| Part Ⅰ: Maternal exposure to bisphenol A during pregnancy affect puberty onset in advance,key gene promoter methylation of hypothalamic Pc G protein and related hormone levels in female offspringObjectives To explore the effects of maternal exposure to bisphenol A(BPA)during pregnancy on puberty onset in advance,key gene promoter methylation of hypothalamic Pc G protein and hormone levels of initiating puberty;while explore the role of key gene promoter methylation of hypothalamic Pc G protein in exposure to bisphenol A during pregnancy and puberty onset in advance of female offspring..Methods To investigation the effects of BPA exposure during pregnancy,pregnant CD-1 mice were randomly divided into four groups: control group(0 mg/kg),low-dose group(8 mg/kg),middle-dose group(40 mg/kg),and high-dose group(200 mg/kg).BPA-exposed groups were administered with BPA dissolved in corn oil by gavage daily from gestational day(GD)1 to 18,while the control group was administered with corn oil.The gender of each offspring was identified by anal reproductive distance after birth.We observed the vaginal opening of female offspring from postnatal day(PND)21 to 33,and distinguished the estrous stage through vaginal smears.Female offspring weighed at PND 1,2,3,7,14,21,28,and 33.All female offspring were sacrificed and hypothalamus tissue werecollected on PND 34.Serum Kisspeptin-10 and Gonadotropin-releasing hormone(Gn RH)levels in female offspring were measured by radioimmunoassay(RIA).serum follicle stimulating hormone(FSH),luteinizing hormone(LH),estradiol(E2)levels in female offspring was measured by enzyme-linked immunosorbent assay(ELISA).The m RNA levels of Eed and Ezh in the hypothalamus were measured by RT-PCR,and the Eed and Ezh methylation levels were measured by pyrosequencing.Results The total number of offspring,the number of females offspring and the number of males offspring in the BPA-exposed group did not reach statistical significance compared with the control(P<0.05).The proportion of female offspring in 40 and 200 mg/kg BPA-exposed group were lower than that in 8mg/kg BPA-exposed group(P<0.05),while there was no significant difference in the proportion of female offspring between the BPA-exposed groups and the control group(P>0.05).Maternal exposure to BPA during pregnancy did not change significantly body weight in female offspring compared with the control group(P>0.05).However,the vaginal opening time of each BPA-exposed group was earlier than that in the control group(P<0.05).Additionally,significant difference of estrus cycle duration was found between 8 mg/kg group and control group(P<0.05).Furthermore,the methylation levels of Eed1 and Eed2 in female offspring were significantly higher in BPA-exposed groups(P<0.05),the methylation levels of Ezh2 in BPA-exposed groups increased significantly(P<0.05),but Ezh1 methylation levels did not change(P>0.05).For another,the m RNA expression levels and methylation levels of Eed1 and Eed2 in female offspring have also been found in this study.Compared with the control group,for one thing,the m RNA expression levels of Eed1 and Eed2 in female offspring were decreased in BPA-exposed groups(P<0.05),and the expression levels of Ezh2 m RNA were decreased in the low-dose and middle-dose groups while that wasincreased in high-dose group(P<0.05),but the expression level of Ezhl m RNA did not change(P>0.05).The levels of Kisspeptin-10 and Gn RH in female offspring during BPA-exposed groups were significantly higher than that in the control group.Compared with the control group,the level of serum LH in the low-dose group was slightly decrease(P<0.05),however,the levels of serum FSH and E2 in female offspring in BPA-exposed groups did not change(P>0.05).In the control group,8 mg/kg,and 40 mg/kg BPA-exposed groups,Eed1,Eed2,and Ezh2 gene methylation and Kisspeptin-10 levels in puberty in advance of female offspring were higher than that in not puberty in advance.And in the 200 mg/kg BPA exposure group,Eed1,Ezh2 methylation,Kisspeptin-10,and Gn RH levels in puberty in advance of female offspring were higher than that in not puberty in advance.The difference was statistically significant(P<0.05).The correlation coefficient between the methylation of Eed1,Eed2,Ezh2 genes,hormone levels,and puberty in advance in the BPA-exposed groups were higher than that in the control group.Additionally,BPA had no direct influence on puberty in advance,and the path coefficient did not reach a significant level(P>0.05);BPA through methylation of Eed2,and Ezh2 had an indirect effect on puberty in advance,and the path coefficient reached a significant level(P<0.05).Conclusions These findings showed maternal exposure to BPA during pregnancy affected puberty onset in advance.Furthermore,the mediating effect of methylation of Eed and Ezh genes on maternal BPA exposure during pregnancy and puberty in advance of female offspring was demonstrated.The above results suggests the mechanism that maternal exposure to BPA during pregnancy induced puberty onset in advance is related to the methylation of Eed and Ezh genes.Part Ⅱ: Critical period study on the effects of maternal bisphenol A exposure during pregnancy on puberty