Fig. 1.

Changes in the diameter of cultured secondary follicles. (A–H) The images of cultured follicles. A and B: a high-growth follicle. C and D: low-growth follicle. E and F: no-growth follicle. G and H: reduced-growth follicle. Panels A, C, E and G are images from culture day 0, and panels B, D, F and H are images of culture day 3. The follicle diameters are as follows: A, 116 μm; B, 135 μm; C, 110 μm; D, 115 μm; E, 110 μm; F, 110 μm; G, 111 μm; and H, 106 μm. (H) On culture day 3, differences in follicle size appeared, and we separated follicles into four groups dependent on the rate of diameter increase (high-growth, low-growth, no-growth and reduced-growth groups). There were significant differences among each group except between the no-growth and reduced-growth groups (P < 0.05). (J–M) The images of follicles cultured for 4 days and stained with YO-PRO (green) and Hoechst 33258 (blue). K and M are the bright-field images of J and L, respectively. J and K are reduced-growth follicles. L and M are high-growth follicles. (N) Granulosa cell proliferation of each group was measured by an EdU labelling kit. Statistical significance is indicated by an asterisk (**P < 0.01, ***P < 0.001). Values are presented as means ± standard deviation. The number of samples per experiment is indicated under the group names. Scale bars show 100 μm.