Fig 1: Sertoli cell ablation of E12.5 testes in AMH-TRECK transgenic (Tg) mice. (A) Testis/ovary explants isolated from Tg and wild-type littermates at E12.5 were treated with diphtheria toxin (DT; 200 ng/ml) for 24 h and then recovered for 24 h in FCS-DMEM. (B) Hematoxylin and Eosin staining of DT-treated wild-type and Tg testis explants, showing degenerated testis cords, including pyknotic nuclei and necrotic cell debris (arrows), but histologically normal surface epithelia/subepithelia (SE; lower panels), in the Tg explant. Dashed lines indicate testis cords. (C,D) Anti-SOX9 (a marker of Sertoli cells and mesonephric tubules), -AMH (a Sertoli cell marker), -3βHSD (a Leydig cell marker) and -NR2F2 (a marker for stromal cells) immunostaining (brown) of wild-type and Tg testes/ovaries with (+) or without (−) DT treatment, showing a marked reduction of SOX9- and AMH-positive signals in DT-treated Tg testes (C). 3βHSD- and NR2F2-positive signals were unaltered in DT-treated Tg testes (D). Broken lines indicate the border between gonad and mesonephros. (E) Morphometric analysis showed a significant reduction in testis size (A-P or D-V axes) in DT-treated Tg testes compared with the two control testis samples (**P<0.01; one-way ANOVA followed by Dunnett's test). Box and whisker plots show medians, interquartile ranges (boxes), and minima and maxima (whiskers). Numbers in parentheses are the numbers of samples. Arrowheads indicate the area magnified on the right in B, and in the insets in C and D. MT, mesonephric tubule. Scale bars: 100 µm in B (top left images); 25 µm in B (top right and lower images); 200 µm in C and D.
Fig 2: Exogenous FGF9 is sufficient to repress FOXL2 inducibility in DT-treated Tg testis explants initiated at E12.5. (A) Schematic of exogenous addition of a candidate ligand (PDGF, AMH, DHH or FGF9) or agonist (BW245C for PGD2) to a 4-day culture of DT-treated Tg testes. (B-D) RT-qPCR analysis showing significant reductions of Foxl2 (B,C), and Gng13 and Lgr5 (D) in the presence of FGF9 (*P<0.05, **P<0.01; one-way ANOVA for FGF9, AMH and BW245C in B and C; paired Student's t-tests for others in B; unpaired Student's t-tests in D). Data are mean±s.e.m. In B, expression levels were normalized to the corresponding non-additive control of DT-treated Tg testes (broken horizontal line; set as 1.0). C shows relative Foxl2 expression levels in DT-treated Tg testes cultured with FGF9 (0, 1, 10 or 50 ng/ml). Actb was used as an endogenous reference. Numbers in parentheses are numbers of samples. (E) Anti-FOXL2 immunostaining of DT-treated Tg testis explants in the presence or absence of FGF9. FOXL2-positive signals near the surface epithelia and mesonephros are repressed in Tg testes by exogenous FGF9. Lower panels are high-magnification images of the regions surrounded by broken rectangles in the upper panels. (F,G) Morphometric analysis of FOXL2-positive cell density [number per 10,000 µm2 in serial sections (each 40 µm apart) of a whole explant; n=3 per group] in DT-treated Tg testes cultured with or without FGF9. FOXL2-positive cells in the surface subepithelial region (SE) and the non-surface regions, including the peri-mesonephric site (non-SE), are enumerated in G (total FOXL2-positive cell density is shown in F). Data are presented as a box and whisker plot (median, center line; whiskers, sample distribution range; *P<0.05 by unpaired Student's t-test). Each point represents the data of one section (Mayère et al., 2022). (H) Schematic of the partial sex reversal of E12.5 testis after Sertoli cell ablation. In E12.5 testis, Fgf9 expression in Sertoli cells, together with mesonephric/vascular cell migration, lead to testis differentiation (Harikae et al., 2013b). DT-dependent Sertoli cell ablation of E12.5 Tg testis triggers degeneration of the testis cord structure. In a 4-day culture of these testes without Sertoli cells, FOXL2-positive/NR2F2-negative cell clusters are re-recruited from the surface testis epithelia/subepithelia, similar to the ovarian cords from the female surface epithelia. FOXL2-positive/NR2F2-positive stromal cells appear in the peri-mesonephric region, independently of 3βHSD-positive Leydig cells in the interstitial region. Exogenous FGF9 represses FOXL2 expression in DT-treated Tg testes, implicating FGF9 in male differentiation of the surface epithelia/subepithelia and peri-mesonephric stromal cells. From top to bottom: cortex (C) to medulla (M) sides. MT, mesonephric tubule; SE, surface epithelium. Scale bars: 100 μm in E, upper panels; 25 µm in E, lower panels.
Fig 3: Analysis of apoptotic stroma cells, primordial follicles and growing follicles in non-transplanted human ovarian tissue and xenografts retrieved from a subcutaneous pocket of mice treated with AMH and/or DXR stained with TUNEL. (A) Representative images of follicles in human ovarian xenografts stained with TUNEL. (B) Proportion of stroma cells that were TUNEL-positive in the non-grafted control ovarian tissue (NGC) and the Control transplanted group. (C) Proportion of stroma cells that were TUNEL-positive in tissue retrieved from the control and treated groups. (D) Proportion of follicles that were classed as apoptotic in the non-grafted control ovarian tissue (NGC) and the Control transplanted group. (E) Proportion of follicles that were classed as apoptotic in tissue retrieved from the control and treated groups. (F) Proportion of primordial follicles (PMFs) that were classed as apoptotic in the non-grafted control ovarian tissue (NGC) and the Control transplanted group. (G) Proportion of PMF that were classed as apoptotic in tissue retrieved from the control and treated groups. (H) Proportion of growing follicles (PMF) that were classed as apoptotic in the non-grafted control ovarian tissue (NGC) and the Control transplanted group. (I) Proportion of growing follicles that were classed as apoptotic in tissue retrieved from the control and treated groups. The data are presented as Mean ± S.E.M. Number in the base of the column is the sample number. *P < 0.05.
Fig 4: Analysis of mouse ovarian follicles in SCID mice carrying human ovary xenografts treated with AMH and/or DXR stained with H and E. (A) Representative images of mouse ovarian follicles (scale bar = 25 μm). (I) Primordial follicles. ii-v: Growing follicles (ii: Transitional; iii: Primary; iv: Secondary; v: Antral). (B) Number of morphologically healthy primordial follicles in mouse ovaries after 8 days of treatment with PBS, DXR, AMH or co-treatment (DXR + AMH). (C) Number of growing follicle numbers in mouse ovaries from the Control, DXR, AMH, and DXR + AMH groups at the end of the experiment. (D) Proportion of morphologically healthy growing follicles in mouse ovaries from different groups. The data are presented as Mean ± S.E.M. Number in the base of the column is the sample number. *P < 0.05, **P < 0.01, ****P < 0.0001.
Fig 5: Analysis of follicles in human ovarian tissue xenografts retrieved from a subcutaneous pocket of mice treated with AMH and/or DXR stained with H and E. (A) Proportion of follicles that were primordial follicles in each treatment group. (B) Proportion of primordial follicles that were morphologically healthy. (C) Proportion of follicles that were growing. (D) Proportion of growing follicles that were morphologically healthy. The data are presented as Mean ± S.E.M. Number in the base of the column is the sample number. *P < 0.05. Total follicle numbers (Mean ± S.E.M.): Control 278.6 ± 137.6; AMH 511.5 ± 157.5; DXR 330.2 ± 91.73; DXR + AMH 220.4 ± 105.3.
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