title: 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 (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.
authors.affiliation: Laboratory of Animal Science, College of Agriculture, Kochi University, Nankoku, Kochi 783-8502, Japan. keisuke@cc.kochi-u.ac.jp
authors.forename: Keisuke
authors.initials: K
authors.lastname: Edashige
authors.forename: Yohei
authors.initials: Y
authors.lastname: Yamaji
authors.forename: F W
authors.initials: FW
authors.lastname: Kleinhans
authors.forename: Magosaburo
authors.initials: M
authors.lastname: Kasai
mesh_terms: D000818:Animals; D051400:Aquaporin 3; D020346:Aquaporins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001693:Biological Transport, Active; D002463:Cell Membrane Permeability; D002470:Cell Survival; D015925:Cryopreservation / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D005307:Fertilization in Vitro; D015870:Gene Expression; D005990:Glycerol / Q000493:pharmacokinetics; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D008845:Microinjections; D009865:Oocytes / Q000166:cytology / Q000378:metabolism; D018075:RNA, Complementary / Q000008:administration & dosage / Q000235:genetics; D014867:Water / Q000378:metabolism
chemical_list: C496028:Aqp3 protein, mouse; D020346:Aquaporins; D018076:DNA, Complementary; D018075:RNA, Complementary; D014867:Water; D051400:Aquaporin 3; D005990:Glycerol