CN114009424A - 一种gv期卵母细胞的冷冻保存及解冻方法 - Google Patents
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Abstract
一种GV期卵母细胞的冷冻保存及解冻方法，属于卵母细胞冷冻技术领域。为了解决玻璃化冷冻法冷冻保存GV期卵母细胞存在的冷冻解冻后卵母细胞细胞器受到冷冻损伤而导致受精卵裂率、囊胚发育率低的问题，本发明采用cryoloop为载体对GV期卵母细胞进行冷冻保存，以冷冻基础液为基质，以DMSO和乙二醇为冷冻保护剂，冷冻、解冻过程中添加原花青素，解决了现有卵母细胞冷冻保存解冻后存活率及发育率低的问题。本发明提供的GV期卵母细胞的冷冻保存及解冻方法能够提高GV期卵母细胞冷冻保存效率，而且成本低廉，便于广泛推广，适用于哺乳动物或人的GV期卵母细胞的冷冻保存。
Description
技术领域
本发明涉及一种GV期卵母细胞的冷冻保存及解冻方法，属于卵母细胞冷冻技术领域。
技术背景
妇科肿瘤患者的年轻化，卵巢早衰及不孕症患者日益增多，使卵母细胞冷冻保存成为医学界研究的热点。另外，卵母细胞冷冻保存可以使卵母细胞资源得到充分利用，为体外成熟﹑体外授精和核移植等胚胎工程技术提供丰富﹑廉价的卵母细胞，克服卵母细胞应用在时间和空间的局限，是加快家畜品种改良和胚胎移植技术产业化的重要组成部分。卵母细胞的冷冻也是保护品种资源和拯救濒危动物不可缺少的技术。
由于卵母细胞是哺乳动物体内最大的细胞，其细胞结构特点决定了卵母细胞较其他细胞相对较难冷冻。与胚胎相比，卵母细胞表面积与体积的比值小，水分及其他冷冻保护剂通过细胞膜出入的速度较慢，冷冻过程中易形成冰晶。另外，冷冻可造成卵母细胞皮质颗粒提前释放和透明带硬化，造成多精受精等问题，冷冻还造成卵胞质内线粒体﹑脂滴﹑内质网等的损伤，氧化系统和抗氧化系统异常以及膜电位降低，最终降低卵母细胞受精率及随后的发育潜能。目前较为常用的卵母细胞冷冻保存技术有慢速冷冻法和玻璃化冷冻法，前者因需要昂贵的仪器设备，操作过程复杂，容易形成冰晶，对卵母细胞细胞器损伤更大而逐渐被玻璃化冷冻保存取代。
对于体外获得的未成熟卵母细胞，如果在第一次减数分裂前期(germimalvesicle，GV)冷冻，可以避开成熟卵母细胞必须面对的纺锤体和染色体损伤；相较于成熟卵母细胞体积较小，分化程度低，细胞器少且分布均匀，细胞膜对水的通透性变化小。因此，以生发泡(Germinalvesicle，GV)期卵母细胞为冷冻对象比成熟卵母细胞更具有优势。
尽管目前采用玻璃化冷冻法冷冻保存GV期卵母细胞已经取得很大的进展，但是冷冻解冻后的卵母细胞受精卵裂率、囊胚发育率仍然不理想。
发明内容
本发明为了解决玻璃化冷冻法冷冻保存GV期卵母细胞存在的冷冻解冻后卵母细胞细胞器受到冷冻损伤而导致受精卵裂率、囊胚发育率低的问题，提供了一种GV期卵母细胞的冷冻保存方法，所述方法是将GV期卵母细胞在平衡液中平衡3分钟，然后转移到冷冻保存液中，再立即转移到冷冻环上，并迅速投入液氮中冷冻；
所述平衡液由冷冻基础液、原花青素、乙二醇、二甲基亚砜组成；原花青素的浓度为100-200μM，乙二醇的体积浓度为7.5％，二甲基亚砜的体积浓度为7.5％；
所述冷冻保存液由冷冻基础液、蔗糖、原花青素、乙二醇和二甲基亚砜组成；原花青素的浓度为100-200μM，乙二醇的体积浓度为15％，二甲基亚砜的体积浓度为15％，蔗糖的浓度0.5M；
所述冷冻基础液由培养液和胎牛血清组成；所述培养液为DMEM培养液、TCM199培养液、M16培养液和MEM培养液中的任意一种，所述胎牛血清的添加量为20％(v/v)。
