| Heavy metal Cadmium(Cd),which is widely used in electroplating,chemical,electronics and nuclear industry,have been considered as the representative of Environmental Endocrine Disruptors.The reserve amount of Cd all over the world is huge,and Cd could be released into the environment easily through a variety of ways.Currently,many countries and regions around the world are suffering from varying degrees of Cd contamination.Cd can enter and accumulate in the body through contaminated water,air and food,but it is difficult for Cd to discharge.Cd could result in multiple organ damage,including female reproductive system.Follicle development and steroid hormone synthesis are important process during ovarian development.Previous studies showed that to mature animals,Cd could damage ovarian morphology and steroid hormone synthesis.Furthermore,Cd could affect the expression of several key genes,and then damage the normal physiological status of organs.Besides,the toxic effect of Cd had relationship to DNA methylation/ microRNA regulatory mechanism.Epidemiological studies suggested that there are a large number of people that had been exposed to Cd,and many women had been living in Cd-exposure environment from childhood to adulthood.However,so far,there are few studies that focus on their ovarian development and function,or the damage pattern when compared to those ovaries that exposed to Cd during adulthood.Moreover,the gene expression and the corresponding regulatory mechanism are poorly understood.Based on the above situation,we established an animal model of Wistar rats that exposed to Cd from weaning to maturity,observed ovarian development condition,the expression of key genes that regulated ovarian development,and the changes of gene DNA methylation/microRNA pattern,tried to study the developmental toxicity of Cd and the potential molecular mechanism,then clarify the role of interaction of environment and genetic factor during the above process.The outcome of this study would provide important guiding significance to prevention and control of Cd.Part I: The effect of Cd to ovarian development when exposed from weaning to maturityObjective:To establish the animal model of rats that were exposed to Cd from weaning to maturity,clarify whether Cd could affect ovarian morphological development and the corresponding character.Method:Forty-eight immature weaned female 21-day-old Wistar rats weighing 45.8 ± 2.5 g were utilized in the experiments.After acclimatization,animals were randomly divided into four groups of 12 rats each.CdCl2 was administered by gavage at doses of 0,0.5,2.0,and 8.0 mg/kg body weight once a day,6 times a week until 75-day-old.Weight gain was measured every three days.Animals were sacrificed by cervical dislocation;afterwards,the ovaries were dissected free of adipose tissue and weighed,H&E stain was performed for follicle counting,transmission electron microscope was used to observe follicle apoptotic situation,TUNEL was used to detect the apoptotic percentage;and ultrastructure of granulose cells were observed by transmission electron microscope.Result:1.After exposure,when compared to the control group,the body weights of the 8.0 mg/kg treatment group were significantly decreased(p<0.01),ovarian wet weights were significantly decreased in the 2.0 mg/kg(p<0.05)and 8.0 mg/kg treatment group(p<0.01),ovarian/body weight ratios were significantly decreased in the 2.0 mg/kg(p<0.05)and 8.0 mg/kg treatment group(p<0.05).2.Compared to the control group,the proportion of primordial follicles in the 8.0 mg/kg treatment group was significantly decreased.However,the proportions of atresic follicles were significantly increased in the 2.0 mg/kg(p<0.01)and 8.0 mg/kg treatment groups(p<0.01).The proportions of primary follicle,secondary follicle,mature follicle and corpus luteum show no significant changes in the control and experimental rats(p>0.05).3.When compared with the control group,the percentages of TUNEL-positive follicle with apoptotic granulosa cells and/or oocytes in the 2.0 mg/kg and 8.0 mg/kg group was significantly increased in ovarian sections from rats exposed to Cd(p<0.01)4.Apoptotic bodies could be found in the the 2.0 mg/kg and the 8.0 mg/kg treatment group.In the