onset in advance in female offspringObjectives To study the effects of maternal exposure to BPA during different pregnancy on puberty onset in advance and key gene promoter methylation of hypothalamic Pc G protein,while explore the critical period of maternal BPA exposure during pregnancy.Methods Forty-eight pregnant mice were randomly divided into three groups,including the first trimester pregnancy group,the second trimester pregnancy group and the third trimester pregnancy group,respectively.Each group was divided into control group(corn oil)and BPA-exposed group(8 mg/kg).In the first trimester pregnancy group,pregnant mice of BPA-exposed group were administered with BPA by gavage daily in GD1-6 and controls were administered with corn oil by gavage daily from GD1-6.In the second trimester pregnancy group,pregnant mice of BPA-exposed group were administered with BPA by gavage daily in GD7-12 and controls were administered with corn oil by gavage daily from GD7-12.In the third trimester pregnancy group,pregnant mice of BPA-exposed group were administered with BPA by gavage daily in GD13-18 and controls were administered with corn oil by gavage daily from GD13-18.The gender of each offspring was identified by anal reproductive distance after birth.We observed the vaginal opening of female offspring,and distinguished the estrous stage through vaginal smears from PND 21 to 33,and distinguished the estrous stage through vaginal smears.Female offspring weighed at 1,7,14,21,28,and 33 days after birth.All female offspring were sacrificed and hypothalamus tissue were collected on PND 34.Serum Kisspeptin-10 and Gn RH levels in female offspring were measured by radioimmunoassay(RIA).The m RNA levels of Eed and Ezh in the hypothalamus were measured by RT-PCR,and the Eed and Ezh methylation levels were measured by pyrosequencing.Results Compared with the control group,there was no significant difference in the offspring number,the female offspring number,the male offspring number and the female rate of exposed to BPA during different pregnancy(P>0.05).Compared with the control group,there was no significant difference in body weight gain between the female offspring during different pregnancy(P>0.05).Compared with the control group,the vaginal opening time of the female offspring in BPA-exposed during the second trimester of pregnancy was earlier than that in the control group(P<0.05),While there was no significant difference in vaginal opening between BPA-exposed group and control during the first trimester and the third trimester of pregnancy(P>0.05).The rate of vaginal opening in BPA-exposed female offspring during the second trimester of pregnancy was significantly higher than that of control group(P<0.05).Compared with the control group,there was no significant difference in estrus period and estrous cycle time during three period of pregnancy(P>0.05).The methylation level of Eed1 in female offspring of maternal BPA exposure was significantly higher than that of the control group(P<0.05).Eed2 methylation in BPA-exposed group during the second trimester of pregnancy were increased than control group(P<0.05).Compared with the control group,there was no significant different in Ezh1 methylation levels of maternal BPA exposure during different pregnancy(P>0.05).Compared with the control group,maternal exposure to 8 mg/kg BPA during the second and third trimester of pregnancy increased methylation level ofhypothalamus Ezh2 in female offspring,with statistical significance(P<0.05).The levels of Eed1 in the hypothalamus of BPA-exposed female offspring were lower than that in the normal offspring of pregnant women during three period of pregnancy(P<0.05).Compared with the control group,maternal BPA exposure during the first trimester of pregnancy increased Eed2 expression in female offspring(P<0.05),and the difference was statistically significant;While for Ezh gene expression,the results showed that the expression levels of Ezh1 in female offspring of maternal BPA exposure during the second trimester of pregnancy were lower than that in the control group(P<0.05),while compared with the control group,the expression of Ezh2 of maternal BPA exposure during the first and second trimester of pregnancy were significantly increased(P<0.05).The serum Kisspeptin levels in BPA-exposed group during the first trimester of pregnancy were lower than that in the control group,while the Kisspepitn serum levels in BPA-exposed group were higher than that in the control group during the second trimester and third trimester(P<0.05).The level of Gn RH in the BPA exposed group during the second and third trimester was higher than that in the control group,and the difference was statistically significant(P<0.05).Conclusions In this study,the study that maternal BPA exposure during different pregnancy show that the second trimester of maternal BPA exposure was significantly advanced puberty in famale offspring,and also show that the methylaton degree of puberty-onset genes were increased in second and third trimester pregnancies.This suggests that the critical period of maternal BPA exposure during pregnancy could be the second and third trimester pregnancies. |
