Successful freezing of unfertilized mouse oocytes and effect of cocultures in oviducts on development of in vitro fertilized embryos after thawing

To establish a freeze-thawing method for unfertilized oocytes with a high success rate, we examined several conditions for freeze-thawing. The effects of EDTA and cocultures in oviducts on the development of embryos fertilized in vitro after thawing were also studied. In the first experiment, unfertilized oocytes that were frozen in 1.5 M dimethylsulfoxide (DMSO) supplemented with 0.2 M sucrose by a slow freeze-thawing method showed the best results (fertilization rate, 71.9%; blastocyst rate per frozen oocyte, 18.8%). The proportion of embryos that developed to blastocysts was significantly higher when DMSO was added at 4 degrees C than at room temperature (39.4 vs 19.4%; P < 0.01). The addition of EDTA (10 microM) to the culture medium did not promote embryo development after fertilization in vitro. However, the rate of development of in vitro fertilized embryos to blastocysts after thawing was significantly higher when the embryos were cultured in oviducts in vitro than the rates in control cultures and those cultured with EDTA (blastocyst rate from fertilized oocytes, 71.4 vs 51.0 and 52.8%, respectively; P < 0.01). Unfertilized mouse oocytes can be cryopreserved successfully by a slow freeze-thawing method with the addition of 1.5 M DMSO and 0.2 M sucrose at low temperatures, and coculture with oviducts enhances the development of embryos that are fertilized in vitro after thawing.