Fig. 4: Oocyte mitochondrial dysfunction causes pronuclear DNA modifications and reduces embryo and fetal telomere length.

Mice were exposed to rotenone (150 ppm in chow) for three weeks before ovulation, IVF and assessments of embryos and fetuses (a) Created in BioRender. Gordon, Y. (2025) https://BioRender.com/u42e503. Zygotes were labelled with MitoSox Red (red) and DNA stain Hoechst-3342 (blue) and fluorescence measured (b; n = 18 control, n = 16 rotenone). Levels of NAD(P)H (c) and FAD + + (d) in zygotes 6 h post-IVF; n = 30/group. Mitochondrial mass in zygotes was assessed using MitoTracker Green (e; n = 35 control, n = 39 rotenone). Zygotes (8 h post-IVF) were immuno-labeled with anti-8-oxodG (green) and fluorescence intensity in–– pronuclei quantified (f; n = 10 control, n = 12 rotenone). Zygotes were co-stained with anti-5-methylcytosine (5mC; magenta) and anti-5-hydroxymethylcytosine (5hmC; green) antibodies (g). Fluorescent signal intensity was quantified for 5mC (left) and 5hmC (right) in maternal and paternal pronuclei (n = 15 control, n = 12 rotenone) and presented as the ratio in paternal versus maternal pronuclei. Zygotes and 8-cell embryos were labelled with MMP indicator TMRM (red, and Hoechst-3342 (blue)) and red fluorescence measured (h; zygote: n = 20 control, n = 22 rotenone; 8-cell n = 17 control, n = 8 rotenone). Telomere length (telomere/ Rn18s (2^-ΔΔCt)) in individual MII oocytes, 8-cells, blastocysts (i; MII oocyte: n = 63 control, n = 66 rotenone; 8-cell: n = 86 control, n = 61 rotenone, blastocyst: n = 45 control, n = 69 rotenone), and ICMs (j; n = 42 control, n = 38 rotenone). Quantitative telomere FISH for telomere (green) with DAPI DNA stain (blue) in dissociated ICMs (k; left) and fluorescence per cell quantified (right; n = 88 nuclei from 33 ICMs derived from 3 mice per group). IVF-conceived blastocysts from rotenone-treated (or control) mice were transferred to surrogate females for gestation, and fetal tissues collected at day 18.5 of pregnancy for qPCR telomere length analysis of heart (l; n = 36 control, n = 35 rotenone fetuses). Individual data points are plotted, horizontal lines are mean ± SEM (b-h, k). Violin plots show population distribution, and horizontal lines are mean ± SEM (i, j, l). qPCR data was log transformed for statistical analysis. Statistical tests were two-sided unpaired t-test (b-g, j-l), one-way ANOVA (h) or two-sided unpaired t-test between the same developmental stage and one-way ANOVA between different developmental stages of the same group (i). For (h, i) different letters indicate p < 0.05. For (b-g, j-l) *p < 0.05, **p ≤ 0.009, ****p < 0.0001. For exact P values see Supplementary Table 4. Representative images shown, scale bar: 20 µm (b, f, g, h), 2 µm (k). Source data are provided as a Source Data File.