大豆苷元通过干扰花生四烯酸代谢导致精子生成障碍的研究

Spermatogenic failure which can be induced by endocrine disruptors is the most common cause of male infertility. We found exposure to daidzein (DAI), an endocrine disruptor was widespread and high in the Chinese, and was associated with spermatogenic failure. Our preliminary studies suggest that DAI may disrupt the metabolism of arachidonic acid (AA) and more prostaglandins may be produced which can induce spermatogenic failure. In this project we will conduct metabolomic profiling covering major metabolites in AA metabolism pathway using seminal plasma, and explore the association between AA metabolism and spermatogenic failure in the population, and then combine data of exposure to DAI with data of AA metabolism to delineate the following relationship: daidzein - AA metabolism - spermatogenic failure. Mouse model will be further established by oral exposure to DAI and AA metabolism intervention, and the expressions of key metabolic enzyme genes and key metabolites in AA metabolism will be analyzed in mouse testes, and spermatogenesis-related results will be collected to verify the effects of DAI on spermatogenesis and AA metabolism and to reveal the role AA metabolism plays in DAI-induced spermatogenic failure. This project will provide novel insights into the mechanism underlying DAI-induced spermatogenic failure, and provide new theoretical basis on diagnosis, prevention and treatment of spermatogenic failure.

精子生成障碍是男性不育最常见的病因，内分泌干扰物可引起精子生成障碍。项目组前期在中国人群中发现内分泌干扰物大豆苷元暴露水平高且广泛，与精子生成障碍有显著关联。进一步开展的预实验提示大豆苷元可能通过干扰花生四烯酸代谢进而产生更多具有抑制精子生成效应的前列腺素导致精子生成障碍。本项目拟基于人群分析精浆代谢谱覆盖花生四烯酸代谢通路主要代谢物，分析花生四烯酸代谢与精子生成障碍的关联，再联合大豆苷元内暴露水平数据探讨大豆苷元-花生四烯酸代谢-精子生成障碍的关联。采用小鼠染毒加花生四烯酸代谢干预模型，检测睾丸花生四烯酸代谢关键代谢酶基因表达和关键代谢物，观察精子生成相关指标，确认大豆苷元暴露对精子生成和对花生四烯酸代谢的影响，阐明花生代谢紊乱在大豆苷元致精子生成障碍中的作用和机制。本研究将为大豆苷元致精子生成障碍的机制提供新的认识，为精子生成障碍的诊断、预防和治疗提供新的依据和途径。

本研究拟探讨大豆苷元（DAI）对精子生成障碍的影响，并探讨花生四烯酸（AA）代谢尤其是其效应代谢物前列腺素E2（PGE2）在其中的作用。采用UPLC-QE建立男性生殖多样本类型代谢组学技术；纳入包含精子生成障碍的患者和健康对照共1194人；采用UPLC-MS/MS检测尿液中DAI含量；采用代谢组学技术分析尿液、精浆、精子、血液的精子生成障碍代谢特征；基于该数据库，采用logistic回归、线性回归分析明确DAI-AA代谢-精子生成障碍的人群关联；依据DAI人群暴露水平建立小鼠青春期到成年期染毒模型，并代谢干预，观察生殖指标并开展交配实验，采用DGE转录组测序和代谢组学技术分析染毒和代谢逆转组睾丸，明确分子机制。结果：1.建立了15分钟可同时检测270种代谢物的快速、灵敏、准确、稳定的代谢组学方法，覆盖65条代谢通路。2.基于大样本代谢组学分析揭示了精子生成相关的多样本类型代谢生物标志物库，其中尿液中PGE2和精浆中AA代谢物显著提高精子生成障碍风险。3.人群中DAI高暴露显著提高精子生成障碍风险。4. DAI暴露与尿液代谢组关联分析发现，AA代谢为富集度最高的通路。DAI暴露与精子生成障碍PGE2尿液含量显著正相关，精浆中亦得到验证。5.小鼠模型发现高剂量DAI（100mg/kg/day）显著降低精子密度并在代谢逆转组恢复。交配实验发现，高剂量DAI暴露显著降低每只怀孕鼠的活胎数量并在代谢逆转组恢复。转录组和代谢组整合分析结合GC-2细胞验证发现高剂量DAI暴露导致睾丸PGE2上升，AA代谢中Pla2g6和Ptges3表达上升，延胡索酸下降、Ass1和Pck1表达下调，并在代谢逆转组恢复。本研究揭示了高剂量DAI暴露增加精子生成障碍的风险。DAI通过上调睾丸Pla2g6酶和Ptges3酶基因表达而提高睾丸中的PGE2水平, PGE2降低了睾丸中的延胡索酸，影响了其代谢下游Pck1和Ass1酶基因表达导致三羧酸循环、糖异生和尿素循环紊乱而影响精子生成导致精子生成障碍。