The m6A modification mediates the imbalance of mitochondrial homeostasis and apoptosis induced by Benzo[b]fluoranthene in mouse spermatocytes: mitophagy versus mitochondrial damage.

INTRODUCTION Mitochondrial homeostasis is intimately associated with the toxic effects of exogenous chemicals, as well as the onset and progression of various diseases. Benzo[b]fluoranthene (BbF) is ubiquitously distributed across various environmental media. The association between BbF exposure and male reproductive damage has been recently discovered. However, the relevant mechanisms remain unexplored. OBJECTIVES To investigate the male reproductive toxicity and mechanisms of BbF. METHODS We established BbF exposure models in both mice and GC-2 mouse spermatocyte cell lines to investigate BbF-associated reproductive toxicity. By integrating transcriptomic sequencing, bioinformatics analysis, and experimental validation, we demonstrated that disruption of mitochondrial homeostasis was the central mechanism. Furthermore, through N6-methyladenosine (m6A) sequencing combined with functional validation, we revealed the regulatory role of m6A modification in modulating mitochondrial homeostasis. RESULTS BbF exposure triggered mitochondrial homeostasis disruption in spermatocytes and contributed to male reproductive toxicity. Specifically, BbF impaired mitochondrial biogenesis and oxidative phosphorylation via the p53/PGC-1α/TFAM signaling pathway. As a compensatory response, PINK1/Parkin-associated mitophagy was activated, thereby partially alleviating cellular damage. To further investigate the regulatory mechanisms of m6A modification in the aforementioned mitochondrial dysfunction process, we observed that BbF exposure resulted in reduced expression of YTHDF2 in spermatocytes. The downregulation of YTHDF2 slowed the degradation of Trp53 mRNA, leading to elevated levels of both Trp53 mRNA and p53 protein, thereby inducing the p53/PGC-1α/TFAM-mediated mitochondrial damage. On the other hand, we demonstrated that METTL3 exerted a crucial protective effect by regulating mitophagy. Particularly, METTL3 enhanced the m6A modification of Mark4 mRNA, thereby stabilizing the transcript, increasing both mRNA and protein levels of MARK4. Furthermore, the METTL3/MARK4 signaling axis promoted PINK1/Parkin-associated mitophagy, which served as a protective mechanism. CONCLUSION As a result of the confrontation between YTHDF2/p53/PGC-1α/TFAM-mediated mitochondrial damage and PINK1/Parkin-associated mitophagy regulated by METTL3/MARK4, BbF disrupted mitochondrial homeostasis, ultimately leading to mitochondrial dysfunction and spermatocyte apoptosis.