Institute of Molecular Medicine and Genetics, Georgia Health Sciences University, Augusta, Georgia, United States of America.
PLoS One. 2011;6(11):e27604. doi: 10.1371/journal.pone.0027604. Epub 2011 Nov 16.
The objective of this study was to elucidate the toxicity of widely used penetrating cryoprotective agents (CPAs) to mammalian oocytes. To this end, mouse metaphase II (M II) oocytes were exposed to 1.5 M solutions of dimethylsulfoxide (DMSO), ethylene glycol (EG), or propanediol (PROH) prepared in phosphate buffered saline (PBS) containing 10% fetal bovine serum. To address the time- and temperature-dependence of the CPA toxicity, M II oocytes were exposed to the aforementioned CPAs at room temperature (RT, ∼23°C) and 37°C for 15 or 30 minutes. Subsequently, the toxicity of each CPA was evaluated by examining post-exposure survival, fertilization, embryonic development, chromosomal abnormalities, and parthenogenetic activation of treated oocytes. Untreated oocytes served as controls. Exposure of MII oocytes to 1.5 M DMSO or 1.5 M EG at RT for 15 min did not adversely affect any of the evaluated criteria. In contrast, 1.5 M PROH induced a significant increase in oocyte degeneration (54.2%) and parthenogenetic activation (16%) under same conditions. When the CPA exposure was performed at 37°C, the toxic effect of PROH further increased, resulting in lower survival (15%) and no fertilization while the toxicity of DMSO and EG was still insignificant. Nevertheless, it was possible to completely avoid the toxicity of PROH by decreasing its concentration to 0.75 M and combining it with 0.75 M DMSO to bring the total CPA concentration to a cryoprotective level. Moreover, combining lower concentrations (i.e., 0.75 M) of PROH and DMSO significantly improved the cryosurvival of MII oocytes compared to the equivalent concentration of DMSO alone. Taken together, our results suggest that from the perspective of CPA toxicity, DMSO and EG are safer to use in slow cooling protocols while a lower concentration of PROH can be combined with another CPA to avoid its toxicity and to improve the cryosurvival as well.
本研究旨在阐明广泛使用的渗透保护剂（CPAs）对哺乳动物卵母细胞的毒性。为此，将小鼠中期 II（M II）卵母细胞暴露于在含 10%胎牛血清的磷酸盐缓冲盐水（PBS）中制备的 1.5 M 二甲基亚砜（DMSO）、乙二醇（EG）或 1,3-丙二醇（PROH）溶液中。为了研究 CPA 毒性的时间和温度依赖性，将 M II 卵母细胞在室温（RT，约 23°C）和 37°C 下分别暴露于上述 CPAs 15 或 30 分钟。随后，通过检查暴露后卵母细胞的存活、受精、胚胎发育、染色体异常和处理卵母细胞的孤雌激活来评估每种 CPA 的毒性。未处理的卵母细胞作为对照。在 RT 下将 M II 卵母细胞暴露于 1.5 M DMSO 或 1.5 M EG 15 分钟不会对任何评估标准产生不利影响。相比之下，在相同条件下，1.5 M PROH 会导致卵母细胞退化（54.2%）和孤雌激活（16%）显著增加。当在 37°C 下进行 CPA 暴露时，PROH 的毒性作用进一步增加，导致存活率降低（15%）和无受精，而 DMSO 和 EG 的毒性仍然不显著。然而，通过将 PROH 的浓度降低至 0.75 M 并将其与 0.75 M DMSO 结合以将总 CPA 浓度降低至保护水平，可以完全避免 PROH 的毒性。此外，与单独使用 DMSO 相比，将较低浓度（即 0.75 M）的 PROH 和 DMSO 结合使用可显著提高 M II 卵母细胞的冷冻保存。综上所述，我们的研究结果表明，从 CPA 毒性的角度来看，DMSO 和 EG 在缓慢冷却方案中更安全，而可以将较低浓度的 PROH 与另一种 CPA 结合使用以避免其毒性并提高冷冻保存效果。
