囊泡分选蛋白Vps34在卵母细胞中作用机制的研究

Vacuolar protein sorting 34 (Vps34), which was initially described as a key endosomal trafficking regulator, is the only PI3K with phosphatidylinositol kinase activity identified in yeast. In mammals, Vps34 is classified as the sole Class Ⅲ phosphoinositide 3-kinase, whose substrate specificity is limited to phosphatidylinositol and its product in cells is PtdIns3P, a phospholipid central for membrane trafficking processes. Vps34 has been found to play a critical role in endosomal transport, autophagy, and nutrient sensing. The precise role of Vps34 in different tissues is unclear for a long time. It was only recently that the physiological roles of mammalian Vps34 has been investigated in mouse gene knockout studies. However we know little about its function in the oocyte so far. In the present study, we have generated Vps34 conditional knockout mice. With oocyte-specific expression of the Cre recombinase, VASA-Cre, DF9-Cre and ZP3-Cre, Vps34 was deleted in the different developmental stages of oocytes. We report here that ablation of Vps34 resulted in infertility in mice with a blockade of autophagy and a disorder of lysosome maturation in oocyte. Future study will be focused on the mechanisms of Vps34 on maintaining normal oocyte functions.

Vps34最早于酵母中发现发挥囊泡分选作用，具有磷脂酰肌醇激酶活性，是目前酵母中唯一的磷脂酰肌醇3-激酶（PI3K）。在哺乳动物中，Vps34是唯一一种第三类磷脂酰肌醇3-激酶（PIK3C3)，只能利用磷脂酰肌醇作为底物，催化形成PI(3)P，是细胞内PI(3)P合成的主要来源。研究显示Vps34参与调节内吞、自噬和mTOR的激活。由于Vps34在各组织细胞中发挥的作用不同，本项目通过Cre-Loxp重组酶系统引入VASA-Cre、GDF9-Cre和ZP3-Cre工具鼠实现Vps34在卵母细胞不同发育阶段的特异性敲除，通过条件性小鼠的表型分析，研究Vps34在卵母细胞发育不同阶段所发挥的功能以及作用机制。初步研究结果表明，ZP3-Cre Vps34 cKO小鼠不育，表现为早期胚胎发育阻滞，囊胚无法形成，卵母细胞内囊泡转运过程异常。未来将对Vps34在卵母细胞中作用的分子机制进行深入研究。

Vps34最早于酵母中发现发挥囊泡分选作用，具有磷脂酰肌醇激酶活性，是目前酵母中唯一的磷脂酰肌醇3-激酶（PI3K）。在哺乳动物中，Vps34是唯一一种第三类磷脂酰肌醇3-激酶（PIK3C3)，只能利用磷脂酰肌醇作为底物，催化形成PI(3)P，是细胞内PI(3)P合成的主要来源。研究显示Vps34参与调节内吞、自噬和mTOR的激活。由于Vps34在各组织细胞中发挥的作用不同，本项目通过Cre-Loxp重组酶系统引入VASA-Cre、GDF9-Cre和ZP3-Cre工具鼠实现Vps34在卵母细胞不同发育阶段的特异性敲除，通过条件性小鼠的表型分析，研究Vps34在卵母细胞发育不同阶段所发挥的功能以及作用机制。结果表明，VPS34在卵母细胞成熟过程中起着关键作用。在机制上，它不仅调控RAB7的激活，还参与RAB7介导的retromer在内体晚期上的募集和RAB7在线粒体自噬起始阶段的转位，这是一个创新的发现。自噬/线粒体自噬的异常导致母体蛋白降解失调，最终导致卵母细胞受精后胚胎发育停滞。本研究将有助于解读卵母细胞内膜转运过程对于卵母细胞发育成熟以及受精后早期胚胎发育的影响，为临床筛选优质卵母细胞和胚胎提供新的评价标准。