Diethylstilboestrol exposure does not reduce testosterone production in human fetal testis xenografts

In rodents, in utero exposure to exogenous estrogens including diethylstilboestrol (DES) results in major suppression of steroidogenesis in fetal testes. Whether similar effects occur in the human fetal testis is equivocal. Based on the results of the rodent studies, we hypothesised that exposure of human fetal testes to DES would result in a reduction in testosterone production. We show, using a xenograft approach, that testosterone production is not reduced in human fetal testis following DES exposure. Human fetal testes (15-19 weeks' gestation, n = 6) were xenografted into castrate male nude mice which were then treated for 35 days with vehicle or 100 µg/kg DES three times a week. For comparison, similar treatment was applied to pregnant rats from e13.5-e20.5 and effects on fetal testes evaluated at e21.5. Xenograft testosterone production was assessed by measuring host seminal vesicle (SV) weights as an indirect measure over the entire grafting period, and single measurement of serum testosterone at termination. Human fetal testis xenografts showed similar survival in DES and vehicle-exposed hosts. SV weight (44.3 v 26.6 mg, p = 0.01) was significantly increased in DES compared to vehicle-exposed hosts, respectively, indicating an overall increase in xenograft testosterone production over the grafting period, whilst serum testosterone at termination was unchanged. In contrast intra-testicular testosterone levels were reduced by 89%, in fetal rats exposed to DES. In rats, DES effects are mediated via Estrogen Receptor α (ESR1). We determined ESR1 protein and mRNA expression in human and rat fetal testis. ESR1 was expressed in rat, but not in human, fetal Leydig cells. We conclude that human fetal testis exposure to DES does not impair testosterone production as it does in rats, probably because ESR1 is not expressed in human fetal Leydig cells. This indicates that DES exposure is likely to pose minimal risk to masculinization of the human fetus

Effects of environmental Bisphenol A exposures on germ cell development and Leydig cell function in the human fetal testis

<div><p>Background</p><p>Using an organotypic culture system termed human Fetal Testis Assay (hFeTA) we previously showed that 0.01 μM BPA decreases basal, but not LH-stimulated, testosterone secreted by the first trimester human fetal testis. The present study was conducted to determine the potential for a long-term antiandrogenic effect of BPA using a xenograft model, and also to study the effect of BPA on germ cell development using both the hFETA and xenograft models.</p><p>Methods</p><p>Using the hFeTA system, first trimester testes were cultured for 3 days with 0.01 to 10 μM BPA. For xenografts, adult castrate male nude mice were injected with hCG and grafted with first trimester testes. Host mice received 10 μM BPA (~ 500 μg/kg/day) in their drinking water for 5 weeks. Plasma levels of total and unconjugated BPA were 0.10 μM and 0.038 μM respectively. Mice grafted with second trimester testes received 0.5 and 50 μg/kg/day BPA by oral gavage for 5 weeks.</p><p>Results</p><p>With first trimester human testes, using the hFeTA model, 10 μM BPA increased germ cell apoptosis. In xenografts, germ cell density was also reduced by BPA exposure. Importantly, BPA exposure significantly decreased the percentage of germ cells expressing the pluripotency marker AP-2γ, whilst the percentage of those expressing the pre-spermatogonial marker MAGE-A4 significantly increased. BPA exposure did not affect hCG-stimulated androgen production in first and second trimester xenografts as evaluated by both plasma testosterone level and seminal vesicle weight in host mice.</p><p>Conclusions</p><p>Exposure to BPA at environmentally relevant concentrations impairs germ cell development in first trimester human fetal testis, whilst gonadotrophin-stimulated testosterone production was unaffected in both first and second trimester testis. Studies using first trimester human fetal testis demonstrate the complementarity of the FeTA and xenograft models for determining the respective short-term and long term effects of environmental exposures.</p></div

IGF-1 treatment does not protect MCF-7 cells from 4OH-tamoxifen-induced cell death.

<p>(A) MCF-7 cells were co-transfected with cDNAs coding for the PH domain of Akt fused to luciferase (Luc-Akt-PH) and YFP fused to a membrane localization sequence. 48 h after transfection, cells were pre-incubated for 6 h in the absence or presence of PUGNAc+GlcN. Cells were incubated for 10 min with coelenterazine and then stimulated with 100 nM IGF-1. Light acquisition at 480 nm and 530 nm started immediately after IGF-1 addition. A typical experiment is shown. (B) PUGNAc+GlcNAc and IGF-1-induced BRET (BRET above basal at the plateau). Results are the means ± S.E.M. of at least 5 independent experiments. Statistical analysis was performed using ANOVA followed by Tukey’s post-test. *, P< 0.05; **, P< 0.01; NS, not significant. (C) MCF-7 cells were cultured in presence of 1% FBS during 24 h in the absence or presence PUGNAc (100 µM), Glucosamine (GlcN, 5 mM), 4-OH-tamoxifen (10 µM) and IGF-1 (100 nM). Cell population growth in each well was evaluated using an Uptiblue-based-assay as the ratio of final fluorescence over the initial one (broken line) in the same well. Each determination corresponds to measurements performed in triplicate wells. Results are the mean±SEM of 5 independent experiments. Statistical analysis was performed using ANOVA followed by Tukey’s post-test. *, P< 0.05; **, P< 0.01; NS, not significant.</p

Inhibition of ERα expression level by O-GlcNAcylation-inducing treatments.

