Dehp On Rana Chensinensis Star, The Influence Of Cyp17 And Cyp19 Expression In Testis

Di-2-ethylhexyl phthalate(DEHP) belongs to the phthalic acid esters(PAEs), is an important esters environmental estrogen, it can lead to the vertebrate reproductive function decline and disrupt normal biological body endocrine and reproductive function. The steroidogenic acute regulatory protein(StAR), cytochrome P450c17enzyme aromatase and cytochrome P450aromatase(P450arom) played a key regulatory role in the process of steroid hormone synthesis. If DEHP can be through the influence of StAR, CYP17and CYP19gene expression to interfere with the synthesis of steroid hormones has not yet been reported. In this study, the adult male Rana chensinensis was exposed to10-7、10-6、10-5、10-4mol·L-1DEHP for10,20,30,40days respectively, use the running water as the blank control group. We use Real-time quantitative PCR methods to detect the expression of StAR、CYP17and CYP19mRNA in the testis, and use immunohistochemistry to detect the expression of StAR、P450c17and P450arom protein in Leydig cells. The results and the conclusions as follows:1. The value of RNA OD was mensured after total RNA’s distillation, the ratio of OD260/OD280between1.8and2.0, and revealed three bars which28S,18S,5S by electrophoretic, indicated the purity of sample is eligible. We successfully amplified273bp sequence of StAR,300bp sequence of CYP17,322bp sequence of CYP19and115bp sequence of β-actin cDNA by RT-PCR, and confirmed that is fragments of Rana chensinensis cDNA sequence by the tool of blast.2. We used Real-time quantitative PCR to detect the StAR mRNA expression, The result displayed that:Compared with the control group, the most of the DEHP concentrations of StAR mRNA expression relative value was significantly higher. Compared with different DEHP concentration group, exposure for20d,30d, StAR mRNA expression relative value reduced along with the increased DEHP concentration, but exposure for40days, the relationship between the relative value and DEHP concentration is irregular.Compared with the control group, the most DEHP treatment groups of CYP17 mRNA expression values decline were significantly. At the same DEHP concentrations, the CYP17mRNA expression relative value decreased along the exposure time; In different DEHP concentrations, the regularity of the relationship between the relative value and DEHP concentration is not strong.Compared with the control group, the CYP19mRNA expression relative values were significantly higher, the CYP19mRNA expression relative value changes with exposure time, the regularity is not the law and also with the concentration of DEHP were not evident.3. We used immunohistochemistry to detect the expression of StAR、CYP17and CYP19, It showed that the positive signals of mRNA mostly expressed in Leydig cells of testis, a small quantity of positive effects expressed in Sertoli cells.