Matilla Elvira, Martín-Cano Francisco Eduardo, González-Fernández Lauro, Sánchez-Margallo Francisco Miguel, Álvarez Ignacio Santiago, Macías-García Beatriz
Department of Cell Biology, School of Life Sciences, University of Extremadura, Avda. de Elvas, 6006, Badajoz, Spain.
Department of Physiology, Faculty of Nursing and Occupational Therapy, University of Extremadura, Avda. de la Universidad s/n, 10003, Cáceres, Spain.
BMC Vet Res. 2019 Jan 17;15(1):31. doi: 10.1186/s12917-018-1743-2.
Vitrification is the safest method to cryopreserve oocytes, however the process alters mitochondrial function resulting from increased reactive oxygen species (ROS) production. Our aim was to alleviate ROS stress in vitrified mice oocytes using N-acetylcysteine (NAC at 1 mM), to improve the oocyte's developmental competence.
Hence, four experimental groups were compared: fresh oocytes (F-C), vitrified oocytes (V-C), NAC addition prior to oocyte vitrification (V-NAC-Pre) and NAC addition after vitrification (V-NAC-Post). V-NAC-Pre and V-NAC-Post exhibited higher levels of mitochondrial polarization compared to vitrified oocytes (36.5 ± 3.1, 37.7 ± 1.3 and 27.2 ± 2.4 measured as the spatial coefficient of variation/oocyte respectively, mean ± SEM; p < 0.05). However, ROS production increased in vitrified oocytes added with NAC compared to the vitrified control (1124.7 ± 102.1 [V-NAC-Pre] and 1063.2 ± 82.1 [V-NAC-Post] vs. 794.6 ± 164.9 [V-C]; arbitrary fluorescence units/oocyte, mean ± SEM; p < 0.05). ATP significantly decreased in V-NAC-Pre compared to V-NAC-Post oocytes (18.5 ± 6.9 vs. 54.2 ± 4.6 fmol/oocyte respectively, mean ± SEM; p < 0.05), and no differences were observed between V-NAC-Post, F-C and V-C groups. Blastocyst rates derived from F-C oocytes was higher than those derived from V-NAC-Pre (90.7 ± 1.8 vs. 79.1 ± 1.8, respectively, mean % ± SEM,; p < 0.05) but similar to those derived from V-NAC-Post (90.7 ± 1.8, mean % ± SEM, p > 0.05). Total blastomere count of blastocysts derived from V-NAC-Post after in vitro fertilization (IVF) was higher than embryos produced from V-C.
The addition of NAC after vitrification improves the quality of vitrified mature murine oocytes while its addition prior to vitrification is advised against.
玻璃化是冷冻保存卵母细胞最安全的方法，然而该过程会因活性氧（ROS）生成增加而改变线粒体功能。我们的目的是使用N-乙酰半胱氨酸（1 mM的NAC）减轻玻璃化小鼠卵母细胞中的ROS应激，以提高卵母细胞的发育能力。
因此，比较了四个实验组：新鲜卵母细胞（F-C）、玻璃化卵母细胞（V-C）、卵母细胞玻璃化前添加NAC（V-NAC-Pre）和玻璃化后添加NAC（V-NAC-Post）。与玻璃化卵母细胞相比，V-NAC-Pre和V-NAC-Post表现出更高水平的线粒体极化（分别以空间变异系数/卵母细胞衡量为36.5±3.1、37.7±1.3和27.2±2.4，平均值±标准误；p<0.05）。然而，与玻璃化对照组相比，添加NAC的玻璃化卵母细胞中ROS生成增加（[V-NAC-Pre]为1124.7±102.1和[V-NAC-Post]为1063.2±82.1，而[V-C]为794.6±164.9；任意荧光单位/卵母细胞，平均值±标准误；p<0.05）。与V-NAC-Post卵母细胞相比，V-NAC-Pre中的ATP显著降低（分别为18.5±6.9与54.2±4.6 fmol/卵母细胞，平均值±标准误；p<0.05），并且在V-NAC-Post、F-C和V-C组之间未观察到差异。F-C卵母细胞衍生的囊胚率高于V-NAC-Pre衍生的囊胚率（分别为90.7±1.8与79.1±1.8，平均值%±标准误；p<0.05），但与V-NAC-Post衍生的囊胚率相似（90.7±1.8，平均值%±标准误，p>0.05）。体外受精（IVF）后V-NAC-Post衍生的囊胚的总卵裂球计数高于V-C产生的胚胎。
玻璃化后添加NAC可提高玻璃化成熟小鼠卵母细胞的质量，而不建议在玻璃化前添加。
