BackgroundFetal exposure to environmental estrogens may contribute to hypofertility and/or to testicular germ cell cancer. However, many of these xenoestrogens have only a weak affinity for the classical estrogen receptors (ERs,) which is 1,000-fold less potent than the affinity of 17β-estradiol (E2). Thus, several mechanisms have been suggested to explain how they could affect male germ cell proliferation at low environmental relevant concentrations.ObjectivesIn this study we aimed to explore the possible promoting effect of bisphenol A (BPA) on human testicular seminoma cells. BPA is a well-recognized estrogenic endocrine disruptor used as a monomer to manufacture poly carbonate plastic and released from resin-lined food or beverage cans or from dental sealants.Methods and resultsBPA at very low concentrations (10−9 to 10−12 M) similar to those found in human fluids stimulated JKT-1 cell proliferation in vitro. BPA activated both cAMP-dependent protein kinase and cGMP-dependent protein kinase pathways and triggered a rapid (15 min) phosphorylation of the transcription factor cAMP response-element–binding protein (CREB) and the cell cycle regulator retinoblastoma protein (Rb). This nongenomic activation did not involve classical ERs because it could not be reversed by ICI 182780 (an ER antagonist) or reproduced either by E2 or by diethylstilbestrol (a potent synthetic estrogen), which instead triggered a suppressive effect. This activation was reproduced only by E2 coupled to bovine serum albumin (BSA), which is unable to enter the cell. As with E2-BSA, BPA promoted JKT-1 cell proliferation through a G-protein–coupled nonclassical membrane ER (GPCR) involving a Gαs and a Gαi/Gαq subunit, as shown by the reversible effect observed by the corresponding inhibitors NF449 and pertussis toxin.ConclusionThis GPCR-mediated nongenomic action represents—in addition to the classical ER-mediated effect—a new basis for evaluating xenoestrogens such as BPA that, at low doses and with a high affinity for this GPCR, could interfere with the developmental programming of fetal germ cell proliferation and/or differentiation when they cross the placenta.

Clinical and experimental studies have suggested that estrogens, the archetype of female hormones, participate in the control of male germ cell proliferation and that fetal exposure to environmental estrogens may contribute to hypofertility and/or to testicular germ cell cancer. However, the underlying mechanisms remain to be elucidated. 17beta-Estradiol (E2) conjugated to BSA was able to stimulate human testicular seminoma cell proliferation by triggering a rapid, nongenomic, membrane-mediated activation of ERK1/2 and cAMP-dependent protein kinase A (PKA). Both ERK1/2 and PKA participated in this promoting effect. This activation was associated with phosphorylation of the transcription factor cAMP response element-binding protein and the nuclear factor retinoblastoma protein. Enhanced proliferation together with ERK activation could be reversed by pertussis toxin, a G protein inhibitor. Estrogen receptors (ERs) in JKT-1 were characterized by immunofluorescence, subcellular fractioning, and Western blot. JKT-1 cells did not express ERalpha but ERbeta, which localized to the mitochondria and the nucleus but not to the membrane. Moreover, neither ICI-182,780, a classical ER antagonist, nor tamoxifen, a selective ER modulator, could reverse the 17beta-estradiol-BSA-induced promoting effect. Estrogens contribute to human testicular germ cell cancer proliferation by rapid activation of ERK1/2 and PKA through a membrane nonclassical ER. This nongenomic effect represents a new basis for understanding the estrogenic control of spermatogenesis and evaluating the role of fetal exposure to xenoestrogens during malignant transformation of testicular germ stem cells.

BackgroundTesticular germ cell tumours are the most frequent cancer of young men with an increasing incidence all over the world. Pathogenesis and reasons of this increase remain unknown but epidemiological and clinical data have suggested that fetal exposure to environmental endocrine disruptors (EEDs) with estrogenic effects, could participate to testicular germ cell carcinogenesis. However, these EEDs (like bisphenol A) are often weak ligands for classical nuclear estrogen receptors. Several research groups recently showed that the non classical membrane G-protein coupled estrogen receptor (GPER/GPR30) mediates the effects of estrogens and several xenoestrogens through rapid non genomic activation of signal transduction pathways in various human estrogen dependent cancer cells (breast, ovary, endometrium). The aim of this study was to demonstrate that GPER was overexpressed in testicular tumours and was able to trigger JKT-1 seminoma cell proliferation.ResultsWe report here for the first time a complete morphological and functional characterization of GPER in normal and malignant human testicular germ cells. In normal adult human testes, GPER was expressed by somatic (Sertoli cells) and germ cells (spermatogonia and spermatocytes). GPER was exclusively overexpressed in seminomas, the most frequent testicular germ cell cancer, localized at the cell membrane and triggered a proliferative effect on JKT-1 cells in vitro, which was completely abolished by G15 (a GPER selective antagonist) and by siRNA invalidation.ConclusionThese results demonstrate that GPER is expressed by human normal adult testicular germ cells, specifically overexpressed in seminoma tumours and able to trigger seminoma cell proliferation in vitro. It should therefore be considered rather than classical ERs when xeno-estrogens or other endocrine disruptors are assessed in testicular germ cell cancers. It may also represent a prognosis marker and/or a therapeutic target for seminomas.

