Heat stress enhances the expression of METTL3 to mediate N6-methyladenosine modification of SOS2 and NLRP3 inflammasome activation in boar Sertoli cells

Heat stress negatively affects pig production by disrupting the immune homeostasis of Sertoli cells (SCs), which compromises sperm quality, culminating in male infertility. Herein, we aimed to study the mechanism by which the NLRP3 inflammasome is activated by heat stress through N6-methyladenosine (m⁶A) modification regulation in SCs. Initially, it was found that heat stress (44°C, 30 min) markedly activated ERK1/2 signaling, which subsequently promoted NLRP3 inflammasome activation and inflammatory cytokine release from SCs. Then, using an m⁶A dot-blot assay, m⁶A Sequencing, and methylated RNA immunoprecipitation, we found that heat stress augmented the level of m⁶A modification in SCs, and METTL3 augmented the m⁶A modification of mRNA encoding SOS Ras/Rho guanine nucleotide exchange factor 2 (SOS2), a key activator of the ERK pathway. Furthermore, YTHDF1 recognized and bound to the m⁶A-modified SOS2 mRNA to enhance its translation efficiency, ultimately triggering ERK1/2 signaling activation. In vivo experiments demonstrated that heat stress-induced decline in semen quality in mice was associated with elevated levels of m⁶A modifications in the testis and NLRP3 inflammasome activation. However, the damage caused by heat stress could be attenuated by intraperitoneal injection of S-Adenosylhomocysteine (SAH), a specific methyltransferase inhibitor. Our results emphasize the critical roles of m⁶A in regulating NLRP3 inflammasome activation under heat stress, identifying a novel therapeutic avenue to address heat stress.

ERK signaling; Heat stress; N6-methylation; NLRP3 inflammasome.