Effect And Mechanism Of TCDD Exposure During Pregnancy On The Transgenerational Damage Of Ovarian Function In Adult Female Rats

ObjectivesTo investigate the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD)during pregnancy exposure on ovarian function impairment and the underlying mechanisms that affect follicular development and function in adult female SD rats,which provides a new and effective theoretical basis for the prevention and control of diseases such as premature ovarian failure.Methods1.The establishment of transgenerational animal model:The healthy Sprague-Dawley rats were mated at a ratio of 2:1 male to female at 8:00 pm.At 8:00 am the next morning,the paired female was checked for positive vaginal embolism.The positive vaginal embolism was noted as gestation day 0(GD0).Pregnant female rats(F0)were randomly divided into three groups(10 rats/group):control group,100 ng/kg TCDD treatment group(low dose group)and 500 ng/kg TCDD treatment group(high dose group),and oral gavage was performed daily from GD 8 to 14.Rats born in F0 generation were F1 generation.One of the females in each nest of F1 was randomly selected and mated with healthy male rats at the age of 8 weeks old.The pups born in F1 generation were F2 generation.In the same way,the F3 generation was obtained after the F2 generation was mated.2.Vaginal opening time and estrus cycle monitoring:On postnatal day 21(PND21)of F3,the ratio of male to female and the date of female vaginal opening in each litter were recorded.When growing up to 8 weeks of age,a vaginal cell smears were performed to observe the estrous cycle and continuously monitor 10 cycles.3.Measurement of related indicators of ovarian function:Female rats were weighted,anesthetized,blood collected and sacrificed during the estrus around the PND70,and both ovaries were removed.One side of the ovary was weighed after being taken out,and was fixed in 4%paraformaldehyde fixed fluid.The other ovary was stored in the RNA store solution at-80?.According to body weight and organ wet weight,the organ coefficient of uterus and ovary was calculated.Serum Lutemizing hormone(LH),Estradiol(E2)and Follicle stimulating hormone(FSH)concentrations were determined by ELISA.Continuous sections of the ovary after H&E staining were used for histopathological observation and TUNEL was used to detect apoptosis of follicular granulosa cells.The expression level of IGF2/H19 mRNA in ovarian follicles was determined by using RT-PCR and the expression level of IGF2 protein was measured by through Western blot.4.Statistical analysis of experimental data was performed using IMB SPSS Statistics 25.0 software.One-way ANOVA were used to analyze the difference among groups.LSD method was performed to test multiple comparison.The significance level was ?=0.05.Results1.Sex ratio and organ coefficient:We did not found there is difference between control group and TCDD-treated groups in the ratio of male to female in F3 generation of female SD rats.The ovarian weight was significantly lower in the 100 ng/kg TCDD group than the control group(P<0.05),while the ovarian organ coefficient in the 500 ng/kg TCDD group was significantly higher than the control one(P<0.05)in F3 generation rats.2.Vaginal opening time and estrous cycle:Compared with the control group,the vaginal opening time of the F3 generation adulthood female rats was not significantly earlier.The control group,100 ng/kg TCDD group and 500 ng/kg TCDD group were 34.65�0.57 days,34.0�0.68 days and 32.8�1.01 days respectively.The length of the estrous cycle did not presented significant difference,but it was obviously in a disordered state,and while the abnormal rates were 20%,54%and 65%respectively.3.Serum hormone levels:The serum LH level was increased of the F3 female rats in the 500 ng/kg TCDD group,and no changes were observed about the FSH and E2 levels in TCDD treatment groups,comparing with the control rats..4.H&E staining results:The results of H&E staining showed that compared with the control group,the follicle tissue morphology of TCDD treated groups in F3 generation rats was abnormal.The number of primordial follicles in the TCDD treated groups was significantly decreased(P<0.05),while no significant difference were observed in the number of secondary follicles and corpus luteum(P<0.05).In addition,the apoptosis rate of ovarian granulosa cells in the TCDD treated groups was significantly increased(P<0.05)5.Ovarian IGF2/H19 expression level:Compared with the control group,after exposure to TCDD during pregnancy,the expression level of H19 mRNA in the ovary increased significantly(P<0.05),and the expression level of IGF2 protein was only reduced(P<0.05)in the 500 ng/kg TCDD group in the ovary of adult SD rats in the F3 generation.ConclusionsExposure to TCDD during pregnancy can induce the effects of follicular development and ovarian function damage in adult female rats,which can persist until the F3 generation.TCDD inhibits follicular development and ovarian reserve by interfering with ovarian steroid hormone secretion and inducing granulosa cell apoptosis,and its mechanism is related to ovarian IGF2/H19 signaling pathway in the ovary.