壬基酚对P450arom和MIS基因表达的影响

Effects of p-nonylphenol on the expression of P450arom and MIS genes

[目的]研究壬基酚(p-nonylphenol,NP)对细胞色素P450芳香化酶基因(P450arom)和苗勒氏管抑制物基因(MIS)表达的影响,探索其对鱼类雌激素样作用的分子机制。[方法]日本比目鱼在性分化过程中能随环境因子改变而发育为不同性别,在幼鱼孵化后第30天到第100天性分化敏感期于27℃水温下,给予幼鱼投食NP 100μg/g饲料。分别对各组比目鱼生殖腺做病理组织切片,判断其性别和雌雄比例,并通过原位杂交方法结合RT-PCR分析第100天青年期鱼生殖腺中P450arom和MISmRNA的表达,观察NP对比目鱼性分化标志基因的作用。[结果]NP对27℃水温饲养下的比目鱼幼鱼有一定程度的雌性化诱导作用,成年雌性的百分比为30%,高于27℃水温对照组(0%);比目鱼幼鱼出生后第100天时对其生殖腺进行原位杂交分析显示,P450arom的mRNA在各组的雌鱼卵巢中有明显表达,但在雄鱼精巢中未见表达;而MIS的mRNA在各组的雌鱼卵巢中不表达,却在雄鱼精巢中能见到很明显的表达。RT-PCR分析也显示,P450芳香化酶的mRNA仅仅在雌鱼卵巢中表达,而MIS也仅仅在雄鱼精巢中表达,证实了原位杂交实验的结果。[结论]在日本比目鱼体内,NP染毒可以诱导P450芳香化酶mRNA表达并抑制MIS的mRNA的表达,从而对生殖腺性分化期的幼鱼发挥雌激素样干扰作用。

[ Objective] To elucidate the effect of p -nonylphenol （NP） on cytochrome P450 aromatase （P450arom） gene and Mullerian inhibiting substance （MIS） gene in fish gonad, exploring the mechanism of its estrogenic action. [ Methods] Treatment with NP to Japanese flounder, a teleost that exhibits temperature -dependent sex determination, was carried out by providing the larvae with an artificial diet mixed with 100μg/g of NP at a masculinizing temperature （27℃） from 30 to 100 days after hatching. Phenotypic sex was determined at 100 （juvenile stage） and 300 dah （adult stage） by histological observation. Sex ratio, P450arom and MIS mRNAs expression in the genetically female flounder treated with NP were examined by in situ hybridization and RT- PCR analyses. [ Results] Treatment with 100μg/g diet of NP induced feminization of the flounder larvae reared at 27℃ with female percentages of 30%. The result of in situ hybridization and RT - PCR analyses showed that feminization of the larvae by NP was accompanied by up - regulation of P450arom mRNA expression and down - regulation of MIS mRNA expression in female gonad at 100 days after hatching. [ Conclusion] In Japanese flounder, treatment of NP can induce the expression of P450arom gene and suppress the expression of MIS gene, excerting its estrogenic disruption on gonadal sex differentiation in fish.