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.
Recent studies have suggested that growth factors and hormones play important roles in cell prolif-eration and differentiation during early embryonic development. In the present study, we examined the expression and localization of insulin in the mouse oocytes and one-cell stage embryos by quantitative ELISA, RT-PCR, Western blot and immunofluorescence. In the mouse oocytes and one-cell stage em-bryos, expression of insulin was uniformly distributed in the cytoplasm. We also examined the expres-sion, activity and localization of mTOR (mammalian target of rapamycin) and p70S6K. The expression of mTOR and p70S6K was not significantly different at the cell cycle of mouse one-cell stage embryos. mTOR and S6K were distributed evenly in the cytoplasm at G1, G2 and M phase phase, but at S phase, the distribution of mTOR and S6K was around the pronucleus. At different phases, the activity of mTOR fluctuated. We also used the PI3K specific inhibitor-Wortmannin to investigate the cleavage rate of eggs. The result showed that the rate obviously decreased. When the mTOR specific inhibitor Rapa-mycin was used, the first mitotic division of the mouse one-cell stage embryo was delayed. These re-sults suggested that insulin was expressed both in mouse oocytes and one-cell stage embryos, and may play functional roles in regulation of mouse early embryogenesis by activating the signal pathway of PI3K/PKB/mTOR/S6K.
YU BingZhi, ZHANG Zhe, DENG Xin, XU XiaoYan, FENG Chen, LI YanXiao, CUI Cheng, SU WenHui, ZHAO HongMei & YU DaHai Department of Biochemical and Molecular Biology, China Medical University, Shenyang 110001, China
