Artificial expression of aquaporin-3 improves the survival of mouse oocytes after cryopreservation
Artificial expression of aquaporin-3 improves the survival of mouse oocytes after cryopreservation
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DOI：
10.1095/biolreprod.101.002394
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发表时间：
2003-01-01
影响因子：
3.6
通讯作者：
Kasai, M
中科院分区：
文献类型：
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作者：
Edashige, K;Yamaji, Y;Kasai, M
哺乳动物细胞的成功冷冻保存需要水和冷冻保护性溶质通过质膜的快速运输。水通道蛋白-3是一种水/溶质通道，可以运输水和甘油等中性溶质。在这项研究中，我们研究了在小鼠卵母细胞中人工表达水通道蛋白-3是否可以改善水和甘油的通透性以及卵母细胞冷冻后的存活率。将小鼠未成熟卵母细胞注射水通道蛋白3cRNA，培养12h后，以10%甘油在25℃的PB1培养液中的相对体积变化为指标，测定成熟卵母细胞的渗透系数(P-Gly)和渗透系数(P-Gly)。注射CRNA的卵母细胞的平均+/-SD值(3.09+/-1.22um min(-1)atm(-1)和3.69+/-1.47×10(-3)cm/min；卵母细胞数=25)显著高于未注射的卵母细胞(0.83+/-0.02um min(-1)atm(-1)和0.07+/-0.02×10(-3)cm/min)；N=13)和注水卵母细胞(0.87+/-0.10um min(-1)atm(-1)和0.08+/-0.02×10(-3)cm/min；n=20)。在以甘油为基础的溶液中冷冻保存后，注射CRNA的卵母细胞(n=27)存活了74%，而注射水的卵母细胞(n=10)无一存活。对冷冻后存活的卵母细胞进行体外受精，其穿透率为40%(n=48)，卵裂率为31%(n=70)，表明卵母细胞仍具有受精能力。这是第一个表明在细胞中人工表达水/溶质通道可以提高细胞在冷冻后的存活率的报告。这种方法可能使冷冻保存一直难以冷冻保存的细胞成为可能。
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 25degreesC. Mean +/- SD values of L-p and P-GLY of cRNA-injected oocytes (3.09 +/- 1.22 mum 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 mum 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 mum 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.