García-Martínez Tania, Martínez-Rodero Iris, Roncero-Carol Joan, Yánez-Ortiz Iván, Higgins Adam Z, Mogas Teresa
Dept. of Animal Medicine and Surgery, Autonomous University of Barcelona, ES-08193, Cerdanyola del Vallès, Spain.
School of Chemical, Biological and Environmental Engineering, Oregon State University, Corvallis, OR, 97331-2702, USA.
Theriogenology. 2022 May;184:110-123. doi: 10.1016/j.theriogenology.2022.02.024. Epub 2022 Mar 10.
The cryopreservation of mammalian oocytes and embryos has become an integral part of assisted reproduction in both humans and veterinary species. However, the methods used to cryopreserve bovine oocytes still have significant shortcomings. A wide variety of approaches has been used to try to improve and optimize methods of cryopreservation. However, these procedures employed are not always designed to specifically take account of the osmotic tolerance response of the cells according to the temperature and time of cryoprotectant (CPA) addition. When these properties are considered, optimal procedures for the addition of CPAs can be designed proactively. Based on in silico and in vitro osmotic observations, we propose shorter dehydration-based protocols at different temperatures (25°C vs. 38.5°C) towards defining an improved cryopreservation method. In vitro matured oocytes were exposed to equilibration solution (ES) at 25°C and 38.5°C and effects of optimized exposure times for each temperature were determined prior to vitrification/warming on oocyte spindle configuration, DNA fragmentation, and further embryo development. Upon exposure to standard ES (7.5% dimethyl sulfoxide + 7.5% ethylene glycol in TCM199 medium + 20% fetal bovine serum), original oocyte volume was recovered within 2 min 30 s at 38.5°C and 5 min 30 s at 25°C. In vitro matured oocytes were then exposed to the aforementioned cryoprotectants at both temperature/duration conditions and vitrified/warmed. While similar percentages of oocytes exhibiting a normally configured spindle and DNA fragmentation were observed in the fresh control group and oocytes vitrified at 38.5°C, significantly higher apoptosis rate and lower percentages of normal spindle configuration were observed in oocytes vitrified at 25°C when compared to control fresh oocytes. Similar cleavage rates and blastocyst yields were observed in the vitrified/38.5°C and fresh controls, while these rates were lower in vitrified/25°C. These results revealed that the limitation of the exposure time of the oocytes to the ES to the point of osmotic equilibrium volume recovery could be a more efficient approach to prepare them for vitrification. Therefore, exposure time to ES to 2 min 30 s at 38.5 °C appears to improve the quality of vitrified/warmed oocytes by protecting spindle integrity and reducing DNA fragmentation thus improving blastocyst rates and embryo quality.
哺乳动物卵母细胞和胚胎的冷冻保存已成为人类和兽医物种辅助生殖中不可或缺的一部分。然而，用于冷冻保存牛卵母细胞的方法仍存在显著缺陷。人们已采用多种方法来尝试改进和优化冷冻保存方法。然而，所采用的这些程序并不总是专门设计来根据冷冻保护剂（CPA）添加的温度和时间具体考虑细胞的渗透耐受反应。当考虑这些特性时，可以主动设计添加CPA的最佳程序。基于计算机模拟和体外渗透观察，我们提出在不同温度（25°C与38.5°C）下基于更短脱水的方案，以确定一种改进的冷冻保存方法。将体外成熟的卵母细胞在25°C和38.5°C下暴露于平衡溶液（ES），并在玻璃化/复温之前确定每个温度下优化暴露时间对卵母细胞纺锤体构型、DNA片段化以及进一步胚胎发育的影响。在暴露于标准ES（TCM199培养基中7.5%二甲基亚砜 + 7.5%乙二醇 + 20%胎牛血清）后，在38.5°C时原始卵母细胞体积在2分30秒内恢复，在25°C时在5分30秒内恢复。然后将体外成熟的卵母细胞在两种温度/持续时间条件下暴露于上述冷冻保护剂并进行玻璃化/复温。虽然在新鲜对照组和在38.5°C下玻璃化的卵母细胞中观察到具有正常构型纺锤体和DNA片段化的卵母细胞百分比相似，但与对照新鲜卵母细胞相比，在25°C下玻璃化的卵母细胞中观察到显著更高的凋亡率和更低的正常纺锤体构型百分比。在玻璃化/38.5°C组和新鲜对照组中观察到相似的分裂率和囊胚率，而在玻璃化/25°C组中这些率较低。这些结果表明，将卵母细胞暴露于ES直至渗透平衡体积恢复的暴露时间限制可能是一种更有效的为其玻璃化做准备的方法。因此，在38.5°C下将暴露于ES的时间控制在2分30秒似乎可以通过保护纺锤体完整性和减少DNA片段化来提高玻璃化/复温卵母细胞的质量，从而提高囊胚率和胚胎质量。
