载脂蛋白E介导双酚A干扰稀有鮈鲫精巢胆固醇平衡致精子发生异常的机制研究

Applicant's previous study found that bisphenol A (BPA) could cause sperm volume decrease, membrane damage and fertilizing ability decline in rare minnow Gobiocypris rarus, but the mechanism is not clear. Cholesterol plays an important role in the process of spermatogenesis, our study also showed that BPA can significantly affect the level of cholesterol in testis, as well as the expression of apolipoprotein E (ApoE) in both liver and testis. ApoE plays an important role in cholesterol transport and distribution. Based on this, we hypothesize that BPA affects spermatogenesis by affecting ApoE expression, and the altered ApoE affects the cholesterol transport between the liver and the testis, and the distribution of testicular cholesterol. The present study intends to adopt a long-term exposure of BPA at environmental relevant concentration to study the relationship between ApoE, testicular cholesterol and spermatogenesis. Moreover, we explore the relationship between ApoE and testicular cholesterol by intervening in vivo ApoE, and relationship between testicular cholesterol and spermatogenesis by intervening in vivo cholesterol levels. The mechanism of BPA regulating ApoE expression was further explored by CHIP and DNA methylation detection. The results of the present study will systematically elucidate the mechanism by which ApoE mediates BPA disrupting the balance of testicular cholesterol, leading to abnormal spermatogenesis. This study will provide a theoretical basis for the reproductive effects of BPA and its homologues on fish.

申请者前期研究发现双酚A（BPA）能够造成稀有鮈鲫精子体积减小、膜破损及授精能力下降，但是作用机制尚不明确。胆固醇在精子发生过程中起着重要作用，而我们发现BPA能显著改变稀有鮈鲫精巢胆固醇含量及肝脏和精巢中载脂蛋白E（ApoE）的表达。ApoE在胆固醇转运与分配过程中发挥着重要作用。据此我们提出假说：BPA通过干扰ApoE的表达，影响肝脏与精巢间胆固醇转运以及精巢胆固醇分布，从而影响精子发生。本项目拟采用环境相关浓度BPA长期暴露，来研究ApoE、精巢胆固醇以及精子发生三者之间的变化关系，通过分别干预体内ApoE和胆固醇的含量，来探究ApoE与精巢胆固醇平衡以及胆固醇与精子发生的关系，并利用CHIP与DNA甲基化检测，深入研究BPA调控ApoE的机制。研究结果将系统阐明ApoE介导BPA干扰稀有鮈鲫精巢胆固醇平衡致精子发生异常的机制，为BPA和其他内分泌干扰物对鱼类的生殖影响提供理论依据。

研究团队前期研究发现双酚A（BPA）能够造成稀有鮈鲫精子膜破损及授精能力下降，但是作用机制尚不明确。胆固醇是性激素的合成原料，也是细胞膜的重要成分，与细胞膜的脆性及流动性相关，在精子发生过程中起着重要作用。而我们也发现BPA能显著改变稀有鮈鲫精巢胆固醇含量及其转运蛋白载脂蛋白E和A1（ApoE和ApoA1）基因的表达。因而本项目从胆固醇转运的角度研究BPA影响鱼类精子发生的机制。首先研究了BPA作用下稀有鮈鲫精巢胆固醇平衡及精子发生异常之间的关系，然后研究了BPA对关键基因ApoE和ApoA1的表达影响及调控机制，最后通过载脂蛋白和胆固醇干预验证ApoE和ApoA1与胆固醇紊乱的关系以及胆固醇与精子形态功能之间的关系。研究结果表明BPA可通过雌激素受体相关受体（Esrrg）介导干扰精巢中ApoE表达，通过Esrrg和DNA甲基化修饰共同调控肝脏ApoE的表达，通过雌激素受体（Esrs）和DNA甲基化干扰精巢中ApoA1的表达，最终导致精巢和肝脏中ApoE的升高和紊乱，诱导精巢中ApoA1积累，进一步影响稀有鮈鲫肝脏和精巢之间的胆固醇转运以及逆转运，导致精巢FC的含量减少和分布紊乱, 最终导致精子膜破损率升高、精子膜流动性及受精率下降。本研究结果系统阐明ApoE和ApoA1介导BPA干扰稀有鮈鲫精巢胆固醇平衡致精子发生异常的机制，为BPA和其他内分泌干扰物对鱼类的生殖影响提供理论依据。