Birth of offspring after transfer of Mongolian gerbil (Meriones unguiculatus) embryos cryopreserved by vitrification
Birth of offspring after transfer of Mongolian gerbil (Meriones unguiculatus) embryos cryopreserved by vitrification
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DOI：
10.1002/mrd.20226
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发表时间：
2005-04-01
影响因子：
2.5
通讯作者：
Ogura, A
中科院分区：
文献类型：
--
作者：
Mochida, K;Wakayama, T;Ogura, A
蒙古沙鼠（Meriones unguiculatus）已被用作许多研究领域的实验物种，包括神经学，肿瘤学和寄生虫学。胚胎冷冻保存已成为保护宝贵遗传资源的有效手段，但其在蒙古沙鼠身上的应用尚未见报道。在本研究中，我们研究了玻璃化冷冻保存的蒙古沙鼠胚胎的体外和体内发育能力。在收集当天，用乙二醇（EG）为基础的EFS20和EFS40溶液（分别含有20%和40% EG），在含有30% (w/v) Ficoll 70和0.5M糖的PB1中玻璃化受精胚胎。首先，我们比较了一步和两步玻璃化方法。一步法将胚胎直接转移到玻璃化液（EFS40）中，而两步法将胚胎连续暴露于EFS20和EFS40中，然后玻璃化。液化（解冻）后，两步法玻璃化的晚期两细胞胚胎（第3天收集）在体外发育到桑葚胚期的比率显著高于一步法玻璃化的胚胎（65% vs. 5%, P < 0.0001）。然后，我们检查了同样的两步方法是否适用于早期两细胞胚胎（第2天收集）、四细胞胚胎（第4天）、桑葚胚（第5天）和囊胚（第6天）。液化后，各组胚胎形态正常的比例为87% ~ 100%，分别有23%和96%的胚胎从早期的2细胞和4细胞胚胎发育到致密的桑葚胚期。移植到雌性受体后，分别有3%（4/123）、1%（1/102）、5%（4/73）和10%（15/155）从玻璃化和液化的早期两细胞胚胎、晚期两细胞胚胎、桑葚胚和囊胚发育为足月子代。这表明蒙古沙鼠胚胎可以安全地冷冻保存使用基于鸡蛋的玻璃化溶液。(C) 2005 Wiley-Liss, Inc。
The Mongolian gerbil (Meriones unguiculatus) has been used as a laboratory species in many fields of research, including neurology, oncology, and parasitology. Although the cryopreservation of embryos has become a useful means to protect valuable genetic resources, its application to the Mongolian gerbil has not yet been reported. In this study, we investigated the in vitro and in vivo developmental competence of Mongolian gerbil embryos cryopreserved by vitrification, In vivo-fertilized embryos were vitrified on the day of collection using the ethylene glycol (EG)-based solutions EFS20 and EFS40, which contained 20% and 40% EG, respectively, in PB1 containing 30% (w/v) Ficoll 70 and 0.5M sucrose. First, we compared one-step and two-step vitrification protocols. In the one-step method, the embryos were directly transferred into the vitrification solution (EFS40), whereas in the two-step method, the embryos were exposed serially to EFS20 and EFS40 and then vitrified. After liquefying (thawing), late two-cell embryos (collected on day 3) vitrified by the two-step method showed significantly better rates of in vitro development to the morula stage compared to those vitrified by the one-step method (65% vs. 5%, P < 0.0001). We then examined whether the same two-step method could be applied to early two-cell embryos (collected on day 2), four-cell embryos (day 4), morulae (day 5), and blastocysts (day 6). After liquefying, 87%-100% of the embryos were morphologically normal in all groups, and 23% and 96% developed to the compacted morula stage from early two- and four-cell embryos, respectively. After transfer into recipient females, 3% (4/123), 1% (1/102), 5% (4/73), and 10% (15/155) developed to full-term offspring from vitrified and liquefied early two-cell embryos, late two-cell embryos, morulae, and blastocysts, respectively. This demonstrates that Mongolian gerbil embryos can be safely cryopreserved using EG-based vitrification solutions. (C) 2005 Wiley-Liss, Inc.