Patrick Fénichel

Low doses of bisphenol A promote human seminoma cell proliferation by activating PKA and PKG via a membrane G-protein-coupled estrogen receptor.

Background Fetal 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. Objectives In 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 results BPA 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. Conclusion This 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.

Disruption of Autophagy at the Maturation Step by the Carcinogen Lindane Is Associated with the Sustained Mitogen-Activated Protein Kinase/Extracellular Signal–Regulated Kinase Activity

Macroautophagy (hereafter referred to as autophagy) has emerged as a key tumor suppressor pathway. During this process, the cytosolic constituents are sequestered into autophagosomes, which subsequently fuse with lysosomes to become autolysosomes where their contents are finally degraded. Although a reduced autophagy has been shown in human tumors or in response to oncogenes and carcinogens, the underlying mechanism(s) remain(s) unknown. Here, we show that widely used carcinogen Lindane promotes vacuolation of Sertoli cells. By electron and immunofluorescent microscopy analyses, we showed that these structures are acid autolysosomes, containing cellular debris, and labeled by LC3, Rab7, and LAMP1, markers of autophagosomes, late endosomes, and lysosomes, respectively. Such Lindane-induced vacuolation results from significant delay in autophagy degradation, in relation with a decline of the lysosomal activity of aryl sulfatase A. At molecular level, we show that this defect in autolysosomal maturation is independent of mammalian target of rapamycin and p38 inhibitions. Rather, the activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway is required for Lindane to disrupt the autophagic pathway. Most importantly, we provide the first evidence that sustained activation of ERK pathway is sufficient to commit cell to autophagic vacuolation. Taken together, these findings strongly support that the aberrant sustained activation of ERK by the carcinogen Lindane disrupts the maturation of autophagosomes into functional autolysosomes. Our findings therefore suggest the possibility that high constitutive ERK activity found in all cancers may provide a malignant advantage by impeding the tumor suppressive function of autophagy.

Control of the autophagy maturation step by the MAPK ERK and p38: lessons from environmental carcinogens.

Macroautophagy (hereafter referred to as autophagy) is the major degradative pathway of long-lived proteins and organelles that fulfils key functions in cell survival, tissue remodeling and tumor suppression. Consistently, alterations in autophagy have been involved in a growing list of pathologies including toxic injury, infections, neurodegeneration, myopathies and cancers. Although critical, the molecular mechanisms that control autophagy remain largely unknown. We have recently exploited the disruption of autophagy by environmental carcinogens as a powerful model to uncover the underlying signaling pathways. Our work published in Cancer Research revealed that the sustained activation of the MAPK ERK pathway by the carcinogen Lindane or the MEK1+ oncogene alters autophagy selectively at the maturation step resulting in the accumulation of large defective autolysosomes. Consistent with our findings, a similar defect is observed with other common xenobiotics such as dichlorodiphenyltrichloroethane and biphenol A that specifically activate ERK. Conversely, Pentachlorophenol that activates both ERK and p38, fails to induce autophagic vacuolation. In addition, evidence is provided that abrogation of p38 by SB203580 is sufficient to interfere with the normal autophagic maturation step. Altogether, these findings underscore the critical role played by MAPK ERK and p38 in the tight control of the autophagy process at the maturation step. Addendum to: Disruption of Autophagy at the Maturation Step by the Carcinogen Lindane is Associated with the Sustained Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase Activity E. Corcelle, M. Nebout, S. Bekri, N. Gauthier, P. Hofman, P. Poujeol, P. Fénichel and B. Mograbi Cancer Res 2006; 66:6861-70

Impaired Gap Junction Connexin43 in Sertoli Cells of Patients with Secretory Azoospermia: A Marker of Undifferentiated Sertoli Cells

Gap junctions are intercellular channels formed of connexins (Cx) at appositional plasma membranes between adjacent cells that have been involved in the control of cell proliferation and differentiation. Altered Cx expression is implicated consistently in several human diseases and in tumorigenesis. Although Cx43 plays a critical role in Sertoli cell control of spermatogenesis, there is no evidence of its altered expression in human testicular pathologies. We show here that Cx43 mRNA expression was significantly reduced in testes of infertile patients with secretory azoospermia (p < 0.05) compared with testes displaying normal spermatogenesis (excretory azoospermic patients). In Sertoli cell-only syndrome, in situ hybridization and immunohistochemistry analyses indicated that Cx43 mRNA and protein were undetectable in Sertoli cells but were still present in the interstitial compartment. In a rat model of Sertoli cell-only syndrome, the lack of Cx43 in Sertoli cells was associated with an impairment of gap junction intercellular communication between adjacent Sertoli cells. These results reveal that Cx43 mRNA and protein expression are markedly impaired in Sertoli cells of infertile patients. This defect could be a new functional marker of undifferentiated Sertoli cells and could be related to the increased risk of testicular cancer recently described in the population of infertile men.

