哺乳动物精子发生减数分裂染色体行为的分子调控

The pairing, synapsis and recombination between homologous chromosomes and their dimorphism between sex chromosomes and autosomes are typical features and core events of meiosis during spermatogenesis in mammals. However, the molecular mechanisms that underlie and coordinate these events remain poorly understood. Here, we propose that the difference in RAD51 recruitment during homologous repair after formation of programmed double-stranded DNA breaks (DSBs) is one of the main causes for dimorphism of the chromosome behavior between the sex chromosomes and autosomes. In this project, we will use the two animal models that are generated by ourselves, Rad51ap2 (RAD51-associated protein 2) KO mice, which are the only known mouse model with defects in RAD51 recruitment on pseudoautosomal region (PAR) of the sex chromosomes only, as well as Tex15 (Testis-expressed sequence 15 protein) KO mice, which may be the only mouse model with defects in RAD51 recruitment only on autosomes till now. Combined with the advanced techniques including CRISPR/Cas9 gene editing, we will explore why and how RAD51AP2 only regulates recruitment of RAD51 on PAR. We will determine experimentally whether TEX15 only regulates recruitment of RAD51 on autosomes. If it indeed regulates the recruitment of RAD51 on autosomes only, we will study why and how TEX15 works. This project will hopefully uncover molecular mechanisms underlying the behaviors of meiotic chromosomes, and their dimorphism between the sex chromosomes and autosomes, and propose new models for the regulation and coordination of the chromosome behaviors during male meiosis in mammals. Our findings are also expected to provide new insights into the causes, diagnosis and treatment of infertility, spontaneous abortions and birth defects induced by meiotic abnormalities in human males.

不尽相同的性染色体和常染色体配对、联会和重组是哺乳动物精子发生减数分裂的典型特征和核心事件，但其调控机制不清楚。我们假设：减数分裂DNA双链断裂的同源修复中单链DNA上RAD51募集机制的不同决定了哺乳动物精子发生中性染色体和常染色体减数分裂行为的差异。本项目将利用我们建立的也是迄今已知唯一的仅表现为性染色体假常染色体区（PAR）和可能只表现为常染色体上RAD51募集缺陷的动物模型Rad51ap2和Tex15敲除小鼠，结合CRISPR/Cas9等技术，研究揭示RAD51AP2为什么只调节及如何调节精子发生性染色体PAR区RAD51的招募，探讨TEX15是否以及为何只调节和如何调节常染色体上RAD51的募集，以期揭示哺乳动物精子发生中性染色体和常染色体减数分裂行为及其差异的调控机制，提出减数分裂染色体行为调控的新理论，为减数分裂异常所致精子发生异常相关疾病的病理机制阐释和诊治，提供新思维。

性染色体和常染色体配对、联会和重组是哺乳动物精子发生减数分裂的典型特征和核心事件，但其调控机制不清楚。在本项目中，我们对中科大人类生殖疾病资源库中精母细胞发育停滞患者进行全外显子测序和生物信息学分析后，在患者中筛选到了RAD51AP2的突变，分析发现RAD51AP2在小鼠睾丸特异表达，而且其在染色体轴上的定位依赖于SPO11所介导的DSBs，并与重组蛋白RAD51存在相互作用，提示其很可能参与减数分裂重组过程。为了确证该患者复合杂合突变是否为致病突变，我们利用CRISPR/Cas9技术制备了Rad51ap2敲除、Rad51ap2突变以及携带类似患者复合杂合突变的基因修饰小鼠。研究发现这三种修饰小鼠表型一致，精子发生均存在异常；具体表现为生精小管内可见减数分裂I中期停滞，多数中期细胞存在落后染色体，并发生凋亡。减数分裂前期进程分析发现Rad51ap2敲除鼠常染色体配对、联会和重组过程未见明显异常，早粗线期XY可正常配对、联会，然而中粗线期和双线期精母细胞存在高比例的XY分离。进一步分析发现敲除鼠重组相关蛋白（如RAD51、RPA）持续位于分离的XY PAR区，提示DSB不能正常修复；深入研究发现敲除小鼠中重组关组蛋白（如RAD51）持续位于分离的XY PAR区，表明PAR区DSB修复受阻。随后，我们发现RAD51AP2通过C端与RAD51相互作用，而在缺失C端4个氨基酸（SRPI）的突变鼠RAD51AP2丧失与RAD51相互作用，说明RAD51AP2与RAD51的相互作用对于RAD51AP2募集到染色体轴上发挥功能至关重要。因此，我们从不育患者中发现了RAD51AP2突变，并确定了其在精母细胞中的定位，揭示了RAD51AP2特异参与减数分裂性染色体PAR区DSB的修复。相关工作的继续有望揭示哺乳动物减数分裂性染色体和常染色体DSB修复的差异及其调控机制，为深入理解性染色体重组过程提供了新见解。此外，在项目执行过程中，我们按期撰写年度进展报告和中期报告，并整理相应研究进展以论文形式予以发表，目前共发表带有本项目资助论文20篇，培养研究生3名。