| Objective:To investigate the effect of intrauterine TCDD exposure during Gonadal sex differentiation on the reproductive health of female offspring and the imprinted gene cluster Igf2/H19,providing laboratory and theoretical basis for the epigenetic mechanism of fetal basis of adult disease.Methods:SD（Sprague Dawley）rats were treated with TCDD at doses of 100 ng/kg bw/d and 500 ng/kg bw/d during gestational day 8-14（GD8-14）and corn oil as control group.On postnatal day 21（PND21）,the pups were separated and fed alone away from the mother rats.The number of litters,the proportion of males to females,and the opening time of the vagina were observed.After vaginal opening,two vaginal cytology smears were performed at 8:00 and 20:00 daily to observe the estrus cycle,and 10 estrous cycles were continuously monitored for approximately 45 days.At about PND70,the rats were sacrificed during the estrous period and the body weight and ovary weight of the rats were weighed.Serum E2 and FSH（Follicle-stimulating hormone）levels were detected by ELISA（Enzyme Linked Immunosorbent Assay）.Serial sections of ovarian tissue were stained with HE to record the number of various types of follicles and observe the pathological changes of the ovary.Apoptosis of follicular cells was detected by TUNEL.Real-time fluorescent quantitative PCR was used to detect the expression of Igf2/H19 mRNA in the ovary,and Western blot was used to detect the expression level of IGF2 protein.Bisulfite Sequencing PCR（BSP）was used to detect the methylation level of the differentially methylated region of the imprinted gene cluster Igf2/H19Results:1.Intrauterine TCDD exposure of SD rat had no significant effect on sex ratio of offspring.The sex ratio in the 500ng/kg dose group had an increasing tendency,but the difference was not statistically significant（P>0.05）.Intrauterine TCDD exposure caused a slight delay in vaginal opening time.In the 100 ng/kg dose group,about half of the pups had disordered estrus cycle,and 75%in the 500 ng/kg dose group.2.Exposure to intrauterine TCDD resulted in a significant reduction in the number of ovarian primordial follicles in adult SD rat offspring（P<0.05）,and a significant increase in secondary follicles and corpus luteum（P<0.05）.3.Apoptosis of follicle granulosa cells was significantly increased（P<0.05）.The apoptosis rate of granulosa cells in follicles was higher than 10%in the control group,and the apoptosis rate of corpus luteum follicle cells was also increased.There was a statistical difference between the treatment group and the control group.（P<0.05）.4.The level of serum E2 in female offspring of SD rat caused by intrauterine TCDD exposure was significantly lower than that in the control group（P<0.05）,and serum FSH level was only increased in the 500ng/kg dose group（P<0.05）.5.Exposure to intrauterine TCDD led to downregulation of Igf2/H19 mRNA expression,but only 500ng/kg dose group had statistically significant difference（P<0.05）;IGF2 protein expression also decreased,but the difference was not statistically significant.6.The methylation levels of Igf2-DMR2 and H19-ICR in the high-dose group were higher than those in the low-dose group,but the difference was not statistically significant.Conclusions:During the period of sex differentiation,intrauterine TCDD exposure resulted in abnormal follicle development in female offspring,and affected the expression of Igf2/H19. |
