dbcAMP通过Cdc25B蛋白调控小鼠1-细胞期受精卵发育

Effect of dbcAMP via Cdc25B on One-cell Stage of Fertilized Mouse Oocytes

为了研究PKA激活剂dbcAMP通过调控小鼠Cdc25B蛋白S149和S321位点磷酸化状态影响小鼠1-细胞期受精卵的发育,将质粒pBSK-Cdc25B-WT、pBSK-Cdc25B-S149A、pBSK-Cdc25B-S321A和pBSK-Cdc25B-S149A/S321A体外转录成mRNA;显微注射入S期受精卵中,在2 mmol/LdbcAMP的M16培养基中培养,观察其对受精卵发育、MPF活性及CDC2-pTyr15磷酸化状态的影响.结果显示,在有dbcAMP存在时,各组受精卵卵裂时间延迟,但Cdc25B-S/A-mRNAs注射组受精卵卵裂率明显高于Cdc25B-WT-mRNA注射组,MPF活性提前达到高峰;CDC2-pTyr15磷酸化状态和MPF活性变化相一致.因此,在小鼠1-细胞期受精卵有丝分裂过程中,PKA对小鼠Cdc25B蛋白S149位点与S321位点的磷酸化修饰是控制受精卵G2/M转换的重要方式.

To study the role of dbcAMP in regulating the phosphorylation status of S149 and S321 sites of Cdc25B on one-cell stage of fertilized mouse eggs,pBSK-Cdc25B-WT,pBSK-Cdc25B-S149A,pBSK-Cdc25B-S321A and pBSK-Cdc25B-S149A/S321A were transcribed into mRNA in vitro,then microinjected into mouse fertilized eggs at S phase and cultured in M16 medium in the presence of 2 mmol/L dbcAMP.The division of fertilized eggs,MPF activity and phosphorylation of CDC2-pTyr15 were analyzed.The dividing rates of the fertilized eggs in Cdc25B-S/A-mRNAs injected group was significantly higher than those of Cdc25B-WT-mRNA injected group,and the peak of MPF activity appeared earlier in the Cdc25B-S/A-mRNAs injected groups than the Cdc25B-WT-mRNA injected group.Cdc2-pTyr15 phosphorylation correlated with MPF activity.We concluded that PKA regulated the early development of mouse embryos by phosphorylation of S149 and S321 of Cdc25B,which played an important role in the regulation of G2/M transition in the mitotic cell cycle of fertilized mouse oocytes.