Takeo Toru, Sztein Jorge, Nakagata Naomi
Division of Reproductive Engineering, Center for Animal Resources and Development (CARD), Kumamoto University, Kumamoto, Japan.
Methods Mol Biol. 2019;1874:243-256. doi: 10.1007/978-1-4939-8831-0_14.
In the last few years sperm cryopreservation was rapidly established as the technique to efficiently manage production, preservation, and transportation of genetically engineered mice. However, occasionally, the reduced fertility of the frozen-thawed mouse sperm can make it difficult to revitalize the mouse by in vitro fertilization (IVF). In particular, the frozen-thawed sperm of C57BL/6 mice, widely used as the background of choice for genetically engineered strains, show very low fertility after freezing and thawing. To overcome this problem, we have developed a new protocol for sperm cryopreservation and IVF with frozen-thawed C57BL/6 sperm as well as other mouse strains. This protocol has the following three modifications: (1) addition of L-glutamine to the sperm cryoprotectant, (2) addition of methyl-β-cyclodextrin to the sperm preincubation medium, and (3) addition of reduced glutathione to the fertilization medium. These modifications greatly enhanced the fertility of frozen-thawed C57BL/6 sperm, resulting in a stable fertilization rate >80% in IVF. Our results indicate that this robust protocol for sperm cryopreservation may improve the archiving and distributing system for genetically engineered mice.
在过去几年中，精子冷冻保存迅速成为高效管理基因工程小鼠的生产、保存和运输的技术。然而，偶尔，冷冻解冻后的小鼠精子生育力降低可能会使通过体外受精（IVF）使小鼠恢复活力变得困难。特别是，广泛用作基因工程品系首选背景的C57BL/6小鼠的冷冻解冻精子在冷冻和解冻后显示出非常低的生育力。为了克服这个问题，我们开发了一种新的精子冷冻保存和使用冷冻解冻的C57BL/6精子以及其他小鼠品系进行IVF的方案。该方案有以下三点改进：（1）在精子冷冻保护剂中添加L-谷氨酰胺，（2）在精子预孵育培养基中添加甲基-β-环糊精，（3）在受精培养基中添加还原型谷胱甘肽。这些改进极大地提高了冷冻解冻的C57BL/6精子的生育力，导致IVF中的稳定受精率>80%。我们的结果表明，这种强大的精子冷冻保存方案可能会改善基因工程小鼠的存档和分发系统。
