Laboratory of Animal Science, College of Agriculture, Kochi University, Nankoku, Kochi 783-8502, Japan.
Cryobiology. 2011 Feb;62(1):8-14. doi: 10.1016/j.cryobiol.2010.11.003. Epub 2010 Nov 27.
As a step to develop a cryopreservation method for zebrafish oocytes, we investigated the cryobiological properties of immature oocytes at stage III by examining their ability to mature and to develop into hatching embryos after fertilization. When oocytes were chilled at -5°C for 30min, the maturation rate decreased, but the rates of fertilization and hatching were not significantly different from those of controls. When oocytes were exposed to hypotonic solutions for 60min at 25°C, the rates of maturation, fertilization, and hatching decreased in a solution with 0.16Osm/kg or below. When oocytes were exposed to hypertonic solutions (containing sucrose) at 25°C for 30min, the maturation rate decreased in solution with 0.51Osm/kg, whereas the hatching rate decreased with lower osmolality (0.40Osm/kg). In an experiment on the toxicity of cryoprotectants (∼10%, at 25°C), it was found that glycerol and ethylene glycol were toxic both by the assessment of maturation and hatching. Propylene glycol, DMSO and methanol were less toxic by the assessment of maturation, but were found to be toxic by the assessment of hatching. Methanol was the least toxic, but it was less effective to make a solution vitrify than propylene glycol. Therefore, a portion of methanol was replaced with propylene glycol. The replacement increased the toxicity, but could be effective to reduce chilling injury at -5°C. These results clarified the sensitivity of immature oocytes to various cryobiological properties accurately, which will be useful for realizing cryopreservation of zebrafish oocytes.
为了开发斑马鱼卵母细胞的冷冻保存方法，我们通过研究未成熟卵母细胞在 III 期的冷冻生物学特性，来评估其受精后成熟和孵化的能力。当卵母细胞在-5°C下冷却 30 分钟时，其成熟率下降，但受精率和孵化率与对照组相比无显著差异。当卵母细胞在 25°C 下暴露于低渗溶液中 60 分钟时，在渗透压为 0.16Osm/kg 或以下的溶液中，其成熟率、受精率和孵化率均下降。当卵母细胞在 25°C 下暴露于高渗溶液（含蔗糖）30 分钟时，0.51Osm/kg 的溶液中成熟率下降，而孵化率则随渗透压降低而下降（0.40Osm/kg）。在一项关于冷冻保护剂毒性的实验（25°C 时约为 10%）中，发现甘油和乙二醇通过成熟和孵化评估均具有毒性。丙二醇、DMSO 和甲醇通过成熟评估的毒性较小，但通过孵化评估发现具有毒性。甲醇毒性最小，但使溶液玻璃化的效果不如丙二醇。因此，用一部分甲醇代替丙二醇。这种替代增加了毒性，但可以有效地降低在-5°C 下的冷却损伤。这些结果准确地阐明了未成熟卵母细胞对各种冷冻生物学特性的敏感性，这对于实现斑马鱼卵母细胞的冷冻保存将是有用的。
