银鲫卵母细胞体外诱导成熟技术的建立
DEVELOPMENT OF IN VITRO MATURATION TECHNOLOGY FOR GIBEL CARP OOCYTES
查看参考文献35篇
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文摘
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研究以银鲫为材料,根据银鲫(Carassius auratus gibelio)卵母细胞生发泡(Germinal vesicle, GV)边移程度及剥离GV中减数分裂前期染色体的凝集状态,将银鲫Ⅳ时相的卵母细胞分为GV0、GV1、GV2和GV3四个时期;并进一步比较了分别处于这4个时期银鲫卵母细胞体外诱导培养的成熟率、卵裂率和孵化率。结果表明, GV1期之后的卵母细胞均可有效进行体外诱导成熟,可正常受精发育,由于GV1期卵母细胞有较长时间用于显微操作,因此GV1期卵母细胞被选为进行体外诱导的最早时期的卵母细胞。以GV1期卵母细胞为研究材料,摸索了银鲫卵母细胞体外诱导成熟的适宜条件:取GV1期的Ⅳ时相卵母细胞,放置于pH 8.5、加有1μg/mL孕酮激素(17α, 20β-dihydroxy-4-pregnen-3-one, DHP)的格氏平衡盐溶液(Gey's balanced salt solution, GBSS)中,在23℃培养箱中体外诱导12h后,将滤泡膜剥离后再进行人工体外授精,其所获胚胎的孵化率可达55.5%。此外,将体外转录合成的带GFP标签的h2af1o mRNA注射到GV1期卵母细胞,发现经显微操作和体外诱导后不仅可以通过GFP绿色荧光信号活体观察GVBD、受精、卵裂和早期胚胎发育的全过程,而且诱导成熟的卵子仍可正常受精和胚胎发育。研究建立的银鲫卵母细胞体外诱导成熟技术为银鲫和其他鱼类卵母细胞发育过程研究及其相关基因和细胞显微操作提供了技术平台。 |
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其他语种文摘
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In vitro oocytes maturation is a key and efficient technology for gene and cellular micromanipulation in oocytes of fish. In this study, we used gibel carp oocytes as studying materials to develop an efficient in vitro induction maturation technology. Based on previously established germinal vesicle (GV) isolation and premeiotic chromosome preparation methods, four different stage oocytes including GV0, GV1, GV2 and GV3 were firstly distinguished by GV migration and premeiotic chromosome condensation status, and the maturation rate (%), cleavage rate (%) and hatching rate (%) were analyzed. GV1, GV2 and GV3 oocytes had the capacity for in vitro induction maturation, and GV1 oocytes were the optimal stage oocytes because they have longer time for micromanipulation. We found that the optimal induction conditions for GV1 oocytes in vitro were 12 hours at 23℃ in pH 8.5 Gey's balanced salt solution(GBSS) with 1 μg/ml DHP (17α, 20β-dihydroxy-4-pregne-3-one), by which up to 55.5% hatching rate was reached from these stripped follicle membrane oocytes. Moreover, we injected the GFP-zfh2af1o mRNA into the GV1 oocytes, and found that the whole process of in vitro maturation, fertilization and embryo development of the micromanipulated GV1 oocytes could be tracked by the expression of GFP, and normal fertilization and embryo development could be produced from the induced mature eggs. Therefore, we established an efficient platform for studying oocyte development and gene and cellular micromanipulation in this fish. |
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来源
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水生生物学报
,2017,41(5):984-991 【核心库】
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DOI
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10.7541/2017.123
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关键词
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银鲫
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卵母细胞成熟
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体外诱导
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生发泡
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显微注射
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地址
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中国科学院水生生物研究所, 淡水生态与生物技术国家重点实验室, 武汉, 430072
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语种
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中文 |
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文献类型
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研究性论文 |
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ISSN
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1000-3207 |
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基金
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中国科学院战略性先导科技专项
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文献收藏号
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CSCD:6068753
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