Artificial Expression of Aquaporin-3 Improves the Survival of Mouse Oocytes after Cryopreservation1

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 (L(P)) and glycerol permeability (P(GLY)) of matured oocytes were determined from the relative volume changes in 10% glycerol in PB1 medium at 25 degrees C. Mean +/- SD values of L(P) and P(GLY) of cRNA-injected oocytes (3.09 +/- 1.22 micro m 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 micro m 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 micro m 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.

Role of membrane transport of water and glycerol in the freeze tolerance of the rice stem borer, Chilo suppressalis Walker (Lepidoptera: Pyralidae)

“…It has already been reported that some water channels, such as human aquaporin 3, transport not only water but also glycerol (Ishibashi, et al 1994). Moreover, success in cryopreservation when aquaporin 3 is artificially expressed has been reported for mouse oocytes (Edashige et al 2003) and zebrafish embryos (Hagedorn et al 2002).…”

“…Deletion of the relevant genes raised sensitivity to freezing, whereas their overexpression improved freeze tolerance (Tanghe et al 2002). In mammals, artificial expression of aquaporin-3 improves the survival of mouse oocytes after cryopreservation using glycerol-based solution (Edashige et al 2003). Previously, there had been no report of mouse oocytes surviving after cryopreservation using glycerol-based solutions because of the low permeability of mouse oocytes to glycerol.…”

Developmental Ability of Vitrified Mouse Oocytes Expressing Water Channels

“…At one site, the oocyte was held with a holding pipette connected to a micromanipulator, and at the other, AQP3 cRNA or water was injected with an injection pipette connected to another micromanipulator on an inverted microscope. Injected oocytes were cultured in modified Eagle's medium supplemented with 10% fetal calf serum, 50 μg/ml sodium pyruvate, 2 mM glutamine, 60 μg/ml penicillin G and 50 μg/ml streptomycin for 6 h to obtain maturing oocytes and for 12 h to obtain mature oocytes (metaphase II) [8]. Then, the cumulus cells were completely removed from oocytes by pipetting in PB1 medium containing 0.5 mg/ml hyaluronidase.…”

“…However, the tolerance of mammalian oocytes to cryopreservation is lower and remains less practical than that of embryos. Increasing the permeability of oocytes would improve their tolerance to vitrification and make their cryopreservation more practical.Previously, we injected AQP3 cRNA into mouse oocytes at the germinal vesicle stage, cultured the oocytes until they matured to metaphase II to express AQP3 and examined whether the exogenous expression enhanced the tolerance of the oocytes to vitrification with a glycerol-based solution [8]. The permeability of AQP3 cRNA-injected oocytes to water and glycerol increased and a high proportion of the oocytes survived vitrification, whereas no water-injected oocytes survived.…”

“…The relative volume was obtained from V=S 3/2 . The water-permeability and glycerol-permeability of oocytes/embryos were determined by fitting water-and solute-movement using a twoparameter formalism [8,15]. Various constants and parameters are listed in Table 1.…”