or 10 min, followed by 55 cycles consisting of two steps: denaturation at 95uC for 30 s, annealing and extension at 60uC for 60 s and then a final cycle of three steps. The PCR product was electrophoresed on a 3% agarose ” gel in 16 TAE buffer and stained with the SYBRH Safe DNA gel stain. All experiments were performed in triplicate and the data shown are representative. Using Western blotting, we compared the expression of GPER between tumoural and normal tissues. Seminomas showed significantly higher GPER protein expression than normal peri-tumoural tissues. No significant difference was observed between non-seminomas and normal peritumoural tissues. These results were confirmed by analysing mRNA levels by RT-PCR, which revealed significantly higher GPER mRNA levels only in seminomas 
but not in non-seminomas, compared to normal peri-tumoural tissues. Analysis of mRNA by RT-PCR revealed “1659636 that both JKT-1 and NCCIT cells expressed GPER. These 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 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 get DHA 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 Hum
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