Expression of aquaporin-3 improves the permeability to water and cryoprotectants of immature oocytes in the medaka (Oryzias latipes)

The permeability of the plasma membrane plays a crucial role in the successful cryopreservation of oocytes and embryos. Several efforts have been made to facilitate the movement of water and cryoprotectants across the plasma membrane of fish oocytes/embryos because of their large size. Aquaporin-3 is a water/solute channel that can also transport various cryoprotectants. In this study, we tried to improve the permeability of immature medaka (Oryzias latipes) oocytes to water and cryoprotectants by artificially expressing aquaporin-3. The oocytes were injected with aquaporin-3 cRNA and cultured for 6–7 h. Then, hydraulic conductivity (LP) and cryoprotectant permeability (PS) were determined from volume changes in a hypertonic sucrose solution and various cryoprotectant solutions, respectively, at 25 °C. The LP value of the cRNA-injected oocytes was 0.22 ± 0.04 μm/min/atm, nearly twice larger than that of intact or water-injected oocytes (0.14 ± 0.02 and 0.14 ± 0.03 μm/min/atm, respectively). PS values of intact oocytes for ethylene glycol, propylene glycol, and DMSO were 1.36 ± 0.34, 1.97 ± 0.20, and 1.17 ± 0.52 × 10−3 cm/min, respectively. The permeability to glycerol could not be calculated because oocytes remained shrunken in the glycerol solution. On the other hand, cRNA-injected oocytes had significantly higher PS values (glycerol, 2.20 ± 1.29; ethylene glycol, 2.98 ± 0.36; propylene glycol, 3.93 ± 1.70; DMSO, 3.11 ± 0.74 × 10−3 cm/min) than intact oocytes. When cRNA-injected oocytes were cultured for 12–14 h, 51% matured to the metaphase II stage, and 43% of the matured oocytes were fertilized and hatched following in vitro fertilization and 14 days of culture. Thus, the permeability of medaka oocytes to water and cryoprotectants was improved by the artificial expression of aquaporin-3, and the oocytes retained the ability to develop to term.