磷脂酶Cζ的结构和表达异常导致受精障碍的机制研究

During fertilization of mammalian oocyte, calcium oscillations is necessary for the oocyte activation. Phospholipase Cζ(PLCζ) is the key factor of inducing the calcium oscillations through the hydrolysis of PIP2. Among all the ICSI treatments of infertility, about 1-5 % patients failed because the eggs are not activated. And these failure may be caused by the abnormal expression or inactivation of PLCζ. There have not been any research to elucidate the mechanism of PLCζ from the protein structure in the world until now, and there is also no report about infertility therapy using the recombinant PLCζ proteins. In this study, we attempted to obtain recombinant human PLCζ protein via the prokaryotic expression system and then used structural biology and X-ray crystal diffraction techniques to resolve the 3-D structure of proteins PLCζ. This structure will clarify the molecular mechanisms of its binding to PIP2 which triggered the calcium oscillations. By injecting the recombinant human PLCζ protein into unfertilized eggs after ICSI, can make a prediction for its clinical application as an potential infertility drug. Meanwhile we will research the PLCζ mutant incidence in northwest of Hubei Province by high-throughput sequencing, and find the best sperm freezing conditions which maintaining the highest activity of PLCζ by western blot and peptide fingerprint analysis. These will provide a theoretical and experimental basis for optimizing clinical operations to achieve higher pregnancy rate.

哺乳动物受精需要通过钙离子振荡激活卵子，磷脂酶Cζ(PLCζ)亚型是催化PIP2引起钙离子振荡的关键因子。在对不孕症进行ICSI治疗中，约1-5%的患者由于卵子不激活而导致受精失败，这可能是由于PLCζ的表达异常或活性缺失导致的。目前从分子结构上阐明PLCζ作用机制的研究在国际上还是空白，利用重组PLCζ蛋白治疗这类不孕症也未见报道。本课题通过构建PLCζ基因的原核表达质粒，得到重组人PLCζ蛋白，利用结构生物学和X-射线衍射技术解析出PLCζ蛋白质的三维结构，阐明其结合PIP2引发钙离子振荡的分子机制；通过将重组人PLCζ蛋白注入ICSI后未受精卵子中并统计其受精率的方法，研究其作为不孕症治疗候选药物的可行性；同时通过高通量测序，western blot，肽指纹图谱分析等方法，研究PLCζ在本地区突变发生率，并确定使PLCζ保持最高活性的精子冷冻条件，为获得更高妊娠率提供理论和实验基础

哺乳动物受精需要通过钙离子振荡激活卵子，磷脂酶Cζ(PLCζ)亚型是催化PIP2引起钙离子振荡的关键因子。在对不孕症进行ICSI治疗中，约1-5%的患者由于卵子不激活而导致受精失败，这可能是由于PLCζ的表达异常或活性缺失导致的。目前从分子结构上阐明PLCζ作用机制的研究在国际上还是空白，利用重组PLCζ蛋白治疗这类不孕症也未见报道。本课题通过构建PLCζ基因的原核表达质粒，得到了重组人PLCζ蛋白，纯度在90%以上，进行晶体筛选未能见到蛋白质晶体形成；通过对多次ICSI不受精的男性患者的外周血DNA进行基因测序，找到了一个位于第3号外显子上的突变Q316R，该突变可能会导致蛋白EF区不能水解PIP2生成IP3，从而无法引起卵子钙离子振荡，最终导致不孕。通过在五种不同的冷冻条件下检测蛋白的降解情况，发现只有在液氮速冻的条件下PLCζ蛋白的降解程度最低，从而以此条件作为最佳的精子冷冻条件，为获得更高妊娠率提供理论和实验基础。此外，课题组还发现高浓度的FSH可能会导致棕色卵子的产生，并降低了最终的受精率和妊娠率。