Fig. 1: Age-associated decline of ovarian blood vessel starts since middle aged.
a Schematic representation illustrating the methodology employed for achieving high-resolution 3D imaging of ovarian blood vessels. b Images of 3D ovarian blood vessels in adult Tek-CreERT2;mTmG mice, showing the reconstructed images are flexible to analyze the ovarian blood vessels from whole organ level to the subcellular level. Whole-mount, single-slice (white frame), Follicle (blue frame), Tip cells (yellow frame, arrowheads) (c) High-resolution 3D images of ovarian blood vessels at different ages. Showing a notable decrease of blood vessel density (left) and a reduction in the number of tip cells (right, arrows) with reproductive aging. d Quantifying the number of tip cells showed a notable decline of angiogenesis with reproductive aging (4 M: n = 7 mice, 8 M: n = 6 mice, 12 M: n = 7 mice). e Quantifying the density of blood vessels revealed a substantial decrease with reproductive aging (4 M: n = 3 mice, 8 M: n = 4 mice, 12 M: n = 5 mice). f Detecting the age-associated blood vessel changes in ovary, heart, liver, lung, and kidney, showing vascular density at 4 months, 8 months, and 12 months (green: PODXL). g Quantification of relative vascular density in organs indicating no significant changes in the heart, liver, lung, and kidney, but a marked decline in ovaries since middle age (Ovary: 4 M: n = 3 mice, 8 M: n = 4 mice, 12 M: n = 4 mice, Heart: n = 4 mice, Liver: n = 4 mice, Lung: n = 4 mice, Kidney: n = 4 mice). h Illustration of the methodology employed to assess blood vessel permeability following Evans blue injection (upper) and the distribution of Evans blue (arrowheads) in ears (middle) and ovaries (bottom) subsequent to tail vein injection at 4, 8, and 12 months. i Quantification of Evans blue signal demonstrating a significant reduction in vascular permeability in ovaries with reproductive aging (n = 3 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, * P < 0.05, n.s. (not significant) P ≥ 0.05. Scale bars: 10 μm (c), 50 μm (f), 500  μm (h). Source data are provided as a Source Data file including exact P values.
