FF-MAS通过Mos/MEK/ERK1/2信号通路调控猪卵母细胞减数分裂的机制研究

Follicle fluid-meiosis activating sterol (FF-MAS) is gaining extensive attention due to the effect of inducing the resumption of meiosis in mammalian oocyte. But there is not a clear understanding of the signaling pathways mediating its action. Based on the experimental result that when the Mos/MEK/ERK1/2 signaling pathway was blocked, the action of FF-MAS on meiotic resumption was inhibited. Accordingly we speculate that the Mos/MEK/ERK1/2 signaling pathway mediates the FF-MAS inducing meiosis of the oocyte. On the basis of our preceding work that FF-MAS promotes nuclear maturation and cytoplasmic maturation of oocytes, different levels of FF-MAS endogenous accumulation will be offered in in vitro maturation system of porcine oocytes, and the expression, positioning, and activity of the key signal molecules and the substrates of ERK1/2 will be detected in FF-MAS-induced meiosis of porcine oocytes. By applying specific inhibitor to block ERK1/2 signal pathway, maturation rate of oocytes and the activity of ERK1/2 will be detected with the aim to research the role of Mos/MEK/ERK1/2 signaling pathway in the process of FF-MAS-induced meiosis of oocytes. The aim of this study is to reveal the signal transduction mechanism of FF-MAS-induced meiotic resumption of oocytes, which will lay a theory fundation for the application of FF-MAS in in vitro maturation system of oocytes.

卵泡液促减数分裂甾醇（FF-MAS）因其诱导卵母细胞减数分裂作用而备受关注，但对于调控其作用的信号通路尚未明确。基于Mos/MEK/ERK1/2信号通路阻断致使FF-MAS的作用受到抑制，因而推测“Mos/MEK/ERK1/2信号通路介导了FF-MAS诱导的卵母细胞减数分裂”。申请者拟在前期研究的FF-MAS促进卵母细胞核成熟、胞质成熟基础上，在猪卵母细胞体外成熟体系给予不同水平的FF-MAS內源积累，检测FF-MAS诱导的猪卵母细胞减数分裂过程中关键信号分子的表达、定位、活性及ERK1/2作用的底物；用特异性抑制剂阻断ERK1/2信号通路，检测卵母细胞成熟率及ERK1/2的活性，研究Mos/MEK/ERK1/2信号通路在FF-MAS诱导卵母细胞减数分裂过程中的作用，揭示FF-MAS诱导卵母细胞减数分裂恢复的信号转导机制，为FF-MAS在卵母细胞体外成熟培养体系的应用奠定理论基础。

卵泡液促减数分裂甾醇（FF-MAS）因其诱导卵母细胞减数分裂作用而备受关注，但对于调控其作用的信号通路尚未明确。课题组在猪卵母细胞体外成熟体系添加不同浓度的AY9944 A-7或Ketoconazole，分别通过抑制FF-MAS的代谢和产生，增加或减少FF-MAS的內源积累，检测了FF-MAS对猪卵母细胞减数分裂恢复及体外成熟的诱导作用；分析了FF-MAS诱导的猪卵母细胞减数分裂过程中关键信号分子的表达；用特异性抑制剂阻断ERK1/2信号通路，检测了卵母细胞成熟率及ERK1/2的活性，研究了Mos/MEK/ERK1/2信号通路在FF-MAS诱导卵母细胞减数分裂过程中的作用。.主要研究结果如下：.（1）添加20或30 µmol/L AY9944 A-7，显著提高了猪卵母细胞的成熟率，其中30 µmol/L AY9944 A-7的效果最佳。Ketoconazole抑制卵母细胞体外成熟，而且Ketoconazole的浓度越大，抑制作用越强，由此证实内源FF-MAS的增加促进了猪卵母细胞减数分裂恢复及体外成熟。.（2）猪COCs培养于添加不同浓度AY9944 A-7的体外成熟培养液中44 h后，猪卵母细胞c-mos、MEK、ERK1、ERK2等基因的mRNA相对表达量均呈现不同程度的增加。相反，添加不同浓度Ketoconazole培养44 h后，这些基因的mRNA相对表达量均显著降低。.（3）猪COCs在含有30 μmol/ L AY9944 A-7或30 μmol/ L Ketoconazole的IVM培养基中培养12、24、36或44 h，猪卵母细胞中c-mos，MEK，ERK1和ERK2 mRNA和蛋白质的相对表达受内源性FF-MAS的影响，并且c-mos，MEK，ERK1 / 2的蛋白质表达趋势和mRNA表达趋势基本一致。此外，FF-MAS诱导的卵母细胞减数分裂过程中c-mos，MEK，ERK1和ERK2表达的动力学与MAPK途径介导的动力学基本一致。.（4）用MEK 特异性抑制剂 U0126阻断Mos/MEK/ERK1/2信号通路后，猪卵母细胞的体外成熟率显著降低，ERK1 / 2以及 pERK1/2的表达量显著降低。.综上所述，MAPK信号通路参与了FF-MAS诱导的猪卵母细胞体外成熟。该结果为FF-MAS在卵母细胞体外成熟培养体系的应用奠定理论基础。