Ma Yongshun, Pan Bo, Yang Haoxuan, Qazi Izhar Hyder, Wu Zhenzheng, Zeng Changjun, Zhang Ming, Meng Qingyong, Zhou Guangbin
Institute of Animal Genetics and Breeding, College of Animal Science and Technology, Sichuan Agricultural University, Chengdu 611130, PR China.
Institute of Animal Genetics and Breeding, College of Animal Science and Technology, Sichuan Agricultural University, Chengdu 611130, PR China; Department of Veterinary Anatomy & Histology, Shaheed Benazir Bhutto University of Veterinary and Animal Sciences, Sakrand 67210, Sindh, Pakistan.
Cryobiology. 2018 Apr;81:206-209. doi: 10.1016/j.cryobiol.2018.02.011. Epub 2018 Feb 22.
The present study aimed to investigate the effect of vitrification on the expression of fertilization related genes (CD9 and CD81) and DNA methyl transferases (DNMT1 and DNMT3b) in bovine germinal vesicle (GV) oocytes and their resulting metaphase Ⅱ (MⅡ) stages after in vitro maturation culture. GV oocytes were vitrified using the open-pulled straw method; after warming, they were cultured in vitro. The vitrified-warmed GV oocytes and more developed MII oocytes were used to calculate the maturation rates (first polar body extrusion under a stereomicroscopy), and to detect mRNA expression (qRT-PCR). Fresh GV oocytes and their in vitro-derived MII oocytes served as controls. The results showed that both the maturation rate (54.23% vs. 42.93%) and the relative abundance of CD9 mRNA decreased significantly (p < 0.05) in bovine GV oocytes after vitrification, but the expression of CD81 and DNMT3b increased significantly. After in vitro maturation of vitrified GV oocytes, the resulting MII oocytes showed lower (p < 0.05) mRNA expression of genes (CD9, CD81, DNMT1 and DNMT3b) when compared to the control group (MII oocytes). Altogether, vitrification decreased the maturation rate of bovine GV oocytes and changed the expression of fertilization related genes and DNA methyl transferases during in vitro maturation.
本研究旨在探讨玻璃化冷冻对牛生发泡（GV）期卵母细胞及其体外成熟培养后形成的中期Ⅱ（MⅡ）期卵母细胞中受精相关基因（CD9和CD81）以及DNA甲基转移酶（DNMT1和DNMT3b）表达的影响。采用开放式拉长细管法对GV期卵母细胞进行玻璃化冷冻；解冻后，将其进行体外培养。利用玻璃化冷冻-解冻后的GV期卵母细胞以及发育程度更高的MⅡ期卵母细胞计算成熟率（在体视显微镜下观察第一极体排出情况），并检测mRNA表达（定量逆转录聚合酶链反应）。新鲜的GV期卵母细胞及其体外培养获得的MⅡ期卵母细胞作为对照。结果显示，玻璃化冷冻后牛GV期卵母细胞的成熟率（54.23%对42.93%）和CD9 mRNA的相对丰度均显著降低（p<0.05），但CD81和DNMT3b的表达显著增加。玻璃化冷冻的GV期卵母细胞体外成熟后，所形成的MⅡ期卵母细胞与对照组（MⅡ期卵母细胞）相比，基因（CD9、CD81、DNMT1和DNMT3b）的mRNA表达较低（p<0.05）。总之，玻璃化冷冻降低了牛GV期卵母细胞的成熟率，并改变了体外成熟过程中受精相关基因和DNA甲基转移酶的表达。
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