control group and the 0.5 mg/kg treatment group,granulose cells showed no obvious toxic changes;however,granulosa cells in the 2.0 mg/kg treatment group exhibited obvious ultrastructure change,such as loosely arranged,different degrees of shrinkage,undulation of nuclear membrane.In the 8.0 mg/kg treatment group,except for the similar manifestation to the 2.0 mg/kg treatment group,chromatin condensation could also be found,and the naked nucleus and the red blood cells were seen under the microscope.Part II: Ovarian SCF/c-kit gene expression and the corresponding changes of DNA methylation/microRNA in rats exposed to cadmium from weaning to maturityObjective:The aim of this study was to determine the effect of Cd to ovarian development-related SCF/c-kit gene expression and the corresponding changes of DNA methylation/microRNA,and understood the role and significance of SCF/c-kit gene in ovarian developmental dysfunction which was caused by Cd.Method:Animal model was built as in Part I,the mRNA and protein expression of SCF/c-kit were analyzed using Real-time PCR and Western blot,DNA methylation levels on CpG islands of gene promoter region were detected by Bisulfite sequencing PCR(BSP),c-kit related microRNA expression was detected by Real-time PCR,according to the testing result of microRNA target prediction tools,the expression of microRNAs that might regulate SCF gene was also detected by Real-time PCR,and the target was validated by luciferase assay.Result:1.When compared with the control group,SCF mRNA expresson in the 2.0 mg/kg and the 8.0 mg/kg treatment groups was significantly decreased(p<0.01),while the protein expression in the 2.0 mg/kg treatment group(p<0.05)and 8.0 mg/kg treatment group(p<0.01)was significantly decreased;2.The total methylation percentage on CpG islands of SCF gene promoter region showed no significance between groups(Fisher's exact test,p=0.911);3.The expression of microRNAs that might regulate SCF gene(miR-449 a,miR-132 and miR-320): when compared with the control group,miR-449 a expresson in the 2.0 mg/kg and the 8.0 mg/kg treatment groups was significantly increased(p<0.05);the expression of miR-132 and miR-320 among groups showed no significance;4.The target validation of SCF and microRNAs: after cotransfect into 293 T cell with wild type SCF and miR-449 a,miR-132,miR-320,respectly,the activity of luciferase showed no significant changes(p>0.05);5.When compared with the control group,c-kit mRNA in Cd-exposed groups was decreased significantly(p<0.01),and the protein expression was also decreased in the 2.0 mg/kg(p<0.05)and the 8.0 mg/kg treatment group(p<0.01);6.The total methylation percentage on CpG islands of c-kit gene promoter region showed no significance between groups(? 2=6.813,p=0.078);7.c-kit regulated microRNA(miR-193,miR-221 and miR-222): when compared with the control group,miR-193,miR-221 and miR-222 expression in the 2.0 mg/kg and the 8.0 mg/kg treatment group was significantly increased(p<0.01);Part III: Follicle development and steroid hormone synthesis related gene expression and the corresponding changes of DNA methylation/microRNA in rats exposed to cadmium from weaning to maturityObjective:The aim of this study was to determine the effect of Cd to follicle development and steroid hormone synthesis related gene expression and the corresponding changes of DNA methylation/microRNA,and understood the role and significance of these genes in follicle developmental and steroid hormone synthesis dysfunction which was caused by Cd.Method:Animal model was built as in Part I,the mRNA and protein expression of follicle development related gene: Figl?,H1 foo,AMH and steroid hormone synthesis related gene: StAR,CYP11A1,3?-HSD,CYP17A1,CYP19A1 were analyzed using Real-time PCR and Western blot,BSP was carried out to detect DNA methylation level of Figl?,CYP17A1 and CYP19A1 gene,whose promoter region contain CpG islands,according to the testing result of microRNA target prediction tools,the expression of microRNAs that might regulate CYP17A1 and CYP19A1 gene was also detected by Real-time PCR.Result:1.When compared with the control group,Figl? mRNA expresson in all of the Cd-exposed groups were significantly decreased(p<0.01),while the protein expression in the 2.0 mg/kg treatment group(p<0.05)and 8.0 mg/kg treatment group(p<0.01)was significantly decreased;the total methylation percentage on CpG islands of Figl? gene promoter region in the 8.0 mg/kg treatment group was significantly increased(? 