Disruption of steroidogenesis and calcium signaling drives VX-induced reproductive toxicity in zebrafish: A spatial metabolomic and transcriptomic study.

VX is recognized as a highly toxic nerve agent, yet its reproductive toxicity and the underlying mechanisms remain poorly understood. In this study, male zebrafish were exposed to VX at concentrations of 19.36, 77.43, and 154.86 μg/L for 4 days to systematically evaluate its reproductive toxicity. The results showed that VX at concentrations above 19.36 μg/L completely inhibited spawning and significantly reduced the survival and hatching rates of fertilized eggs. Histopathological examination revealed testicular damage, including loss of the seminiferous epithelium, with apoptosis increasing in a dose-dependent manner. Transmission electron microscopy further demonstrated disrupted testicular cellular structures and extensive cell membrane damage in the exposed groups. Biochemical analyses indicated that VX significantly decreased testicular SOD and CAT activities as well as testosterone (T) levels, while increasing estradiol (E2) content. DESI-MSI visually confirmed the dose-dependent accumulation and spatial distribution of VX in the testis. Integrated spatial metabolomics and transcriptomics analyses revealed that VX disrupts steroid hormone biosynthesis and calcium signaling pathways, leading to imbalanced sex hormone secretion, dysregulated calcium homeostasis, and oxidative stress. These disruptions ultimately cause spermatogenic cell damage, impaired spermatogenesis, and reduced reproductive capacity. By applying an innovative strategy integrating DESI-MSI-based spatial metabolomics with transcriptomics, this study provides the first comprehensive evidence for VX-induced reproductive toxicity in zebrafish at both the spatial and molecular levels, providing novel insights for the ecological risk assessment of this agent.