双酚A对斑马鱼精巢性激素生成酶基因表达的影响

Alterations in the mRNAs of Enzymes Responsible for Sex Steroidogenesis in the Testis of Zebrafish Exposed to Bisphenol-A

为了解环境雌激素对鱼类精巢发育的影响,将成年雄性斑马鱼(Danio rerio)暴露于不同浓度双酚A(0、500、1 000、2 000、4 000μg·L-1)下21 d,并在此基础上,进一步研究了暴露在相同浓度(BPA2 000μg·L-1)不同暴露时间下各基因表达的动力学模式。用荧光定量PCR(qRT-PCR)方法检测试验鱼精巢性激素合成相关细胞色素P450酶类基因(CYP17、CYP11B和CYP19A)以及雌、雄激素受体和促卵泡激素受体(ERα、ERβ、AR和FSHR)基因的表达,用常规组织学方法研究试验鱼精巢结构的变化。结果表明,BPA促进了精巢内源雌激素合成相关酶类基因CYP19A和雌激素受体ERαmRNA、ERβmRNA的表达,且BPA浓度为2 000μg·L-1时3者的表达量显著上调,同时随着暴露时间的延长,具有明显的时间累积效应。1 000μg·L-1BPA可导致斑马鱼精巢CYP17 mRNA的表达量显著下调,BPA2 000μg·L-1暴露12 d引起了CYP11B mRNA表达下调,而随着暴露时间的延长有所回升,但它对精巢AR mRNA的表达却无明显影响。同时,BPA 500μg·L-1引起了FSHR基因的显著上调,在时间动态学上,呈上升趋势。在组织学上,2 000μg·L-1BPA引起斑马鱼精巢生精小管内精子浓缩严重,精原细胞(Sg)和精母细胞(PS和SS)数目都有所减少,BPA 4 000μg·L-1组斑马鱼精巢退化。因此,BPA可诱导精巢内源雌激素合成和雌激素受体的表达,提高雌激素效应,通过抑制CYP17基因的表达,一定程度上抑制雄激素的合成,从而引起斑马鱼精巢组织的破坏。同时,据实验数据推测,雄激素的下调可能启动了下丘脑-垂体-性腺(HPG)轴的负反馈调节机制,而此作用机制还有待进一步研究。

In order to elucidate the effect of different concentrations of an environmental estrogen on testicu- lar development in fish, male aduk zebrafish （Danio rerio） were exposed to 0, 500, 1 000, 2 000 and 4 000 μg·L-1 bisphenol A （BPA） for21 days. Then, another group of male zebrafish were exposed to 2 000 μg·L-1 BPA for the time-course gene expression analysis. The mRNAs of CYP450 enzymes responsible for sex steroid synthe- sis （CYP17, CYPllB, CYPI9A）, estrogen receptor a （ERa）, estrogen receptor 13 （ER13）, androgen receptor （AR）, and follicle-stimulating hormone receptor （FSHR） were quantified using real-time polymerase chain reactions （qRT-PCR）. Histological alterations in the testis were subsequently investigated. Results showed that, BPA ex- posure upregulated testicular CYP19A , ERa and ER[3 mRNAs. At 2 000 μg·L-1 BPA, the expression of all three genes was significantly upregulated. With the prolongation of BPA exposure, the effect on gene expres- sion became more pronounced. BPA at 1 000 μg·L-1 significantly decreased the expression of CYP17 gene, while CYPllB mRNA was decreased in the fish treated with 2 000 μg·L-1 BPA for 12 days, and upregulated thereafter. But BPA had no effect on the AR mRNA. Meanwhile, 500 μg·L-1 BPA markedly increased the ex- pression of FSHR gene, and a significant increase in FSHR mRNA expression was observed as exposure time extended. Histologically, in the fish treated with 2 000 μg·L-1 BPA, sperms were found to be densely con- gregated in seminiferous tubules. Spermatogonia, primary spermatocytes and secondary spermatocytes were reduced. Testis degradation was observed in the fish exposed to 4 000 μg·L-1 BPA. These results indicated that BPA can induce both the synthesis of endogenous estrogen and the expression of ER gene to enhance its estrogenic effects. Also, BPA can inhibit the synthesis of androgen to some extent by suppressing CYP17 gene expression, ultimately leading to the impairment of testicular structure and impediment of testicular devel- opment. Based on the results, it is postulated that the downregulation of androgenic hormone may have acti- vated the negative feedback mechanism for the hypothalamus-pituitary-gonad （HPG） axis