小鼠受精卵微注射Cdc25b mRNA可提高卵裂率并促进受精卵发育

Microinjection of Cdc25b mRNA into Mouse Fertilized Eggs Increases Division Rate and Promotes Mitosis

为了观察Cdc25B蛋白及PKA/Cdc25B信号途径在小鼠受精卵发育中的作用,将突变型和野生型Cdc25b转录成mRNA,显微注射到小鼠受精卵中,放入含有或不含有dbcAMP的M16中,相差显微镜下观察受精卵卵裂情况;用蛋白激酶活性测定方法检测MPF的活性;利用Western印迹检测Cdc2-Tyr15的磷酸化状态.结果显示,未加dbcAMP的Cdc25b-S321A mRNA注射组与Cdc25b-WT组相比,能够提前使受精卵发生G2/M期转变,导致卵裂,并明显提高卵裂率;MPF的活性测定和Cdc2-Tyr15磷酸化状态的检测结果也显示,Cdc25b-S321A组先于Cdc25b-WT组提前激活MPF.此外,Cdc25b-S321A mRNA注射组可以有效恢复由PKA引起的受精卵G2期阻滞,显著增加卵裂率;MPF的活性测定和Cdc2-Tyr15磷酸化状态的检测结果也显示,在PKA持续激活的情况下,对比于Cdc25b-WT组,Cdc25b-S321A组提前激活MPF.因此,在小鼠受精卵发育过程中PKA主要通过磷酸化Cdc25B的321位丝氨酸,从而调控MPF的激活与失活来控制有丝分裂进程.

To investigate the role of PKA/Cde25B signal pathway in the development of mouse fertilized eggs, Cdc25b mutant mRNA and Cdc25 b-WT mRNA were mieroinjeeted into mouse fertilized eggs at one-cell stage in the presence/absence of dbcAMP （PKA）, respectively. The change of MPF activity as well as the phosphorylation status of Cde2-Tyrl5 was detected in experimental groups and control groups by protein kinase activity assay and Western blots, respectively. In contrast to the Cdc25 b-WT mRNA mieroinjeetion group, the fertilized eggs injected Cdc25b-S321A mRNA entered mitosis in advance and increased the percentage of cleavage significantly in the absence of dbeAMP. At the same time, we also found that MPF in Cdc25b- $321A group was activated prior to Cdc25b-WT group by detecting the MPF activity and Cde2-Tyrl5 phosphorylation status, respectively. In addition, when injected a variety of Cdc25b mRNA into mouse fertilized eggs incubated in the presence of dbeAMP, strong overexpression of Cdc25 b-S321A mRNA overcame the G2 arrest induced by dbeAMP. On the other hand, MPF in Cdc25b-S321A group was activated precede Cdc25b-WT group by measuring MPF activity and Cdc2-Tyrl5 phosphorylation status, respectively. Taken together, we demonstrated in mouse fertilized eggs that PKA plays a critical regulatory role in cell cycle progression, by phosphorylating the Cdc25B S321.