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The modulation of the tolerance of oocytes to cryopreservation by artificial expression of water channels -vertebrates and invertebrates-

通过水通道的人工表达调节卵母细胞对冷冻保存的耐受性-脊椎动物和无脊椎动物-

基本信息

批准号：
16380190
负责人：
KASAI Magosaburo
金额：
$ 9.6万
依托单位：
Kochi University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2006
项目状态：
已结题

项目摘要

The permeability of the plasma membrane to water and cryoprotectants is an important factor determining the tolerance of cells to cryopreservation and suitable condition for cryopreservation. There are two pathways for the movement of water and cryoprotectants through the plasma membrane, i.e., simple diffusion across the plasma membrane and facilitated diffusion via channel processes. In this study, we tried to clarify the relationship between the permeability of the plasma membrane and cryo-sensitivity in oocytes and embryos of various species.As a model for mammals, we used mice, and the permeability to water and cryoprotectants of oocytes and embryos at various stages were examined from the viewpoint of the expression of a water/cryoprotectant channel. It was found that in embryos at the morula stage, water and some cryoprotectants moved rapidly through the plasma membrane predominantly by facilitated diffusion via aquaporin 3 (AQP3), a water/cryoprotectant channel, whereas in oocy … More tes and embryos at early cleaved stages, water and cryoprotectants moved slowly through the plasma membrane predominantly by simple diffusion.In fish and amphibians, the cryopreservation of oocytes and embryos has not been realized. We tried to find suitable conditions for the cryopreservation of the oocytes from the viewpoint of membrane permeability. We used medaka and zebrafish as models for fish, and Xenopus laevis as a model for amphibians. In medaka and zebrafish, the permeability to water and cryoprotectants of the plasma membrane was higher in immature oocytes than mature oocytes. The high permeability of immature oocytes was increased further by the artificial expression of AQP3 by injecting AQP3 cRNA into them. Similar increase in the permeability to water and cryoprotectants was observed in AQP3 cRNA-injected immature Xenopus oocytes. However, considering the large volume of the oocyte in these animals, further increase in membrane-permeability would be necessary for successful cryopreservation of oocytes.In addition, we tried to cryopreserve insect embryos, derived from parthenogenetically activated oocytes of Nasonia vitripennis. Removal of the wax layer of Nasonia embryos was effective to make them permeable to water and cryoprotectants (ethylene glycol and DMSO). When wax-removed embryos were vitrified with ethylene glycol-based solutions, many embryos survived, and some (〜30%) of them developed to larvae. Therefore, the conservation of various strains of Nasonia vitripennis by the cryopreservation of embryos would become possible. Less

质膜对水和冷冻保护剂的渗透性是决定细胞对低温保存耐受性和低温保存适宜条件的重要因素。水和冷冻保护剂通过质膜的运动有两种途径，即通过质膜的简单扩散和通过通道过程的促进扩散。在本研究中，我们试图阐明不同物种卵母细胞和胚胎的质膜通透性与低温敏感性之间的关系。作为哺乳动物模型，我们使用小鼠，从水/冷冻保护剂通道表达的角度研究了不同阶段卵母细胞和胚胎对水和冷冻保护剂的渗透性。研究发现，在桑葚胚期，水分和一些低温保护剂主要通过水通道蛋白3 （AQP3）快速通过质膜，而在卵泡期和卵泡裂胚期，水分和低温保护剂主要通过简单扩散通过质膜。在鱼类和两栖动物中，卵母细胞和胚胎的冷冻保存尚未实现。我们试图从卵母细胞膜通透性的角度寻找适合卵母细胞低温保存的条件。我们用水母和斑马鱼作为鱼类的模型，用爪蟾作为两栖动物的模型。在medaka和斑马鱼中，未成熟卵母细胞对水和低温保护剂的渗透性高于成熟卵母细胞。通过向未成熟卵母细胞中注入AQP3 cRNA，人工表达AQP3，进一步提高其高通透性。在注射AQP3 crna的未成熟爪蟾卵母细胞中，观察到对水和冷冻保护剂的渗透性也有类似的增加。然而，考虑到这些动物的卵母细胞体积大，进一步增加膜通透性是成功冷冻保存卵母细胞的必要条件。此外，我们还尝试了低温保存昆虫胚胎，这些胚胎来自于孤雌激活的玻璃嫩鼻虫卵母细胞。去除蜡层可以有效地使纳索蝇胚对水和低温保护剂（乙二醇和二甲基亚砜）具有渗透性。当去蜡胚用乙二醇基溶液玻璃化时，许多胚存活，其中一些（约30%）发育成幼虫。因此，利用胚胎低温保存方法保存不同品系的玻璃喙蝇是可能的。少