Characterisation and role of integrins during gametic interaction and egg activation.

It has recently been proposed that some of the processes induced by fertilisation in mammals may be mediated by integrins. By performing immunofluorescence labelling and Western blots with antibodies directed against some of the alpha and beta subunits of integrins, we show here the presence of some of these proteins in human and hamster oocytes. Among them, alpha 2 and alpha 5 were also present on in vitro preparations of sea urchin egg cortices. In addition, antibodies raised against these two proteins were the most effective at inhibiting attachment and fusion of human spermatozoa with hamster oocytes. We suggest that alpha 2 and alpha 5 integrin chains may be common mediators in adhesion-fusion mechanisms triggered by fertilisation. Using similar techniques, we show that eggs are rich in three cytoskeletal proteins known to be linked to the beta chain of integrins: talin, vinculin and alpha-actinin. Moreover, we found that talin and alpha-actinin were associated with proteins phosphorylated on tyrosine after fertilisation in sea urchin eggs. We suggest that integrins might be involved during fertilisation and trigger egg activation through cytoskeletal structures.

Exposure to selected endocrine disruptors and neonatal outcome of 86 healthy boys from Nice area (France)

In utero and lactational exposure to endocrine disruptors is thought to be potentially harmful on fetal and infant development. Data of exposure in France is scarce. This is a prospective study with (1) collection of 84 cord bloods (CB) and 69 milks from 86 mothers delivering healthy boys (gestational age >or= 34 weeks) at two maternity wards in Southern France, between 2002 and 2005 and (2) screening for 15 xenobiotics with anti-androgenic and/or estrogenic effects: DDE, 7 PCBs, dibutylphthalate and its metabolite mBP, HCB, lindane, linuron, procymidone and vinclozoline. Correlations were made with delivery and neonatal outcomes. All CB and milks were contaminated by one or more xenobiotics (mainly PCBs, DDE, HCB, and phthalates) with good correlation between CB and milk concentrations. Compared to other geographical areas, exposure was usually in the lower bracket. Milk [PCB180] was associated with lower birth weight. Infant head circumference correlated negatively with [HCB] and positively with [mBP] in CB. There was a similar but not significant trend for birth weight and length. [DDE] in milk was higher in older mothers and in women born in Africa. In utero and lactational exposure is ubiquitous in our area. Contamination of milk with HCB, mBP, and PCB 180 showed weak correlations with infant growth. This snapshot of exposure in an area with no major industry will serve for further monitoring.

Cord Blood Thyroid Tests in Boys Born With and Without Cryptorchidism: Correlations with Birth Parameters and In Utero Xenobiotics Exposure

BACKGROUND In utero exposure to environmental chemicals can result in reproductive toxicity via endocrine disruption mechanisms. Whether some of those contaminants also have an impact on fetal thyroid function or pathways, and, thus, potentially on neuropsychological development, is still debated. METHODS We used samples from a cord blood (CB) and milk bank, established for a research on cryptorchidism and xenobiotic exposure to compounds known for their anti-androgenic and/or estrogenic activity, to study CB thyroid tests and their correlation with CB and milk xenobiotics concentrations in boys born in Nice area. RESULTS No difference was found in thyroid tests between 60 cryptorchid boys and 76 matched controls (median thyroid stimulating hormone 5.97 vs. 6.55 mUI/L, free thyroxine [fT4] 13.1 vs. 12.9 pmol/L, free triiodothyronine [fT3] 1.9 vs. 2.1 pmol/L), with no influence of season of birth, gestational age, maternal smoking, or mode of delivery (except for higher fT4 in control boys born vaginally). FT4 was correlated with fetal growth only in cryptorchid boys. Since we had previously shown differences between cryptorchid and controls exposure, we studied correlations of thyroid tests with xenobiotics in control boys only. All tested CB or maternal milk was contaminated by one or more selected xenobiotics, mainly polychlorinated biphenyls (PCBs), dichloro diphenyl dichloroethylène (DDE), dibutylphthalate, hexachlorobenzene, and bisphenol A. We found a significant negative correlation between fT4 and concentrations of PCB118, PC180, and DDE in milk (respectively r = -0.342, p < 0.03, r = -0.296, p = 0.031, r = -0.315, p = 0.016), persisting after adjustment for mode of delivery. There was a significant positive correlation of fT3 with milk concentrations of PCB138, PCB153, ΣPCB, and dibutylphthalate (respectively r = 0.31, p = 0.016, r = 0.28, p = 0.029; r = 0.34, p = 0.0079 and r = 0.272, p = 0.0295), with a trend for PCB180 (r = 0.259, p = 0.061). There was no correlation of thyroid stimulating hormone with any of the measured xenobiotics, except for a weak negative trend with CB bisphenol A (r = -0.25, p = 0.077). CONCLUSIONS CB thyroid tests are within normal range in cryptorchid boys, similar to controls. Our data in controls suggest a possible weak correlation between in utero exposure to some xenobiotics (PCBs, DDE) and fT3 and fT4 CB concentrations, with usually negative correlations with fT4 and positive with fT3 concentrations, which we speculate could suggest an impact on deiodinases.

