Octylphenol Rana Testis P450c17 (of Cyp17) Expression

With the development of industrialization, environmental endocrine disruptors (EDCs) were continuously released into the environment, which had a huge impact on the survival of humans and wildlife by diet, exposure and others. Octylphenol (OP) was an important phenolic environmental estrogen, found in water, soil and daily consumables widely, the estrogenic activity of OP was 10-4 times bigger than estradiol. Experiments had proved that OP could decrease sperm quality and quantity, disorder the balance of sex hormones and action, and affect the ability of male reproductive. Cytochrome P450c17 was a key enzyme in the steroidogenesis path, which was associated with the testosterone synthesis. There were abundant reports about OP interfering cytochrome P450c17, whereas, there was few investigations of OP exposed to the expression of P450c17 mRNA and protein in amphibian. This experiment took Rana chensinensis exposed in water including OP, discussed the effects of OP to the expression of P450c17 mRNA and protein in Leydig cells of Rana chensinensis, explained the mechanism that OP effected steroid hormone synthesis, provided a theoretical basis for environmental pollutants control.In this study, we take male Rana chensinensis as experiment animals. Animals had been exposed in 10-6,10-7,10-8 mol/L OP for 10d,20d,30d,40d,17β-estradiol(E2) was taken as positive comparison; then testis were got from Rana chensinensis. The expression of P450c17 protein in Leydig cells was detected by immunohistochemistry; the expression of CYP17(P450c17)mRNA was tested by in situ hybridization technique and quantitative real-time PCR, the experimental results were as follows,1. We extracted total RNA successfully from the testis of Rana chensinensis. The value of RNA OD (ratio of OD260/OD280) were betweenl.7 and 2.0. We successfully cloned a 300bp fragment of the CYP17, and had submitted to NCBI library, got a number "HQ610611". Compared with CYP17 mRNA of Rana dybowskii, Rana rugosa and Xenopus laevis, the comparability were 97%,97%, 96% respectively. We made use of the fragment sequence to synthesis RNA probe.2. In situ hybridization results showed that:CYP17 mRNA were mainly expressed in Leydig cells, sertoli cells and spermatogenic cells were a little expressed.The result were in line with the immunohistochemical findings.3. Detecting the photodensity of immunohistochemical electropositive signals, the results showed that:exposing 10d, compared with the control, the relative intensity of P450c17 protein treated with OP groups changed little(p>0.05); exposing 20,30d, compared with the control, the relative intensity of 1016,10-7 mol/L OP groups decreased, the change was significant(p<0.05), the relative intensity of 10-7,10-8mol/L OP groups were higher than 10-6 mol/L OP group, the change was significant(p<0.05); exposing 40d, compared with the control, the relative intensity of 10-6,10-7 mol/L OP groups decreased, the change was significant(p<0.05), the changes between 10-8 mol/L OP group with 10-7mol/L OP group and between 10-7mol/L OP group with 10-6mol/L OP group were no significant(p>0.05).Comparing the different exposing time groups in the same treated concentration, with the 10-6 mol/L OP group, the relative intensity of P450c17 protein exposed 20d were lower than group exposed 10d, the group exposed 30d were lower than group exposed 20d, the changes were significant(p<0.05); with 10-7mol/L OP group, the relative intensity of P450c17 protein exposed 20d were lower than group exposed 10d, the change was significant(p<0.05), the change between the group exposed 30d and 20d was not significant (p>0.05); with 10-8mol/L OP group, the changes between the group exposed 20d and 10d, between the group exposed 30d and 20d, were not significant (p>0.05)In brief, after exposing OP, the relative expression of P450c17 protein decreased and had a downtrend with increasing OP dose and exposing time, presenting dose and time accumulative effect. The relative expression of P450c17 protein exposed 30d was the lowest; exposing 40d, the changes among different dose groups were not significant, the dose accumulative effect was weaken by time accumulative effect.4. The expression of CYP17 mRNA was detected by real-time PCR. The results showed that: exposing 10,20d, compared with the control, the relative expression of P450c17 protein treated with OP groups changed significant(p<0.01), the relative expression of 10-7,10-8mol/L OP groups were higher than 10-6mol/L OP group, the change was significant(p<0.05), the change between 10-8 mol/L OP group with 10-7mol/L OP group was not significant(p>0.05); exposing 30,40d, compared with the control, the relative expression of P450c17 protein treated with OP groups changed significant (p<0.01), the changes between 10-8mol/L OP group with 10-7mol/L OP group and between 10-7mol/L OP group with 10-6mol/L OP group were not significant(p>0.05).Comparing the different exposing time groups in the same exposed concentration, with the 10-6 mol/L OP group, the relative expression of P450c17 protein exposed 20d were lower than the group exposed 10d (p<0.05), the change between the group exposed 30d and 20d was not significant (p>0.05); with 10-7,10-8mol/L OP group, the changes between the group exposed 20d and 10d, between the group exposed 30d and 20d, were not significant (p>0.05).In brief, after exposing OP, the relative expression of CYP17 mRNA decreased and had a downtrend with increasing OP dose and exposing time, which was consistent with the expression of P450c17 protein. However, exposing between 20d and 30d, the expression of CYP17 mRNA was lowest, which was earlier than the expression of P450c17 protein.