基于代谢组学研究策略的钼暴露致精子生成障碍的研究

Heavy metal pollution is becoming increasingly serious, and it may affect spermatogenesis. However, previous studies are limited in a small number of heavy metals with known toxicity. By using the profiling analysis for 52 kinds of urinary heavy metals in combination with OPLS-DA technique, in the preliminary experiment of this project, we found molybdenum was the heavy metal with the top contribution to spermatogenesis failure from the overall perspective of all detected heavy metals based on 500 pairs of cases of spermatogenesis failure and controls. There are few studies on male reproductive toxicity of molybdenum, and the mechanism is not clarified. The preliminary study using metabolomics analysis suggested that molybdenum might cause spermatogenesis failure by disrupting adenosine metabolism. Based on the above population, this project will use integrated metabolomics analysis of seminal plasma and urine in combination with mediating effect analysis to clarify the relationship of molybdenum exposure-adenosine metabolism-spermatogenesis failure. Independent population of high exposure to molybdenum will next be used to verify the relationship. Then, we will establish the mouse models with molybdenum treatment according to the actual exposure levels in humans, with or without the intervention of adenosine metabolism covering upstream NT5E enzyme, adenosine and downstream adenosine A1 receptor, in which we will carry out general toxicity evaluation, reproductive outcome evaluation and metabolic mechanism study to clarify the role of molybdenum exposure in spermatogenesis failure and underlying mechanism. This study will provide the basis on diagnosis, prevention and treatment of spermatogenesis failure from the new perspective of the control of molybdenum exposure.

重金属污染日趋严重可能影响精子生成，既往研究局限于少数已知毒性重金属。本项目前期实验基于500对精子生成障碍的病例和对照采用52种重金属的暴露谱分析结合OPLS-DA技术发现，钼为综合所有检测的重金属暴露后对精子生成障碍贡献最大的重金属。钼的男性生殖毒性研究还很少，机制未阐明。预实验代谢组学分析提示钼可能通过干扰腺苷代谢导致精子生成障碍。本项目拟基于上述人群，采用精浆和尿液的代谢组学联合分析结合中介效应分析阐明钼暴露-腺苷代谢-精子生成障碍的关联，并采用钼高暴露独立人群验证。建立符合人群实际暴露水平的钼暴露结合系统的腺苷上下游代谢干预（包含上游NT5E酶、腺苷和下游腺苷A1受体）的小鼠模型，对各组开展一般毒性评价、生殖结局评价和代谢机制研究，阐明钼暴露致精子生成障碍的作用和机制。本研究将从防控钼暴露的全新角度，为精子生成障碍的诊断、预防和治疗提供新的依据和途径。

本项目探讨了钼暴露对精子生成的影响，并研究了腺苷代谢在其中的作用和机制。纳入包含精子生成障碍的患者和健康对照共962人；采用ICP-MS检测尿液中重金属暴露谱的含量；采用UPLC-Q Exactive分析尿液和精浆的代谢组；分析钼暴露-腺苷代谢-精子生成障碍的人群关联。依据钼人群暴露水平建立多年龄阶段小鼠染毒模型，并开展腺苷代谢干预（上游代谢酶 NT5E 干扰、腺苷分解和下游腺苷 A1 受体干扰），观察生殖指标，分析关键下游分子，阐明分子机制。结果：1. 在检测的52种重金属中，钼为最显著的且含量最高的与精子生成障碍相关的元素，其显著提高精子生成障碍的风险。尿液中检测出代谢物366种，精浆中检测出代谢物370种，覆盖氨基酸代谢、糖代谢、脂质代谢、核酸代谢和辅酶代谢。发现尿液钼和尿液腺苷、腺苷/AMP的比值具有显著的正相关。尿液中腺苷剂量依赖地提高精子生成障碍的风险，精浆腺苷与一次射精总数、精子密度、精子活力具有显著的负相关。进一步结合第二阶段钼高暴露人群验证，本研究证明了钼暴露-腺苷代谢-精子生成障碍的关联。另外还发现地表水体中钼的广泛存在，含量为ng/ml级别，是人体摄入钼的重要来源。2.采用8、11、56周龄小鼠染毒钼，发现睾丸钼元素水平剂量依赖上升，病理学检查发现其特异性地损伤了睾丸组织。精子数量和前进性精子比例下降，精子畸形率上升。小鼠睾丸中精母细胞和支持细胞均存在凋亡的加剧。交配实验发现钼导致妊娠毒性。睾丸中Bax、Caspase-9、Caspase-3的基因表达和氧化型谷胱甘肽染毒后上升。上述效应一定程度被腺苷代谢逆转所恢复。3.采用体外实验，发现钼染毒对GC-2细胞增殖无明显影响，促进细胞凋亡并导致凋亡基因表达上升，上述效应在腺苷代谢逆转组被恢复。本项目阐明了钼通过腺苷代谢致精子生成障碍的作用和机制，为钼暴露的男性生殖安全性评价和钼的防控提供了新的科学依据。