hese results were confirmed by Western blotting, which revealed the expected 42-kDa band for the GPER protein. The JKT-1 cells showed significantly higher GPER protein levels 12409010 than the NCCIT cells, whereas GPER mRNA expression was higher in the NCCIT cells, suggesting post-translational regulation of GPER expression in these cells. E2-BSA stimulates JKT-1 cell proliferation by interacting with GPER After 24-h exposure at a physiological intratesticular concentration of 1029 M, E2 induced a significant decrease in cell proliferation whereas E2-BSA at the same concentration stimulated JKT-1 cell proliferation; testosterone-BSA, at the same concentration, had no effect on JKT-1 cell proliferation . As we previously reported that this E2-BSA specific effect was not inhibited by ICI-182,780, a pure ER antagonist, but was reversed by Pertussis toxin, a G protein inhibitor, we hypothesize that E2-BSA directly interacted with GPER to induce JKT-1 cell proliferation. G1, a GPER-selective agonist, reproduced the same proliferative effect as that observed with E2-BSA. G15, a GPER-selective antagonist, had no effect alone on 
JKT-1 cell proliferation but completely neutralized the E2-BSA-induced proliferative effect. To confirm the role of GPER in E2BSA signalling, we performed GPER silencing in the JKT-1 cells using GPER siRNA, which led to a 98% GPER silencing confirmed by Western blotting and RT-PCR. Whereas transfection with control siRNA had no effect on JKT-1 cell proliferation after incubation with E2 and E2-BSA, GPER silencing had no effect on proliferation of the JKT-1 cells incubated with E2 but it completely neutralized the E2-BSA-induced proliferative effect, similar to co-incubation with G15, confirming that GPER mediated the effects of E2-BSA on JKT-1 cell proliferation. One may notice that the inhibition of the proliferative effect of E2-BSA obtained by G15 and GPER siRNA was in both cases Statistical analysis All data were analysed using the StatViewH5 software. Results of the cell count and densitometric analysis are expressed as percentages of variation compared with the control. A non-parametric MannWhitney U test was used for statistical analysis. All probabilities were twosided and P,0.05 was considered statistically significant. Results GPER immunolocalization in normal and tumoural testes Human testicular tissues were studied by immunofluorescence to determine whether GPER was expressed in normal testis and seminomas. Both normal and tumoural testes showed an intense Overexpression of GPR30 in Human Seminoma associated with an E2-like suppressive effect. The limited release of free E2 was likely involved as tested by addition with ICI. Discussion Several research groups have recently shown that GPER, an orphan GPCR with no evident physiological ligand, mediates a rapid E2-dependent activation of DM-1 signal transduction pathways in various human estrogen-dependent cancer cells and displays E2 binding typical of a membrane oestrogen receptor. We report here for the first time a characterization of GPER in normal and malignant human testicular germ cells. GPER was overexpressed in seminomas, was localized at the membrane of seminoma cells and was able to mediate the promotive effect on seminoma cell proliferation observed in vitro 1346650 with E2-BSA. GPER was expressed by somatic and germ cells in normal adult human testes. In seminiferous tubules, Sertoli cells were stained for GPER, similar to the adult mouse Sertoli cell line 42GPA9 previ
