Artificial expression of aquaporin-3 improves the survival of mouse oocytes after cryopreservation

Abstract

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 × 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 × 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 × 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.