本发明还提供了一种GV期卵母细胞的解冻方法，所述方法是将冷冻保存的卵母细胞从液氮中取出立即放到解冻液Ⅰ中3分钟，再转移到解冻液Ⅱ中3分钟，之后转移到基础液中3分钟，即完成冷冻卵母细胞的解冻；
所述解冻液Ⅰ由冷冻基础液、原花青素和蔗糖组成，解冻液Ⅰ中原花青素的浓度为100-200μM，蔗糖的浓度为0.5M；
所述解冻液Ⅱ由冷冻基础液、原花青素和蔗糖组成，解冻液Ⅱ中原花青素的浓度为100-200μM，蔗糖的浓度为0.25M；
所述冷冻基础液由培养液和胎牛血清组成；所述培养液为DMEM培养液、TCM199培养液、M16培养液和MEM培养液中的任意一种，所述胎牛血清的添加量为20％(v/v)。
进一步地限定，解冻过程均在37℃环境中进行。
进一步地限定，所述基础液由冷冻基础液和原花青素组成，原花青素的浓度为100-200μM。
进一步地限定，在上述GV期卵母细胞的冷冻保存方法或上述GV期卵母细胞的解冻方法中，所述GV期卵母细胞为哺乳动物或人的GV期卵母细胞。
本发明的有益效果：
本发明提供的GV卵母细胞的冷冻保存及解冻方法可有效降低卵母细胞冷冻解冻后细胞器损伤，从而提高卵母细胞经体外受精或孤雌激活后的胚胎发育率。采用本发明所述GV期卵母细胞的冷冻保存及解冻方法获得的GV卵母细胞，经体外成熟后受精，囊胚率提高了12.8％，孤雌激活囊胚率提高了5.1％。
本发明GV期卵母细胞冷冻保存及解冻方法能够降低玻璃化冷冻引起的卵母细胞细胞器损伤，提高卵母细胞冷冻保存效率，而且成本低廉，便于广泛推广，适用于哺乳动物或人的GV期卵母细胞的冷冻保存。
具体实施方式
实施例1：GV期卵母细胞的冷冻保存方法
冷冻保存方法为将GV期卵母细胞在平衡液中平衡3分钟，然后转移到冷冻保存液中，再立即转移到冷冻环上，并迅速投入液氮中冷冻。
其中，平衡液由冷冻基础液、原花青素、乙二醇、二甲基亚砜组成；原花青素的浓度为100μM，乙二醇的体积浓度为7.5％，二甲基亚砜的体积浓度为7.5％；
冷冻保存液由冷冻基础液、蔗糖、原花青素、乙二醇和二甲基亚砜组成；原花青素的浓度为100μM，乙二醇的体积浓度为15％，二甲基亚砜的体积浓度为15％，蔗糖的浓度0.5M；
冷冻基础液由培养液和胎牛血清组成；培养液为DMEM培养液、TCM199培养液、M16培养液和MEM培养液中的任意一种，胎牛血清的添加量为20％(v/v)。
本实施方式中GV期卵母细胞可为哺乳动物或人的GV期卵母细胞。
实施例2：GV期卵母细胞的冷冻保存方法
冷冻保存方法为将GV期卵母细胞在平衡液中平衡3分钟，然后转移到冷冻保存液中，再立即转移到冷冻环上，并迅速投入液氮中冷冻。
其中，平衡液由冷冻基础液、原花青素、乙二醇、二甲基亚砜组成；原花青素的浓度为200μM，乙二醇的体积浓度为7.5％，二甲基亚砜的体积浓度为7.5％；
冷冻保存液由冷冻基础液、蔗糖、原花青素、乙二醇和二甲基亚砜组成；原花青素的浓度为200μM，乙二醇的体积浓度为15％，二甲基亚砜的体积浓度为15％，蔗糖的浓度0.5M；
冷冻基础液由培养液和胎牛血清组成；培养液为DMEM培养液、TCM199培养液、M16培养液和MEM培养液中的任意一种，胎牛血清的添加量为20％(v/v)。
本实施方式中GV期卵母细胞可为哺乳动物或人的GV期卵母细胞。
实施例3：GV期卵母细胞的解冻方法
冷冻保存解冻方法为将冷冻保存的卵母细胞从液氮中取出立即放到解冻液Ⅰ中3分钟，再转移到解冻液Ⅱ中3分钟，之后转移到基础液中3分钟，即完成冷冻卵母细胞的解冻，解冻过程均在37℃环境中进行。
其中，解冻液Ⅰ由冷冻基础液、原花青素和蔗糖组成，解冻液Ⅰ中原花青素的浓度为100μM，蔗糖的浓度为0.5M；
解冻液Ⅱ由冷冻基础液、原花青素和蔗糖组成，解冻液Ⅱ中原花青素的浓度为100μM，蔗糖的浓度为0.25M；
