蛋白激酶Pkmyt1对小鼠1-细胞期受精卵发育的抑制作用

doi:10.13481/j.1671-587x.20150104

吉林大学学报(医学版) ›› 2015, Vol. 41 ›› Issue (01): 16-20.doi: 10.13481/j.1671-587x.20150104

蛋白激酶Pkmyt1对小鼠1-细胞期受精卵发育的抑制作用

刘超¹, 孟智超², 任丽莉², 刘乙蒙², 郭淼², 孙弋雅², 肖建英²

1. 辽宁医学院基础医学院发育生物学教研室, 辽宁锦州 121001;
2. 辽宁医学院基础医学院生物化学与分子生物学教研室, 辽宁锦州 121001

收稿日期:2014-06-18 发布日期:2015-01-30
通讯作者: 肖建英,教授,硕士研究生导师(Tel:0416-4673259,E-mail:xiaojianying@lnmu.edu.cn) E-mail:xiaojianying@lnmu.edu.cn
作者简介:刘超(1978-),女,辽宁省铁岭市人,副教授,理学博士,主要从事分子生殖和分子肿瘤学方面的研究。
基金资助:
国家自然科学基金资助课题(81270698,31371173,81401199)

Inhibitory effect of protein kinase Pkmyt1 on development of one-cell-stage mouse embryos

LIU Chao¹, MENG Zhichao², REN Lili², LIU Yimeng², GUO Miao², SUN Yiya², XIAO Jianying²

1. Department of Developmental Biology, School of Basic Medical Sciences, Liaoning Medical University, Jinzhou 121001, China;
2. Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Liaoning Medical University, Jinzhou 121001, China

Received:2014-06-18 Published:2015-01-30

摘要/Abstract

摘要：

目的: 通过在小鼠1-细胞期受精卵内过表达Pkmyt1-wild type(WT) (野生型) mRNAs,研究蛋白激酶Pkmyt1对小鼠受精卵早期发育的影响,为进一步阐明Pkmyt1对哺乳动物受精卵早期发育调控的机制奠定基础。方法: 构建pcDNA3.1/myc- Pkmyt1-WT质粒。超排卵方法获得小鼠1-细胞期受精卵,实验分为未注射组、TE缓冲液注射组和Pkmyt1-WT mRNAs注射组,收集各组卵细胞后采用Western blotting法检测Pkmyt1蛋白表达和Cdc2-pTyr15磷酸化状态;相差显微镜观察受精卵的形态变化并计数卵裂率。结果: pcDNA3.1/myc-Pkmyt1-WT质粒构建成功。与未注射组(0.414±0.016)和TE缓冲液注射组(0.441±0.013)比较,受精卵显微注射Pkmyt1-WT mRNAs 4 h后,Pkmyt1蛋白表达水平(1.112±0.025)增高(P<0.01)。与未注射组(60.81%±3.27%)和TE缓冲液注射组(61.79%±4.08%)比较,Pkmyt1-WT-mRNA注射组卵裂率(34.2%±3.25%)显著降低 (P<0.01)。Pkmyt1-WTmRNA注射组在注射hCG后29.0 h检测到微弱的磷酸化信号,29.5 h检测不到任何磷酸化信号。结论: Pkmyt1过表达后磷酸化Cdc2-pTyr15可抑制小鼠1-细胞期受精卵的卵裂率,提示Pkmyt1负调控小鼠受精卵有丝分裂的进程。

关键词: 蛋白激酶Pkmyt1, 蛋白激酶Wee1, 有丝分裂, 小鼠受精卵

Abstract:

Objective To study the influence of protein kinase Pkmyt1 on the development of one-cell stage mouse embryos by the means of overexpression of Pkmyt1-wild type (WT) mRNAs, and to pay foundation for further study on the mechanism of the early development of mouse embryos. Methods pcDNA3.1/myc-Pkmyt1-WT was constructed and one-cell-stage mouse embryos were collected after superovulation, and the embryos were cultured in vitro and divided into control group(no injection), TE buffer microinjection group and Pkmyt1-WT mRNAs-injected group.The protein expression levels of Pkmyt1 and the phosphorylation status of Cdc2-pTyr15 were observed by Western blotting method.The morphological changes and cleavage rates of mouse embryos were observed under phase-contrast microscope. Results pcDNA3.1/myc-Pkmyt1-WT was constructed successfully.The Pkmyt1 protein expression level was increased at 4 h after Pkmyt1-WT-mRNAs microinjection into one-cell stage mouse embryos (1.112±0.025)compared with no injection group(0.414±0.016) and TE buffer injection group(0.441±0.013)(P<0.01).The cleavage rate of one-cell-stage mouse embryos in Pkmyt1-WT mRNAs group (34.2%±3.25%) was significantly lower than those in no injection group(60.81%±3.27%) and TE buffer injection group(61.79%±4.08%)(P<0.01).The cdc2-pTyr15 phosphorylation signal in Pkmyt1-WT-mRNA-injected group was detected at 29.0 h after hCG injection, and there was no signal at 29.5 h. Conclusion Overexpression of Pkmyt1 phosphorylates Cdc2-pTyr15, thus inhibits the cleavage rate of one-cell-stage mouse embryos, suggesting that Pkmyt1 negatively regulates mitosis during early embryogenesis in mouse embryos.

Key words: protein kinase Pkmyt1, protein kinase Wee1, mitosis, mouse embryos

中图分类号:

Q132

刘超, 孟智超, 任丽莉, 刘乙蒙, 郭淼, 孙弋雅, 肖建英. 蛋白激酶Pkmyt1对小鼠1-细胞期受精卵发育的抑制作用[J]. 吉林大学学报(医学版), 2015, 41(01): 16-20.

LIU Chao, MENG Zhichao, REN Lili, LIU Yimeng, GUO Miao, SUN Yiya, XIAO Jianying. Inhibitory effect of protein kinase Pkmyt1 on development of one-cell-stage mouse embryos[J]. Journal of Jilin University Medicine Edition, 2015, 41(01): 16-20.

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蛋白激酶Pkmyt1对小鼠1-细胞期受精卵发育的抑制作用

Inhibitory effect of protein kinase Pkmyt1 on development of one-cell-stage mouse embryos

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