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Embryo |
a Laboratory of Animal Science, College of Agriculture, Kochi University, Nankoku, Kochi 783-8502, Japan
b Department of Physics, Indianapolis University and Purdue University Indianapolis, Indianapolis, Indiana 46202
Successful cryopreservation of mammalian cells requires rapid transport of water and cryoprotective solutes across the plasma membrane. Aquaporin-3 is known as a water/solute channel that can transport water and neutral solutes such as glycerol. In this study we examined whether artificial expression of aquaporin-3 in mouse oocytes can improve water and glycerol permeability and oocyte survival after cryopreservation. Immature mouse oocytes were injected with aquaporin-3 cRNA and were cultured for 12 h. Then the hydraulic conductivity (LP) and glycerol permeability (PGLY) of matured oocytes were determined from the relative volume changes in 10% glycerol in PB1 medium at 25°C. Mean ± SD values of LP and PGLY of cRNA-injected oocytes (3.09 ± 1.22 µm min-1 atm-1 and 3.69 ± 1.47 x 10-3 cm/min, respectively; numbers of oocytes = 25) were significantly higher than those of noninjected oocytes (0.83 ± 0.02 µm min-1 atm-1 and 0.07 ± 0.02 x 10-3 cm/min, respectively; n = 13) and water-injected oocytes (0.87 ± 0.10 µm min-1 atm-1 and 0.08 ± 0.02 x 10-3 cm/min, respectively; n = 20). After cryopreservation in a glycerol-based solution, 74% of cRNA-injected oocytes (n = 27) survived as assessed by their morphological appearance, whereas none of the water-injected oocytes survived (n = 10). When cRNA-injected oocytes that survived cryopreservation were inseminated in vitro, the penetration rate was 40% (n = 48) and the cleavage rate was 31% (n = 70), showing that oocytes retain their ability to be fertilized. This is the first report to show that artificial expression of a water/solute channel in a cell improves its survival after cryopreservation. This approach may enable cryopreservation of cells that have been difficult to cryopreserve.
2 Correspondence. FAX: 81 88 864 5219; keisuke{at}cc.kochi-u.ac.jp
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