原发性纤毛运动障碍候选新基因SPEF2突变通过破坏精子鞭毛组装导致男性不育的分子机制研究

原发性纤毛运动障碍（PCD）伴随的男性不育表型易被忽视。我们前期在5个PCD家系10名男性不育患者中筛选到SPEF2双等位基因突变，患者精子完全不动，鞭毛中央微管丢失，小鼠模型有相似表型，提示SPEF2是PCD伴男性不育新基因，但导致不育的机制不清楚。我们设想，该基因缺陷通过特定转录本破坏精子鞭毛内运输途径引起鞭毛组装异常，最终导致男性不育。为此，我们拟以患者精液样本及模式小鼠为材料，结合患者表型，通过辅助生殖技术、电镜与转录本分析研究该疾病生殖干预策略及该基因生物学特性；通过蛋白质组学筛选与人及小鼠鞭毛组装相关的蛋白，并采用IF和Co-IP等技术探讨所筛选蛋白与其它鞭毛组装分子的相互作用；通过基因拯救技术恢复模式鼠表型及相应分子事件，进一步明确该基因的功能。本研究将增加我们对SPEF2如何参与精子鞭毛组装功能的了解，并为其缺陷导致的PCD伴男性不育提供管理新经验、诊断新思路和治疗新靶点。

Primary ciliary dysplasia (PCD) is a disorder in which the associated male infertility phenotype is easily be neglected. We have previously identified biallelic SPEF2 mutations in 10 male PCD individuals from 5 unrelated families. The spermatozoa of the patients were completely immobile, and ultrastructural analysis of patients’ spermatozoa showed absence of flagellar central pairs. The PCD phenotypes have been validated by Spef2 knock-out mouse model, indicating that SPEF2 is a new disease-causing gene for PCD with male infertility. However, the molecular mechanism resulting in infertility is unclear. In the study, we speculate that SPEF2 deficiency leads to male infertility through disrupting the functions of intra-flagellar-transport (IFT) proteins and flagellum assembly by a specific transcript. To prove this hypothesis, we will take advantage of the Spef2 knock-out and knock-in mouse models, combining with the patient's phenotype and assisted reproductive technology, to study the biological characteristics of SPEF2 gene through transmission electron microscopy and transcription analysis. Differential expressed proteins during flagellum assembly in patients and knock-in mice will be obtained by proteome analyses. The functional network of the candidate proteins involved in flagellar assembly will be investigated in vivo and vitro, using immunofluorescence, RNA interference and Co-IP methods. Finally, the role of SPEF2 in flagellum assembly will be further clarified by restoring phenotypes and corresponding molecular events of knock-in mice using gene rescue experiment. Overall, this study will improve our understanding about the essential role that SPEF2 plays during sperm flagellum assembly, and provide new management expertise, new diagnostic ideas and new therapeutic targets for patients caused by SPEF2 mutations.