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. 2019 Sep 26;10(11):825–831. doi: 10.1007/s13238-019-00654-0

Figure 2.

Figure 2

Neo-meiosis and functional test of oocytes developed from Fragilis+cells. (A) Immunofluorescence of SCP1 (red) and SCP3 (green, appear as yellowish by merge with SCP1) showing pachytene spread of E12.5 Fragilis+ cells obtained from aggregates with fetal E12.5 somatic cells 6 days following transplantation, compared with those of E17.5 ovaries. Right panel, Percentage of synaptonemal complex elements based on count of spread at pachytene (n = 22). Scale bar = 5 μm. (B) Representative immunofluorescence of MLH1 foci at pachytene stage by co-immunostaining of SCP3 (red) and MLH1 (green). Right panel, Statistics of MLH1 foci represents 20 spread per each group. Scale bar = 5 μm. (C) Morphology of reconstituted ovaries 28 days following transplantation of E12.5 Fragilis+ cells aggregated with E12.5 gonadal somatic cells into kidney capsules of ovariectomized mice (n = 8). Scale bar = 1 mm (upper panel). Middle panel, Follicles are shown in sections by H&E staining. Scale bar = 100 μm. Bottom panel, Co-immunostaining of GFP (green) and Vasa (red) in reconstituted ovaries. BF, bright-field. Nuclei were stained in blue by Hoechst. Scale bar = 100 μm. (D) Morphology of grafts 28 days following kidney capsule transplantation of 6-week Fragilis+ cells aggregated with E12.5 gonadal somatic cells (n = 3). No follicles but GFP+ somatic cells are seen. (E) Morphology of GV oocytes isolated from reconstituted ovaries developed from E12.5 Fragilis+ cells aggregated with E12.5 gonadal somatic cells, and 2-cell embryos following in vitro maturation (IVM) and fertilization (IVF). (F) Immunofluorescence of SCP1 (red) and SCP3 (green) in aggregates obtained from 6-week old Fragilis+ cells with fetal E12.5 gonadal somatic cells 6 days following transplantation. Scale bar = 10 μm. (G–I) Transcriptome of Fragilis+ and Fragilis− cells sorted from E12.5 ovaries and Fragilis+ cells from 6-week old mouse ovaries. (G) TSNE of global gene expression profiles of Fragilis+ cells sorted from fetal ovaries (E12.5 Fra+), Fragilis− cells sorted from fetal ovaries (E12.5 Fra−) and Fragilis+ cells sorted from 6-week ovaries (6W Fra+). (H) Scatter plots comparing transcriptome among these three cell populations. Parallel diagonal lines indicate threshold in expression difference. Red, up-regulated genes in E12.5 Fra+ cells; blue, down-regulated genes in E12.5 Fra− or in 6W Fra+ cells. (I) Heatmap highlighting gene expression profile of germ cells, proliferation and gonad somatic cells