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. 2018 Apr 19;10(6):1782–1792. doi: 10.1016/j.stemcr.2018.03.016

Figure 1.

Figure 1

FGF2 Expands GFRA1+ Spermatogonia Phenotypically Distinct from Those Induced by GDNF

(A) Flow chart of biodegradable gelatin microsphere (BGM) experiments. Mock-, FGF2-, or GDNF-adsorbed BGMs were bilaterally injected into the interstitium of 7-week-old mouse testes. BrdU was administered via water ad libitum until mice were sacrificed. At 10 days after injection, testes were analyzed by immunofluorescence staining. Results were obtained independently from three mice.

(B and C) Cluster formation and proliferation of GFRA1+ spermatogonia induced by BGM transplantation. (B) Immunofluorescence staining. GFRA1 and incorporated BrdU are visualized by cyan and red, respectively. Nuclei were counterstained with Hoechst 33,342 (gray). (C) Classification of large GFRA1+ clusters by the number of constituting spermatogonia. Mock-BGMs, n = 5 testes; FGF2-BGMs, n = 6 testes; GDNF-BGMs, n = 4 testes. A total of 1,434 tubules with 261 large GFRA1+ clusters carrying 2749 GFRA1+ spermatogonia were analyzed.

(D–G) Characterization of BGM-induced large GFRA1+ clusters. (D and E) PLZF expression in large GFRA1+ clusters. (D) Immunofluorescence staining. GFRA1 is visualized by cyan, while PLZF is visualized by green. Nuclei were counterstained with Hoechst 33,342 (gray). (E) Quantitative analysis of PLZF expression in large GFRA1+ clusters. FGF2-BGMs, n = 5 testes; GDNF-BGMs, n = 4 testes. A total of 957 tubules with 190 large GFRA1+ clusters carrying 2,111 GFRA1+ spermatogonia were analyzed. In this experiment, one FGF2-BGM-treated testis, which did not carry large GFRA1+ clusters, was excluded from the statistical analysis. (F and G) RARG expression in large GFRA1+ clusters. (F) Immunofluorescence staining. GFRA1 is visualized by cyan, while RARG is visualized by red. Nuclei were counterstained with Hoechst 33,342 (gray). (G) Quantitative analysis of RARG expression in large GFRA1+ clusters. FGF2-BGMs, n = 6 testes; GDNF-BGMs, n = 4 testes. A total of 1,347 tubules with 214 large GFRA1+ clusters carrying 2,147 GFRA1+ spermatogonia were analyzed.

Results are presented as means ± SEM. Scale bars, 50 μm (B, D, and F). See also Figures S1 and S2; Table S1.