冷冻基础液由培养液和胎牛血清组成；培养液为DMEM培养液、TCM199培养液、M16培养液和MEM培养液中的任意一种，胎牛血清的添加量为20％(v/v)；
基础液由冷冻基础液和原花青素组成，原花青素的浓度为100μM。
本实施方式中GV期卵母细胞可为哺乳动物或人的GV期卵母细胞。
实施例4：GV期卵母细胞的解冻方法
冷冻保存解冻方法为将冷冻保存的卵母细胞从液氮中取出立即放到解冻液Ⅰ中3分钟，再转移到解冻液Ⅱ中3分钟，之后转移到基础液中3分钟，即完成冷冻卵母细胞的解冻，解冻过程均在37℃环境中进行。
其中，解冻液Ⅰ由冷冻基础液、原花青素和蔗糖组成，解冻液Ⅰ中原花青素的浓度为200μM，蔗糖的浓度为0.5M；
解冻液Ⅱ由冷冻基础液、原花青素和蔗糖组成，解冻液Ⅱ中原花青素的浓度为200μM，蔗糖的浓度为0.25M；
冷冻基础液由培养液和胎牛血清组成；培养液为DMEM培养液、TCM199培养液、M16培养液和MEM培养液中的任意一种，胎牛血清的添加量为20％(v/v)；
基础液由冷冻基础液和原花青素组成，原花青素的浓度为200μM。
本实施方式中GV期卵母细胞可为哺乳动物或人的GV期卵母细胞。
实施例5：小鼠GV期卵母细胞的冷冻保存及解冻
将小鼠GV期卵母细胞在平衡液中平衡3分钟，然后转移到冷冻保存液中，再立即转移到冷冻环并迅速投入液氮中冷冻保存；
将冷冻保存的小鼠卵母细胞从液氮中取出立即放到解冻液Ⅰ中3分钟，再转移到解冻液Ⅱ中3分钟，之后转移到基础液中3分钟，即完成冷冻卵母细胞的解冻。
其中，平衡液、冷冻保存液、冷冻基础液、解冻液Ⅰ、解冻液Ⅱ和基础液的成分如实施例1和实施例3所示。
配制卵母细胞体外成熟液：45mL的TCM199培养液(购自GIBCO公司)中加入5mL胎牛血清和5000IU青霉素及5000μg链霉素，轻微搅拌混匀，再加入2.5IU的FSH、2.5IU的LH、50μg的17β-雌二醇、1.21mg的丙酮酸钠和500ng的EGF，轻轻混匀，静置2～3小时后，用Φ0.22μm的过滤器抽滤消毒后，分装到1.5mL离心管中，4℃下储存，备用。
卵母细胞体外成熟：将解冻的小鼠GV期卵母细胞放入37℃预热2小时的卵母细胞体外成熟培养液中，然后置于CO2体积浓度为5％的环境内成熟培养14小时或24小时。
卵母细胞线粒体测定：冷冻解冻卵母细胞经体外成熟后，脱去卵丘细胞，用0.5μMMitotracker Red(分子探针，USA)和10μg/ml Hoechst 33342染色，染色后用培养液清洗卵母细胞并固定于载玻片中，激光共聚焦扫描测定结果。
卵母细胞皮质颗粒测定：冷冻解冻卵母细胞经体外成熟后，脱去卵丘细胞，应用链蛋白酶去除卵母细胞透明带，卵母细胞经甲醛固定后，用PBS加0.1％BSA，0.75％甘氨酸和0.2％叠氮化钠阻滞，用阻滞液中添加0.1％Triton X-100进行穿透，卵母细胞经清洗后，用1μg/mlFITC，10μg/ml PI染色，清洗后固定于载玻片中，激光共聚焦扫描测定结果。
卵母细胞孤雌激活：小鼠卵母细胞采用SrCl2激活，卵母细胞先在含10mM SrCl2和CB的无钙CZB中培养2.5h，再在含有CB的含钙CZB中培养3.5h，之后转移至CZB中继续培养，培养48小时至4-细胞胚胎期转移至含糖CZB中培养至囊胚。
卵母细胞体外受精：选取性成熟(10～12周龄)昆明白雄鼠，已通过交配实验证明其有受精能力，颈椎脱臼法处死，从附睾尾和输精管收集精子，将收集的精子团块置入1mL含10mg/mL BSA的T6液滴内，用口吸管轻轻吹打，使精子团分散开，于37℃、5％CO2及饱和湿度的CO2培养箱内中孵育1.5小时左右，进行获能；在此期间用细胞计数板检测精子密度；再将IVM后14小时的卵母细胞在受精液(T6+20mg/mL BSA)中洗3次，移入已经平衡过夜的受精滴(20枚/40μL)中，加入适量体积的获能精子，使精子密度在1×106左右。培养6小时后，用无糖CZB液洗去卵母细胞周围附着精子，选取含两原核和第二极体的受精卵，置于无糖CZB中培养。
