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. 2023 Jan 23;9:7. doi: 10.1038/s41526-023-00248-5

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — a Illustration of the strategy to induce labeling of the GCs in Foxl2-CreER^T2;mTmG follicles. With a low dosage of tamoxifen treatment, membrane-localized red fluorescent protein (mT) switches to green-fluorescent protein (mG) in GCs of Foxl2-CreER^T2;mTmG follicles, which allows for imaging of the cell outline of GCs under high-resolution imaging system. b The standard to separate the GCs in growing follicles. The inner layer of GCs was defined as the layer which directly connected to the oocyte by GC-TZPs (I-GCs, green); the middle layers of GCs was distributed in the middle multilayers position of follicle (M-GCs, pink); The outer layer of GCs was defined as the layer adjacent to the theca cells (O-GCs, blue). c Images of Foxl2-CreER^T2;mTmG follicles showing the morphology of GCs in different regions in the NG group. I-GCs (green box) exhibited cuboidal shape with tree root-like GC-TZPs oriented toward the oocyte. Rounded M-GCs (red box) exhibited extended random cellular projections. Similar shaped cell with I-GCs, cuboidal O-GCs (blue box) extended a few cellular projections toward to M-GCs. Scale bars, 15 μm. d In the SMG group, both the polarity and the communicating structures on GCs were abnormal, showing a failure of GC-TZPs on I-GCs (green boxes), and dramatically reduced cellular projections on M-GCs (red boxes) and O-GCs (blue boxes). I-GCs and O-GCs exhibited a loss of polarity shape under SMG condition compared to that in the NG group. Scale bars, 15 μm. The cartoon model as shown in Supplementary Fig. 3. e Statistical analysis of GCs showing a significant increase in the proportion of non-polarity in I-GCs and O-GCs under SMG conditions (n = 10) compared to the NG group (n = 10). I-GCs: p value = 0.0000000010, M-GCs: p value = 0.64, O-GCs: p value = 0.00000082. f Loss of cellular projections in all layers of GCs under SMG conditions (n = 20) compared to that in NG conditions (n = 20). I-GCs: p value = 0.00000000000000000000064, M-GCs: p value = 0.00000000059, O-GCs: p value = 0.0000051. Representative images are shown. Data are presented as the mean ± SD. Data were analyzed by two-tailed unpaired Student’s t-test and n.s. P ≥ 0.05, ***P < 0.001. The colors were inverted to black/white (b/w) to highlight GCs in (c, d).