大量研究已经证实生长因子和激素在胚胎早期发育的细胞增殖与分化过程中起着重要的作用.应用定量ELISA,RT-PCR,免疫印迹和免疫荧光的方法检测胰岛素在小鼠受精卵和卵母细胞中的表达和定位.发现胰岛素均匀分布在卵细胞的胞浆中.同时也检测到mTOR(mammalian target of rapamycin)和p70S6K的表达、活性和定位.在小鼠受精卵中mTOR和p70S6K的表达没有明显不同.二者在G1,G2和M期分布在细胞浆,在S期聚集在原核的周围.在不同时期,mTOR的活性是波动的.利用PI3K的特异性抑制剂渥曼青霉素,观察到卵裂率明显减低.当使用mTOR的特异性抑制剂雷帕霉素时,受精卵的第一次有丝分裂延迟.这些结果表明胰岛素存在于小鼠的卵母细胞和受精卵中,并且胰岛素可能通过激活PI3K/PKB/mTOR/S6K的信号传导通路在小鼠的早期胚胎发育中发挥功能作用.
The role of protein kinase B (PKB) in the regulation of early development of fertilized eggs in mouse and its biological function on the development of mouse 1\|cell stage fertilized eggs were studied.The kinase activity of PKB was detected,and mRNA and protein level of PKB both in MⅡ oocytes and 1\|cell stage fertilized eggs were semi\|quantitatively assayed by Western blotting and RT\|PCR.10 μmol/L LY294002,the inhibitor of PKB,was microinjected into 1\|cell stage fertilized eggs of mouse to observe its effect on the development of mouse 1\|cell stage fertilized eggs.The results showed that the activity of PKB increased obviously after the fertilization,the mRNA and protein level of PKB also increased obviously in 1\|cell stage fertilized eggs,while in MⅡ oocytes the mRNA level was not detected.The protein level was lower than that in the 1\|cell stage fertilized eggs.After microinjecting LY294002 the development from 1\|cell stage to 2\|cell stage was inhibited obviously.All the results demonstrate that PKB exists in the fertilized eggs,and the activity,expression and transcription level increase after fertilization.PKB may play a positive role during the early development of fertilized eggs in mouse from 1\|cell to 2\|cell stage,but the mechanism is still unknown.These experiments are helpful for the further study of PKB on the regulation of early development of fertilized eggs.
