Joint Analysis of Genome-wide DNA Methylation and Transcription Sequencing Identifies the Role of BAX Gene in Heat Stress–Induced-Sertoli Cells Apoptosis

The problem of male infertility is a global health crisis and poses a serious threat to the well-being of families. Under heat stress (HS), the reduction of Sertoli cells (SCs) inhibits energy transport and nutrient supply to germ cells, leading to spermatogenesis failure. DNA methylation of genes is a central epigenetic regulatory mechanism in mammalian reproduction. However, it remains unclear how DNA methylation regulates gene expression in heat-stressed SCs. In this study, we investigated whether the decrease in SC levels during HS could be related to epigenetic DNA modifications. The cells exposed to HS showed changes in differential methylation cytosines and regions (DMCs/DMRs) and differential expression genes (DEGs), but not in global DNA methylations. One of the most important biological processes affected by HS is cell apoptosis induced by the intrinsic apoptotic signaling pathway (GO: 2,001,244, P < 0.05) by enrichment in the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). The joint analysis showed that several gene expressions in RNA-seq and WGBS overlapped and the shortlisted genes BAX, HSPH1, HSF1B, and BAG were strongly correlated with stress response and apoptosis. Methylation-specific PCR (MSP) and flow cytometry (FCM) analyzes showed that reduced promoter methylation and enhanced gene expression of BAX with a consequence of apoptosis. The activity of BAX, as well as an increase in its expression, is likely to result in a reduction of SCs population which could further impair ATP supply and adversely affect membrane integrity. These findings provide novel insights into the molecular mechanisms through which stressors cause male reproductive dysfunction and a new molecular etiology of male infertility.