Bisphenol A Chinese Forest Frog Testis Of Aromatase And Star

At present,along with the fast development of industry and agriculture,a large number of chemic contamination that could pollute entironment and endanger human healthy were released to circumstance.Alkylphenol polyethoxylates(APEs) and its ramifications,with the stable chemical character,difficult to degrade,enter into organism easily,disturb natural physiological activity,bring blight to reproductive and endocrine system of animals.Researchs indicate,bisphenol A lead decline of reproductive function,abnormal reproduction and disorder the well-balance of endocrine function in vertebrate.At the same time,there were abundant reports about APEs interfere pivotal enzyme of steroidogenesis in animals.Whereas,there were few investigations of BPA to aromatase,StAR in steroidogenesis of Rana chensinensis.This experiment takes R.chensinensis as study object, discuss the effects of BPA to CYP19 mRNA,P450arom,StAR in Leydig cells of R. chensinensis,The aim of this experiment is to reveal the mechanism of environmental chemical pollutants endanger the endocrine of amphibian,provide theoretical basis for decline of reproductive function in amphibian which caused by BPA.We take male R.chensinensis as experiment animals.Animals had been treated in 10^-5,10^-6,10^-7M BPA for 10d,20d,30d,17β-estradiol(E₂)as positive comparison,then testis were got from R.chensinensis.Expressions of CYP19 m RNA are tested by in situ hybridizationand technique.The relative intensity of P450arom,StAR are tested by immunohistochemical technique.The results and the conclusions are listed as follows:1.The value of RNA OD were mensured after total RNA's distillation,the ratio of OD₂₆₀/OD₂₈ between1.9 and 2.2,and appeared three bars which 28S,18S,5S by electrophoretic examination,it shows,the purity of sample is eligible.We successfully expanded 322bp sequence of CYP19cDNA, compare with P450arom mRNA of Rana pipiens and Rana rugosa the comparability were 96%,95% respectively.We primarily confirm the sequence of that obtained is one fragment of CYP19cDNA. Plasmid were distilled and sequenced after the fragment were transformed successfully,as a result,we ensure the insert fragment is aim fragment,at last,we synthesized RNA probe by linearization of plasmid.2.We used the probe that was synthesized before to carry through reaction of in situ hybridizationand in BPA,E₂ and coutrol groups,the electropositive signals of CYP19 mRNA presented amethyst grain matters in Leydig cells and sustentacular cells of R.chensinensis,located in cytoplasm,the electropositive signals of CYP19 mRNA mostly expressed in Leydig cells,a small quantity of electropositive signals expressed in sustentacular cells.Detecting the relative intensity of CYP19 mRNA expression in Leydig cells of R.chensinensis by in situ hybridizationand technique, the result shows:along with different treated times in 10⁺⁵M,10^-7M BPA treatment groups the relative intensity of CYP19 mRNA expression change little.Following different treated times,the relative intensity of CYP19 mRNA expression higher than control group in 10^-6M BPA,10^-8M E₂ groups and the expression reach most in BPA,E₂ group after treated 20d.Following the treated times increased,the relative intensity of CYP19 mRNA expression increased,the expression higher than control group also in 10^-9M E₂ group,CYP19 mRNA expression was enhanced along with treated times.In same treated times,the relative intensity of CYP19 mRNA expression were increased with concentration increasing in 10^-6,10^-7M BPA and 10^-8,10^-9M E₂.It shows that the expression of CYP19 mRNA has relationship with BPA concentration,the number of CYP19 mRNA molecule expression has significant relation with time and concentration.3.Detecting the expression of P450arom by immunohistochemical technique,the electropositive signals of P450arom mostly expressed in Leydig cells,a small quantity of electropositive signals expressed in sustentacular cells.Detecting the relative intensityof P450arom expression in Leydig cells.Along with different treated times in 10^-5M,10^-7M BPA treatment groups the relative intensity of P450arom expression change little.Following different treated times,the relative intensity of P450arom expression higher than control group,in 10^-6M BPA,10^-8M E₂ groups and the expression reach most in BPA,E₂ group after treated 20d.Following the treated times increased,the relative intensity of P450arom expression increased,the expression higher than control group also in 10^-9M E₂ group,P450arom expression was enhanced along with treated times. In same treated times,the relative intensity of P450arom expression were increased with concentration increasing in 10^-6,10^-7M BPA and 10^-8,10^-9M E₂.It shows that the expression of P450arom has relationship with BPA concentration,the number of P450arom protein expression has significant relation with time and concentration.We obtained similar outcome with CYP19 mRNA in situ hybridization.4.Detecting the expression of StAR by immunohistochemical technique,the electropositive signals of StAR mostly expressed in Leydig cells,a small quantity of electropositive signals expressed in sustentacular cells.Detecting the relative intensity of StAR expression in Leydig cells, we obtained similar outcome with P450arom in immunochistochemistry.Only one dissimilar result appeared,along with different treated times the relative intensity of StAR expression change evidently in 10^-5M BPA group,and the expression of StAR lowerr than control group,the expression of StAR were reduced.It shows that the expression of StAR has relationship with BPA concentration,the number of StAR protein expression has significant relation with time and concentration at same time.5.E₂ as positive comparison in this experiment,it shows:along with different treated times the relative intensity of CYP19 mRNA,P450arom,StAR expression evidently higher than BPA group,the estrogen activity of E₂ bigger than BPA.These results show:BPA can effects the expression of pivotal enzyme in steroidogenesis,P450arom and StAR in Leydig cells of R.chensinensis.We presume BPA disturb endocrine activity of animals by regulate the expression of P450arom,STAR.