Single-nucleus RNA Sequencing reveals the mechanism of cigarette smoke exposure on diminished ovarian reserve in mice

The systematic toxicological mechanism of cigarette smoke (CS) on ovarian reserve has not been extensively investigated. Female 8-week-old C57BL/6 mice at peak fertility were exposed to CS or indoor air only for 30 days (100 mice per group) and the effects of CS on ovarian reserve were assessed using Single-Nucleus RNA Sequencing (snRNA-seq). In addition, further biochemical experiments, including immunohistochemical staining, ELISA, immunofluorescence staining, transmission electron microscopy, cell counting kit-8 assay, flow cytometry analysis, senescence-associated beta-galactosidase staining, and western blotting, were accomplished to confirm the snRNA-seq results. We identified nine main cell types in adult ovaries and the cell-type-specific differentially expressed genes (DEGs) induced by CS exposure. Western blot results verified that downregulation of antioxidant genes (Gpx1 and Wnt10b) and the steroid biosynthesis gene (Fdx1) occurred in both ovarian tissue and human granulosa cell-like tumor cell line (KGN cells) after CS exposure. Five percent cigarette smoke extract (CSE) effectively stimulated the production of reactive oxygen species (ROS), DNA damage, cellular senescence and markedly inhibited KGN cell proliferation by inducing G1-phase cell cycle arrest. Moreover, down-regulation of Gja1, Lama1 and the Ferroptosis indicator (Gpx4) in granulosa cells plays a significant role in ultrastructural changes in the ovary induced by CS exposure. These observations suggest that CS exposure impaired ovarian follicle reserve might be caused by REDOX imbalance in granulosa cells. The current study systematically determined the damage caused by CS in mouse ovaries and provides a theoretical basis for early clinical prediction, diagnosis and intervention of CS exposure-associated primary ovarian insufficiency (POI), and is of great significance in improving female reproductive health.