ChronicDietary Exposure to Environmental Levels ofGlyphosate Increases the Risk of Reproductive Dysfunction in MaleMice

Extensive use of glyphosate (GLY) has aroused significant public concerns due to its adverse effects on reproductive health, but the toxic mechanism remains unclear. This study was designed to elucidate the effects of GLY on male reproductive health by <i>in vivo</i> and <i>in vitro</i> studies. Data showed that chronic environmental levels of GLY reduced sperm quality and spermatogenic cell number in mice. Transcriptomic and metabolomic analyses revealed that GLY downregulated key glycolytic rate-limiting enzymes HK2 and PFK1, and decreased lactate abundance, while a strong positive correlation between sperm quality and lactate level was established. Further assays showed that GLY inhibited the expression of four glycolytic key proteins and disrupted the HK2 mitochondrial localization. Molecular docking and immunoprecipitation assays confirmed that HK2 binds to VDAC1 on the mitochondrial outer membrane, which was inhibited by GLY. This inhibition was significantly alleviated by VBIT-4, an inhibitor of VDAC1 oligomerization. Notably, oxidative stress-mediated VDAC1 oligomerization due to excessive mitochondrial fission was identified as a key mechanism by which GLY inhibits glycolysis in Sertoli cells. In summary, this study uncovers a novel metabolic regulatory mechanism by which GLY impairs spermatogenesis via the VDAC1-HK2-glycolysis axis, providing critical insights for preventing GLY-induced reproductive toxicity and related environmental risks.