Effects Of Exposure To Bisphenol A During Pregnancy On Ovarian Development And Its Mechanism In Female Offspring Rats

ObjectivesBisphenol A（BPA）is widely used in the production of plastic products and coatings as a raw material for the synthesis of polycarbonate and epoxy resin.It can enter the human body through digestive tract,respiratory tract and skin contact.Studies have shown that BPA exposure can cause steroid hormones disturbances and histopathological changes of ovary and uterus,which induces female reproductive dysfunction,ultimately leading to reduced fertility and even infertility.Embryonic and fetal periods are critical periods for human reproductive development.And BPA can pass through the placental barrier,so the adverse effect of BPA is particularly evident when the exposure takes place during pregnancy.Maternal exposure to BPA during pregnancy can cause neurodevelopmental disorders,endocrine hormone disorders and male reproductive dysfunction in offspring.However,the research on the female reproductive dysfunction induced by prenatal BPA exposure is few,and the mechanism is unclear.Epigenetic regulation has emerged as an important mechanism of reproductive toxicity induced by BPA.Moreover,the Wnt/β-catenin signaling pathway plays an important role in embryonic development.Therefore,this study investigated the effects of prenatal BPA exposure on ovarian reproductive function of offspring rats at different developmental stages,and explored its effects on Wnt/β-catenin signaling pathway and its DNA methylation modification in ovarian tissue,in order to provide a theoretical basis for the prevention and early intervention of BPA-related reproductive diseases.Methods1.A total of 30 SPF female SD rats and 15 male SD rats,aged 10 weeks,were selected.Female rats were mated with males（2:1）overnight.Vaginal smears were collected the next morning to determine pregnancy of rats.The day of sperm detection under the microscope was defined as gestation day 0（GD 0）.A total of 30 pregnant rats were randomly divided into 5 groups:0,0.05,0.5,5,50 mg/kg/d groups,with 6pregnant rats in each group.The BPA-exposed groups received different doses of BPA by oral gavage once daily from GD 5 to GD 19,the control group received equivalent corn oil,and the infected volume is 5 ml/kg.Female pups were anaesthetized and sacrificed at postnatal day 21（PND 21）and PND 56,and biological samples were collected and related indexes were detected.The body and organ weights of female pups were recorded,and the serums and testes were separated and stored at-80℃immediately for further analysis.2.The general condition of female offspring at PND 21 and PND 56 were detected and compared.3.HE staining was used to evaluate the histopathological changes of ovary induced by BPA.4.The levels of P and E₂ in serum of PND 56 days were measured by ELISA.5.The expression levels of Cyp11a1,Cyp17a1,Cyp19a1,estrogen receptor ERα（Esr1）and ERβ（Esr2）m RNA in ovarian tissues at PND 21 and PND 56 were determined by real-time quantitative PCR（RT-PCR）.6.The expression levels of DNMT 1,DNMT 3a,DNMT 3b,WNT4 andβ-catenin were analyzed by RT-PCR and Western blot.7.Methylation specific PCR（MSP）was used to analyze the Dkk1 promotor methylation.And Dkk1 m RNA expression was analyzed by RT-PCR.Results1.Effects of prenatal BPA exposure on ovarian function,Wnt/β-catenin signaling pathway and its DNA methylation modification in female offspring rats at PND 21（1）Compared with the control group,the body weight of female offspring was significantly increased in 0.05 mg/kg group and significantly decreased in 5 mg/kg group（P<0.05）.The anogenital distance（AGD）in 50 mg/kg group was significantly shortened（P<0.05）.The wet weight of ovary in 50 mg/kg group was significantly decreased（P<0.05）,but there was no significant difference in ovarian organ coefficient（P>0.05）.The wet weight of uterus and ovary and organ coefficients in 5mg/kg group were significantly increased（P<0.05）.（2）The pathological results showed that there were medium size follicles,including primary and secondary follicles in the ovaries of female pups at PND 21 in the control group.After exposure to BPA during pregnancy,the number of follicles in the ovaries increased and the cavity became larger,but the structure was fuzzy.In the 50 mg/kg group,obvious damage of ovarian structure could be observed.（3）Compared with the control group,the m RNA levels of Cyp11a1 and Cyp17a1 in 0.05,5 and 50 mg/kg groups were significantly decreased（P<0.05）.The expression of Cyp19a1 m RNA was not significantly different（P>0.05）.The m RNA level of estrogen receptor Esr1 was significantly increased in 0.05 mg/kg group,and significantly decreased in 5 and 50 mg/kg groups（P<0.05）.The m RNA level of estrogen receptor Esr2 in 50 mg/kg group was significantly decreased（P<0.05）.