The Role Of Histone H3 Methylation In Cadmium-induced Injury Of Male Reproduction

Part 1 Cadmium exposure alters H3 methylation modification in mouse males[Purpose] To explores the reproductive toxicity and histone H3 methylation modification of adult C57 mice after 5-week exposure to cadmium at different doses.[Methods] Adult male C57 mice were randomly divided into 5 groups.The influence of Cd on the body weight,testes,and sperm quality of male C57BL/6J mice were examined by intraperitoneally injecting Cd Cl2 to mice with different concentrations of 0,0.25,0.5,1.0,and 2.0 mg/kg of body weight for 35 days.Sperm quality,testicular histomorphology,testicular enzyme activity and the expression of histone proteins H3K4me3,H3K9me3,H3K27me3 and H3K36me3 were assessed in different groups.[Results] Exposure to cadmium for 5 consecutive weeks leads to continuous accumulation of cadmium in the testes,affecting body weight and reducing testicular enzyme activity.Then,the morphology of the testis tissue is destroyed,resulting in a significant decrease in sperm count and vitality.The expression levels of histones H3K4me3,H3K9me3 and H3K27me3 enhanced with the increase of cadmium exposure dose.[Conclusions] Cadmium exposure induces male reproductive injury by affecting growth and development,reducing the activity of testicular cells and germ cells,and reducing the quality of semen.During this process,the expression levels of histones H3K4me3,H3K9me3 and H3K27me3 in testicular cells were increased.Histone H3 methylation modification may play an important role in cadmium-induced damage to male reproductive function.Part 2 The regulation of histone H3 methylation modification on spermatogenic cell line transcriptome[Purpose] To investigate specific role of certain histone H3 lysine methylation in mouse germ cell line GC-2.[Methods] we ectopically expressed H3.3 K4 M,K9M,K27 M,K36M,K79 M mutants in GC-2 cells respectively,and analyzed changes of transcriptome including expression of endogenous retroviruses.[Results] Almost all mutants significantly affected the global transcriptional program in GC-2 cells.PCA show that the expression of H3.3 K4 M,K9M and K36 M,but not H3.3 K27 M,significantly changed cell identity.MA plot show that overexpression of H3.3 K4 M led to downregulation of 252 genes and upregulation of 107 genes,implying that H3K4 methylation is mainly involved in gene activation.In contrast,more genes were upregulated in H3.3 K9M(164 vs 43 genes),K27M(185 vs 46 genes),and K36M(275 vs 44 genes)-overexpressed cells,showing that methylation of these lysine residues mainly participates in gene silencing.Notably,Acta2 and Krt16 were misregulated in all mutant groups.The weighted gene co-expression network analysis(WGCNA)was performed to explore the co-expression patterns among genes across cells expressing histone H3.3 K-to-M mutants and found that found that a significant number of genes,such as Foxo1,Tspyl1,Stag3,Stx2,Bmi1,Tdrp,Cdh2,and Eno4,play crucial roles in reproduction,particularly male reproduction.Moreover,the H3.3 K-to-M transgenes,especially H3.3 K36 M,impacted the expression of endogenous retrovirus ERVK.[Conclusions] The decreased methylation level of H3K4/9/27/36 significantly affected the transcriptome of GC-2 cells.Methylation at different sites had different effects on the gene subsets of GC-2 cells.Histone H3 methylation modification can regulate transcriptome of spermatogenic cell line in mice.