Fig. 2: Ovarian blood vessel decline decreases the follicle developmental potential in the middle-aged ovaries.

a Age-related changes in tip cells (arrows) on growing follicles during reproductive aging. b Quantification of the tip cells on growing follicles at different ages, showing a significantly reduced angiogenesis with age in the ovaries (4 M: n = 7 mice, 8 M: n = 10 mice, 12 M: n = 7 mice). c Live imaging tracking the growth of blood vessels on follicles at 4, 8, and 12 months in vitro culture. d Statistical analysis of the relative growth rate of follicle blood vessels over 24 h in vitro culture, showing a significant decrease at 8 and 12 months compared to 4 months (4 M: n = 8 follicles from 3 mice, 8 M: n = 11 follicles from 3mice, 12 M: n = 10 follicles from 3 mice). e Relative growth rate of follicle diameter over 24 h in vitro culture, demonstrating consistent growth ability of follicles across different ages (4 M: n = 8 follicles from 3 mice, 8 M: n = 11 follicles from 3 mice, 12 M: n = 10 follicles from 3 mice). f Evans blue signal (left) and quantification of EB intensity (right) in the follicles at different ages, revealing a significant decrease in dye penetration in aging follicles (4 M: n = 21 follicles from 3 mice, 8 M: n = 9 follicles from 3 mice, 12 M: n = 15 follicles from 3 mice). g BrdU staining detection of proliferative granulosa cells in antral follicles at different ages (red: BrdU, blue: Hoechst). h Quantification of BrdU⁺ GCs indicating a significant decreased GC proliferation in antral follicles with age (n = 8 follicles from 3 mice). i BrdU staining detection of proliferative GCs after 24 h of PMSG treatment (red: BrdU, blue: Hoechst) at different ages (left). j Quantification of BrdU⁺ GCs showing significantly decreased responses of PMSG in follicle development at 8 months and 12 months compared to that at 4 months (n = 4 mice). Data are presented as the means ± SD. Data were analyzed by 2-tailed unpaired Student’s t-test without adjustments; *** P < 0.001, ** P < 0.01, n.s. P ≥ 0.05. Scale bars: 20 μm (a), 100 μm (c and f), 50 μm (g and i). Source data are provided as a Source Data file including exact P values.