Fig. 4: Vascular senescence leads to the blood vessel decline in middle-aged ovaries.

a In situ hybridization results showing the expressions of Vegfa in ovaries at 4 months and 12 months, highlighting the granulosa cell (GCs) as the primary cells secreting VEGFA. b Relative Vegfa expression levels in GCs demonstrating increased Vegfa expressions with age (n = 3 mice). c Western blot detections revealing a significant increase in VEGFA expression in ovaries with age from 4 months to 12 months (n = 3 mice). d UMAP plot illustrating that young (2 months, red plots) and middle-aged (10 months, blue plots) ovarian vascular endothelial cells (oVEs) share similar cell populations, including capillary (C0), artery (C1), vein (C2), and Ada+ endothelium(C4), with proliferating endothelium (C3) only present in young oVEs. e UMAP plot (left) displaying the cellular atlas of young (green frame) and middle-aged oVEs (blue frame), emphasizing changes in cell type proportions between the two age groups (right). f Immunostaining results (left) showing the ratio of PODXL⁺BrdU⁺ endothelium in follicles (upper, arrows) and livers (bottom, arrowheads) (green: PODXL, red: BrdU). Quantification of the PODXL⁺BrdU⁺ endothelium ratio (right) revealing a significant decrease in oVEs with reproductive aging (4 M: n = 5 mice, 8 M: n = 3 mice, 12 M: n = 5 mice). g Relative telomere length detection indicating a significant decrease in oVEs during reproductive aging (n = 3 mice). h Gene Ontology analysis of differential genes in capillaries (upper), arteries (middle) and veins (bottom) of young and middle-aged oVEs. i Violin plot displaying angiogenesis-related genes including End1 and Tspo significantly decreasing and Flt1 significantly increasing in middle-aged oVEs, indicating reduced angiogenesis with reproductive aging (upper); oxidative stress-related genes including Gpx1, Gsr and Prdx5 significantly decreasing in middle-aged oVEs, suggesting increased oxidative stress with reproductive aging (bottom). Data shown as median, interquartile range (IQR), and 1.5x IQR (Young: n = 1992 cells, Middle-age: n = 488 cells). Data are presented as the means ± SD. Data were analyzed by 2-tailed unpaired Student’s t-test without adjustments (b, c, f, g) and Wilcoxon rank sum test with adjustments (i); *** P < 0.001, ** P < 0.01, * P < 0.05. Scale bars: 200 μm (a), 25 μm (f). Source data are provided as a Source Data file including exact P values.