Dominant negative effect of connexin33 on gap junctional communication is mediated by connexin43 sequestration

Gap junctional intercellular communication is involved in the control of cell proliferation and differentiation. Connexin33, a member of the multi-gene family of gap junction proteins, exerts an inhibitory effect on intercellular communication when injected into Xenopus oocytes. However, the molecular mechanisms involved remain to be elucidated. Our results show that connexin33 was only expressed within the seminiferous tubules in the testis. In contrast to the majority of connexins, connexin33 was unphosphorylated. Immunoprecipitation experiments revealed that connexin33 physically interacted with connexin43, mainly with the phosphorylated P1 isoform of connexin43 but not with connexin26 and connexin32, two other connexins expressed in the tubular compartment. In Sertoli cells and COS-7 cells, connexin43 was located at the plasma membrane, whereas in connexin33 transfected cells, the specific association of connexin33/43 was sequestered in the intracellular compartment. High-resolution fluorescent deconvolution microscopy indicated that the connexin33/43 complex was mainly found within early endosomes. Sequestration of connexin33/43 complex was associated with a complete inhibition of the gap junctional coupling between adjacent cells. These findings provide the first evidence of a new mechanistic model by which a native connexin, exerting a dominant negative effect, can inhibit gap junctional intercellular communication. In the testis, connexin33 could exert a specific role on germ cell proliferation by suppressing the regulatory effect of connexin43.

Delayed diagnosis of Sheehan's syndrome in a developed country. A retrospective cohort study.

BACKGROUND Little is known about Sheehans syndrome (SS), even though it is believed that its incidence is low. The aims of this study were to determine the clinical features and diagnostic delay of SS and to ascertain whether early signs could have allowed earlier diagnosis. SUBJECTS AND METHODS All patients with SS diagnosed in reference units in the southeast of France between 1980 and 2011 were recruited for this study. Data on obstetrical history, clinical symptoms suggestive of hypopituitarism, early signs, hormone analysis, and magnetic resonance imaging were collected. RESULTS Of the 40 women found to have SS, 39 were studied. Mean delay in the diagnosis of SS was 9 ± 9.7 years. We found that four of the 35 assessable patients were diagnosed with agalactia, 16 of the 29 assessable ones with amenorrhea, 19 of the 39 with hypothyroidism, eight with acute adrenal insufficiency, and 15 with asthenia. Among the patients for whom there was a diagnostic delay of more than 1 year (n=28), seven had headaches during the postpartum period, all assessable patients had agalactia, six of the 22 assessable ones had amenorrhea, seven of 28 had hypothyroidism, and 12 of 28 had asthenia. CONCLUSION Most signs of SS are aspecific and classical signs such as agalactia and amenorrhea are often difficult to detect, which can explain the long diagnostic delay. We suggest that all women failing to lactate after postpartum hemorrhage (PPH) should be evaluated by measuring prolactin levels and women with signs such as amenorrhea and asthenia, even several years after PPH, should undergo a blood test including assessment of thyroxine, TSH, 0800  h ACTH-cortisol, and IGF1 levels.

Genetic Variants of GPER/GPR30, a Novel Estrogen-Related G Protein Receptor, Are Associated with Human Seminoma

Testicular germ cell tumors (TGCTs) are the most common solid cancers in young men, with an increasing incidence over several years. However, their pathogenesis remains a matter of debate. Some epidemiological data suggest the involvement of both environmental and genetic factors. We reported two distinct effects of estrogens and/or xeno-estrogens on in vitro human seminoma-derived cells proliferation: (1) an antiproliferative effect via a classical estrogen receptor beta-dependent pathway, and (2) a promotive effect via a non-classical membrane G-protein-coupled receptor, GPR30/GPER, which is only overexpressed in seminomas, the most common TGCT. In order to explain this overexpression, we investigated the possible association of polymorphisms in the GPER gene by using allele-specific tetra-primer polymerase chain reaction performed on tissue samples from 150 paraffin-embedded TGCT specimens (131 seminomas, 19 non seminomas). Compared to control population, loss of homozygous ancestral genotype GG in two polymorphisms located in the promoter region of GPER (rs3808350 and rs3808351) was more frequent in seminomas but not in non-seminomas (respectively, OR = 1.960 (1.172–3.277) and 7.000 (2.747–17.840); p < 0.01). These polymorphisms may explain GPER overexpression and represent a genetic factor of susceptibility supporting the contribution of environmental GPER ligands in testicular carcinogenesis.