Fig. 4: Nicotine exposure-induced modifications in the testicular microenvironment are partially reversible.

A The classification of all metabolites with quantitative information. B Principal component analysis (PCA) plot of the three mice groups (CON, NIC, and CES) based on targeted metabolomic data (n = 4 mice per group). Volcano plots exhibiting differential metabolites in NIC (C) and CES (D) mice compared to CON mice. Metabolites with a fold change (FC) >1.5 and a p-value < 0.05 were considered differential. E Heatmap of all differentially expressed metabolites (DEMs) across the three groups (NIC and CES mice compared to CON mice). F KEGG pathway analysis of the increased metabolites in NIC mice relative to CON mice. Quantification of ATP levels (G, n = 8 mice per group) and ADP-to-ATP ratio (H, n = 4 mice per group) in the testicular samples from the mice three groups. Immunofluorescence images (I) and statistical analysis (J) of testicular samples stained with DHE probe and DAPI across the three groups (n = 6 mice per group). Scale bar = 100 μm. Immunohistochemistry images (K) and statistical analysis (L) of mice testes stained with hypoxia-inducible factor 1 alpha (HIF-1α) across the three groups (n = 6 mice per group). Scale bar = 200 μm. The red arrows indicate hypoxic somatic and germ cells. Data are represented as mean ± SEM. All statistical analyses were performed using ANOVA followed by Bonferroni’s multiple comparisons test, ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; ns not significant.