| 研究実績の概要 |
We have clarified that vitrification of porcine oocytes at the immature stage does not damage the energy and redox balance and cytoplasmic maturation in oocytes, and gap junctional communications between oocytes and cumulus cells. We found no evidence for altered mRNA and Ca2+ levels in vitrified oocytes. However, we revealed that vitrification triggers premature meiotic resumption and causes sub-lethal damages in the nucleolus and microfilaments from which some oocytes could recover during subsequent IVM culture. Supplementation of the IVM medium with the antiapoptotic agent resveratrol improved the developmental competence of vitrified oocytes and therefore it is recommended to enhance the recovery process. The use of alternative cryoprotectant agents (CPAs) such as polyethylene glycol and Supercool X-1000 did not improve the efficacy of the vitrification system. However, the detrimental effects of the vitrification protocol could be reduced by determining the optimum duration and temperature for CPA treatment (30 sec and 25 C, respectively), omitting cytochalasin B from the system and the replacement of bovine serum albumin with polyvinyl pyrrolidone. These modifications significantly improved the survival rates to above 80% and the developmental competence of vitrified oocytes to the blastocyst stage to over 30%. In conclusion, by the above-mentioned modifications of both the vitrification and the IVM protocols, we could improve the efficacy of the oocyte cryopreservation system on the level of embryo development according to the original aim of the project.
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