Fig. 1

TEC transplantation restores spermatogenesis after busulfan treatment. a Quantification of Gdnf mRNA in Sertoli cells and TECs, and GDNF levels by ELISA in conditioned media from Sertoli cells or TECs without or with FGF-2 (20 ng ml⁻¹) treatment for 3 days. Data are representative of 3 experiments. *P < 0.05, ***P < 0.001, Bars and error bars show the mean ± s.e.m. b Murine GFP⁺Thy1.2⁺SSCs were co-cultured with TECs or testicular cells depleted of CD31+ TECs but containing all other cell populations in the testes. After 3 weeks, SSC colonies were absent in CD31-depleted testicular cell co-cultures with Sertoli and PTM cells present, while a significant number of GFP⁺ Thy1.2⁺ SSC colonies were present in co-cultures with TECs alone. Data are representative of two independent experiments. Two-way ANOVA test. c Immunofluorescence staining for CD31 (for ECs) on testis sections from wild-type mice 5 weeks after vehicle or busulfan treatment. Microvessel density (MVD) are quantified on the right, n = 3–5. d Western blot analysis of GDNF expression in whole testis lysates harvested from WT mice 5 weeks after PBS or busulfan treatment. Actin was probed as a loading control and each lane is an individual testis. e Whole testes from mice after treatment with PBS, busulfan or busulfan plus transplantation of either mouse LuECs or TECs. Bright-field image: bar = 1 cm. Fluorescent images (bar = 300 μm) show testis from WT mice treated with busulfan with and without transplanted GFP⁺ TECs. f Images of testis sections harvested from WT mice after busulfan treatment with and without transplantation of GFP⁺ TECs or GFP⁺ LuECs. Sections were stained with H&E and immunostained for the stem cell markers, PLZF (red) or DDX4 (white) or for GFP (green) to detect transplanted ECs. Quantification of seminiferous tubules showing spermatogenesis per high-powered field (HPF) and PLZF⁺ cells per HPF is shown on right, n = 3–4. Immunofluorescence images of testis sections from WT mice after indicated treatments were immunostained for CD31 (red), GFP (green) and DAPI (blue). Quantification of MVD and vessel length is shown on the right, n = 3. Data are from three independent experiments and presented as the mean ± s.e.m. Bar = 50 μm. *P < 0.05. ***P < 0.001. Two-tailed unpaired T-test