Fig. 5

GDNF expression in TECs is regulated by CaN–NFAT signaling. a Immunofluorescence images of testis sections immunostained with the stem cell markers DDX4 and Oct4 and DAPI to stain DNA. Testes were harvested from 1-week-old or 4-week-old Euploid or Ts65Dn mice. Quantification of DDX4⁺ and Oct4⁺ cells relative to DAPI per high-powered field (HPF) is shown on the right, ***P < 0.0001. Bar = 50 μm. Two-way ANOVA test. b Quantification of GDNF levels by ELISA in conditioned media from control iPS-derived ECs (C-iPS EC) and Down syndrome iPS-derived ECs (DS-iPS-ECs) after 2 ng ml⁻¹ FGF-2 treatment in the presence or absence of cyclosporin A (CsA). Values are mean ± s.e.m, n = 4. c Quantification of GDNF levels by ELISA in conditioned media from murine TECs, LuEC and LiEC after 2 ng ml⁻¹ FGF-2 treatment in the presence or absence of CsA. Values are mean ± s.e.m., n = 4. d H&E-stained cross-sections from testes of age-matched Euploid, Ts65Dn or Ts65Dn mice 5 weeks after implantation with GFP⁺ TECs isolated from syngeneic wild-type mice (Ts65Dn+GFP⁺ TECs). Red dotted line encircles mature sperm (yellow arrow) in the lumen of the seminiferous tubules. Immunofluorescence images with GFP and DAPI to stain DNA of testis sections from Ts65Dn+GFP⁺TECs mice. e Western blot analysis of EGR-1 protein expression in TECs and LuECs untreated or after FGF-2 treatment, expression of constitutively active NFATc1 (caNFATc1) or an empty vector control. Actin was probed as a loading control. f Quantification of nuclear translocation of EGR1 and NFATc1 in TECs after treatment with conditioned media (CM), 2 ng ml⁻¹ FGF-2±CsA. Values are mean ± s.e.m. f Chromatin immunoprecipitations (ChIP) of TECs expressing caNFATc1 with anti-NFATc1 mAb or of TECs with anti-EGR-1 antibody. NFATc1 or EGR-1 was immunoprecipitated and DNA probed by PCR for NFATc1 consensus sites on the Egr1 promoter or EGR1 consensus sites on the Gdnf promoter. IgG pulldown was used as a control. h Schematic of GDNF regulation via FGF-2-CaN-NFATc1-EGR-1 in TECs