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

• 基础研究 • 上一篇    下一篇

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. 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. 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:

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