双酚A对稀有鮈鲫精巢载脂蛋白E基因的影响及调控机制

Effect and mechanism of bisphenol A on apolipoprotein E gene in rare minnow (Gobiocypris rarus)

摘要:

目的 双酚A (BPA)是一种具有雌激素效应的塑化剂，对雄性生殖影响显著，载脂蛋白E (ApoE)对精巢中甘油三酯和胆固醇等脂质的运输过程有重要作用。本研究探讨BPA对精巢ApoE基因的影响及调控机制，以期找到BPA所引起的雄性生殖问题与脂代谢之间的潜在联系。方法 将稀有鮈鲫暴露于BPA (15 μg/L) 1、3和5周，通过实时荧光定量 (qPCR)、免疫印迹 (WB)及免疫荧光 (IF)检测BPA对ApoE基因表达和分布的影响，通过染色质免疫共沉淀 (ChIP)和DNA甲基化检测探究BPA对ApoE的调控机制。结果 qPCR、WB及IF结果显示，BPA可以显著影响精巢ApoE基因的表达和分布。ChIP结果显示，ApoE的启动子区存在一个雌激素相关受体γ (Esrrg)阳性作用位点，ApoE mRNA的表达变化与Esrrg的募集变化一致。DNA甲基化检测结果显示，ApoE的启动子区处于未甲基化状态，BPA处理对其无显著影响。结论 BPA可以通过调控稀有鮈鲫精巢ApoE基因启动子区域的Esrrg的募集直接干扰ApoE基因表达，BPA可能通过影响稀有鮈鲫精巢脂质转运影响其生殖功能。本研究结果将为后续从脂质代谢角度探究环境内分泌干扰物对雄性生殖的影响提供参考。

Abstract:

Bisphenol A (BPA), a plasticizer with estrogenic activity, has significant effects on male reproduction. Apolipoprotein E (ApoE) plays an important role in the transport process of lipids, such as triglycerides and cholesterol in spermatozoa. In this study, we investigated the effects of BPA on the ApoE gene in testis and its regulatory mechanism, to elucidate the potential relationship between BPA-induced male reproductive problems and lipid metabolism. Rare minnows were exposed to BPA (15 μg/L) for 1, 3 and 5 weeks. The effects of BPA on ApoE gene expression and distribution were assessed using real-time fluorescence quantification (qPCR), immunoblotting (WB), and immunofluorescence (IF). Additionally, the regulatory mechanisms of BPA on ApoE were explored using chromatin immunoprecipitation (ChIP) and DNA methylation assays. Results from qPCR, WB, and IF demonstrated that BPA significantly affected ApoE gene expression and distribution in spermatozoa. ChIP analysis revealed an estrogen-associated receptor γ (Esrrg) positive effect site in ApoE promoter region, with changes in ApoE mRNA expression correlation with Esrrg recruitment. DNA methylation assays showed that the ApoE promoter region was unmethylated, and that BPA treatment did not significantly alter its methylation status. These findings indicated that BPA directly interferes with ApoE gene expression by regulating Esrrg recruitment in the ApoE gene promoter region. Consequently, BPA may affect the reproductive function of rare minnow by disrupting testicular lipid transport. Our results provided a reference for future research on the effects of environmental endocrine disruptors on male reproduction from the perspective of lipid metabolism.