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.

Alternative Splicing during Fiber Development in G. hirsutum.

Cotton is a valuable cash crop in many countries. Cotton fiber is a trichome that develops from a single epidermal cell and serves as an excellent model for understanding cell differentiation and other life processes. Alternative splicing (AS) of genes is a common post-transcriptional regulatory process in plants that is essential for plant growth and development. The process of AS during cotton fiber formation, on the other hand, is mainly unknown. A substantial number of multi-exon genes were discovered to be alternatively spliced during cotton fiber formation in this study, accounting for 23.31% of the total number of genes in Gossypium hirsutum. Retention intron (RI) is not necessarily the most common AS type, indicating that AS genes and processes during fiber development are very temporal and tissue-specific. When compared to fiber samples, AS is more prevalent at the fiber initiation stages and in the ovule, indicating that development stages and tissues use different AS strategies. Genes involved in fiber development have gone through stage-specific AS, demonstrating that AS regulates cotton fiber development. Furthermore, AS can be regulated by trans-regulation elements such as splicing factor and cis-regulation elements such as gene length, exon numbers, and GC content, particularly at exon-intron junction sites. Our findings also suggest that increased DNA methylation may aid in the efficiency of AS, and that gene body methylation is key in AS control. Finally, our research will provide useful information about the roles of AS during the cotton fiber development process.

Arachidonic acid impairs the function of the blood-testis barrier via triggering mitochondrial complex-ROS-P38 MAPK axis in hyperthermal Sertoli cells.

The death of Sertoli cells (SCs) under condition of heat stress (HS) affects spermatogenesis and is associated with impaired function of the blood-testis barrier (BTB). The fatty acid arachidonic acid (AA) is essential for the maintenance of cellular function. However, excessive release of AA during HS may adversely affect the reproductive function. The molecular mechanisms through which AA modulates the BTB in SCs are unclear. In this study, we found that 100 µM AA damaged testicular morphology and accelerated SC apoptosis during HS, reducing the stability of tight junction proteins (TJPs), shown by measurement of the levels of Claudin 11, 5, Occludin, and trans-epithelial electrical resistance (TEER). It was also found that AA adversely affected TJPs by increasing the accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA), activating p38 mitogen-activated protein kinases (P38 MAPK) and reducing mitochondria DNA (mtDNA) and the expression of mitochondrial complexes I and III. In contrast, pretreatment with SB203508 (a P38 MAPK inhibitor), Rotenone (an inhibitor of complex I) and Antimycin A1 (an inhibitor of complex III) reversed TJPs degradation induced by AA. Interestingly, pretreatment of cells with 10 µM Baicalein, a 12/15 lipoxygenase (12/15-LOX) -dependent inhibitor of AA production, protected against AA-induced TJPs degradation, restored mitochondrial function, and reduced apoptosis. These results suggested an intriguing link between the induction of TJPs degradation induced by AA overload and mitochondrial antioxidant function during HS, which was found to be regulated by the mitochondrial complex-ROS-P38 MAPK axis.