Fig. 3: Nicotine exposure inhibits spermatogenesis and increases apoptosis in the testis.

A Hoechst fluorescence-based gating on individual spermatogenic populations. Before isolating spermatogenic cells, the main cell population was selected, and adherent or inactive cells were excluded. The proportions of meiosis II spermatocytes (B) and round spermatids (C) in CON and NIC mice (n = 7 mice per group). Detection of gene expression levels of DEGs using qPCR (n = 5 mice per group) in meiosis II spermatocytes (D) and round spermatids (E). F KEGG pathway analyses of upregulated DEGs in eSTid_2 and eSTid_4. Immunofluorescence images (G) and statistical analysis (H) of testicular samples stained with cleaved Caspase-3 and DAPI across the three groups (n = 8 mice per group). Scale bar = 100 μm. I Immunofluorescence images of testicular samples stained with TUNEL, PNA, and DAPI. The white arrows indicate different types of germ cells. Scale bar = 50 μm. Data are represented as mean ± SEM. All statistical analyses were performed employing a two-tailed unpaired t-test or ANOVA followed by Bonferroni’s multiple comparisons test, ***p < 0.001; **p < 0.01; *p < 0.05; ns not significant.