| IntroductionBisphenol A (BPA，CAS:80-05-7) name2,2-two (4-hydroxyphenyl) propane，is recognized as one of several environmental estrogens. BPA is a monomer of thesynthetic polymer material that is used in the production of plasticizers, pesticides,paint, and other chemical products. BPA is used in a wide variety of products, such asbaby bottles, lunch boxes, toys, water pipes, and other materials. BPA monomers canleach from a plastic container that holds food or beverages, and the release process isaccelerated under conditions of high temperature, strong acidity, or strong alkalinity.Human exposure to BPA is extensive; epidemiological studies have shown that thechemical could be detected in the urine of more than90%of tested individuals. Thechemical structure of BPA is similar to that of estradiol (E2); it can therefore mimicestrogenic actions in the body.The use of BPA in baby products has already been banned in the European Union,the United States, China, and many other countries. However, the prepubertal periodis also an important time in child development, and BPA exposure will occur duringthis period through a variety of pathways. Children are especially vulnerable to higherconcentrations of BPA as a consequence of food and drinking water contamination.Although environmental concentration compared with estrogens in vivo is low，thehypothalamus-pituitary-gonadal axis of children is not yet mature during thepre-puberty stage, and the levels of sex hormones in the body are relatively low;therefore, children’s reproductive systems are more sensitive to environmentalestrogens than those of adults. We speculated that exposure to high concentrations ofBPA during the prepubertal period may affect the normal developmental process of ovarian follicles, and the resulting toxic effects may be mediated by the regulatorygenes involved in follicular development. This study explored the roles andmechanisms of genes that are associated with ovarian development in the toxic effectsof short-term BPA exposure on rat ovarian development. We accomplished this aim bystudying changes in expression among follicle development-promoting genes, such asKitlg, Figla and H1foo, and follicular development inhibitory genes, such as AMH.This study could provide an important experimental evidence for the reproductivesystem developmental effect of children exposure to BPA.Part Ⅰ: Effect of bisphenol A on ovarian development and function inprepubertal rat.Objective: The aim of this study was to determine whether BPA could affect ovariandevelopment and function in prepubertal rat.Methods: In this study,28-day-old female Wistar rats were exposed to BPA throughdaily intraperitoneal injections (at rates of10mg/kg,40mg/kg, and160mg/kg) forone week. The effects of BPA on ovarian structure and function were assessed bymeasuring the weight of the ovaries, counting ovarian follicles, apoptosis in ovarianfollicles, ratios of vaginal opening and first estrus, assaying sex hormone levels in theserum, and the ultrastrueture of ovarian tissue use of transmission electronmicroscopy.Results:1. Prepubertal rats exposed to BPA for7days, no significant difference wasobserved in body weights between the BPA exposure groups and the control group,whereas the ovarian weights in the BPA exposure groups decreased with increasingBPA doses. The ovarian weights and ovary coefficients of the rats from the40mg/kgand160mg/kg BPA groups were significantly different from those of the controlgroup (P<0.01)2. The ratios of vaginal opening and first estrus have no significant differencebetween the BPA exposure groups and the control group（P＞0.05）.3. The levels of serum E2in rats from each BPA group decreased with increasingdoses of BPA, but the results were not significantly different from that of the rats in the control group (P0.05). However, the levels of serum P4in rats from the BPAgroups were significantly different from that of the control group rats (P<0.01); inparticular, the decreases of serum P4in the40mg/kg and160mg/kg BPA groupswere significantly different from that of the control group, respectively (P<0.01).4. The number of follicles in each BPA exposure group decreased with increasingBPA doses; in comparison to the control group, significant changes were observed inthe10mg/kg,40mg/kg, and160mg/kg BPA groups (P<0.05, P<0.01, and P<0.01,respectively); In addition, the constituent ratios of primordial follicles, primaryfollicles/preantral follicles, antral follicles, and corpus luteum (CL) all decreased inresponse to increasing BPA concentrations, but the constituent ratio of atretic folliclesincreased. These changes in the40mg/kg and160mg/kg BPA groups relative to thecontrol group were statistically significant (P<0.01).5. Compered with control group, the apoptosis ratios of preantral follicles, antralfollicles, and corpus luteum (CL) all increased in