UHRF1基因在精子发生中的功能及其机理

Azoospermia is defined as the absence of sperm in at least two different ejaculate samples. Approximately, 1% of all men suffer from azoospermia-caused infertility. The impaired meiosis has been reportedly contribute to development of azoospermia. In meiosis prophase I, some key events happen, including chromosome synapsis and meiotic recombination, establishing accurate separation of homology chromosomes. However, molecular basis and regulatory web are largely unknown. .UHRF1（ubiquitin-like, PHD and RING finger containing 1 encodes a multifunctional protein. Three functional domains of UHRF1 have been characterized to interact with DNMT1, a methyltransferase that is in charge of maintenance of DNA methylation in mammals. However, the role of UHRF1 in meiosis has not been reported. We have established a strain of mice whose UHRF1 was knocked out selectively in testis. Our preliminary study showed that spermatogenesis of UHRF1-/- mice was severely impaired, including small sizes of testis (about 40% of control) and the absence of detectable sperm in epididymis. We also observed an accumulating number of Zygotene cells and reduced number of Pachytene cells in UHRF1-/- testis. In addition, the abnormal styles of homologous chromosome synapsis were observed in the cultured UHRF1-/- spermatocyte. These results, taken together, strongly suggest that UHRF1 play a key role in meiosis I prophase. .In order to explore the mechanism of role of UHRF1 in meiosis, we next plan to comparatively study the gene expression profiles and DNA methylomes of different stages of meiosis I prophase including Leptotene, Zygotene and Pachytene. The important genes will be screened based on the above analysis, and their localization and expression level in prophase will be studied via the immunostaining assays. Finally, given our UHRF1-/- mice actually represents an animal model of azoospermia, we will also study association of UHRF1 SNP and clinical azoospermia and oligozoospermia.

无精子症约占所有男性1%，减数分裂异常则是该病的重要原因。减数分裂I前期同源染色体的精确配对、联会和重组奠定了其正确分离的基础。但对其调控机制了解甚少。UHRF1蛋白能与DNA甲基化转移酶1发生相互作用，因此参与了基因组DNA甲基化调控。但UHRF1在精子发生中的作用和机制尚未见报道。我们建立了UHRF1睾丸条件性敲除小鼠模型，初步研究发现其附睾中没有精子，减数分裂偶线期停滞，少数同源染色体配对或联会异常等，提示UHRF1在减数分裂Ⅰ中具有关键作用。我们计划探讨UHRF1敲除对前期各阶段（细线期、偶线期、粗线期和双线期）基因表达谱与DNA甲基化谱的影响，发现其调节精子发生的分子通路；在此基础上，在无精子和少精子症患者中，对UHRF1的全外显子进行测序，检测相应的突变在人精子与DNA甲基化的关系，以期阐明UHRF1在精子发生的作用及其分子机制，为人类精子发生障碍的研究提供新思维。

无精子症约占所有男性1%，减数分裂异常则是该病的重要原因。DNA的表观遗传学修饰在雄性减数分裂中发挥重要功能。UHRF1能识别DNA甲基化和羟甲基化。甲基化在生精细胞减数分裂中的功能已多次被报道。但UHRF1调控DNA羟甲基化的生理学意义是什么，目前相关文献甚少。本课题组很早就发现了UHRF1缺失会导致DNA-5hmC发生改变。为研究5hmC在生精过程中的作用，我们借助UHRF1基因敲除小鼠重点做了以下两个内容的研究：一、UHRF1敲除后生精细胞在减数分裂前期过程中出现的具体发育缺陷；二、UHRF1敲除引起的基因表达水平的改变其具体机制是如何发生的。结果是：一、UHRF1缺失导致生精细胞发生了偶线期阻滞，联绘配对失败，同源重组异常。二、UHRF1缺失导致基因组水平5-羟甲基化的异常升高，TSS位点的5-羟甲基化升高抑制了基因的表达。UHRF1缺失，联绘中轴蛋白Sycp1定位错误，同源染色体的配对失败。DMC1则滞留在染色体轴上，γH2AX出现在常染色体位置，故UHRF1缺失影响了DSB的修复，交换蛋白MLH1位点也明显减少。同时在RNA-seq样本中，我们发现了和染色体配对、DSB修复以及遗传信息交换相关的基因都显著下调。进一步分析，发现UHRF1缺失导致羟甲基化上调，TSS位点的5hmC上调抑制了基因的表达。通过动物模型，我们证明了UHRF1在精子发生中的功能。在少精症临床样本中，我们在UHRF1启动子区域发现了多个SNP位点，这些位点很有可能影响了UHRF1的表达量，进而影响了精子发生。有意思的是，我们在少弱精临床样本中发现重要基因AChE启动子区域发现5hmC异常升高。本研究首次在精子发生中揭示了5hmC对基因表达的调控功能。在我们的另一份BPA暴露人群和精子质量的研究中，我们发现暴露人群的精子质量差于正常人群，但是暴露人群精子DNA-5hmC却显著上升，特别是在基因的位置。同时，伴随着DNA-5hmC的上升，一些和减数分裂密切相关的基因表达下调。综上所述，我们通过动物模型发现UHRF1通过抑制5hmC促进基因的表达。同时我们在动物、人群样本等不同水平揭示了5hmC在精子发生中的功能。