ously established in our laboratory, and as already reported in Zebrafish and primary immature rat Sertoli cells. We found that spermatogonia and spermatocytes expressed GPER 12409010 while amazingly Rago et al. reported a negative staining in human germ cells, likely due to use of abnormal granulomatous testes. Moreover, our results are in agreement 4 Overexpression of GPR30 in Human Seminoma with the one reported with a mouse spermatogonial cell line GC-1 and as 8673721 reported in adult Zebrafish, Croaker and rat testicular germ cells. Thus, these data illustrate the 
wide conservation of GPER but don’t assume the precise role of GPER in testicular germ cells differentiation and proliferation. Although male GPER KO mice are not infertile, their precise gonadal phenotype remains unexplored. In fact, it is possible that this orphan GPCR may only interfere with oestrogen and/or xeno-oestrogen activation during normal and/or pathological regulation of germ cell proliferation and apoptosis, as shown using G1 in rat pachytene spermatocytes and round spermatids and in human seminoma cells in our study. GPER is a G protein-coupled seven transmembrane spanning receptor that induces signalling through the Gs or Gi protein, strongly suggesting the plasma membrane as GPER’s site of action. However, the precise location of GPER remains controversial as GPER is localized at the plasma membrane of different targeted and transfected cells but is expressed predominantly in the endoplasmic reticulum in other reports. One explanation could be the different antibodies used, which triggered different epitopes and/or cell trafficking of the protein, which is described as highly unusual in human embryonic kidney HEK293 cells with an accumulation in the peri-nuclear space after endocytosis from the plasma membrane. It could also be cell model dependent. Similar to that in HEK-293 cells, we found double localization of GPER at the membrane and in the cytoplasm in JKT-1 seminoma cells. Moreover, membrane localization of GPER was supported by its co-localization with E2-BSA-FITC, which does not cross the membrane, and its ability to trigger a very rapid signal transduction induced by E2-BSA, a membrane impermeable estrogen. Phosphorylation of extracellular regulated kinases and the cAMP response element-binding protein occurred as soon as 5 to 15 min and was reversed by pertussis toxin, a G protein inhibitor. In 2011, both Franco et al. and Rago et al. showed by immunohistochemistry that GPER was present in testicular germ cell tumours, including seminoma. Our study TAK 438 free base chemical information confirmed this expression and demonstrated that GPER was overexpressed in seminomas but not in non seminoma tumours by using a robust quantitative approach of mRNAs and proteins levels by Q-PCR and Western blotting related to b-actin, considered as a housekeeping gene, and compared with the peri-tumoural normal testicular tissue for each patient. Eventhough some abnormal expression could be already observed in peritumoural tissues, this method excludes inter-individual variations of GPER expression as each patient represents its own control. This overexpression was also confirmed by comparison between JKT-1 seminoma cells and NCCIT choriocarcinoma cells. GPER overexpression has been linked to the development of advanced breast cancer, high-grade endometrial tumours and poor prognosis for ovarian cancer. GPER overexpression in seminomas may also represent a prognosis factor for testicular germ cell tumours and perhap