Influence Of Dioxin On The Expression Of PR And DNMT-1of Eutopic Endometrium In Mice

Objective: Endometriosis (EMS) is called “benign cancer” because it isa benign estrogen-dependent disease but owns malignant behavior. Thedisease could cause the women dysmenorrhea, infertility and others. Themobility rate of it increased in recent years, but the occurent mechanism is notclarified absolutely. There has been a lot of research indicated that the risk ofgetting EMS is related to TCDD and the exposure in prenatal may be increasethe risk of the disease in adulthood. We have studied that TCDD indeedimproves the occurence and the development of EMS especially on the overyby the influence on the immunity system, cytokine signaling,hormoneregulation and others. We also have found that the expression of progesteronereceptor is less in eutopic endometrium of the EMS model. Hypermethylationof the CpG islands in the promoter region is related to the silence of the geneand the DNA methyl-transferase-1(DNMT-1)is very important in the progress.There have been studies indicated that TCDD owns the methylation. So thisexperiment will test the relationship between the expression of progesteronereceptor A(PR-A), progesterone receptor B(PR-B)and DNMT-1in eutopicendometrium of the model,and we also will investigate whether thehypermethylation of the CpG islands in promoter region of PR-A and PR-Bgene would down-regulated the expression of them in the model of EMSexposed in TCDD.Methods:1Establishment of EMS Models exposed in TCDD60SPF C57BL/6female mice were mated with30C3H male micerandomly and observed their plugs.The days of appearing plugs considered tobe the gestational day.On the8th gestational day the pregnant female micewere divided into two groups according to the exposed dosage of TCDD primary control group(0ug/kg,)and primary exposed group(3ug/kg,).21daysafter birth the female offspring in the primary control group were divided intotwo groups according to the received dosage of TCDD(ug/kg):0/0and0/3groups. the female offspring in the primary exposed group were divided intothree groups according to the received dosage of TCDD(ug/kg):3/0,3/3and3/10groups. There were12female offspring in each group.49days after birththe offspring received3ug/kg again except the0/0group.70days after birthautotransplantation of endometrium was used to establish the mouseendometriosis model,after the surgery the treatment groups were exposured to3ug/kg TCDD at3,6,9weeks and at12weeks after surgery the mice wereeuthanized.In this way the female offspring were divided into5groupsaccording to the exposed dosage of TCDD in their development and to becalled0/0(control),0/3(puberty+adult-hood),3/0（fetus period+adulthood），3/3（fetus period+puberty+adulthood，general dose exposure），3/10（fetusperiod+puberty+adulthood, high dose exposure）.2Detection of PR-A,PR-B and DNMT-1We used immunohistochemical method to detect the expressions ofDNMT-1, PR-A,and PR-B and methylation-specific PCR (MSP)methodswere used to explore the methylation of CpG islands in promoter region ofPR-A and PR-B gene.3SPSS19.0statistical software package was used to analyze alldata.Measurement was noted by formulas of ⅹ±s and analysis them byone-way ANOVA and enumeration data was analyzed by Fisher exact test.We considered P<0.05to be significant for all statistical analysis.Results:1The expression of DNMT-1,PR-A,PR-B of eutopic endometrium inmice between different groups.The expression of DNMT-1between0/0and0/3and3/0were notsignificant different(P>0.05，）.But the expression of which between0/0and3/3was significant different（P=0.000<0.05）and the expressions of which in3/3was higher than that in0/0. At the same time the expression of which between3/10and3/3was significant different（P=0.000<0.05）and theexpression of which in3/10was higher than that in3/3.The expression of PR-B between0/0and0/3and3/0was not significantdifferent(P>0.05）.But the expression of which between0/0and3/3wassignificant different（P=0.000<0.05）and the expression of which in3/3waslower than that in0/0. At the same time the expression of which between3/10and3/3was significant different（P=0.000<0.05）and the expression of whichin3/10was lower than that in3/3.But there were not significant differences in the expression of PR-Abetween different groups.2The methylation of CpGs Islands of PR-A gene and PR-B gene ineutopic endometrium of the EMS model of mice between different groups.The methylation of CpGs Islands of PR-B gene between0/0,0/3and3/0was not significant differen（tP>0.05）.But the methylation of CpGs Islands ofPR-B gene in0/0was significant lower than3/3（P=0.014<0.05，）and themethylation of which in3/10was significant higher than in3/3(P=0.019<0.05). The methylation of which of PR-A was not significantdifferent between different groups.3In the same group, the methylation of CpG islands of PR-B was moresignificant than that of PR-A except0/0group, and the expression of PR-A ishigher than that of PR-B, this tendency was more significant with theincreased dosage of TCDD.Conclusions:1TCDD perhaps up-regulates the expression of DNMT-1and thiscould lead the expression of PR-B decreased. The intensive function ofTCDD is increased following the added dosage.2TCDD improves the expression of DNMT-1and the methylation ofCpG islands of PR-B gene and the intensive function of TCDD is increasedfollowing the added dosage. These are related to the decreased expression ofPR-B.3Different dose of TCDD exposure to fetus、puberty and adulthood stage mice promotes the occurrence of EMS in adulthood by altering the influenceon the local eutopic endometrium to hormone regulation and the physiologicalfunctions of eutopic endometrium.