DNA Methylation Differences And Nucleotide Polymorphisms Of Genes Related To Testicular Development In Male Rattus Norvegicus

Rattus norvegicus is a worldwidely distributed species of rodent pests.Previously,we found inhibition of gonadal development during autumn and winter in Rattus norvegicus caraco,a subspeceies distributed in Harbin,rather than in Rattus norvegicus norvegicus,another subspeceies distributed in Zhanjiang.By genomic and sperm methylome sequencing,we detected differentially methylated regions?DMRs?near genes?Fshr,Gsk3?,Pitx2,Esr1,Ar,etc.?related to gonadal development.The coexisted transcription factor binding sites?TFBSs?related to gonadal development on both DMRs and promoter implied the potential regulatory functions of these DMRs and their possible relation with gonadal development.In this study,we characterized their molecular features in a large group of sperm samples?57 samples from R.n.caraco,and 41 from R.n.norvegicus?.The DNA polymorphisms of these DMRs have been tested using PCR sequeicng.Then,the regulatory activity of dominant haplotypes of each individual DMR has been tested using luciferase gene reporter system,and their methylation levels have been tested using bisulfite sequencing.The main findings are as follows.1.Chi-square testing for the allele frequency of haplotypes/genoptypes revealed that DNA sequences of each DMR were significantly divergent between two subspecies.2.The promoter activity of selected promoter sequence of each gene has been identified by double luciferase gene reporter system?P < 0.01?.The dominant haplotypes of two subspecies showed the significantly different regulatory activities,which may be related to gain/loss of TFBSs at SNP loci and their different methylation states.3.Independent t test showed the significant differences of sperm methylation levels between two subspecies?P < 0.01?in three DMRs of Pitx2,Gsk3? and Esr1?DMR₄8?rather than other three DMRs of Fshr,Ar and Esr1?DMR₄9?.The further analysis revealed methylation divergence between two subspecies when genetic background was the same,and methylation divergence among different haplotypes when environmental background was the same.These results indicate methylation changes depended on both DNA sequence and environmental conditions,and their effect on gene expression should be studied further.In conclusion,our results indicated the DNA sequence of DMR can act as a regulatory element in293 T cells,which may particapted in regulation of testis development by regulating the expression of target genes.The allele frequencies of haplotypes were significantly diverged between two subspecies and displayed the divergent regulatory acitivties,which may be related to gain/loss of TFBSs at SNP loci and their different methylation states.The methylation changes depended on both DNA sequence and environmental conditions,and their synergentic changes may be the determining factors of phenotypic difference of testis development between two subspecies.