Mechanistic Study on METTL3-Mediated m6A Modification of PDE4D in Zearalenone-Induced Testicular Injury in Mice
Zearalenone (ZEA) is a non-steroidal estrogenic mycotoxin that causes marked testicular injury and male reproductive toxicity, but the involvement of RNA N6-methyladenosine (m6A) modification in this process remains unclear. In this study, we used Balb/c mice and mouse Sertoli TM4 cells to investigate whether ZEA-induced testicular damage is mediated by m6A dysregulation. Global m6A levels and the expression of m6A regulators were first examined, revealing that ZEA exposure significantly decreased overall m6A methylation and downregulated the methyltransferase METTL3 in mouse testes and TM4 cells. We then performed MeRIP-seq (m6A-seq) combined with RNA-seq on testes from control and ZEA-treated mice to profile ZEA-induced changes in the m6A epitranscriptome and transcriptome. Integrated analysis identified 615 differential m6A peaks and 529 differentially expressed genes, with 76 genes showing both altered m6A modification and mRNA expression. KEGG pathway enrichment indicated prominent involvement of the cAMP signaling pathway, tight junctions and inflammatory signaling. Among these candidates, phosphodiesterase 4D (PDE4D) emerged as a key m6A-regulated target. ZEA increased PDE4D expression while reducing its m6A level, whereas METTL3 overexpression enhanced m6A modification on PDE4D, promoted PDE4D mRNA decay, restored cAMP content and reactivated the cAMP–PKA–CREB anti-inflammatory pathway. Functionally, METTL3 overexpression or PDE4D knockdown alleviated ZEA-induced inflammatory cytokine upregulation, disruption of blood-testis barrier–related junction proteins and TM4 cell proliferation arrest. These data demonstrate that ZEA impairs testicular function at least in part by suppressing METTL3-mediated m6A modification of PDE4D and inhibiting the cAMP–PKA–CREB pathway, providing an epitranscriptomic mechanism for ZEA-induced male reproductive toxicity. Overall design: This submission includes total testicular RNA samples from male Balb/c mice assigned to vehicle control and zearalenone (ZEA)–exposed groups (n = 3 biological replicates per group). Testes were collected from individual mice after in vivo exposure, and RNA from each mouse was used to generate both m6A-enriched (MeRIP-seq) and matched input (RNA-seq) libraries. Vehicle-treated samples serve as reference controls for ZEA-treated samples to assess the effects of ZEA exposure on testicular m6A methylation and gene expression.