自噬在甲醛暴露致大鼠生精损伤中的作用及分子机制研究

Formaldehyde is a ubiquitous environmental pollutant. As an important chemical for global economy, formaldehyde output has reached more than 46 billion pounds annually in worldwide. Over the last 20 years, China has experienced rapid economic growth and a simultaneous rise in demand for formaldehyde. In 2004, China surpassed the United States as the largest formaldehyde producer and consumer in the world. China’s actual FA output has reached a staggering 12000 kt in 2007, about 4000 times the amount five decades earlier. Coinciding with the growth, formaldehyde pollution has also increased considerably in China. A substantial body of evidence that consistently indicated the adverse effects of formaldehyde on male reproduction in recent years. However, the molecular and cellular mechanisms underlying this phenomenon remain elusive. .Autophagy is an evolutionarily conserved self-degradation process which provides a membrane-dependent mechanism for the sequestration and lysosomal turnover of subcellular components and organelles. Increasing evidence demonstrated that deregulated autophagy may critically influence vital cellular processes and thereby may play a critical role in the pathogenesis of many human diseases. However its role and characterization remains unclear in male reproductive injuries..Our earlier study has found that markedly enhanced autophagy occurred in testicular tissues of formaldehyde exposure rats. Moreover, the alterations of autophage were consistent with the spermatogenesis injuries. Therefore, the present study was designed to further take autophagy as the breakthrough point, to investigate the role and molecular mechanisms of autophagy in spermatogenesis injuries induced by formaldehyde exposure. Specific strategies include: ① At first, Transmission electron microscopy, MDC, Real-time PCR and Western blot were used to detect autophagy phenomenon in formaldehyde exposure rat models and various spermatogenetic cell models. Afterward, we will further confirm the role of autophagy in male reproductive toxicity via inhibition of autophagy by using autophagy inhibitors or RNAi technique respectively. ② With AMPK/mTOR/ULK1 signaling pathway as the penetration point, we will verify the key upstream molecular switches of autophagy in formaldehyde exposure rat models and various spermatogenetic cell models by using various techniques such as Real-time PCR，Western blot，HPLC，Pathway inhibors or RNAi etc .③ On the basis of our previous Chip screening, Immunoprecipitation (IP), Laser confocal microscopy, Bioinformatics and ChIP techniques will be used to further analysis whether autophagy regulate the expression of spermatogenesis specific genes such as SPO11 and TEX15 to trigger spermatogenic impairment. These experiments will help us to identify the downstream pathways which autophagy induced spermatogenesis injuries. In conclusion, this study will not only lay the foundation for clarifying the male reproductive toxicity of formaldehyde, but also provide new clues and evidence for prevention and treatment male reproductive toxicity of formaldehyde.

甲醛是目前全球最主要的环境污染物之一，已有研究显示甲醛具有雄性生殖毒性，但具体分子机制不明。我们前期研究发现甲醛暴露引发大鼠睾丸自噬水平显著增高，且与生精损伤密切相关。本课题拟在前期基础上，以自噬为突破点，深入探索自噬在甲醛诱发生精损伤中的作用和分子机制。具体策略为：①在甲醛暴露大鼠和生精细胞模型中利用电镜，MDC，Real-time PCR，Western blot等手段观察自噬改变；继而抑制自噬，揭示自噬在生精损伤中的作用；②以AMPK/mTOR/ULK1通路为切入点，利用体内外实验验证调控自噬发生的上游分子开关；③在前期芯片筛查基础上，利用IP，激光共聚焦、生物信息学，ChIP等技术分析自噬是否通过调控SPO11、TEX15基因表达进而导致生精异常，明晰自噬引发生精损伤的下游靶点。本课题将为阐明甲醛雄性生殖毒性奠定基础，为进一步防治甲醛雄性生殖毒性提供新的线索和依据。

在众多的环境污染物中，甲醛以其来源广，毒性大，污染时间长等特点已成为全球最主要的环境污染物之一。尽管人群研究和动物实验已证明甲醛具有雄性生殖毒性，但其分子毒理机制尚不明确。本研究以自噬为突破点，从分子、细胞和动物整体水平综合研究自噬调节在甲醛诱发生精损伤中的作用和分子机制。 我们（1）构建不同时间、剂量甲醛暴露大鼠模型和生精细胞模型，利用透射电镜、Western blotting、 Real-time PCR、MDC等观察自噬，发现睾丸组织和生精细胞自噬水平的增加具有时间和剂量依赖性，且与睾丸生精损伤密切相关；（2）动物水平和细胞水平均显示随着甲醛暴露水平的增高，AMPK/mTOR/ULK1通路激活，是调控自噬发生的上游分子开关；（3）甲醛暴露会引起精子发生特异基因SPO11、TEX15表达下降，其下降与自噬水平负相关，与睾丸生精小管直径和生精上皮高度下降正相关。抑制自噬能通过SPO11、TEX15途径部分改善精子发生，SPO11 、TEX15可能是自噬发挥作用的下游靶点。此外，本研究在完成原有计划的同时，（4）我们还增加构建了甲醛暴露大鼠差异circRNA表达数据库，对自噬的上游调控circRNA进行筛查与通路机制验证，进一步探索rno_circRNA_016194/rno-miR-449a-5p/Atg4b调控网络在甲醛暴露致大鼠睾丸组织自噬中的调控作用；（5）我们还增加构建了非梗阻性无精症（NOA）患者睾丸组织差异circRNA表达数据库，探索其对NOA患者睾丸组织自噬的调控作用和分子机制，相关研究结果也为后续研究探索方向。总之，该课题通过揭示自噬在甲醛诱发生精损伤中的作用及分子机制，为阐明甲醛雄性生殖毒性奠定基础，也为进一步防治精子发生障碍提供新的思路、线索和实验依据。本课题共发表论文17篇，其中SCI收录论文14篇（其中ESI高被引1篇），中文核心期刊论文3篇。培养博士研究生3名（毕业1名），硕士研究生5名（毕业2名）。进行本科生开放实验项目6期，培训本科生21人次。研究成果大会报告和会议交流4次。研究结果为甲醛雄性生殖毒性及机制的研究提供了丰富的资料和数据，亦为环境与男性生殖健康研究提供了实验依据和下一步的研究方向。