Zhou Guang-Bin, Hou Yun-Peng, Jin Fang, Yang Qi-En, Yang Zhong-Qiang, Quan Guo-Bo, Tan Hong-Ming, Zhu Shi-En
Laboratory of Animal Embryonic Biotechnology, College of Animal Science and Technology, China Agricultural University, Beijing, P.R. China.
Anim Biotechnol. 2005;16(2):153-63. doi: 10.1080/10495390500263831.
This study was performed to pursue the optimal condition for the cryopreservation of mouse morulae by a two-step OPS method and to investigate the feasibility of the optimal condition for vitrification of embryos at other developmental stages. First, the mouse morulae were vitrified in OPS using one-step procedure-that is, embryos were vitrified after direct exposure to EDFS30 (15% ethylene glycol (EG), 15% dimethyl sulfoxide (DMSO), Ficoll and sucrose), or two-step method-that is, embryos were first pretreated in 10%E + 10%D (10% EG and 10% DMSO in mPBS) for 30 sec, then exposed to EDFS30 for 15 to 60 sec, respectively. After vitrification and warming, the embryos were morphologically evaluated and assessed by their development to blastocysts, expanded/hatched blastocysts, or to term after transfer. The result showed that all the vitrified-warmed morulae had similar blastocyst rate compared to that of control (91.7% vs. 100%), and the highest developmental rate to expanded blastocysts (100%) or hatched blastocysts (62.3%) was observed when the morulae were pretreated with 10%E + 10%D for 0.5 min, exposed to EDFS30for 25 sec before vitrification and warming in 0.5 M sucrose for 5 min. After transfer, the survival rate (33.1%) in vivo of the vitrified morulae was higher (P > 0.05) than that of the fresh embryos (24.6%). Secondly, embryos at different stages were cryopreserved and thawed following the above program. Most (93.4 to 100%) of the embryos recovered after vitrification were morphologically normal at all the developmental stages. The blastocyst rates of the vitrified one-cell (52.5 to 66.7%) and the two-cell (63.3 to 68.9%) embryos were lower (P < 0.05) than those of the vitrified four-cell embryos (81.7 to 86.4%), the eight-cell embryos (90.0 to 93.3%), morulae (96.7 to 100%), and the expanded blastocysts rate (98.3 to 100.0%) of the vitrified early blastocysts. The highest survival rate in vivo of vitrified embryos were from the early blastocysts (40.4%), which was similar to that of fresh embryos (48.6%). The data demonstrate that the optimal protocol for the cryopreservation of morulae was suitable for the four-cell embryos to early blastocyst stages and that the early blastocyst stage is the most feasible stage for mouse embryo cryopreservation under our experimental conditions.
本研究旨在探寻采用两步法OPS对小鼠桑椹胚进行冷冻保存的最佳条件，并研究该最佳条件用于其他发育阶段胚胎玻璃化的可行性。首先，采用一步法程序，即将小鼠桑椹胚直接暴露于EDFS30（15%乙二醇（EG）、15%二甲基亚砜（DMSO）、菲可和蔗糖）中进行玻璃化，或采用两步法程序，即先将胚胎在10%E + 10%D（mPBS中10%EG和10%DMSO）中预处理30秒，然后分别暴露于EDFS30中15至60秒，对小鼠桑椹胚进行玻璃化。玻璃化和复温后，对胚胎进行形态学评估，并根据其发育至囊胚、扩张/孵化囊胚或移植后发育至足月的情况进行评价。结果显示，与对照组相比，所有玻璃化-复温后的桑椹胚具有相似的囊胚率（91.7%对100%），当桑椹胚在10%E + 多10%D中预处理0.5分钟、在玻璃化和在0.5M蔗糖中复温5分钟前暴露于EDFS30中25秒时，观察到其发育至扩张囊胚（100%）或孵化囊胚（62.3%）的最高发育率。移植后，玻璃化桑椹胚的体内存活率（33.1%）高于新鲜胚胎（24.6%）（P>0.05）。其次，按照上述程序对不同阶段的胚胎进行冷冻保存和解冻。玻璃化后复苏的大多数胚胎（93.4%至100%）在所有发育阶段形态正常。玻璃化的单细胞（52.5%至66.7%）和二细胞（63.3%至68.9%）胚胎的囊胚率低于玻璃化的四细胞胚胎（81.7%至86.4%）、八细胞胚胎（90.0%至93.3%）、桑椹胚（96.7%至100%）以及玻璃化早期囊胚的扩张囊胚率（98.3%至100.0%）（P<0.05）。玻璃化胚胎的体内最高存活率来自早期囊胚（40.4%），与新鲜胚胎（48.6%）相似。数据表明，桑椹胚冷冻保存的最佳方案适用于四细胞胚胎至早期囊胚阶段，并且在我们的实验条件下，早期囊胚阶段是小鼠胚胎冷冻保存最可行的阶段。