2=8.446,p=0.038)2.When compared with the control group,h1 foo mRNA expresson in all of the Cd-exposed groups were significantly decreased(p<0.01),while the protein expression in the 8.0 mg/kg treatment group was significantly decreased(p<0.01);3.When compared with the control group,AMH mRNA expresson in the 8.0 mg/kg treatment group was significantly increased(p<0.01),while the protein expression in the 2.0 mg/kg treatment group(p<0.05)and the 8.0 mg/kg treatment group(p<0.01)were significantly increased;4.When compared with the control group,StAR mRNA expresson in the 8.0 mg/kg treatment group was significantly decreased(p<0.01),however,the protein expression of StAR in the 0.5 mg/kg treatment group was significantly increased(p<0.01),while significantly decreased in the 2.0 mg/kg treatment group and the 8.0 mg/kg treatment group(p<0.01);5.When compared with the control group,CYP11A1 mRNA expresson in all of the Cd-exposed groups were significantly decreased(p<0.01),while the protein expression in the 2.0 mg/kg and 8.0 mg/kg treatment group were significantly decreased(p<0.01);6.When compared with the control group,3?-HSD mRNA expresson in all of the Cd-exposed groups were significantly decreased(p<0.01),however,the protein expression of 3?-HSD in the 0.5 mg/kg treatment group was significantly increased(p<0.01),while significantly decreased in the 2.0 mg/kg treatment group(p<0.05)and the 8.0 mg/kg(p<0.01)treatment group;7.When compared with the control group,CYP17A1 mRNA expresson in the 8.0 mg/kg treatment group was significantly decreased(p<0.05),while the protein expression in the 2.0 mg/kg treatment group and the 8.0 mg/kg treatment group(p<0.01)were significantly decreased;the total methylation percentage on CpG islands of CYP17A1 gene promoter region showed no significance between groups(? 2=4.394,p=0.222);in microRNAs that might regulate CYP17A1,expression level of let-7a and miR-98 in all of the Cd-exposed groups showed no significance(p>0.05);8.When compared with the control group,CYP17A1 mRNA expresson in the 2.0 mg/kg and 8.0 mg/kg treatment group was significantly decreased(p<0.01),while the protein expression in all of the Cd-exposed groups were significantly decreased(p<0.01);the total methylation percentage on CpG islands of CYP19A1 gene promoter region showed no significance between groups(? 2=6.157,p=0.104);in microRNAs that might regulate CYP17A1,expression level of miR-27 a in all of the Cd-exposed groups showed no significance(p>0.05),while miR-133 a in the 8.0 mg/kg treatment group was significantly increased(p<0.05).Conclusion:1.Cd exposure from weaning to maturity could affect ovarian development,resulting in a significant decrease in ovarian wet weight,ovarian/body weight ratios,and primordial follicles,in addition to an increase in atresic follicles,an increased level of follicle apoptosis as Cd concentration increased,and damage granulose cells ultrastructure,the manifestation of Cd exposure from weaning to maturity is similar to that of Cd exposure during maturity.2.Downregulation of SCF/c-kit mRNA and protein expression could be an important underlying reason for ovarian developmental dysfunction caused by Cd,miR-449 a,miR-132 and miR-320 participate in c-kit protein expression downregulation caused by Cd,however,methylation of CpG islands did not participate in SCF/c-kit expression regulation caused by Cd,and miR-449 a,miR-132,miR-320 did not participate in SCF post-transcriptional control;3.Cd exposure from weaning to maturity could interrupt mRNA and protein expression of follicle development related gene: Figl?,H1 foo,AMH and steroid hormone synthesis related gene: StAR,CYP11A1,3?-HSD,CYP17A1,CYP19A1;Figl? gene promoter region methylation participate in Figl? mRNA expression downregulation,however,CYP17A1 and CYP19A1 gene promoter region methylation level did not change after Cd exposure;miR-133 a might participate in CYP19A1 protein expression downregulation. |
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