低剂量辛基酚通过干扰腺苷代谢致精子生成障碍的作用及其机制研究

Spermatogenic failure is the most common cause of male infertility which could be induced by endocrine disrupting chemicals (EDCs). Widespread exposure of alkylphenols has been found in Chinese men, and among the alkylphenols detected in Chinese male urine, the exposure level of 4-t-octylphenol (4-t-OP) was the highest which was significantly associated with spermatogenic failure in our previous study. Further, results of preliminary studies indicated that low-dose 4-t-OP may disturb adenosine metabolism and apoptosis of spermatogenic cell which can induce spermatogenic failure. This project will be based on the analysis of metabolic profile in seminal plasma which covers the major metabolites in adenosine metabolism pathway in large population and the internal exposure level of 4-t-OP in population to further confirm the relationships among the exposure of 4-t-OP, adenosine metabolism and spermatogenic failure. Mice treated with low-dose 4-t-OP, with or without adenosine metabolic intervention in testis will be conducted to detect the internal exposure level of 4-t-OP, the expression of key metabolic enzymes and the concentrations of major metabolites involved in adenosine metabolism pathway, the parameters of apoptosis and parameters of spermatogenesis in mouse testis which will confirm the effects of low-dose 4-t-OP on spermatogenesis and adenosine metabolism and further clarify the role of adenosine metabolism in low-dose 4-t-OP induced spermatogenic failure and the underlying mechanism. This project will provide new insights about the mechanism underlying low-dose 4-t-OP induced spermatogenic failure and new theoretical basis of diagnosis, treatment and prevention of spermatogenic failure.

精子生成障碍是男性不育最常见的病因，环境内分泌干扰物可以引起精子生成障碍。项目组前期研究发现烷基酚类物质在中国男性中暴露广泛，其中辛基酚暴露水平最高，且与精子生成障碍密切相关。进一步预实验结果提示低剂量辛基酚可能通过干扰腺苷代谢引起生精细胞异常凋亡导致精子生成障碍。本项目拟基于覆盖腺苷代谢通路的大样本人群精浆代谢谱分析，联合辛基酚内暴露水平数据，进一步明确辛基酚暴露—腺苷代谢—精子生成障碍的关联。并采用低剂量辛基酚染毒小鼠加睾丸腺苷代谢干预模型，检测小鼠辛基酚内暴露水平，检测小鼠睾丸腺苷代谢关键代谢酶表达及关键代谢物，检测睾丸凋亡指标，观察精子生成相关指标，确认低剂量辛基酚暴露对精子生成和腺苷代谢的影响，阐明腺苷代谢紊乱在低剂量辛基酚致精子生成障碍中的作用及机制。本研究将从代谢组学角度为低剂量辛基酚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、治疗和预防提供新的依据。

男性不育中仍有约40%～75%找不到原因，称为特发性男性不育症。精子生成障碍是导致男性不育的最常见原因。作为一种环境内分泌干扰物（Endocrine Disrupting Chemicals，EDCs），辛基酚（OP）应用广泛，大量研究表明OP具有生殖毒性。本课题基于人群病例对照研究，使用超高效液相色谱串联质谱（UPLC-MS/MS）检测特发性不育男性和健康对照尿液中OP含量，采用UPLC Q-Exactive代谢组学检测平台对OP检出和OP未检出的特发性不育男性进行精浆代谢组学分析，结合其精液参数，分析OP内暴露，腺苷代谢及精液参数三者之间的相关性。结果显示：OP暴露会增加特发性男性不育发病风险（P<0.05），且OP暴露会降低特发性男性不育患者的腺苷（Adenosine）代谢（P<0.05）。进一步在动物实验和体外实验中探讨腺苷代谢在低剂量辛基酚致精子生成障碍的分子机制。选择SPF级成年雄性ICR小鼠为OP染毒模型，随机分为3组，按照0、0.01μg/kg/day和1μg/kg/day剂量染毒。结果显示：OP染毒后，小鼠体重、睾丸脏器系数、睾丸病理（HE染色）均未发生显著改变；染毒组中凋亡的精母细胞数量和圆形精子数量显著减少（P<0.05）；染毒组小鼠睾丸中Adenosine含量显著降低（P<0.05），提示低剂量OP可能通过凋亡途径对精子生成产生影响，Adenosine代谢可能在此过程中扮演重要角色。体外（GC-2细胞系）染毒剂量分别为0、10-11M和10-8M。结果显示：OP染毒后细胞增殖、细胞周期均未发生显著改变，细胞凋亡受到明显抑制（P<0.05）；染毒后细胞线粒体膜电位显著上升（P<0.05）；凋亡相关基因表达情况分析显示：染毒后Bax,caspase-9和caspase-3表达显著降低，Bcl-2表达显著上升，提示OP可能通过线粒体凋亡途径抑制GC-2细胞凋亡；染毒后细胞Adenosine含量显著降低（P<0.05），在细胞染毒的同时在培养基中加入Adenosine干预，结果显示OP染毒引起的凋亡抑制及线粒体膜电位的变化得到恢复。综上所述，OP暴露会增加特发性男性不育的发病风险，且腺苷代谢在低剂量OP致精子生成障碍中扮演重要角色，OP可能通过抑制腺苷代谢来进一步影响生殖细胞凋亡进而干扰精子生成。该研究为OP致生殖毒性及其机制的研究提供了新的依据。