钙离子信号对非细胞体系中小鼠卵母细胞游离生发泡减数分裂启动的影响

本文构建了包括HeLa裂解液和游离小鼠卵母细胞生发泡的实验体系,用于研究Ca2+及其下游信号对小鼠卵母细胞减数分裂启动的影响.游离的卵母细胞生发泡可以在M期细胞裂解液中发生减数分裂启动,表现为染色质的凝集.进一步的研究表明,Ca2+信号的存在对G2期细胞裂解液促进减数分裂启动是至关重要的,G2期中期的细胞裂解液只有经Ca2+诱导后才具有启动生发泡减数分裂的作用,而G2期晚期无论Ca2+存在与否均诱发减数分裂的启动,但是G2期早期的裂解液元启动减数分裂的作用.卵母细胞的体外培养实验分析也表明,抑制CaM和CaMKII的活性可以阻止GVBD和抑制第一极体的释放.免疫沉淀及Western Blotting结果显示,HeLa细胞裂解液中的MPF从G2期中期到M期均存在,且Cdc2亚基的Tyr由磷酸化向去磷酸化转变.结果进一步证明,卵母细胞减数分裂的启动可能是通过一种Ca2+/CaM依赖的途径来推动的.

STUDY ON THE ROLE OF CALCIUM SIGNAL DURING MATURE RESUMPTION OF ISOLATED MOUSE GERMINAL VESICLES IN A CELL FREE SYSTEM

A cei - free system including HeLa cell lysate of synchronized metaphase or G2-phase and isolated germinal vesicles (GV) from mouse oocytes was used to study the role of calcium and its downstream mediator during mature resumption. The isolated GVs could resume meiotic maturation in the lysate prepared from M phase HeLa cell, which marked by chromatin condensation. And this process was not affected by calcium chelating agent. But calcium in lysate from G2 phase cells was critical to meiotic maturation. Only in mid-G2 phase cell lysate (released from nocodazole for about 20-23 h) chromatin condensation could be induced by calcium. Calcium had no effect on the cell lysate prepared from earlier (18 - 20h) and later (24h) G2 phase cells. Further studies showed that down stream mediator CaM and CaMKII might also involove in this process. Inhibition the function of CaM and CaMKII could block GVBD and first polar body extrusion of DOs cultured in vitro. The target of calcium signal might be MPF because MPF was existed from mid-G2 phase to metaphase and the tyrosine phosphorylation level of Cdc2 subunit was significantly dephosphorylated in M phase. Our results further confirmed that the resumption of meiosis maturation was promoted in a calcium/ CaM depended pathway.