Effect of ERα gene inactivation on <i>in vitro</i> testicular response to BPA.

<p>Testes from homozygous 12.5 dpc (ERα−/−), heterozygous (ERα+/−) and wild-type (ERα+/+) ERα-deficient fetuses were cultured on floating filters for 48 h. One testis from each animal was cultured in control medium and the other one in medium containing 10⁻⁵ M BPA. Values are means ± SEM of testosterone secreted in the medium during the 2 days of culture by BPA-treated testes referred to testosterone secreted by the respective contralateral control testes; n = 6 (ERα+/+), 13 (ERα+/−) and 5 (ERα−/−), * p<0.05 in the statistical comparison between BPA-treated and control testes using the Wilcoxon’s parametric paired t test.</p

Effect of BPA on testosterone secretion by human fetal testes as a function of their developmental stage.

<p>Individual (normalized to D0 and expressed as the percentage of the control explants) values at D2 of culture for the samples of the experiment described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051579#pone-0051579-g002" target="_blank">Figure 2</a> are presented. GW: gestational week.</p

Effect of BPA treatment on testicular histology.

<p>Histological sections of human, rat and mouse fetal testes removed at 11 gestational week (GW), 14.5 day post conception (dpc) and 12.5 dpc respectively after one day of culture in control medium (D0) followed by 3 days of culture in the absence (control) or presence (BPA) of 10⁻⁵ M BPA. At the end of the culture, testes were fixed in Bouin’s fluid and hematoxylin/eosin staining of the histological sections was performed. The testicular architecture and morphology were not affected by BPA-treatment. Black arrows: Sertoli cells; white arrows: Leydig cells; arrowhead: gonocytes.</p

Effect of BPA on testosterone secretion by rat and mouse fetal testes.

<p>Testes were removed from 14.5 dpc rat and 12.5 dpc mouse fetuses and cultured for one day in control medium (D0). Then, for each fetus, one testis was kept in control medium and the other one in medium supplemented with various concentrations of BPA as indicated for 3 days (D1 to D3). Values were calculated and expressed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051579#pone-0051579-g001" target="_blank">Figure 1</a>; Rat: n = 9 for 10⁻¹² M, n = 12 for 10⁻⁸ M, n = 9 for 10⁻⁷ M, and n = 7 for 10⁻⁵ M. Mouse: n = 9 for 10⁻¹² M, n = 15 for 10⁻⁸ M, n = 17 for 10⁻⁷ M and n = 10 for 10⁻⁵ M. *p<0.05, ** p<0.01, *** p<0.001 in the statistical comparison between BPA-treated and control testes using the Wilcoxon’s non-parametric paired test.</p

Effect of BPA on testosterone secretion by human fetal testes.

<p>One testis per fetus (between 6.5 and 10.5 gestational week) was cut in different pieces that were cultured as separated explants in different wells (4 to 12 wells according to the age of the fetus). After 24 h of culture in control medium (D0), half of the explants were cultured in the absence (control) and the other half in the presence of BPA at concentrations ranging from 10⁻¹² M to 10⁻⁵ M for 3 days (D1 to D3). The daily testosterone secretion in treated and untreated samples was measured by radioimmunoassay and the values at D1 to D3 were normalized to the D0 secretion of the same well. The D1 to D3 mean normalized secretion of treated samples are expressed as the percentage of that of the controls (untreated samples). Means ± SEM of these percentages from n fetuses are presented. n = 5 for 10⁻¹² M BPA, n = 7−8 for the other BPA concentrations. *p<0.05, ** p<0.01 in the statistical comparison between BPA-treated and control testes using the the Wilcoxon’s non-parametric paired test.</p

Effect of DES on testosterone secretion by human, rat and mouse fetal testes.

