ERK1/2 and protein kinase A through a membrane GPCR unrelated to classical ERs. 1 Overexpression of GPR30 in Human Seminoma GPR30, an orphan GPCR, mediates the E2-induced proliferative effects in an ER-negative SKBr3 breast cancer cell line. It has recently been renamed as G protein-coupled oestrogen receptor . GPER is widely expressed in various cell types and cancer cell lines and is overexpressed in endometrial cancers, aggressive breast cancers and ovarian cancers. Although the actual physiological ligand of GPER remains unknown, we considered that it could be a good candidate for mediating the proliferative effect of E2-BSA and of some xeno-oestrogens such as bisphenol A, which are able in vitro to stimulate seminoma cell proliferation. We aimed to investigate GPER expression in normal and malignant human testicular germ cells and its ability to trigger in vitro seminoma cell proliferation. Materials and Methods Cell culture The JKT-1 cell line, a kind gift from Dr. Kinugawa, was established from a human pure testicular seminoma developed from the testis of a 40-yr-old man. It was recently verified that the JKT-1 cells maintained in our laboratory still expressed 946128-88-7 specific embryonic stem cell markers. The JKT-1 cells were maintained in DMEM supplemented with 2% sodium pyruvate and 10% FBS in a humidified 5% CO2 atmosphere at 37uC. The NCCIT cell line was developed from a human testicular embryonic carcinoma and obtained from the American Type Culture Collection. These TGCT adherent cells were grown in RPMI-1640 medium supplemented with 15% FBS and were maintained in a humidified 5% CO2 atmosphere at 37uC. The 42GPA9 murine Sertoli cell line was maintained in DMEM supplemented with 2% sodium pyruvate and 10% FBS in a humidified 5% CO2 atmosphere at 32uC. The mouse spermatogonial GC-1 cell line with specific features common to type B spermatogonia was maintained in DMEM supplemented with 10% FBS in a humidified 5% CO2 atmosphere at 37uC. charged glass slides. All immunostaining procedures were performed after the sections were baked dry, de-waxed using xylene substitute and rehydrated in a graded ethanol series. Testis sections were washed with PBS for 5 min and heated at 90uC for 5 min in pre-heated citrate buffer. The slides were then incubated with rabbit anti-GPER Ab in PBS, 5% FBS, 0.2% gelatin for 1 h at room temperature and subsequently at 4uC overnight. Coverslips were then 
incubated with Texas RedTM-conjugated donkey anti-rabbit Ab for 2 h at room temperature before the nuclei were stained with Hoescht 33258. In order to specifically identify Sertoli cells in seminiferous tubules, a mouse monoclonal anti-vimentin clone V9 Ab was used. Seminoma cells were identified on tumoural sections by using a mouse monoclonal antibody against placental alkaline phosphatase. Image acquisition and analysis were performed on C3M Cell Imaging Facility sections using a confocal laser scanning microscope. Cell proliferation assay After 48 h, the JKT-1 cells were washed and oestrogen starved overnight in phenol red-free DMEM supplemented with 1% charcoal-stripped FBS. We then added E2, freshly prepared E2-BSA devoid of free E2, which is removed by filtration, ICI-182,780, G1, G15 or ethanol at 1029 M concentration and incubated them for 24 h. We harvested the cells using trypsin and counted them using the Vi-CELL software. Results are expressed as percentages of variation compared with the control. GPER silencing The JKT-1 cells were transfected
