of Korea; Graduate School of Veterinary Biosecurity and Protection, Chungbuk National University, Cheongju, Republic of Korea.
Theriogenology. 2021 Apr 1;164:84-92. doi: 10.1016/j.theriogenology.2021.01.009. Epub 2021 Jan 27.
Copper (Cu) ions have redox activity and act as cofactors of enzymes related to respiration, radical detoxification, and iron metabolism. In this study, we aimed to examine the effects of copper (II) chloride dihydrate (CuCl2·2H2O) on porcine oocytes during in vitro maturation (IVM) and subsequent embryonic development following parthenogenetic activation (PA). Nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels, cumulus expansion, the mRNA expression levels of various genes, and developmental competence were analyzed. During IVM, the maturation medium was supplemented with various concentrations of Cu (0, 0.7, 1.4, and 2.8 μg/mL). After 42 h of IVM, Cu supplementation significantly increased the number of oocytes in the metaphase II stage. Further, the 1.4 μg/mL Cu group showed significantly higher intracellular GSH levels than the control group. However, Cu supplementation increased intracellular ROS levels regardless of their concentration. Additionally, the mRNA levels of Has-2, the cumulus cell expansion-related gene, were higher in all the Cu-treated groups than in the control group. The cumulus cell expansion index was higher in the 0.7 and 1.4 μg/mL Cu groups than in the other groups. In the 0.7 μg/mL Cu group, the mRNA expression levels of PCNA, Zar1, and NPM2, which are related to developmental competence, were significantly higher than those in the control group. Moreover, increased levels of Sod1 transcript, correlated with the antioxidative response, were observed in the 0.7 and 1.4 μg/mL Cu groups. The apoptosis rate in Cu-treated cumulus cells and oocytes was decreased compared to that in the corresponding control groups. Upon evaluation of subsequent embryonic development after PA, the 0.7 μg/mL Cu group showed significantly improved cleavage and blastocyst formation rate compared to the control group. In conclusion, our results suggest that Cu supplementation at appropriate concentrations in IVM medium improves porcine oocyte maturation and the subsequent embryonic potential of PA embryos by reducing oxidative stress and apoptosis.
铜（Cu）离子具有氧化还原活性，可作为与呼吸、自由基解毒和铁代谢相关的酶的辅助因子。在本研究中，我们旨在研究二水合氯化铜（CuCl₂·2H₂O）对猪卵母细胞体外成熟（IVM）及孤雌激活（PA）后后续胚胎发育的影响。分析了核成熟、细胞内谷胱甘肽（GSH）和活性氧（ROS）水平、卵丘扩展、各种基因的mRNA表达水平以及发育能力。在IVM期间，成熟培养基中添加了不同浓度的铜（0、0.7、1.4和2.8μg/mL）。IVM 42小时后，添加铜显著增加了处于中期II期的卵母细胞数量。此外，1.4μg/mL铜组的细胞内GSH水平显著高于对照组。然而，无论铜的浓度如何，添加铜都会增加细胞内ROS水平。此外，所有铜处理组中与卵丘细胞扩展相关的基因Has-2的mRNA水平均高于对照组。0.7和1.4μg/mL铜组的卵丘细胞扩展指数高于其他组。在0.7μg/mL铜组中，与发育能力相关的PCNA、Zar1和NPM2的mRNA表达水平显著高于对照组。此外，在0.7和1.4μg/mL铜组中观察到与抗氧化反应相关的Sod1转录本水平升高。与相应对照组相比，铜处理的卵丘细胞和卵母细胞的凋亡率降低。在评估PA后的后续胚胎发育时，0.7μg/mL铜组的卵裂率和囊胚形成率与对照组相比显著提高。总之，我们的结果表明，在IVM培养基中添加适当浓度的铜可通过降低氧化应激和凋亡来改善猪卵母细胞成熟及PA胚胎的后续胚胎发育潜力。
