The Effects Of Bpa On DNA Methylation Of Ovarian Steroidogenic Genes In Rare Minnow Gobiocypris Rarus

As an endocrine disrupt chemical(EDC),BPA is widely used to produce polycarbonate plastic and epoxy resins.For the the similar structure with estradiol(E2),BPA has has weak estrogenic effects and can disrupt normal endocrine system.It has been suggested that BPA could disturb the process of steroidogenesis,wheras the the potential mechanism of the regulation is barely understood.To explore the effects and the regulation mechanism of BPA on steroidogenesis,we used female Gobiocypris Rarus as test model and exposed them with 15 ?g/L BPA for 7 and 14 days.After BPA exposure,the histological changes of ovaries were observed,the serum steroid hormones levels were detected by using ELISA kits,the mRNA expressions of steriodogenic genes,estrogen receptor genes and DNA methylation related genes were determined by RT-PCR,the 5' flanking region of cyp11a1 was cloned,the ERE-ER interactions of steroidogenic genes were measured by CHIP assays,and the DNA methylation levels of steriodogenic genes were tested by bisulfite sequencing.The main results of the present study were as following:1.Although there were histological changes of ovaries,the GSI and serum E2 levels were significantly increased after 14 days BPA exposure.The results suggested that BPA could perturb the process of steroidogenesis.2.After 7 days BPA exposure,the transtript expressions of ovarian star,cyp11a1,cyp17a1,hsd11b2 and esr1 were significantly down-regulated in female G.rarus,while the the transtript expression of ovarian cyp19a1 a was obviously up-regulated.After 14 days BPA exposure,the mRNA levels of cyp11a1 and cyp17a1 were still markablely decreased,while the mRNA levels of hsd3 b and esr2 a were distinctly increased.Those results might revealed that BPA could affect the the process of steroidogenesis by disturbing the expressions of steriodogenic and estrogen receptor genes.3.We obtained 2059 bp 5' flanking sequence of cyp11a1 by cloning.We predicted ERE sites in the 5' flanking regions of star,hsd3 b and hsd11b2.Later,we confirmed that ERE site(-643 ~-662bp)in star,ERE site(-201 ~-223bp)in hsd3 b and ERE site(-201 ~-223bp)in hsd11b2 were shown the capacity to bind to ER by CHIP assays.Additionally,BPA obviously suppressed the ER-EREs binding of star and hsd11b2 at day 7,while stimulated the ER-ERE binding in hsd3 b at day 14.These results suggested that BPA could disturb the expression of steriodogenic genes by affecting on the ER-ERE interactions of star,hsd3 b and hsd11b2.4.After 7 days BPA exposure,the DNA methylation levels in 5' flanking region of star gene at-719 bp CpG site,cyp11a1 gene at-1923 bp CpG site,and hsd11b2 gene at-1788 bp were significantly increased,while the DNA methylation level of cyp17a1 at-72 bp CpG site was remarkablely decreased.After 14-day BPA treatment,the DNA methylation level in 5' flanking region of cyp11a1 gene at-1722 bp CpG site,cyp17a1 gene at-35 bp and-51 bp CpG sites,were significantly increased,while the DNA methylation of cyp17a1 gene at-72 bp CpG site was remarkablely decreased.That suggested that DNA methylation involved in the effects of BPA on the expression of steriodogenic genes.5.Following 7 days BPA treated,the transtript expression of dnmt1 was significantly increased.It might be related to the hypermethylation of star,hsd11b2 and cyp11a1 after 7 days BPA exposed.Furthermore,the mRNA levels of mbd2 and mecp2 was significantly up-regulated.It might suggeste that DNA heyermethylation repressed expression of star,cyp11a1 and hsd11b2 via increasing expression of mbd2 and mecp2 at 7-days BPA treated.Taken together,the present study indicated that BPA could perturb steroid biosynthesis by interfering with the expression of ovarian steroidogenic genes in G.rarus.After BPA exposure,DNA methylation mediated the expression alterations of steriodogenic genes.We also confirmed that there were ERE sites in the 5' flanking region of star,hsd11b2 and hsd3 b.As well as,our findings suggested BPA perturbed the binding of transcriptional factor ER to EREs in the 5 flanking of star,hsd11b2 and hsd3 b.