Low Doses Of Bisphenol A Cross-generational Transfer Effect On The Development Of Ovary And Uterus Of Rats And Its Molecular Mechanism

AIMThis study was to investigate the cross-generational transfer effects of maternal exposure of Bisphenol A(BPA) on the development of the ovary and the uterus in Fl-generation SD rats at different stages (gestation and lactation), and identify the sensitive phase, sensitive organ and the lasting time of BPA action. We also studied the effects of BPA on the expression of genes and angiogenesis-related proteins in the ovary of offspring rat, and its molecular mechanisms of action.METHODS1. In accordance with BPA limits in different countries (USA and China:0.05mg/kg, EU:0.5mg/kg, Japan:2.5mg/kg), SD rats were orally administered0.05,0.5,2.5mg/kg/d BPA during gestation(from day7to20after pregnancy) and lactation(from day1to21after labor) period, respectively. Other rats were orally administered0.5mg/kg/d solvent and0.05mg/kg/d diethylstilbestrol as negative and positive control, respectively. The body weights of the offspring were recorded within30days after birth. The vaginal opening time and estrous cycle were observed. At the end of day30(before puberty, PND30) and60(adult, PND60) of birth, the female offspring rats were killed, respectively. Weight and pathological changes of ovary and uterus were recorded, and serum levels of estradiol (E2) and progesterone (P4) were detected by ELISA.2. The vaginal opening time, the organ coefficient of the ovary and the uterus, the pathological histology of the ovary and the uterus, and the hormone levels of serum were used as indicators evaluating the sensitive period, sensitive organ and the lasting time of BPA action.3. In order to investigate the alteration of typical genes and signal pathways in the ovary and the uterus of Fl-generation rats induced by BPA, we screened the differential genes expression profile associated with inflammation and autoimmunity between the groups of negative control and BPA(2.5mg/kg) -treatment using high-throughput microarray technology.4. By western blotting technology, we examined the expression of angiogenesis-related proteins vascular endothelial growth factor(VEGF) and pleiotrophin(PTN) in the ovary of F1-generation rats prenatal exposed to BPA.RESULTS1. Effects of BPA exposure during gestation period on the development of ovary and uterus in F1-generation rats Compared with the negative control, the rats of F1-generation in BPA(0.05,0.5,2.5mg/kg) treatment groups exhibited significantly earlier time of vaginal opening(P<0.05). There were no significant changes on ertrous cyclicity in all groups(P>0.05). The coefficients of the ovary and the uterus were increased significantly in BPA(2.5mg/kg)-treated group(P<0.05), and the levels of serum E2and P4were also increased significantly in BPA(2.5mg/kg)-treated group on PND30(P<0.05). Typical polycystic pathological changes were observed in the ovary of BPA(2.5mg/kg)-treated group on PND30and PND60. No effects of BPA on the organ coefficient of the ovary and the uterus, and the serum sexual hormones levels were observed in the adult offspring rats(PND60). There were no pathological changes in the uterus of all groups.2. Effects of BPA exposure during lactation period on the development of ovary and uterus in Fl-generation rats Compared with the negative control, the rats of Fl-generation in the BPA(2.5mg/kg) treatment group exhibited significantly earlier time of vaginal opening(P<0.05). There were no obviously different in the organ coefficients and the pathological histology of the ovary and the uterus, and the hormone levels of serum in the F1-generation rats in all groups(P>0.05).3. Evaluation of the sensitive period, sensitive organ and the lasting time of BPA action Through overall evaluation of the vaginal opening time, ovary and uterus coefficients, ovary and uterus pathological changes, serum E2and P4levels, we found that gestational exposure to BPA had more severe adverse effects on the development of the ovary and the uterus than that of lactational exposure, and the ovary is more sensitive to BPA than the uterus. Moreover, the pathological changes on the ovary and the uterus induced by BPA before puberty would weaken in adulthood.4. According to the data of high-throughput microarray analysis, there were109pieces of gene significantly affected by BPA (2.5mg/kg) in the ovary, including83pieces of gene up-regulated and26pieces of gene down-regulated, and of which the expression of30pieces of gene were changed more than10times. There were104pieces of gene significantly affected by BPA (2.5mg/kg) in the uterus, including97pieces of gene up-regulated and7pieces of gene down-regulated, and of which the expression of12pieces of gene were changed more than10times. The markedly regulated signal pathways involved cytokine-cytokine receptor interaction, angiogenesis, Jak-STAT, chemokine, MAPK and Apoptosis.5. The results of western blotting showed that the expression of VEGF and PTN were significantly increased in BPA(2.5mg/kg)-treated group in the ovary of Fl-generation rats on PND30(P<0.05), compared with the negative control. However, there was no statistically significant difference in the expression of VEGF and PTN in all groups on PND60.CONCLUSION1. Gestational exposure to BPA had more severe adverse effects on the development of the ovary and the uterus than that of lactational exposure. BPA could dose-dependently advance vaginal opening, increase ovary and uterus coefficients, and serum E2and P4levels in offspring rats before puberty. Furthermore, BPA could induce polycystic ovarian disease in offspring rats on PND30and PND60. Therefore, the relatively safe dose of BPA for the Fl-generation rats is under0.5mg/kg.2. The influences BPA exerted on the development of the ovary and the uterus, and the levels of serum E2and P4before puberty would weaken in adulthood. 3. The ovary of F1-generation rats might be more sensitive to BPA than the uterus.4. The mechanisms of action of BPA may relates to signal pathway, such as Inflammation pathway, angiogenesis pathway, Jak-STAT pathway, MAPK pathway and apoptosis pathway.5. The up-regulation of VEGF and PTN expression induced by BPA(2.5mg/kg) in the ovary of Fl-gereration rats implied that it might be one of the mechanisms by which BPA induce polycystic ovarian disease.