Establishment Of A Mouse Model Of Paternal Reproductive Defects Exposed To Bisphenol A And Its Mechanisms

Background: It is generally accepted that about70%of birth defects are results of theinteraction between genetics and environmental factors. Maternal factors on embryonicdevelopment have been a major point of study on birth defects because embryodevelopment is particularly vulnerable to the external environment. However, paternaleffects on birth defects do not receive the attention it deserves. With the development ofsociety and economy, environmental pollution, unhealthy living habits and otherenvironmental factors make the human semen quality declined, which is likely to increasethe incidence of birth defects through the male germ line.Bisphenol A (BPA) is a kind of environmental endocrine disruptor, which humanexposure is universal. It was reported that BPA exposure has adverse effects on theneurobiology, immune system, endocrine system, and reproductive system. BPA exertsbiological effects on target organs through a variety of mechanisms. BPA can not onlybind to the classic estrogen receptor ERα and ERβ, but also bind to G protein-coupledmembrane estrogen receptor GPR30to response quickly via non-genomic pathways. Inaddition, BPA is also involved in epigenetic mechanisms.Most of animal experiments indicated that high doses of BPA exposure were harmfulto male reproductive system, especially exposure during organ formation anddevelopment stage. However, effects of low-doses of BPA on the male reproduction arestill a controversial issue. Effects of BPA on reproduction of male offspring remain nodefinite conclusions. BPA effects on sperm epigenetic modification such as DNAmethylation, histone methylation and its significance in the transgenerational effects, arescientific problems to be studied.Objective: To establish a mouse model of reproductive birth defects of F1generationthrough paternal abnormal sperms induced by BPA and establish the epigeneticcharacteristics of mouse sperm exposure to BPA. To reveal the possible epigenetic basisof abnormal sperm and descendant reproductive defects induced by BPA.Methods: We first established a mouse model of male mice exposure to BPA and generated the F1and F2descendants. The50mg/kg/day dose of BPA was intraperitonealinjected to3-weeks-old C57BL/6J male mice for7days. After5weeks, mice weresacrificed to obtain samples or to mate with normal female mice to generate the F1offspring; the F1male mice mated with normal female mice to generate the F2offspring.Sperm counts, sperm malformation and testis histology of the F0, F1and F2generationmale mice were examined. Mouse sperm DNA methylation profiles were established byhigh-throughput sequencing MeDIP DNA and bioinformatics analysis. Differential DNAmethylation of genes and some imprinting genes were selected for subsequentverification. Spermatogonial cells of F1mice were separated and further for mRNAmicroarray analysis and bioinformatics analysis. Dnmts mRNA and histone methylationwere evaluated by real-time quantitative PCR, Western blot, and immunohistochemistry.Low doses of5μg/kg/day,50μg/kg/day BPA were gavaged to3-weeks-oldC57BL/6J male mice for5weeks respectively. Then mated with normal female mice togenerate the F1offspring. After enrichment of methylated sperm DNA using MBDmethod, real-time PCR was applied to detect the levels of methylation of gene Tnnt2，Tectb between BPA group and the control group. Histone methylation levels of sperm inthe F1generation were also evaluated by Western blots.Effects of BPA on cell proliferation and epigenetic mechanisms of mousespermatogonial cell line C18-4were also evaluated.Results:1. High dose of BPA exposure has harmful effects on spermatogenesis of malemice and their offspring of the F1and F2generation. BPA can induce epididymal spermcount decreased by20.6%（P<0.01）, decreased by12.1%in the F1generation, decreasedby9.2%in the F2generation compared with the control group respectively. Exposure tohigh dose of BPA increased the sperm abnormality rate in the F1generation, but not inthe F1and F2generation. In addition, exposure to BPA can increase the sex ratio of maleto female of the F1generation.2. High-throughput sequencing of the methylated sperm DNA in the F0sperm werescreened out1,597differential genes, including1112hypomethylated genes, accountingfor69.6%;379hypermethylated genes, accounting for23.7%;106genes partly withhypermethylation and hypomethylation.Methylation rates of DMR at imprinting genes, including Gtl2, H19, Igf2r, Kcnq1, Gnas, Impact, Lit1, Nespas, Mest, Peg3, Peg10, Snrpn, have no difference compared withthe controls（P>0.05）. The methylation level of Magef1gene promoter was significantlyincreased in BPA group（P<0.01）; the methylation levels of Tnnt2gene in sperm of theF0and F1generation were both decreased, while the methylation levels of Magef1genewere both increased after BPA exposure.High dose of BPA exposure reduced the global DNA methylation levels in testis andsperm, and increased the levels of H3K9Me3in testis.3. A total of2091differentially expressed genes were obtained after mRNA microarrayanalysis of the mouse spermatogonia in the F1mice, in which the1546up-regulatedgenes and the545down-regulated genes were induced by BPA. A total of101genes hadthe negative correlation between sperm DNA methylation and mRNA expression ofspermatogonia.4. Low dose of50μg/kg/day BPA induced the sperm number decreased by20.1%（P<0.01）. Low doses of BPA had no noticeable impact on organs index such as testis,epididymis, seminal vesicle and prostate.50μg/kg/day BPA can induce the changes ofmethylation levels of Tnnt2, Tectb genes in the testis and sperm But low dose of50μg/kg/day BPA exposure can result in the reduction of the level of H3K9me3, theincrease of H3K27Me3, H3K36Me3levels in the F1sperm.5.10-9M-10-6M doses of BPA exposure can promote cell proliferation of mousespermatogonial cell line C18-4, while10-5M of BPA significantly inhibit cell growth.10-5M BPA significantly increased the apoptosis rate of C18-4cells（P<0.01）. The globalgenomic DNA methylation level, the levels of H3K27Me3, H3K36Me3weresignificantly reduced by50%exposed to10-5M BPA（P<0.01）.Conclusions:(1) High dose of BPA exposure to pubertal male mice can adversely affectthe spermatogenesis of adult mice and the subsequent F1mice. High dose of BPAexposure can decrease the global DNA methylation levels of the F0testis and sperm, andalso increase the levels of H3K9Me3in the testis. BPA induced the methylation level ofMagef1gene significantly increased. The levels of Tnnt2gene methylation in the spermof F0and F1mice were decreased and the methylation levels of Magef1gene wereincreased by BPA exposure.(2)50μg/kg/day of low dose of BPA has an adverse effect onspermatogenesis in mice, and induced changes in histone methylation levels of F1sperm. (3) BPA exposure on cell proliferation of mouse spermatogonial cell line C18-4had non-monotonic dose response effects.10-5M of BPA can disturb epigenome and histone methylation of the spermatogonial cell.