Impact Of BPA On The Male Reproduction And Offspring Development In Rare Minnow (Gobiocypris Rasus) And The Underlying Molecular Mechanisms

Bisphenol A(BPA)is an important industrial synthetic chemical,which is widely used in the production of polymer plastics and chemical materials.Globally,BPA has been frequently detected in aquatic environments,which might pose a potential threat to the reproduction and development of fish.Numerous studies have shown that BPA possesses reproductive toxicity in mammals by affecting male reproductive function.However,the reproductive toxicity effects of BPA on fish species are largely unknown.In this study,male rare minnows(Gobiocypris rarus)were exposed to 15?g L^-1 BPA for 7,14 and 21 d,respectively.Meanwhile,a positive control group was performed with 25ng L^-1 17?-ethynyl estradiol(EE2).The western blotting,immunohistochemistry,real-time quantitative PCR,genome walking,chromatin immunoprecipitation and high-throughput sequencing were conducted to investigate the histological changes of testis and the regulation mechanism of BPA on testis testosterone(T)level.The effect of BPA on testicular Sertoli cell barrier(SC)integrity was detected.The mechanism of transcriptional regulation induced by inflammatory factors in BPA-induced destruction of the Sertoli cell barrier integrity of testes was further explored.Based on these studies,to further reveal the real environmental problems faced by fish,a chronic BPA exposure experiment(90 d)on male G.rarus was conducted.In addition,BPA exposed male parents were mating with normal females to obtain the first and second generation.mi RNA high-throughput sequencing and other methods were used to further explore the intergenerational inheritance of the paternal effect of BPA.The main results obtained were as follows:1.15?g L^-1BPA induced inflammatory response in the testes and reduced the activation rate of sperm.BPA exposure for 7 d and 14 d resulted in lower testosterone(T)levels and abnormal germ cells proliferation in the testis in rare minnow.Transcriptome analysis showed that 354 transcripts(203 up-regulated and 151 down-regulated)significantly differentially expressed after BPA exposure for 14 d,several of them were enriched in the signaling pathways of cell cycle process,PPAR signaling pathway,the steroid synthesis pathway and estrogen signaling pathway.The content of PPAR?was significantly increased after BPA exposure for 7 d while decreased after BPA exposure for 14 d.However,PPAR?was significantly decreased after BPA exposure for 7 d and 14 d.Estrogen receptor(ER)protein levels were significantly increased after BPA exposure for 7 d and 21 d.The expression of St AR was significantly down-regulated after BPA exposure 7 d and 21 d,but significantly up-regulated after BPA exposure for 14 d.Meanwhile,BPA disrupted the St AR expression by interfering ER enrichments within St AR 5'flanking region.2.BPA destroyed the integrity of testis SC barrier by interfering with the expression or distribution of Claudin-3?CX43?Occludin?N-cadherin??-catenin and ZO-1.The transcripts of CX43 and occludin were decreased and SP1 recruitment in each gene promoters were repressed after BPA exposure.Moreover,the cytokines(TNF?and IL-1?)were significantly increased while the JNK signal pathway was activated.BPA also increased the MMP1 and MMP2 levels in the testes.In addition,estrogenic effect might not entirely explain the mechanism by which BPA disrupted the SC barrier in G.rarus.3.Chronic BPA treatment induced reproductive impairments with decreased fertilization capacity and movement time of sperm.Transcriptome analysis indicated 1421 transcripts that were differentially expressed in response to BPA exposure,which were involved in the biological process of oxidative stress,immune responses and DNA/histone methylation.BPA caused the oxidative stress via significantly increasing hydrogen peroxide(H₂O₂)levels and inhibiting the activities of antioxidant-related enzymes(Catalase,CAT).BPA caused an inflammatory response in the testes by significantly increasing IL-1?levels and inducing infiltration of inflammatory cells.Moreover,exposure to 15?g L^-1 BPA significantly decreased the genomic DNA methylation level.The content of H3K4me3 in testis was significantly decreased in the 15?g L^-1 BPA treatment group and significantly increased in the 225?g L^-1 BPA treatment group.4.Male parent BPA exposure significantly reduced hatchability(7 and 14 d)and increased malformation rate(14 d)in the first generation,but had no significant effect on the second generation.mi RNA transcriptome analysis indicated 28 mi RNAs(13 up-regulated and 15down-regulated)that were differentially expressed in the sperm after BPA exposure,which were involved in the biological process of Wnt signaling pathway,oocyte division,Ca²⁺binding and cell cycle regulation.Among the differentially expressed mi RNAs,8 were related to gonad development regulation and 17 were related to bone development.Meanwhile,facial cartilage staining showed that male parental BPA exposure inhibited facial cartilage development in the first generation.In addition,male parental BPA exposure resulted in abnormal sex ratio of the offspring,which showed a tendency of masculinization.Bone development-related genes Runx1 and bmp2a were significantly down-regulated after fertilization for 24 h and 120 h in the first generation.At the same time,the aca-mi R-16a-5P,which was involved in the targeted regulation of bmp2a,were significantly up-regulated after fertilization for 24 h during embryonic development.Taken together,BPA exposure resulted in the decrease of sperm quality,the occurrence of immune response in the testes.In addition,BPA disrupted the T levels by interfering ER enrichments within St AR 5'flanking region.BPA disrupted the SC barrier integrity by inhibiting SP1 enrichments within CX43 and occludin 5'flanking region through activated cytokines/JNK signaling pathway.MMPs were taken part in the disruption of SC barrier caused by BPA exposure.Meanwhile,chronic BPA exposure had adverse effects on male reproduction.Oxidative stress,inflammatory response and DNA/histone methylation might account for the decreased sperm quality.Male parent exposure to BPA lead to inhibition of facial cartilage development in the first generation by changing mi RNA expression in sperm.