（4）Compared with the control group,the expression level of Dnmt1 m RNA in each dose group had no significant difference（P>0.05）,while the DNMT1 protein was significantly decreased in 0.5,5 and 50 mg/kg groups（P<0.05）.The m RNA levels of Dnmt3a and Dnmt3b in 0.05,5 and 50 mg/kg groups were significantly increased（P<0.05）,while the protein levels of DNMT3a and DNMT3b showed no significant difference（P>0.05）.（5）Compared with the control group,the expression level of Wnt4 m RNA in ovary tissue of offspring rats was significantly decreased in 5 and 50 mg/kg groups（P<0.05）.WNT4 protein expression was significantly decreased in 0.5,5 and 50mg/kg groups（P<0.05）.The m RNA expression level ofβ-catenin（Ctnnb1）was significantly increased in 0.05 mg/kg group,and significantly decreased in 5 and 50mg/kg groups（P<0.05）;The expression ofβ-catenin protein was significantly decreased in 0.5 and 50 mg/kg groups（P<0.05）.（6）Compared with the control group,the methylation level of Dkk1 promoter in ovary tissues of offspring rats showed no significant difference in each dose group（P>0.05）.The m RNA relative expression level of Dkk1（Dickkopf 1）was significantly decreased in 0.5,5 and 50 mg/kg groups（P<0.05）.2.Effects of prenatal BPA exposure on ovarian function,Wnt/β-catenin signaling pathway and its DNA methylation modification in female offspring rats at PND 56（1）Compared with the control group,the results of general toxicity in female rats showed that the vaginal opening was delayed at 0.5 mg/kg group and advanced at50 mg/kg group.The daily weight of vaginal opening decreased in 0.5 and 5 mg/kg groups（P<0.05）.Body weight,wet ovarian weight and Ovarian organ coefficient were no significantly changed（P<0.05）.Uterine wet weight and uterine organ coefficient in 50 mg/kg group were significantly decreased（P<0.05）.（2）The pathological results showed that there were primary follicles,secondary follicles and luteum in the ovaries of offspring rats at PND 56 in the control group,and the boundaries of follicles were clear.After BPA exposure,the ovaries shrinked,the number of primary follicles decreased,and the corpus luteum was insufficiency.（3）Compared with the control group,BPA exposure during pregnancy had no significant effect on serum P level of female offspring rats（P>0.05）.E₂ level decreased with the increase of BPA dose,and there was statistical significance in 0.5、5 and 50 mg/kg groups（P<0.05）.（4）Compared with the control group,there was no significant difference in the m RNA levels of Cyp11a1 in 0.05 mg/kg group was significantly increased（P<0.05）.The m RNA level of Cyp17a1 in 0.05 and 50 mg/kg group was significantly increased（P<0.05）.The m RNA levels of Cyp19a1 was no significant effect.（P>0.05）.The m RNA level of estrogen receptor Esr1 in 50 mg/kg group was significantly increased（P<0.05）.And the level of Esr2 m RNA in 5 and 50 mg/kg groups was significantly increased（P<0.05）.（5）Compared with the control group,the expression level of Dnmt1 m RNA in5 and 50mg/kg groups was significantly decreased（P<0.05）,and the DNMT1 protein was significantly decreased in 0.5,5 and 50 mg/kg groups（P<0.05）.The m RNA and protein expression levels of DNMT3a in 50 mg/kg group were significantly increased（P<0.05）.The expression level of Dnmt3b m RNA in 5 mg/kg group was significantly decreased（P<0.05）,and the DNMT3b protein was significantly decreased in 0.05 and 50 mg/kg groups（P<0.05）.（6）Compared with the control group,the expression level of Wnt4 m RNA and protein in ovary tissues of offspring rats was significantly increased in 50 mg/kg group（P<0.05）.The m RNA expression level ofβ-catenin（Ctnnb1）in 0.5,5 and 50mg/kg groups was significantly increased（P<0.05）,while the level ofβ-catenin protein in 0.5 and 50 mg/kg groups was significantly increased（P<0.05）.（7）Compared with the control group,the methylation level of Dkk1 promoter was increased in 0.05,0.5,5 and 50 mg/kg groups（P<0.05）.The m RNA level of Dkk1 was significantly decreased in 0.05、0.5、5 mg/kg group（P<0.05）.Conclusion1.BPA exposure during pregnancy can lead to the decrease of serum E₂ level,the disturbances of steroid hormone synthase and estrogen receptor expression,and the histopathological damage of ovaries in female offspring rats,resulting in abnormal puberty development and reproductive function damage.2.BPA exposure during pregnancy can cause disordered expression of DNA methyltransferase in ovaries of female offspring rats,resulting in changes in gene methylation levels.3.BPA exposure during pregnancy can activate the Wnt/β-catenin signaling pathway through hypermethylation in the promoter region of Dkk1 gene,affecting the development of ovaries in female offspring rats.However,this mechanism is not obvious in the ovarian tissue of offspring at PND 21,and the specific mechanism needs to be further studied.