Higaki Shogo, Mochizuki Kentaro, Akashi Yuichiro, Yamaha Etsuro, Katagiri Seiji, Takahashi Yoshiyuki
Laboratory of Theriogenology, Department of Veterinary Clinical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
J Reprod Dev. 2010 Apr;56(2):212-8. doi: 10.1262/jrd.09-136e. Epub 2009 Dec 9.
The feasibility of cryopreservation of zebrafish (Danio rerio) primordial germ cells (PGCs) by rapid cooling (i.e., vitrification) of dechorionated whole embryos at the 14- to 20-somite stage was investigated. Initially, we examined the glass-forming properties and embryo toxicities of six cryoprotectants: methanol (MeOH), ethylene glycol (EG), glycerol (GC), dimethyl sulfoxide (DMSO), propylene glycol (PG) and 1,3-butylene glycol (1,3-BG). According to the results of glass-forming and embryo toxicity tests, pretreatment solution (PS) containing 2 or 3 M cryoprotectant and vitrification solution (VS) containing 5 M cryoprotectant and 0.5 M sucrose were prepared using each cryoprotectant. Dechorionated embryos, the PGCs of which were visualized by injection of green fluorescence protein-nos1 3'UTR mRNA, were cooled rapidly by plunging into liquid nitrogen after serial exposure to PS and VS. All embryos cooled with MeOH, PG and 1,3-BG showed ice formation during cooling, and few embryos had live PGCs after warming. Most embryos cooled with GC did not show ice formation; however, few embryos had live PGCs. All embryos cooled with EG and most embryos cooled with DMSO had live PGCs when the embryos did not show ice formation during cooling. Based on the number of live PGCs in fresh embryos, the maximum survival rates of PGCs recovered from embryos cooled with EG and DMSO were estimated to be about 40 and 20%, respectively. The present study indicates that rapid cooling of dechorionated whole embryos, especially using EG-based solutions, could be utilized as a simple and promising tool for cryopreservation of PGCs.
研究了在14至20体节期对去绒毛膜的完整胚胎进行快速冷却(即玻璃化)来冷冻保存斑马鱼(Danio rerio)原始生殖细胞(PGC)的可行性。最初,我们检测了六种冷冻保护剂的玻璃化形成特性和胚胎毒性:甲醇(MeOH)、乙二醇(EG)、甘油(GC)、二甲基亚砜(DMSO)、丙二醇(PG)和1,3 - 丁二醇(1,3 - BG)。根据玻璃化形成和胚胎毒性测试结果,使用每种冷冻保护剂制备了含有2或3 M冷冻保护剂的预处理溶液(PS)以及含有5 M冷冻保护剂和0.5 M蔗糖的玻璃化溶液(VS)。通过注射绿色荧光蛋白 - nos1 3'UTR mRNA使PGC可视化的去绒毛膜胚胎,在依次暴露于PS和VS后,投入液氮中进行快速冷却。所有用MeOH、PG和1,3 - BG冷却的胚胎在冷却过程中都出现了结冰现象,解冻后几乎没有胚胎具有存活的PGC。大多数用GC冷却的胚胎没有出现结冰现象;然而,只有少数胚胎具有存活的PGC。当胚胎在冷却过程中没有出现结冰现象时,所有用EG冷却的胚胎以及大多数用DMSO冷却的胚胎都有存活的PGC。根据新鲜胚胎中存活PGC的数量,估计从用EG和DMSO冷却的胚胎中回收的PGC的最大存活率分别约为40%和20%。本研究表明,对去绒毛膜的完整胚胎进行快速冷却,特别是使用基于EG的溶液,可作为一种简单且有前景的PGC冷冻保存工具。