Fig. 5 Reconstitution of the dormant state using fetal ovaries.

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Fig. 5 Reconstitution of the dormant state using fetal ovaries. Reconstitution of the dormant state using fetal ovaries. (A) Culture of E12.5 gonads under exogenous pressure. Time courses of the culture experiments are shown (top). Images show the results of immunofluorescence analysis of E12.5 ovaries at 21 days of culture with or without exogenous pressure (33.3 kPa). Images show the results of immunostaining of MVH (green) and FOXO3 (red). Nuclei were stained with DAPI (blue). Dashed lines indicate the region of small oocytes characteristically observed under exogenous pressure. The results of immunofluorescence analysis using a frozen section from E12.5 ovaries cultured for 21 days with exogenous pressure (33.3 kPa) are shown at the bottom of the figure. Scale bars, 50 μm. The graph shows the percentage of oocytes with the size indicated under each culture condition. Data representing the means ± SD of three independent experiments are shown. Statistical significance was determined using Tukey’s multiple comparison test. *P < 0.05, **P < 0.01. (B) Culture of E12.5 gonads with LY294002. Time courses of the culture experiments are shown (top). Images show E12.5 ovaries at 16 days of culture without (Control) or with Ly294002 (LY). BF, bright field; SC, stella-CFP. Scale bars, 50 μm. The graph shows the percentage of the oocytes with the size indicated under each culture condition. Data representing the means ± SD of three independent experiments are shown. Statistical significance was determined using Tukey’s multiple comparison test. *P < 0.05, **P < 0.01. (C) Effect of each inhibitor on the dormant state of oocytes. Time courses of the culture experiments are shown (top). The percentages of small (<20 μm) MVH-positive oocytes with nuclear FOXO3 are shown. Data representing the means ± SD of three independent experiments are shown. Statistical significance was determined using Tukey’s multiple comparison test. Comparisons were performed with LY294002. **P < 0.01. P, pressure. (D) Schematic illustration of pathways regulating the localization of FOXO3 in oocytes. Go Nagamatsu et al. Sci Adv 2019;5:eaav9960 Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).