| At present,alkylphenol polyethoxylates(APEs) as industrial material are used broadly in the world.Therefore it is very easy for the APEs to enter organism,disturb the normal hormone effects and have profound and all-pervading influence on the creatural reproduction.Recently,some reports shows that Bisphenol A has caused serious effects on the growth development and propagation of amphibian;it is the one of the main reasons which induces the number of amphibian.Most reports about the effects of BPA on amphibian focus on its estrogenic effects,the report about whether it can induce the apoptosis in spermatogenetic cells of R.chensinensis hasn't been studied.So we take R.chensinensis as experimental animal to investigate the effects of BPA on the apoptosis index and the expression of Bax,Bcl-2 in spermatogenetic cells of R.chensinensis.The aim of this experiment is to reveal the mechanism of environmental chemical pollutants endanger the propagation activity of animal,whether this mechanism is implemented though Bax/bcl-2 pathway. Provide theoretical basis for protecting decreasing propagation ability caused by BPA.We take the R.chensinensis in spermatogonia proliferation stages and in spermatocyte maturation division stage as experiment animals.R.chensinensis in spermatogonia proliferation stages had been treated in 10-7,10-6,10-5mol/L BPA and control group for 3d,5d,7d,R.chensinensis in spermatocyte maturation division stage had been treated in 10-7,10-6,10-5mol/L BPA and control group for 4d,14d,28d.Then testis was got from R.chensinensis at certain days.Apoptosis cells are tested by TUNEL and Methyl Green-Pyronine technique.The relative intensity of Bax and Bcl-2 are tested by immunohistochemical technique.The results and the conclusions are listed as follows:1.Detecting apoptosis cells in spermatogenetic cells of R.chensinensis.by TUNEL and Methyl Green-Pyronine technique.In both spermatogonia proliferation stages and spermatocyte maturation division stage,Spermatogenetic cells have failed into the middle of lumen with cell chromatin pyknosis.Some damaged cells which gather into cluster in soon abscisic status close to the middle of lumen.It shows that BPA induce the Spermatogenetic cells apoptosis.In spermatocyte maturation division stage,some irregular necrosis focus in the lumen can be seen in every BPA treatment group which has been treated for 14d.It shows that BPA induce the cell necrosis of spermatogenetic cells.2.Detecting the expression of Bax,Bcl-2 in spermatogenetic cells of R.chensinensis by immunohistochemical technique,in spermatogonia proliferation stages,apoptotic index in 10-7mol/LBPA treatment group from 3d to 7d change little,but apoptotic index is increased with concentration and time in 10-6mol/LBPA and 10-5mol/L BPA groups.It shows that BPA has accumulation effect in the R.chensinensis testis,apoptotic index of spermatogenetic cells have relation to the BPA accumulate concentration.There exist significant dose-effect relationship between BPA and AI.In spermatocyte maturation division stage,AI of 10-7mol/L BPA treated for 4d is similar with control group.It shows that because the low concentration of BPA and organism's regulating ability,the effects of BPA has been reduced,so the AI of damage cells hasn't increased obviously.In 10-5mol/L treated for 28d,although the AI is higher than it in control group,but lower in 14d BPA treatment group.It shows that organism not only start the apopotosis pathway but also the cell necrosis pathway to exclude the damage cells.So the AI is decreasing.3.Comparing the AI and its relative BPA concentration in these two stages,in spermatogonia proliferation stages,AI of spermatogenetic cells in 10-6mol/L treated for 7d is 8.24±1.03%,while in spermatocyte maturation division stage,AI reach to 12.96±1.66%when treated with 10-6 mol/L BPA for 4d.It shows that compared to spermatogonia,spermatocyte's AI is higher in relative low BPA accumulate concentration.It suggested that sensitivity of the two kinds of cells is different. Spermatocyte is much more sensitive.4.Detecting the expression of Bax,Bcl-2 by immunohistochemical technique,In spermatogonia proliferation stages and in spermatocyte maturation division stage,the results shows that,the expression of Bax is increasing with concentration and time while Bcl-2 expression is decreasing with the concentration and time in 10-7 mol/L,10-6 mol/L BPA.It shows that the expression of Bax has relationship with BPA concentration;the number of protein expression has significant relation with time and concentration.5.In spermatogonia proliferation stages,the results suggested that the expression of Bax is the biggest in 10-5 mol/LBPA treated for 7d,while Bax is the highest in10-5 mol/LBPA after treated for 4d in spermatocyte maturation division stage.In spermatogonia proliferation stages the expression of Bcl-2 is the lowest in 10-5 mol/L treated for 5d while the lowest expression of Bcl-2 after treated for 14d in spermatocyte maturation division stage.It suggests that the spermatocyte is easy to regulate the expression of Bax in relative low BPA concentration,while the spermatogonia are easy to regulate the expression of Bcl-2.6.Analyzing the linear regression relation of AI and the ratio of Bax/Bcl-2 expression,it suggests that in these two stages,significant linear correlation exist between AI and the ratio of Bax/Bcl-2 expression.It suggests that the relative expression of Bax and Bcl-2 is the key factor which determinates whether cell apoptosis happens.After treated with BPA,organism can increase the expression and decrease the expression of Bcl-2 to regulate the cell apoptosis.It shows that cell apoptosis induced by BPA is though Bax/Bcl-2 pathway. |
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