Aoto Toshihiro, Totsuka Yoshikazu, Takahashi Riichi, Ueda Masatsugu
The YS Institute, Utsonomiya, Tochigi, Japan.
J Am Assoc Lab Anim Sci. 2007 May;46(3):41-6.
Methods for generating genetically engineered mice have progressed, and the number of valuable mouse strains has increased rapidly, requiring methods for managing and maintaining these strains. Sperm cryopreservation and assisted-reproduction techniques, such as intracytoplasmic sperm injection (ICSI), can contribute greatly; however, the number of possible progeny is limited due to the finite number of cryopreserved preparations. The ability to refreeze and reuse sperm preparations would extend the utility of each cryopreserved sperm preparation. The purpose of this study was to develop a reproduction protocol involving ICSI that yielded live progeny after repeated freezing and thawing of a cryopreserved sperm preparation. We used mouse sperm subjected to repeat freezing and thawing in TYH medium for in vitro fertilization. Three inbred strains of laboratory mice--C57BL/6J, BALB/cA, and C3H/HeN--were reproduced by ICSI after reuse of a sperm preparation that had been frozen and thawed repeatedly. In particular, C57BL/6J progeny could be reproduced from spermatozoa frozen and thawed 10 times. From these results, we conclude that the reuse of cryopreserved spermatozoa can extend the opportunities for reproduction of progeny from cryopreserved sperm and can increase the utility of cryopreserved preparations as bioresources. Our results broaden management options regarding bioresource banking, particularly for mice.
基因工程小鼠的制备方法不断进步，有价值的小鼠品系数量迅速增加，这就需要对这些品系进行管理和保存的方法。精子冷冻保存和辅助生殖技术，如胞浆内单精子注射（ICSI），能发挥很大作用；然而，由于冷冻保存制剂数量有限，可能产生的后代数量也受到限制。重新冷冻和再利用精子制剂的能力将扩展每个冷冻保存精子制剂的效用。本研究的目的是开发一种涉及ICSI的繁殖方案，该方案在冷冻保存的精子制剂反复冻融后能产生存活后代。我们使用在TYH培养基中反复冻融的小鼠精子进行体外受精。三种近交系实验小鼠——C57BL/6J、BALB/cA和C3H/HeN——在反复冻融的精子制剂再利用后通过ICSI进行繁殖。特别是，C57BL/6J后代可以从冻融10次的精子中繁殖出来。从这些结果中，我们得出结论，冷冻保存精子的再利用可以扩大冷冻保存精子产生后代的繁殖机会，并可以提高冷冻保存制剂作为生物资源的效用。我们的结果拓宽了生物资源库的管理选择，特别是对于小鼠。