response to increasing BPAconcentrations.6. The ovarian tissue ultrastrueture of steatosis and secondary lysosomes allincreased in response to increasing BPA concentrations, compered with control group.Part Ⅱ: Effect of ovarian follicle development-related genes expression inprepubertal rat exposed to bisphenol A.Objective: The aim of this study was to determine whether BPA could affect ovarianfollicle development-related genes expression in prepubertal rat.Methods: In this study,28-day-old female Wistar rats were exposed to BPA throughdaily intraperitoneal injections (at rates of10mg/kg,40mg/kg, and160mg/kg) forone week. The mRNA and protein expression levels of follicle development-relatedgenes: Stem cell factor(Kitlg), Factor In the Germline alpha (Figla), oocyte-specifichistone H1variant (H1foo), and anti-Mullerian hormone (AMH) were analyzed usingreal-time quantitative PCR, western blots and immunohistochemistry.Results:1. The real-time PCR revealed that Kitlg gene mRNA expression levels in the160mg/kg BPA group significantly decreased (P<0.01) in comparison to the control. The mRNA expression levels of the Figla gene in all of the BPA groups significantlydecreased (P<0.05) relative to the control. And the mRNA expression levels of theH1foo gene in all of the BPA groups significantly decreased (P<0.01) relative to thecontrol, but the mRNA expression levels of the AMH gene in the160mg/kg BPAgroup significantly increased (P<0.01) relative to the control.2. Western blot analysis revealed that protein expression levels of KITLG in eachBPA group significantly decreased relative to the control, with P<0.05for the10mg/kg BPA and40mg/kg group and P<0.01for the160mg/kg groups, and theprotein expression levels of FIGLA in the160mg/kg BPA group significantlydecreased (P<0.05) relative to the control, and the protein expression level of H1FOOin each BPA group significantly decreased relative to the control, with P<0.05for the10mg/kg BPA group and P<0.01for the40mg/kg and160mg/kg groups, but theprotein expression level of the AMH gene in each BPA group significantly increasedrelative to the control, with P<0.05for the10mg/kg BPA group and P<0.01for the40mg/kg and160mg/kg groups.Part Ⅲ: Study on epigenetic regulation mechanism of toxicity during ovarianfollicle development in prepubertal rat exposed to bisphenol A.Objective: The aim of this study was to determine whether BPA could affect DNAtotal methylation status and gene promoter region DNA methylation status of ovarianfollicle development-related genes in prepubertal rat.Methods: In this study,28-day-old female Wistar rats were exposed to BPA throughdaily intraperitoneal injections (at rates of10mg/kg,40mg/kg, and160mg/kg) forone week. Study on global methylation status, and gene promoter region DNAmethylation status, to explore the epigenetic regulation mechanisms underlying BPAtoxicity during ovarian development.Results:1. Compered with control group,the OD and IOD of combined5-mC scores ofovarian granulosa cell increased gradually in response to increasing BPAconcentrations. These changes in the40mg/kg and160mg/kg BPA groups relative tothe control group were statistically significant (P<0.05). 2. All genes test results from the Methprimer software showed only that there isone CpG island in the promoter region of the Kitlg gene, with35methylationcites(Cs). and then use the bisulfite sequencing polymerase chain reaction (BSP)method to test the methylation status of Kitlg gene. The sequencing results showedthat levels of methylation cites increased with increasing doses of BPA. in particular,the increases of methylation cites in the160mg/kg BPA groups was significantlydifferent from that of the control group (P<0.05). and7Cs exists methylation status inthe control group, but demethylation in BPA exposure group;16Cs show nomethylation status in the control group,but obvious methylation in BPA exposuregroup.Conclusion:1. Our results indicated that short-term, concentrated BPA exposure during theprepubertal period may have inhibitory effects on ovarian follicle development andhormone secretion, which were observed as the ovarian weight, counting ovarianfollicles were decreased, the constituent ratios of all growing follicles decreased andatretic follicles increased, the apoptosis ratios of follicles increased, and the inhibitionof progesterone secretion.2. The down-regulation of Kitlg, Figla and H1foo genes and the up-regulation ofthe AMH gene may play important roles in the BPA-induced mechanism of toxicitythat affects ovarian development.3. The possible role of DNA methylation patterns change in the regulation ofgene expression in Kitlg, Figla, H1foo and AMH genes are clearly worth considering;the mRNA expression levels of the Kitlg gene decreased significantly in the BPAgroups，may associated with promoter region methylation pattern. |
PDF Full Text Request