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Effect And Molecular Mechanism Of Bisphenola On Rare Minnow Spermatogenesis

Posted on:2018-07-24Degree:DoctorType:Dissertation
Country:ChinaCandidate:Y Y ZhangFull Text:PDF
GTID:1311330512982263Subject:Aquatic biology
Abstract/Summary:PDF Full Text Request
Bisphenol A(BPA)is an impotant industrial chemical that commonly used in the manufacturing of numerous consumer products such as food and beverage containers,baby bottles,cups,glasses,and many other commodities.For the similar structure with endogenous17?-estradiol(E2),BPA could mimic E2 effect and disrupt normal reproduction.It is suggested that BPA exposure was related to the disorder reproduction hormone levels,normal testicular development,as well as decreased sperm quality.However,the mechanism underline these effects was not well understood.Using rare minnow Gobiocypris rarus as test model,we firstly investigated the testicular transcriptome in response to BPA at different concentritions.Based on the transcriptome data,we performed in-depth investigations on the aspect of sex hormone synthesis,germ cell development,sperm morphology and function,and testicular biomarker in the present study.The progress of the present study was as following:1.RNA-seq data showed that following 7 days exposure,BPA at 1,15 and 225 ?g/L all affected lots of testicular gene transcription.BPA at all the three concentrations affected the expression of genes related to testicular sex hormone biosynthesis,blood-testis barrier,proteolysis,lipid transport and metabolism,cell cycle,and cell apoptosis.Moreover,BPA at 1?g/L induced gene expression in rennin angiotensin system pathway and possibly initiate membrane form of estrogen receptor(mER);1 and 15 ?g/L BPA inhibited tRNA processing-related genes expression;15 and 225 ?g/L BPA decreased hemostasis and blood coagulation-related gene expression.2.In the transcriptome data,we found that the sex hormen levels were significantly afected by BPA.To investigate the mechanism,we cloned and analyzed the 5'-flanking of sex hormone synthesis-related genes(star,hsd3 b,hsd11b2,cyp11 a,cyp17a1 and cyp19a1a),we found that potential binding sites for sexual steroid receptors(Esrs and Ar)and transcription factors(Nr5as and Foxl2)were exsited in their promoter region.Following BPA exposure,significant correlations were found between the expression profiling of testicular star and esr1,hsd3 b and nr5a1 a,hsd11b2 and nr5a1a/esr2 b,cyp11a and nr5a1a/nr5a1 b,cyp17a and esr2b/ar,cyp19a1 a and foxl2/esrs.It suggested that BPA-mediated actions on testicular sexhormone synthesis possibly involve sex steroid hormone receptor signaling and transcription factors such as Nr5 as and Foxl2.3.Up to now,there was no study on the effect of BPA on fish germ cells apoptosis,but our transcriptome data showed that several apoptosis related genes were affected by BPA.Through TUNEL and ultrastructure analysis,we found that the amount of apoptotic spermatocytes significantly increased in a time dependent manner following BPA exposure.The decreased ratio of testicular Bcl2/Bax(anti-apoptosis gene/pro-apoptosis gene)decided the final apoptosis of spermatocytes.Mitochondrial apoptotic pathway including bcl2,bax,casp9,cytc and mcl1 b played important roles in this process.In addition,mRNA expression of meiosis regulation genes(kpna7 and wee2),and genes involved in both apoptosis and meiosis(birc5,ccna1 and gsa1a)were also affected by BPA.So the spermatocyte apoptosis was likely initiated by BPA induced meiosis arrest.4.Through ultrastructure analysis,we found that 15 and 225 ?g/L BPA increased the percentage of sperm with swelling head and neck and damaged membrane.At the same time,sperm movement was affected,and sperm fertilization ability was decreased.Moreover,the influences were aggravated along with the exposure time and concentrations.Our transcriptome data showed that BPA induced the expression of aqps,which play critical roles in spermatid deformation,movement and osmoregulation.Flow cytometry analysis showed that BPA exposed sperm did decrease its tolerance to hypotonic solution.The following IHC and WB results showed that Aqp3 and 8 was distributed in various germ cells in rare minnow,and their expression was induced by BPA.It possibably contribute to sperm paramorphia and quality reduction.The final ChIP analysis showed that BPA could regulate aqp3 and 8expression via ERE in their 5'-flanking region and increased Aqp3 and 8 proteion levels.5.Several vtg subtypes were found in rare minnow testicular transcriptome,Vtg proteins were proved to mainly deposit in spermatogonia and spermatocyte.Following BPA exposure,testicular vtg1 and 2 significantly decreased in a time denpendent manner.Through ChIP and CoIP,we found that BPA could enhance Er-Hsp90 complex formation and decrease Er recruitment in vtg1 promoter,and reduced Er's transcription activation effect on vtgs.Following long time exposure,testicular Vtgs were possibly replenished by the induced hepatic Vtgs.Moreover,it suggested that vtg mRNA is a potential testicular specific biomarker for BPA exposure.Taken together,the present study shows that BPA have extensive impacts on rare minnow testis development.BPA could influence testicular sex hormone synthesis through sex steroid hormone receptor signaling,gonadotropin/gonadotropin receptor pathway,as well as transcription factors.BPA could induce spermatocyte apoptosis,and it was likely initiatedby BPA induced meiosis arrest.BPA could affect sperm morphology,motility and osmoregulation through Aqps,which finaly contribute to the decreased sperm fertilization.Moreover,BPA could inhibit endogenous Vtgs biosynthesis and vtg mRNA is a potential testicular specific biomarker for BPA exposure.
Keywords/Search Tags:bisphenol A, rare minnow, spermatogenesis, transcriptome, sex hormone
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