人类ZP2基因突变导致卵子透明带结构异常及精子结合障碍的分子机制研究

受精失败是一种由精卵融合障碍导致的严重不孕不育疾病，在体外受精治疗中的发生率为5-10%，是目前生殖治疗面临的重大难题。已知精卵结合和穿透障碍是受精失败主因，但其分子机制尚不明确。申请人在前期研究中发现了人类卵子受精失败的新致病基因ZP2，其纯合突变引发卵子透明带（ZP）结构紊乱及精子结合障碍。由于ZP2是ZP的重要成分和一种精子受体，根据预实验结果，我们提出致病机制假说：突变后C-端截短的ZP2蛋白在细胞内滞留，不能正常分泌和参与ZP基质组装，同时阻碍其他ZP蛋白的分泌，从而导致ZP结构和功能缺陷。为了验证这一科学假说，本项目拟通过体外细胞模型及小鼠和人卵模型（生理水平）探讨疾病相关突变对ZP2及其他ZP蛋白分泌和组装ZP的影响；然后，通过构建C-端不同位置截短的突变体揭示该区域中各功能元件的作用。本项目将为受精失败提供新的分子机制，并为ZP2作为受精失败诊断标记的临床应用提供科学依据。

Fertilization failure is a serious disorder which leads to infertility and sterility, and is also a recurrent problem for about 5-10% of routine in vitro fertilization (IVF) cycles. Although fertilization failure occurs at different IVF stages, sperm-zona binding and penetration has been reported as a chief cause. Nevertheless, the molecular mechanisms underlying fertilization failure remain largely unknown. We have recently identified an oocyte-specific gene ZP2 as a novel causative gene for human fertilization failure. Homozygous mutations in ZP2 resulted in a thin zona pellucida (ZP) exhibiting defective sperm-binding (Dai Can et al. Genetics in Medicine. 2018). Since ZP2 is a component of ZP and the primary receptor for sperm, we hypothesize that the mutations lead to a C-terminal truncated ZP2 protein which is unable to secrete and incorporate into the extracellular ZP matrix. Moreover, within cells, the truncated ZP2 prematurely interacts with other ZP proteins and thus partially hinder their secretion and incorporation, which results in a structurally abnormal and dysfunctional ZP. In this study, the effects of ZP2 mutations on secretion and incorporation of ZP2 and other ZP proteins will be evaluated by functional experiments in CHO cells and subsequently assessed in mouse and human oocytes (physiological condition). Next, the role and importance of each element located in the C-terminal region of ZP2 protein would be analyzed by using a series of vectors expressing gradually C-terminal truncated ZP2. This study could provide novel insights into the molecular mechanisms underlying fertilization failure, and significantly, it would offer valid experimental supports for using ZP2 as a diagnostic marker for fertilization failure.