| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2003: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2002: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Research Abstract |
Water-and cryoprotectant-permeability of the plasma membrane of oocytes and embryos plays vital roles on their tolerance to cryopreservation. I studied whether artificial expression of aquaporins, water channels, in mouse oocytes and zebrafish oocytes and embryos can improve water-and cryoprotectant-permeability of them and whether the expression can improve the tolerance of them to cry preservation. Injection of aquaporin-3 cRNA in mouse oocytes significantly increased water-and glycerol permeability, and the tolerance to vitrification with glycerol-based solutions, with which intact oocytes did not survive after vitrification. Many of the cRNA-injected and cryopreserved oocytes were fertilized by in vitro fertilization. These results indicate that artificial expression of water/cryoprotectant channels in the cell can improve the tolerance to cryopreservation. I also studied whether injection of aquaporin cRNA into zebrafish oocytes and embryos can also improve their water-and cryopro
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tectant-permeability. In the case of oocytes, injection of aquqprin-3 cRNA appeared to increase water-and cryoprotectant permeability of the oocytes. In the case of embiyos, however, increased water-and cryoprotectant-permeability could not be elucidated because the aquaporin-7 cRNA-injected embryos did not show typical shrink-swell volume curves. Fish oocytes could be cryopreserved by artificial expression of water/cryoprotectant channels in them.
Water-and cryoprotectant-permeability is an important cryobiological property that influences on cryoinjuries caused by intracellular ice-formation and chemical toxicity of cryoprotectants. If it were possible to deduce which type of injury had occurred during cryopreservation of embryos by their appearance, it would help to optimize cryopreservation protocols. When mouse embryos at the 2-cell stage and the blastocyste stage were injured by intracellular ice formation, they swelled in isotonic PB1 medium just after being thawed and diluted. When blastocytes were injured by chemical toxicity of cryoprotectants during cryopreservation, they showed decompaction after 1 h of culture. In the case of 2-cell embryos, they looked normal after being thawed and diluted. However, they did not develop to the 4-cell stage after 24 h of culture. Thus, it is possible to deduce which type of injury had occurred in cryopreserved embryos from their appearance at recovery and during subsequent culture. This may help to improve cryopreservation protocols for embryos of many species. Less
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