Laboratory of Theriogenology, Department of Veterinary Clinical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan.
Reproduction. 2010 Apr;139(4):733-40. doi: 10.1530/REP-09-0549. Epub 2010 Feb 12.
This study aimed to produce fertile zebrafish (Danio rerio) possessing germ cells (gametes) that originated from cryopreserved primordial germ cells (PGCs). First, to improve the vitrification procedure of PGCs in segmentation stage embryos, dechorionated yolk-intact and yolk-removed embryos, the PGCs of which were labeled with green fluorescent protein, were cooled rapidly after serial exposures to equilibration solution (ES) and vitrification solution (VS), which contained ethylene glycol, DMSO, and sucrose. Yolk removal well prevented ice formation in the embryos during cooling and improved the viability of cryopreserved PGCs. The maximum recovery rate of live PGCs in the yolk-removed embryos vitrified after optimum exposure to ES and VS was estimated to be about 90%, and about 50% of the live PGCs showed pseudopodial movement. Next, to elucidate the ability of cryopreserved PGCs to differentiate into functional gametes, PGCs recovered from the yolk-removed embryos (striped-type) that were vitrified under the optimum exposure to ES and VS were transplanted individually into 218 sterilized recipient blastulae (golden-type). Two days after the transplantation, 7.5% (14/187) of morphologically normal embryos had PGC(s) in the genital ridges. Six (5 males and 1 female) of the 14 recipient embryos developed into mature fish and generated progeny with characteristics inherited from PGC donors. In conclusion, we demonstrated the successful cryopreservation of PGCs by vitrification of yolk-removed embryos and the production of fertile zebrafish possessing germ cells that originated from the PGCs in vitrified embryos.
本研究旨在获得具有来源于冷冻保存原始生殖细胞（PGC）的生殖细胞（配子）的可育斑马鱼（Danio rerio）。首先，为了改进卵黄完整和去卵黄卵裂期胚胎中 PGC 的玻璃化程序，用绿色荧光蛋白标记 PGC 后，将卵黄完整和去卵黄胚胎依次暴露于平衡溶液（ES）和玻璃化溶液（VS）中，迅速冷却，VS 含有乙二醇、DMSO 和蔗糖。去卵黄处理可防止胚胎在冷却过程中形成冰，并提高冷冻保存 PGC 的活力。去卵黄胚胎在最佳 ES 和 VS 暴露后玻璃化的活 PGC 的最大回收率估计约为 90%，约 50%的活 PGC 表现出伪足运动。接下来，为了阐明冷冻保存 PGC 分化为功能性配子的能力，从最佳 ES 和 VS 暴露下玻璃化的去卵黄胚胎（条纹型）中回收 PGC 并单独移植到 218 个无菌受体囊胚（金色型）中。移植后 2 天，形态正常的胚胎中有 7.5%（14/187）在生殖嵴中出现 PGC。在 14 个受体胚胎中，有 6 个（5 个雄性和 1 个雌性）发育成熟，并产生了具有源自玻璃化胚胎 PGC 供体遗传特征的后代。总之，我们通过去卵黄胚胎的玻璃化成功地冷冻保存了 PGC，并获得了具有源自玻璃化胚胎 PGC 的生殖细胞的可育斑马鱼。
