The study found that breathing fine air pollution (PM2.5) harms male mouse sperm cells and messes up their mitochondria, the cell's power plants. As the mitochondria get damaged, the body makes more of a tiny protein called humanin, which seems to act as a stress signal. The pollution also triggers a chain reaction that creates more harmful reactive oxygen species and turns on the aryl hydrocarbon receptor pathway, further hurting energy metabolism.

Fine particulate matter (PM_2.5)-induced male reproductive toxicity arouses global public health concerns. However, the mechanisms of toxicity remain unclear. This study aimed to further investigate toxicity pathways by exposure to PM_2.5<i>in vitro</i> and <i>in vivo</i> through the application of metabolomics and transcriptomics. <i>In vitro</i>, spermatocyte-derived GC-2spd cells were treated with 0, 25, 50, 100 &#x3bc;g/mL PM_2.5 for 48 h. <i>In vivo</i>, the real-world exposure of PM_2.5 for mouse was established. Forty-five male C57BL/6 mice were exposed to filtered air, unfiltered air, and concentrated ambient PM_2.5 in Tangshan of China for 8 weeks, respectively. The results <i>in vitro</i> and <i>in vivo</i> showed that PM_2.5 exposure inhibited GC-2spd cell proliferation and reduced sperm motility. Mitochondrial damage was observed after PM_2.5 treatment. Increased Humanin and MOTS-c levels and decreased mitochondrial respiratory indicated that mitochondrial function was disturbed. Furthermore, nontargeted metabolomics analysis revealed that PM_2.5 exposure could disturb the citrate cycle (TCA cycle) and reduce amino acids and nucleotide synthesis. Mechanically, the aryl hydrocarbon receptor (AhR) pathway was activated after exposure to PM_2.5, with a significant increase in CYP1A1 expression. Further studies showed that PM_2.5 exposure significantly increased both intracellular and mitochondrial reactive oxygen species (ROS) and activated NRF2 antioxidative pathway. With the RNA-sequencing technique, the differentially expressed genes induced by PM_2.5 exposure were mainly enriched in the metabolism of xenobiotics by the cytochrome P450 pathway, of which <i>Cyp1a1</i> was the most significantly changed gene. Our findings demonstrated that PM_2.5 exposure could induce spermatocyte damage and energy metabolism disorder. The activation of the aryl hydrocarbon receptor might be involved in the mechanism of male reproductive toxicity.