体外受精及孤雌激活后的胚胎继续培养7天，培养48小时记录2-细胞胚胎及4-细胞胚胎比率。
实施例6：小鼠GV期卵母细胞的冷冻保存及解冻
本实施例与实施例5的不同点在于常规培养液为M16培养液。
实施例7：小鼠GV期卵母细胞的冷冻保存及解冻
本实施例与实施例5的不同点在于冷冻基础液、平衡液、冷冻液和解冻液中不含原花青素。
实施例5-7解冻后卵母细胞经体外成熟后线粒体分布情况如表1所示。
表1实施例5-7解冻后卵母细胞经体外成熟后线粒体分布情况
由表1展示的结果可知，玻璃化冷冻、解冻保存过程中添加原花青素能够降低卵母细胞成熟后细胞器线粒体的损伤，提高正常线粒体分布的比例。
实施例5-7解冻后卵母细胞经体外成熟后皮质颗粒分布情况如表2所示。
表2实施例5-7解冻后卵母细胞经体外成熟后皮质颗粒分布情况
由表2展示的结果可知，玻璃化冷冻、解冻保存过程中添加原花青素能够降低卵母细胞成熟后细胞器皮质颗粒的损伤，提高卵母细胞皮质颗粒完全迁移比例。
实施例5-7解冻后卵母细胞的受精卵裂率、受精囊胚发育率、孤雌激活卵裂率和孤雌激活囊胚发育率如表3所示。
表3实施例5-7解冻后卵母细胞的受精卵裂率、受精囊胚发育率、孤雌激活卵裂率和孤雌激活囊胚发育率
表3实验结果表明，利用本发明提供的CV期卵母细胞的冷冻保存及解冻方法可有效提高解冻后卵母细胞存活率及卵母细胞经体外受精和孤雌激活后的胚胎发育率。
Claims (5)
1.一种GV期卵母细胞的冷冻保存方法，其特征在于，将GV期卵母细胞在平衡液中平衡3分钟，然后转移到冷冻保存液中，再立即转移到冷冻环上，并迅速投入液氮中冷冻；
所述平衡液由冷冻基础液、原花青素、乙二醇、二甲基亚砜组成；原花青素的浓度为100-200μM，乙二醇的体积浓度为7.5％，二甲基亚砜的体积浓度为7.5％；
所述冷冻保存液由冷冻基础液、蔗糖、原花青素、乙二醇和二甲基亚砜组成；原花青素的浓度为100-200μM，乙二醇的体积浓度为15％，二甲基亚砜的体积浓度为15％，蔗糖的浓度0.5M；
所述冷冻基础液由培养液和胎牛血清组成；所述培养液为DMEM培养液、TCM199培养液、M16培养液和MEM培养液中的任意一种，所述胎牛血清的添加量为20％(v/v)。
2.一种GV期卵母细胞的解冻方法，其特征在于，将冷冻保存的卵母细胞从液氮中取出立即放到解冻液Ⅰ中3分钟，再转移到解冻液Ⅱ中3分钟，之后转移到基础液中3分钟，即完成冷冻卵母细胞的解冻；
所述解冻液Ⅰ由冷冻基础液、原花青素和蔗糖组成，解冻液Ⅰ中原花青素的浓度为100-200μM，蔗糖的浓度为0.5M；
所述解冻液Ⅱ由冷冻基础液、原花青素和蔗糖组成，解冻液Ⅱ中原花青素的浓度为100-200μM，蔗糖的浓度为0.25M；
所述冷冻基础液由培养液和胎牛血清组成；所述培养液为DMEM培养液、TCM199培养液、M16培养液和MEM培养液中的任意一种，所述胎牛血清的添加量为20％(v/v)。
3.根据权利要求2所述的解冻方法，其特征在于，解冻过程均在37℃环境中进行。
4.根据权利要求2所述的解冻方法，其特征在于，所述基础液由冷冻基础液和原花青素组成，原花青素的浓度为100-200μM。
5.根据权利要求1所述GV期卵母细胞的冷冻保存方法，或权利要求2-4所述GV期卵母细胞的解冻方法，其特征在于，所述GV期卵母细胞为哺乳动物或人的GV期卵母细胞。
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