人类无精子症新基因TDRD7突变导致精子形成障碍的分子机制研究

Azoospermia is a type of common disorder which leads to serious deterioation to male reproductive health. On the base of two unrelated consanguineous Chinese families and knockout mice model study, we identified a novel gene TDRD7, one of the components of chromatoid bodies (CBs), leading to human azoospermia, with its loss-of-function mutations leading to azoospermia via arresting of spermatogenesis at the spermiogensis stage, during which a round haploid spermatid develops into an elongated spermatozoon. Since CBs are known to be involved in nucleic acid metabolism in spermiogensis, we hypothesize that TDRD7 function loss leads to lack of properly assembled CBs, which protects and regulates and the mRNA required for acrosome and tail cilia formation. To confirm this hypothesis, Tdrd7 knockout mice is used as the research model, and RNA-Seq, miRNA sequencing and proteomics technology will be performed to screen the differentially expressed genes and miRNAs regulation target Tdrd7 gene in testicular tissue from wild mice and knockout mice. Differentially expressed genes and proteins will be examined to understand their roles and interaction during spermiogensis. Several key genes will be selected to further functional validation. Finally, validation will be performed using molecular biological techniques, such as RNA interference, overexpression, Co-IP, immunofluorescence, et al., in the patients’ fibroblast and HEK 293T cell line. In summary, this study intends to find a complex biological network regulated by TDRD7 during spermiogensis and provide the useful data for the biological studies of spermiogensis.

无精子症是一种严重损害男性生殖健康的常见疾病。我们前期研究发现TDRD7是导致人类无精子症的新基因，其缺失导致精子形成障碍。由于TDRD7是拟染色体的关键组分，在精子形成中参与RNA加工与储存，本研究设想，TDRD7缺失破坏拟染色体结构，使精子形成相关的RNA转录、降解及蛋白调控异常，导致精子形成受阻。为证实此假说，本研究拟以Tdrd7基因敲除鼠为模型，通过RNA和miRNA测序及蛋白质组学技术，筛选野生型鼠和敲除鼠睾丸组织中差异表达基因和Tdrd7基因靶向调控的miRNAs，聚类分析其与精子形成中拟染色体、顶体和鞭毛结构等的调控关系，挑选关键的分子，采用过表达、Co-IP、免疫荧光等技术，在TDRD7缺陷患者成纤维细胞及HEK 293T细胞系上验证。本研究拟通过筛选与精子形成相关的RNA及蛋白质，探讨其变化与TDRD7突变的作用关系，为精子形成过程中的转录后调控网络提供新的生物学知识。

基因突变引起的精子发生障碍是导致男性不育的重要原因，我们前期的研究发现核糖核蛋白（RNP）颗粒组分TDRD7突变可同时导致先天性白内障和男性不育，但具体分子机制不清楚。本研究在前期基础上发现Tdrd7缺失可导致Tdrd7敲除鼠胚胎成纤维细胞自噬体降解受阻，即自噬体不能与溶酶体融合形成自噬溶酶体，进而表现为自噬体异常积累。随后在Tdrd7敲除鼠眼睛和睾丸组织中通过RNA-Seq发现自噬降解受阻的关键调节分子TBC1D20同时在眼睛和睾丸组织中显著下调。并进一步在体内外通过生化实验发现TDRD7可直接结合TBC1D20的mRNA，其缺失可下调TBC1D20的表达导致自噬体降解受阻，最终导致先天性白内障和男性不育。此外，我们对收集到的特发性男性不育患者进行全外筛查，在另一个NOA与CCs的近亲家系中发现了TDRD7纯合移码突变；与此同时，我们还意外的发现了多个导致男性不育的新基因，如，发现RPL10L和M1AP等是导致严重少精子症的新基因，发现DNAH10和SPEF2等基因突变可导致弱畸形精子症。以上研究结果累计发表28篇SCI论文，这些研究结果将为这类患者的遗传咨询和生育治疗提供重要的分子基础。

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