| Bisphenol A(BPA)is an important chemical material and extensively utilized to manufacture various daily necessities.Every year,a large amount of BPA is released into the soil,water and atmosphere.Human and other animals contact and expose to BPA by multiple ways in the environment.Previous studies showed that BPA seriously harm the animal reproductive development and result in reproductive abnormality.Though researchers have carried out in-depth studies of the effects of BPA on animal reproductive development and its molecular mechanisms,there is scarce systematic research on the epigenetic mechanism of BPA involved in affecting the animal reproductive development.Our previous studies showed that BPA exposure at 15?g/L significantly influence the reproductive development of Gobiocypris rarus(G.rarus).In the present study,used the G.rarus were exposed to 15?g/L BPA for 7 to 63 days,to explore the epigenetic mechanism underlying BPA's adverse effects on the reproductive development of the G.rarus parents and their offsprings.The major findings are as follows:1.BPA resulted in differentially significant alterations of the genome DNA methylation levels in gonads of G.rarus,with significant increase in testis at 7d and in ovary at 35 d,respectively.And the gene expressions of dnmts could contribute to the alteration of global DNA methylation in gonads during BPA exposure.After BPA exposure,the negative correlation between the changes of DNA methylation and transcript of cyp19a1 a in gonads suggests that BPA could regulate the transcripts of cyp19a1 a in the ovary by altering DNA methylation level of the gene.2.After the females were exposed to BPA for 21,42 and 63 d,the level of estrogen E2 was significantly increased at 21 d,while decreased at 63 d in ovary.Obvious histopathological changes was found with decreased mature oocytes and increased primary and secondary oocytes after BPA exposure for 63 days.The genome DNA methylation levels and histone trimethylation levels(H3K4me3,H3K9me3 and H3K27me3)were significantly altered in the ovary in BPA exposure from 21 to 63 d.And the transcripts of dnmts(dnmt1 and dnmt3-8)and histone methyltransferase genes(mll2-5,setdb1-2 and ezh2)were also markedly changed under BPA exposure for 21-63 days.The transcriptions of 4 steroidogenesis related genes including star,cyp11a1,cyp17a1 and cyp19a1 a were regulated by DNA methylation of the corresponding gene and/or H3K9me3 to different degrees.These data suggest that epigenetic modulation including DNA and histone methylation could be responsible for the detrimental effects on ovary development upon BPA exposure.3.After the male G.rarus were exposed to BPA for 21 to 63 days,the histological results showed that a small amount of inflammatory cells appeared in testis tissue and the interstitial space among various germ cells was enlarged after BPA exposure for 21 days.In addition,the fertilizing capacity of the sperms was significantly decreased after BPA exposure for 63 days.BPA caused significant increase of the genome DNA methylation levels in testis at 21-63 d exposure.The immunohistochemical analysis showed that 5-methyl cytosine was obviously increased in spermatocytes,spermids and sperms after BPA exposure.The transcripts of dnmt3 and dnmt5-8 were significantly increased in testis after BPA exposure.Besides,after BPA exposure,the histone trimethylation levels(H3K4me3,H3K9me3 and H3K27me3)as well as the transcripts of their corresponding histone methyltransferase genes(mll2-5,setdb1-2 and ezh2)were significantly altered in testis with different patterns.These results suggested that the damage on testis and sperms under BPA exposure could be related to the epigenetic modification of DNA and histone methylations.4.After the females were exposed to BPA for 2 months,the spawning rate,egg number,egg diameter and fertilization rate were all significantly decreased.F1 generation bred from both the females and males exposed to BPA for 2 months showed decreased fertilization rate of embryos and hatching rates,delayed hatching time and increased heart rates.Moreover,the F1 generation exhibited significantly increased abnormality rates with six types of abnormal shapes.The ratio of the females to males was significantly decreased in 5-month old F1 generation,and the ovary weight and the number of mature oocytes of the 5-month old F1 generation were significantly reduced,which suggested the ovary development of offspring was significantly inhibited.The transcripts of 4 steroidogenesis related genes(star,cyp11a1,cyp17a1 and cyp19a1a)were significantly affected in ovaries of 5-month old F1 generation,with increased transcript for cyp19a1 a while decreased transcripts for star,cyp11a1 and cyp17a1.However,for the offspring of the parent fish both exposed to BPA for 2 months and subsequently depurated in fresh water for 3 months,all the indexes above mentioned returned to normal level compared with the controls.These results suggest that the G.rarus parents' BPA exposure could damage embryo development of F1 generation and inhibit the ovary development of F1 generation females,while do not affect testis development of the F1 generation males.Moreover,the negative effects on the offspring of parents' BPA exposure are reversible.5.The levels of genome DNA methylation,H3K9 me,and H3K27me3 as well as the transcripts of dnmts(dnmt1,dnmt5 and dnmt7)and histone methyltransferase genes(setdb1,setdb2 and ezh2)were increased significantly in the ovaries of the 5-month old F1 generation of G.rarus parents exposed to BPA.The DNA methylation levels of star and cyp17a1 were significantly increased in ovaries of the 5-month old F1 generation,while The DNA methylation levels of cyp19a1 a was reduced.In the ovaries of the 5-month old F1 generation,the histone trimethylation levels(H3K4me3 and H3K9me3)for star gene was significantly decreased,while the histone trimethylation levels(H3K9me3 and H3K27me3)for cyp11a1 gene was significantly increased.For cyp17a1 gene,the(H3K9me3 level was significantly increased while H3K27me3 level was significantly decreased in ovaries of the 5-month old F1 generation.For cyp19a1 a gene,the H3K4me3 level was significantly increased,while H3K9me3 and H3K27me3 levels were significantly decreased.These results suggest that the parents' BPA exposure could cause reproductive toxicity in F1 generation and the epigenetic changes in DNA methylation and histone trimethylation could account for the gene expression for steroidogenesis related genes in G.rarus ovary for the offspring with parents' BPA exposure.In conclusion,the genome DNA methylation levels and histone methylation levels were significantly altered in the gonads of G.rarus in BPA exposure and the ovaries of the 5-month old F1 generation of G.rarus parents exposed to BPA.The epigenetic changes in DNA methylation and histone trimethylation could account for the gene expression for steroidogenesis related genes in ovary of G.rarus parents and F1 generation.The G.rarus parents' BPA exposure could damage embryo development and ovary development of F1 generation,and the negative effects on the offspring of parents' BPA exposure are reversible. |
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