EDASHIGE Keisuke Kochi University, College of Agriculture, Associate Professor, 農学部, 助教授 (30175228)
KOSHIMOTO Chihiro University of Miyazaki, Frontier Science Research Center, Associate Professor, フロンティア科学実験総合センター, 助教授 (70295210)
|Budget Amount *help
¥15,000,000 (Direct Cost: ¥15,000,000)
Fiscal Year 2006: ¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 2005: ¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 2004: ¥9,000,000 (Direct Cost: ¥9,000,000)
The permeability of the plasma membrane to water and cryoprotectants is an important factor determining the tolerance of cells to cryopreservation and suitable condition for cryopreservation. There are two pathways for the movement of water and cryoprotectants through the plasma membrane, i.e., simple diffusion across the plasma membrane and facilitated diffusion via channel processes. In this study, we tried to clarify the relationship between the permeability of the plasma membrane and cryo-sensitivity in oocytes and embryos of various species.
As a model for mammals, we used mice, and the permeability to water and cryoprotectants of oocytes and embryos at various stages were examined from the viewpoint of the expression of a water/cryoprotectant channel. It was found that in embryos at the morula stage, water and some cryoprotectants moved rapidly through the plasma membrane predominantly by facilitated diffusion via aquaporin 3 (AQP3), a water/cryoprotectant channel, whereas in oocy
tes and embryos at early cleaved stages, water and cryoprotectants moved slowly through the plasma membrane predominantly by simple diffusion.
In fish and amphibians, the cryopreservation of oocytes and embryos has not been realized. We tried to find suitable conditions for the cryopreservation of the oocytes from the viewpoint of membrane permeability. We used medaka and zebrafish as models for fish, and Xenopus laevis as a model for amphibians. In medaka and zebrafish, the permeability to water and cryoprotectants of the plasma membrane was higher in immature oocytes than mature oocytes. The high permeability of immature oocytes was increased further by the artificial expression of AQP3 by injecting AQP3 cRNA into them. Similar increase in the permeability to water and cryoprotectants was observed in AQP3 cRNA-injected immature Xenopus oocytes. However, considering the large volume of the oocyte in these animals, further increase in membrane-permeability would be necessary for successful cryopreservation of oocytes.
In addition, we tried to cryopreserve insect embryos, derived from parthenogenetically activated oocytes of Nasonia vitripennis. Removal of the wax layer of Nasonia embryos was effective to make them permeable to water and cryoprotectants (ethylene glycol and DMSO). When wax-removed embryos were vitrified with ethylene glycol-based solutions, many embryos survived, and some (〜30%) of them developed to larvae. Therefore, the conservation of various strains of Nasonia vitripennis by the cryopreservation of embryos would become possible. Less