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

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.

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。

哺乳动物受精起始于精子与卵子透明带(zona pellucida, ZP)的结合和穿透，研究数据显示精卵识别和穿透障碍是受精失败的主要原因，在受精失败周期中的占比高达52%。ZP是围绕在哺乳动物卵母细胞外的一层基质，在卵母细胞生长、受精和胚胎发育过程中发挥重要作用。近十年来，越来越多的ZP基因突变被发现与女性不孕和卵母细胞ZP异常有关，但是ZP基因突变与女性不孕以及ZP异常的因果关系有待明确，致病机制有待阐明。本项目以透明带异常的不孕女性及其携带的ZP1-3基因突变为对象，在CHO细胞模型以及患者卵巢和卵母细胞中明确了C端功能元件不是人ZP1-3蛋白细胞外分泌所必需的，C端功能元件缺失的ZP蛋白可以胞外分泌但是不能参与ZP基质组装。后续我们以“胶冻状”透明带卵母细胞为研究对象，对该异常类型ZP的形成机制及其对卵母细胞发育潜能的影响进行了研究。本项目成果为女性不孕和卵子透明带异常患者的表型鉴定、遗传病因筛查、变异效应评估等提供实验依据，为透明带组装和精卵结合机制研究提供了方向和思路。

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