Octylphenol On The Expression Of Star And Cyp19 In The Chinese Forest Frog Testis

With the rapid development of industry and agriculture, content of endocrine disruptors in the water environment was increased continually. Phenol environmental pollutants and its ramifications, with the stable chemical character, difficult to degrade, enter into organism easily, disturb natural physiological activity, and bring blight to reproductive and endocrine system of animals. Octylphenol (4-tert-Octylphenol, OP) can disrupt endocrine function, so that caused the reproductive abnormal dysfunction in animals, but the mechanisms remain unclear. Steroidogenic acute regulatory protein (StAR) and Cytochrome P450 Aromatase (P450arom) are key enzymes in the synthesis of steroid hormone. In the amphibian, OP affect the synthesis of sex hormones by interfering with the activity of two enzymes still requires a lot of experimental evidences. In this experiment, Rana chensinensis was used as the research animals, to study the expression of StAR and P450arom in the testis after exposed to OP, provied experimental evidence to reveal the mechanisms of effects on endocrine and reproductive, contribute to the protection of amphibians and governance environment for evaluation and prediction.In this study, adult male Rana chensinensis was exposed to 10-6,10-7,10-8 mol/L OP for 10,20, 30,40d, and 10-7,10-8 mol/L 17(3-estradiol (E2) was used as positive comparison.We use in situ hybridization and real-time quantitative PCR methods to detect the expression of StAR and CYP19 mRNA in the testis, and use immunohistochemistry to detect the expression of StAR and P450arom. The results and the conclusions as follows:1. The value of RNA OD was mensured after total RNA's distillation, the ratio of OD260/OD280 between 1.8 and 2.0, and revealed three bars which 28S,18S,5S by electrophoretic, indicated the purity of sample is eligible. We successfully amplified 115bp sequence ofβ-actin cDNA by RT-PCR, and confirmed that isβ-actin fragments of Rana chensinensis cDNA sequence by the tool of blast.2. We successfully amplified 273bp sequence of StAR cDNA by RT-PCR (sequence number HQ 610610), and used the probe that was synthesized before to detect the positive reaction use in situ hybridization in OP exposure and control groups. The positive signals of StAR mRNA mostly expressed in Leydig cells of testis, a small quantity of positive effects expressed in Sertoli cells. Immunohistochemical localization StAR protein in cells was consistent with the former results.We used real-time quantitative PCR to detect the StAR mRNA expression.In 10-6mol/L OP group, with the prolonged exposure time, StAR mRNA expression gradually increased, and the expression was significantly increased in 20 and 30 days. In 10-7,10-8 mol/L OP group, it was reached the maximum at 30 days exposure. It is suggested that StAR is a more sensitive acting elements response to the OP, different dose and exposure time for expression of StAR mRNA is more evident.3. We successfully amplified 322bp sequence of CYP19 cDNA by RT-PCR (sequence number HQ 602881), and used the probe that was synthesized before to detect the positive reaction in the testis use in situ hybridization. It was showed that CYP19 mRNA mostly expressed in Leydig cells of testis. a small quantity of positive effects expressed in sertoli cells. Immunohistochemical localization P450arom protein was mainly in Leydig cells of testis.We used real-time quantitative PCR to detect the CYP19 mRNA expression.In 10-8mol/L OP group, exposed to OP in 20 days, CYP19 mRNA expression was significantly increased, and reach to maximum in 40 days. In 10-6 and 10-7 mol/L OP group, CYP19 mRNA expression did not significantly increased in 10 and 20 days, but it was significantly increased in 30 days. In 10-6 mol/L OP group, CYP19 mRNA expression was not increased significantly in 40 days compared with 30 days, but it was markedly increased in 10-7 mol/L OP group. It is suggested that CYP19 mRNA could be induced by OP, in a certain concentration of OP exposure, CYP19 mRNA synthesis increased with prolonged exposure time, thus result in estrogenic effects.4. We chosen E2 group as a positive control in this experiment, in 10-9 and 10-8 mol/L E2 group, StAR and CYP19 mRNA expression was significantly higher than 10-6mol/L OP group. It is showed that estrogen 17β-E2 has greater effects for StAR and CYP19 than OP.