The Mechanism Of Prenatal Di-n-butyl Phthalate Exposure-induced Transgenerational Effect Of Spermatogenic Failure In Rats

Environmental endocrine disruptors(EDCs)are associated with a variety of reproductive diseases.Phthalic acid esters(PAEs)chemicals are widely used in human daily life.It is used as an additive to plastics to enhance the flexibility,transparency and durability.In addition,it also used in agricultural chemicals,coatings,cosmetics,spices,etc.It is now recognized that PAEs is one of the most important environmental pollutants in the world.Dibutyl phthalate(DBP)is one of the largest scope of pollution and hazardous substances of PAEs.Studies have shown that DBP interferes growth and fuction of reproductive system of the males.However,whether DBP exposure results in transgenerational spermatogenic failure and,if so,the potential underlying molecular mechanisms remain unknown.Our aim is to observe the mechenism of prenatal DBP exposure-induced transgenerational failure of spermatogenesis in rats.Applying epigenetics,metabolomics,transcriptome,bioinformatics and molecular mechanism to spermatogenesis failure transgenerational effects causing by DBP.It can provide the theory basis for the risk assessment and safety evaluation,control the use of DBP,improve health quality and promote the reproductive health.Part ? Prenatal di-n-butyl phthalate exposure-induced Whole-genome methylation transgenerational failure of spermatogenesis in ratsObjectiveComparing the whole genome methylation between the offspring from DBP treated and control ancestor.Methods1.Applied of pregnant rat as an animal model to validate that DBP exposure can cause transgenerational spermatogenic failure.2.We used WGBS technology to screen and identificate aberrant DNA methylation gene in DBP exposure-induced offsping.3.We investigated the global DNA methylation level of DBP treated and control offersping using the 5-mC DNA ELISA.Results1.The body weight of F3 generation was significantly decreased.The levels of serum hormones were changed in the adult rats of different generations.The number of sperms and sertoli cells deceased in treated dams compared with its controls.2.Numerous genes in the genomic-wide was aberrant methylated in DBP exposuered offspring.Furthermore,more genes with aberrant hypomethylation than genes with hypermethylation in controls.3.DBP exposuered offspring have a lower global DNA methylation level than controls.4.Aberrant methylation gene mainly enrichment in metabolic pathways.ConclusionTaken together,our data for the first time reveals the prenantal DBP exposure could result in male offspring spermatogenic failure from F1 to F3.The whole genome methylation differences between DBP treated and control offspring,which may unfold the important mechanisms involved in this transgenerational effects.Part ? Metabolomics reveals a role of betaine in prenatal di-n-butyl phthalate exposure-induced epigenetic transgenerational failure of spermatogenesis in ratsObjectiveThrough the study of metabonomics and transcriptome,we intend to find the mechanisims of aberrant expression levels of metabolites between DBP treated and control offspring.Methods1.We established a metabonomics analysis platform using UPLC-MS/MS method.To determine the mechanisms of the transgenerational spermatogenic failure observed in offspring,we performed a non-targeted metabolomic analysis of F1 and F3 generation testes of control and DBP exposed animals.2.To gain molecular insight into the pathogenesis of DBP-induced transgenerational effect of spermatogenesis in rats,we examined the mRNA expression profile in testis of the F3 generation rats using RNA-seq technique.Find a key pathway associated with spermatogenesis.3.Methylation specific PCR(MSP)analysis showed that there were indeed distinct DNA methylation in testis at the Fstl3 promoter CpG islands.Results1.Results of the main analysis identified that betaine metabolism was one of the most important metabolism pathway that was altered in the experimental group.2.Based on RNA-seq,11 genes(Apob,Ar,Brca2,Cyp26b1,Foxa3,Fstl3,Lhcgr,Notch1,Piwil1,Sox9,and Wipf3)were identified to be affected in the spermatogenesis pathway.Our results further showed that Fstl3 expression levels were up-regulated in the rat of F1,F2,and F3 generations who exposed to DBP in FO gestation.3.The methylation pattern of Fstl3 promoter region were significantly hypomethylated in F1 generation DBP-treated rats.Meanwhile,the global meththylation was decreased because of the change of betaine metabolism.ConclusionUsing state-of-the-art metabolic and epigenetic approaches,our study investigated F1 and F3 testis metabolic and epigenetic patterns of the in utero DBP-treated rat dams.Several biologically important observations were made and novel biomarkers of DBP-induced spermatogenesis failure were identified.Increased testicular betaine levels,but reduced BHMT activity,accompanied by global and gene(Fstl3)-specific promotor hypomethylation likely explains the spermatogenesis failure in F1 through F3 generation offspring following exposure to DBP in FO gestation.These datas reveal novel metabolic and epigenetic mechanistic link for the in utero DBP exposure-induced transgenerational spermatogenic failure.Part ? Abnromal miRNA expression in prenatal di-n-butyl phthalate exposure-induced transgenerational failure of spermatogenesis in ratsObjectiveBy comparing testis miRNA expression profile between the F3 generation of DBP treated with controls,we intend to find the miRNA biomarkers associated with DBP exposure.Methods1.MiRNA expression:Using the miRNA microarray technology to detect the differences between F3 generation of DBP exposure testicular tissue and the control group.We chose to further study the miRNAs with 8-fold changes in the two groups;2.Abnormal expression of mirRNA on the influence of key metabolic enzyme gene:Using bioinformatics software TargetScan and PicTar to predict the target genes of miRNA.The dual luciferase reporter gene assay in vitro was conducted to confirm this miRNA binding with the target gene.Results1.Using miRNA expression profiling of 762 miRNAs,We identified 476 expressed miRNAs in F3 generations from control group testicular tissue and 463 in DBP treated offspring(Ct<40);.2.Multiple differentially expressed in testicular tissue more than 8 times:miR-206?miRNA-875?miRNA-199b?miRNA-297c?miRNA-450a?miRNA-499?miRNA-15a?let-7a?miRNA-833a?miRNA-200a?miRNA-423?miRNA-326?miRNA-542?miRNA-10b?miRNA-667.miR-206 and miR-875 are up-regulated,the others are down regulated;3.The most important miRNA is miR-206,which could combine to the 3'UTR of Ehhadh,decreasing the expression of the Ehhadh.ConclusionThe miRNA-206 as a small noncoding RNA can directly combine 3'UTR region,thus affecting metabolic enzyme gene Ehhadh,the metabolic enzyme gene disrupted the two spermatogenesis related metabolic pathways.Abnormal expression of miRNA,as a kind of detection of DBP exposure may extend the effect of a kind of symbol.