<p>Cells were cultured for 24 h in the absence or presence of PUGNAc+GlcN. (A) RNA were extracted and the expression of ERα mRNA was evaluated by RT-qPCR. (B) Cells were lysed and the expression of ERα protein was analysed by western-blot. GAPDH expression level was used as loading control. (C) ERα/GAPDH signals quantified by densitometric analysis of the autoradiograms of western-blots from 6 independent experiments. Statistical analysis was performed using unpaired t test. *, P< 0.05; ***, P< 0.001.</p

Inhibition of OGT expression sensitizes MCF-7 cells to tamoxifen-induced apoptosis.

<p>MCF-7 cells were transfected with control (siNEG) or anti-OGT siRNA (siOGT, sequence: TGGCATCGACCTCAAAGCA). (A) OGT protein expression and global O-GlcNAc levels were analysed 48h later by western-blot. (B) 48h after transfection with siRNA, cells were cultured for 24 hours in absence or presence of 4-OH-tamoxifen (10 µM) and then analysed by FACS after Annexin V-FITC/Propiduim Iodide staining. Statistical analysis was performed using ANOVA followed by Tukey’s post-test *, P< 0.05. Results correspond to the mean±SEM of 5 independent experiments.</p

O-GlcNAc-inducing treatments stimulate the PI-3 kinase/Akt pathway in MCF-7 cells.

<p>(A) Principle of the BRET assay to measure PIP₃ production in living cells. Activation of PI-3 kinase induces the phosphorylation of phosphatidyl-inositol 2 phosphate (PIP₂) into phosphatidyl-inositol 3 phosphate (PIP₃) and subsequent recruitment of Akt to the plasma membrane through its pleckstrin homology (PH) domain. To monitor the production of PIP₃ induced by receptor activation, cells are co-transfected with cDNAs coding for the PH domain of Akt fused to luciferase (Luc-Akt-PH) and YFP fused to a membrane localization sequence. (B) 48 h after transfection, cells were pre-incubated for 6 h in presence of PUGNAc, GlcN or both. Cells were incubated for 10 min with coelenterazine, then light acquisition at 480 nm and 530 nm started. A typical experiment is shown. (C) PUGNAc and GlcNAc-induced BRET (BRET above basal at the plateau). Results are the means ± S.E.M. of 4 to 8 independent experiments. Statistical analysis was performed using ANOVA followed by Dunnet’s post-test. **, P< 0.01 when compared to untreated control. (D) Effect of O-GlcNAcylation-inducing treatment on Akt phosphorylation. MCF-7 cells were incubated in the absence or presence of PUGNAc+GlcN for 6h or 24 h and lysed. The phosphorylation of Akt was evaluated by western-blot using anti-phospho-S473-Akt antibody.</p

Protection of MCF-7 cells from 4-OH-tamoxifen-induced cell death is not abrogated by inhibition of the PI-3 kinase/Akt pathway.

<p>MCF-7 cells were cultured in presence of 1% FBS during 24 hours in the absence or presence PUGNAc (100 μM), Glucosamine (GlcN, 5 mM), 4-OH-tamoxifen (10 μM) and either DMSO (vehicle) or the PI-3 kinase inhibitor LY294002 (LY) at a concentration of 10 or 20 µM. The growth of the cell population in each well was evaluated using Uptiblue-based-assay as the ratio of final fluorescence over the initial one (broken line) in the same well. Each determination corresponds to measurements performed in triplicate wells. Results are the mean±SEM of 3 independent experiments. Statistical analysis was performed using ANOVA followed by Dunnet’s post-test. *, P< 0.05 when compared to corresponding untreated control; NS, not significant.</p

The hexosamine biosynthetic pathway controls O-GlcNAc-modification of proteins.

<p>Cytosolic and nuclear O-GlcNAc glycosylation constitutes a dynamic process that regulates the activity, the localisation or the stability of the modified proteins. O-GlcNAc glycosylation of proteins on serine and threonine residues depends on the flux of glucose through the hexosamine biosynthetic pathway (HBP). A fraction (2 to 5%) of the glucose entering the cell is directed into this pathway for the biosynthesis of UDP-GlcNAc. OGT uses UDP-GlcNAc as a substrate to add GlcNAc on serine or threonine residues, and its activity is tightly dependent on the concentration of UDP-GlcNAc in the cell. These modifications can be reversed by O-GlcNAcase (OGA), which removes the O-GlcNAc moiety from O-GlcNAc-modified proteins. Experimentally, the level of O-GlcNAc in proteins can be increased by incubating cells with glucosamine (which bypasses allosteric inhibition of the rate limiting enzyme GFAT (Glutamine Fructose 6-P amidotransferase)), or with PUGNAc (O-[2-acetamido-2-deoxy-D-glucopyranosylidene] amino-N-phenylcarbamate), which inhibits deglycosylation by OGA.</p

PUGNAc+GlcNAc treatment inhibited the effect of 4-OH-tamoxifen on <i>p21</i> and <i>Egr1</i> gene expression.

<p>Cells were cultured for 24h in absence or presence of 4-OH tamoxifen or in presence of 4-OH-Tamoxifen and PUGNAc+GlcN. (A) RNA were extracted and the level of p21 and Egr1 mRNA was evaluated by RT-qPCR and normalised using Cyclophilin A expression levels. Results are the mean ±SEM of 3 independent experiments. Statistical analysis was performed by ANOVA followed by Dunnet’s post-test. *, P< 0.05; **, P< 0.01 when compared to untreated control.</p