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It is now well established that estrogens participate in the control of normal spermatogenesis and endogenous or environmental estrogens are involved in pathological germ cell proliferation including testicular germ cell tumors. Studying a human testicular seminoma cell line, JKT-1, we show here that 17 -estradiol (10 −12 to 10 −6 M) induced in vitro a significant dose-dependent decrease of cell growth. This antiproliferative effect was maximum after 4 days of exposure at a physiologically intratesticular concentration of 10 −9 M, close to the K d of ER, and reversed by ICI 182780, an ER antagonist, suggesting an ER-mediated pathway. By RT-PCR and Western blot we were able to confirm that JKT-1, like tumoral seminoma cells and normal human testicular basal germ cells, expresses estrogen receptor (ER ), including ER 1 and ER 2, a dominant negative variant, but not ER . Using immunofluorescence and confocal microscopy, ER was observed as perinuclear intracytoplasmic spots in JKT-1 and tumoral seminoma cells without significant translocation of ER into the nucleus, under 17 -estradiol exposure. Double staining observed by confocal microscopy revealed that ER colocalized in JKT-1 cells with cytochrome C, a mitochondrial marker. We report for the first time the expression of a functional aromatase complex in seminoma cells as assessed by RT-PCR, Western blot and enzymatic assay. Seminoma cells are able to respond to estrogens through a possible autocrine or paracrine loop. These preliminary results support estrogen-dependency of human testicular seminoma, the most frequent tumor of young men, and suggest potential pharmacological use. Whether this estrogen control, however, involves an ER -mediated stimulation of cell apoptosis and/or an ER -mediated inhibition of cell proliferation, remains to be further determined.

Testicular germ cell tumours (TGCTs), the most frequent solid tumour of the young men, originate from the primitive germ cells. They share some pluripotency stem-cell markers which may help to distinguish between seminoma, the most frequent TGCTs and non-seminoma tumours, such as embryonal carcinoma, teratocarcinoma or choriocarcinoma. Due probably to the propensity of seminoma to apoptosis, only two cell lines originated from pure testicular seminoma, TCam-2 and JKT-1 have been up to now, established, maintained and proposed as representative models of human testicular seminoma. However, both seem, following recent reports, to be able to drift. Thus, the molecular signature of embryonic stem-cell markers of the JKT-1 cells cultured in our laboratory, were studied by RT-PCR, Western blot and immunofluorescence (IF). JKT-1 cells analysed after 30 passages, expressed placenta alkaline phosphatase but not alphafoetoprotein (alphaFP) nor beta-human chorionic gonadotropin. JKT-1 cells also expressed markers of pluripotency such as NANOG and OCT3/4 and more specific seminoma markers, such as AP2gamma and HIWI. However, protein expression of OCT3/4 and AP2y was weak and these JKT-1 cells expressed SOX2, a marker of embryonal carcinoma and did not express c-KIT usually expressed in most seminoma. Possible derivation through in vitro culture conditions was supported by looking at later passages (61) which showed a decrease of NANOG and HIWI protein expression. JKT-1 cells express a signature of markers which is still near from the one express by seminoma cells, allowing carcinogenetic studies. However, because of their great ability to drift as shown for TCam-2, it is recommended to verify and to precise this molecular signature before reporting functional results.

In a recent issue of Cancer Biology & Therapy, Vitale et al. introduced the paper of Franco et al. reporting the expression of GPER (a seven-transmembrane-spanning receptor also known as GPR30) on testicular germ cell tumors (TGCTs). The paper commented on the possible pathophysiological role of this G-protein-coupled receptor (GPCR) during testicular germ cell carcinogenesis. They also proposed the potential use of selective antagonists of GPER as new-targeted therapy in these tumors. We agree with this hypothesis and would like to highlight here some published results supporting this hypothesis and give some details concerning the role of estrogens and xeno-estrogens in testicular carcinogenesis and the effects of GPER antagonist in human seminoma cells. We are sure that this information we obtained by working for several years on a human seminoma cell line will be appreciated by the readers of Cancer Biology & Therapy.

(breast, ovary, endometrium).