<p>Testes were removed from human fetuses at 6.5 to 10.5 weeks of gestation or from mouse and rat fetuses at 12.5 and 14.5 dpc, respectively. Like for the experiments presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051579#pone-0051579-g001" target="_blank">Figures 1</a> to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051579#pone-0051579-g003" target="_blank">3</a>, human rat and mouse testis explants were cultured in control medium (D0) for 24 h and then in the absence or presence of DES at 10⁻⁶ M or 10⁻⁵ M for the three subsequent days (D1–D3). Testosterone secretion in both treated and untreated samples at D1 to D3 was normalized to the D0 secretion of the same sample. The figure presents mean ± SEM of the D1 to D3 normalized values of treated samples expressed as the percentage of the control (untreated samples) values; n = 6, n = 7–10 and n = 7 for human, rat and mouse testes respectively. *p<0.05, ** p<0.01, *** p<0.001 in the statistical comparison between DES-treated and control testes using the Wilcoxon’s non-parametric paired test.</p

Effect of BPA exposure on germ cell differentiation in first trimester human fetal testis xenografts.

<p>Human fetal testes (9.1–11.3 GW) were xenografted into castrate Nude (host) mice. Host mice received vehicle (Control) or 10μM BPA in the drinking water for five weeks. (A) Histological sections of testes after immunostaining for AP-2γ (gonocytes). Positive (red arrows) and negative (black arrows) germ cells can be identified. Scale bar: 60 μm. (B) Quantification of AP-2γ-positive cells displayed as mean ± SEM (n = 9) on the left panel and as individual values with a line drawn between the control and the corresponding BPA-treated testis from the same fetus on the right panel. (C) Histological sections of testes after immunostaining for MAGE-A4 (prespermatogonia). Positive (red arrows) and negative (black arrows) germ cells can be identified. Scale bar: 60 μm. (D) Quantification of MAGE-A4-positive cells displayed as mean ± SEM (n = 8) on the left part and as individual values with a line drawn between the control and the corresponding BPA-treated testis from the same fetus on the right part. Data analyzed using the Wilcoxon paired-test. *p<0.05, **p<0.01.</p

Effect of BPA exposure on plasma BPA concentration in xenografted mice.

<p>Plasma levels of BPA were quantified by gas chromatography coupled to tandem mass spectrometry (GC-MS/MS) from castrated Nude male mice xenografted with first trimester human fetal testis (9.1–11.3 GW, mean 10.2 ± 0.2 GW; n = 6–7) and exposed to vehicle (Control) or BPA (10 μM in the drinking water) for five weeks. For each fetus, all the pieces from one testis were grafted in a control mouse and all the pieces from the contralateral testis were grafted in a BPA-treated mouse. Statistical analysis was performed using the Mann-Whitney test. *p<0.05, **p<0.01.</p

Effect of BPA exposure on germ cell density in first trimester human fetal testis xenografts.

<p>Human fetal testes (9.1–11.3 GW) were xenografted into castrate Nude (host) mice. Host mice received vehicle (Control) or 10μM BPA in the drinking water for five weeks. (A) Histological sections after haematoxylin-eosin-saffron staining. Germ cells (black arrow) can be easily identified. Scale bar: 20 μm. (B) Quantification of germ cell density displayed as mean ± SEM (n = 9) on the left panel and as individual values with a line drawn between the control and the corresponding BPA-treated testis from the same fetus on the right panel. Data analyzed using the Wilcoxon paired-test. *p<0.05 compared with control condition.</p

Effect of BPA exposure on germ cell apoptosis and proliferation in first trimester human fetal testes cultured using the FeTA system.

<p>Human fetal testes (6–12 GW, mean 8.7 ± 0.6 GW) were cultured using the ex vivo <u>h</u>uman <u>Fe</u>tal <u>T</u>estis <u>A</u>ssay system (hFeTA). After 24 hours in control medium, explants were cultured with 100 ng/mL of LH for the 3 subsequent days in the presence of ethanol vehicle (control explants) or BPA at concentrations ranging from 0.01 to 10 μM. Control and BPA-treated explants were paired samples from the same testis. (A) Histological sections after labeling with anti-cleaved caspase-3 antibody (brown) and anti-AMH antibody (green). Positive (red arrows) and negative (black arrows) germ cells can be identified. Scale bar: 10 μm. (B) Quantification of cleaved caspase-3 positive cells (mean ± SEM; n = 4–8). (C) Histological sections after labeling with anti-Ki-67 antibody (brown) and anti-AMH antibody (green). Positive (red arrows) and negative (black arrows) germ cells can be identified. Scale bar: 50 μm. (D) Quantification of Ki67 positive gonocytes (mean ± SEM; n = 4–8).</p