昼夜节律紊乱中Clock生物钟基因抑制同源重组导致精子数量下降的分子机制研究

申请人青年项目发现昼夜节律紊乱（Circadian Disruption，CD）与人及小鼠精子数量下降有关。自精母细胞以后各阶段生精细胞标志物均表达下调，转录组测序显示同源重组（精母细胞生精过程关键事件）通路变化最为显著（含Nbn、Atm等关键分子），生物钟通路也显著变化（Clock、Creb1等关键分子）。生信分析提示作为CD响应分子的生物钟基因可能调控同源重组通路。据此我们假设：CD通过生物钟基因抑制同源重组，导致精子数量下降。项目拟利用CD小鼠模型全面识别差异表达的生物钟基因和同源重组通路基因；重点针对Clock、Creb1、Btrc、Nbn、Atm等关键分子，用原代生精细胞和动物模型于体内外证实相关基因在CD减少精子数量中的作用，并分析关键分子间相互作用；用人睾丸组织检测两通路共表达情况及其与倒班工作的关系，验证动物和细胞实验结果。项目可为阐明CD致生殖损伤机制和损伤防治提供依据。

Our previous research showed that circadian disruption (CD) was associated with decrease of sperm count in human and mice. The expression of specific markers of spermatogenesis later than spermatocyte stage was decreased. Transcriptome sequencing and RT-PCR showed that the homologous recombination (key process of spermatogenesis in spermatocyte) pathway (including Nbn, Atm, etc.) and the clock genes (including Clock, Creb1, etc.) had substantial change. Bioinformatics analysis suggested that the clock genes may regulate the homologous recombination pathway. Hence we hypothesize that CD may inhibit homologous recombination via clock genes, leading to decrease of sperm count. The plan of the present project is to comprehensively investigate the differential expression of clock genes and the homologous recombination pathway genes in CD mice; to investigate the role of these differentially expressing genes, especially Clock, Creb1, Btrc, Nbn, Atm, etc., in CD-induced decrease of sperm count; to investigate the interfacing of these molecules, to validate the animal/molecular results in the testis tissue of obstructive azoospermic patients. The project may help to illustrate the mechanism of CD-induced reproductive damage and to develop the prevention and treatment strategy.