abcc基因及RA信号通路在环境内分泌干扰物致多代精子发生异常过程中的作用机制研究

The problem of infertility caused by environmental factors is becoming more and more serious in China, especially male reproductive problem caused by plasticizers. Epidemic investigation and experimental results suggest that male reproductive abnormalities caused by phthalates can be intergenerational transmitted and DNA methylation may be one of its important mechanisms. DNA methylation pattern in zebrafish sperm can be transmitted to offspring and participate in the early embryonic development. With this advantage, we selected zebrafish as a model to study the transgenerational effects of phthalates on reproduction. Previous studies showed that the reduction of male reproduction and spermatozoa induced by DBP exposure could be transmitted to offspring, and 16 genes including abcc were found to be modified in expression cross the generation. RA signaling pathway plays an important role in life, including regulating abcc expression and participating in spermatogenesis. Does DBP exposure cause the decrease of abcc expression and sperm count by interfering with RA function? The purpose of this study is to explore the effects of DBP exposure on RA signaling pathway and meiosis in zebrafish testis. To find out the molecular mechanism of DBP-induced male reproductive dysfunction, we will re-perform the DBP exposure test and investigate the expression pattern of important genes in RA signaling pathway. To explore the trans-generation mechanism of this phenomenon, we will perform DNA methylation detection of related important genes. The result of this study may provide a basis understanding for plasticizer-induced infertility, and the scientific basis for prevention.

环境内分泌干扰物（EEDs）之一，邻苯二甲酸酯类（PAEs）暴露导致的雄性生殖异常具有传代现象，DNA甲基化可能是其重要机制之一。斑马鱼精子DNA甲基化谱可以传递至子代并指导子代早胚发育，我们利用这一优势以斑马鱼为模型研究PAEs对生殖功能的跨代影响。前期实验表明，邻苯二甲酸二丁酯（DBP）暴露导致雄性生殖功能降低、精子减少可传递至子代，且两代精巢表达谱比较发现包括abcc在内的16个基因表达改变趋势相同。RA信号通路既调节abcc基因表达又参与精子生成。DBP暴露是否通过干扰RA功能导致abcc表达下降及精子减少？本研究拟通过DBP暴露导致多代雄性生殖功能降低的斑马鱼模型，探究精巢中RA信号通路受DBP暴露的影响及进一步对减数分裂的影响，寻找DBP导致雄性生殖功能下降的分子机理；并检测相关重要基因的DNA甲基化水平，探寻这一现象的传代机制，为塑化剂导致不孕不育的防治提供科学依据。

邻苯二甲酸酯类（PAEs）暴露对雄性生殖系统功能的损害具有传代现象，学者们多认为DNA甲基化是可能的机制之一。斑马鱼精子DNA甲基化可以传递至子代并指导子代早期胚胎发育。前期实验发现邻苯二甲酸二丁酯（DBP）暴露后，2代斑马鱼精巢中abcc基因表达下降。RA信号通路既调节abcc基因表达又参与精子生成。我们选择斑马鱼研究DBP暴露对3代精巢中RA信号通路的影响及进一步对减数分裂的影响，寻找DBP导致雄性生殖功能下降的分子机理。通过荧光定量PCR分析我们发现DBP暴露后，3代斑马鱼精巢中RA转运蛋白rbp4表达降低，受体raraa在暴露后2代斑马鱼中升高，这些变化干扰了RA正常功能，可能是导致生精减少的原因之一；通过荧光定量PCR及免疫组化，我们发现DBP暴露后3代斑马鱼精巢中精原细胞减少，减数分裂期异常双链断裂增加，推测这是导致生精减少的另一个原因；通过焦磷酸测序，我们发现3代斑马鱼精子中RA受体raraa的编码基因DNA甲基化下降，下降趋势随着传代降低，RA降解酶cyp26a1的编码基因DNA甲基化水平升高，升高趋势对着传代增加，推测这两个可传代的DNA甲基化改变是导致1代暴露，多代受损的原因之一。以上研究结果有利于深入理解PAEs导致多代雄性生殖损害机理，为塑化剂导致的不育防治提供理论依据。.