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 plasma testosterone level and seminal vesicle weight in the host mice xenografted with first trimester human testes and on steroidogenic genes expression in the xenografts.

<p>Human fetal testes (9.1–11.3 GW) were xenografted into castrate Nude (host) mice. Host mice received vehicle (Control) or 10 μM BPA in the drinking water for five weeks. (A) seminal vesicle weight displayed as mean ± SEM (n = 6) on the left panel and as individual values with a line drawn between the control and the corresponding BPA-exposed testis from the same fetus on the right panel. B) plasma testosterone concentration in host mice displayed as mean ± SEM (n = 6) on the left panel and as individual values with a line drawn between the control and the corresponding BPA-exposed testis from the same fetus on the right panel. C) Expression of key genes in the steroidogenic pathway (STAR, CYP11A1, CYP17A1 and CYP19) using quantitative RT-PCR standardized to either β-ACTIN (ACTIN) or RPLP0 or CYPA as endogenous control. Results are presented as a percentage of the control value (mean ± SEM; n = 4). Data analyzed by Wilcoxon test. No significant difference between BPA-treated and control mice were identified.</p

Effect of BPA exposure on testosterone plasma level and seminal vesicle weight in the host mice carrying second trimester human fetal testis xenografts.

<p>Human fetal testes (14-18GW) xenografted into castrated Nude (host) mice. Each human fetal testis was grafted into 1–3 host mice which received the same treatment. Host mice received vehicle (Control), BPA (0.5μg/kg or 50μg/kg/d) daily by oral gavage for five weeks. A) seminal vesicle weight as overall mean ± SEM (n = 4) is displayed on the left with individual values on the right. B) plasma testosterone concentration in host mice as overall mean ± SEM (n = 4) is displayed on the left with individual values on the right. Data analysed by Mann-Whitney test. No significant differences were observed for serum testosterone or seminal vesicle weight between BPA-treated mice compared to control.</p

Xenografting first trimester human fetal testis.

<p>Human fetal testes (9.1–11.3 GW) xenografted in castrated Nude (host) mice. Host mice received vehicle (Control) or 10μM BPA in the drinking water. (A) Host mouse six weeks after xenografting demonstrating xenograft tissue below the skin (green circles). (B) Xenografted explants (green circles) in the back muscle of the host mouse at the end of the experiment, in a control and a BPA-treated mouse. Scale bar: 5 mm. (C) Histological section of a xenograft after haematoxylin-eosin-saffron staining, T: testis; M: mouse muscle; C: connective tissue. Scale bar: 100 μm. (D) Histological section of a xenograft showing a blood vessel (BV) in the interstitial tissue. LC: Leydig cell. Scale bar: 10 μm.</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

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

Effects of BPA on testosterone production after 72h of culture of 7–12 GW human fetal testis explants.

<p>A) Dose-dependent effects of BPA—diluted in DMSO or ethanol—with hLH on testosterone production by human fetal testis explants (RTP; RTP, %Ctrl): BPA was diluted in DMSO or ethanol and the media collected after 72 hr of culture were assayed. Results are expressed as normalized production of testosterone of treated samples as the percentage of that of the respective untreated first day of culture sample (RTP) (top) and as the percentage of that of the respective untreated first day of culture sample and control (RTP, %Ctrl) (bottom). Values are mean +/- SEM of testosterone from the respective untreated first day of culture basal sample and control. The number of testes (n) is indicated below the graphic for each condition. Dose-responses were analyzed for significance with a Wilcoxon test. The effects of the vehicle were analyzed with two-way ANOVA. *p<0.05. B) Testosterone production after culture of human fetal testis in the presence of BPA and hCG, hLH or no gonadotrophin (-gonado) supplementing the medium (RTP). n = 7–13 testes for the hCG group, n = 3–20 testes for hLH group and n = 9–21 in the absence of gonadotrophin. *p<0.05, ***p<0.001 (two-way ANOVA followed by a Holm-Sidak test or a Wilcoxon test to compare matched samples). C) Testosterone production represented as a fold change from the respective first day of culture sample and control (RTP, %Ctrl). Results are expressed as normalized production of testosterone of treated samples as the percentage of that of the respective untreated first day of culture sample (RTP) and control (RTP, %Ctrl). n = 7–13 testes for the hCG group, n = 3–20 testes for hLH group and n = 9–21 in the absence of gonadotrophin. *p<0.05, ***p<0.001 (two-way ANOVA followed by a Holm-Sidak test or a Wilcoxon test to compare matched samples).</p

Representative immunostaining of CYP11A1 of human fetal testis explants 11–12 GW.

<p>The 3,3-diaminobenzidine tetrahydrochloride staining appears dark brown in all panels, and sections were counterstained with hematoxylin. Testis cords could be easily delineated in all treated explants except in the sections of explants that have been exposed to BPA 10^-5M without gonadotrophin, in which the boundaries of the testicular cords appeared somewhat blunted. Scale bar corresponds to 100 μm.</p

INSL3 production after culture of 8–12 GW human fetal testis explants in absence (Ctrl) or presence of 10^-8M-10^-5M.

<p>A) INSL3 production after culture of human fetal testis explants in the presence of BPA and hCG, hLH or no gonadotrophin (-gonado) supplementing the medium (RIP). INSL3 concentrations after culture of 8–12 GW human fetal testis in the presence of ethanol (Ctrl) or from 10^-8–10^-5 M BPA. Results are expressed as normalized production of INSL3 of treated samples as the percentage of that of the respective untreated first day of culture sample (RIP). n = 3–9 for hLH group, n = 3–11 testes for hCG group and n = 6–9 for the group without gonadotrophin. Values are means +/- SEM. *p<0.05, **p<0.01, ***p<0.001 (two-way ANOVA, followed by a Holm-Sidak test or a Wilcoxon test to compare matched sample). B) INSL3 production represented as normalized production of INSL3 of treated samples as the percentage of that of the respective untreated first day of culture sample and control (RIP, %Ctrl). INSL3 concentrations after culture of 8–12 GW human fetal testis explants in the presence of ethanol (Ctrl) or from 10^-8–10^-5 M BPA. Results are expressed as normalized production of INSL3 of treated samples as the percentage of that of the respective untreated first day of culture sample and control (RIP, %Ctrl). n = 3–9 for hLH group, n = 3–11 for hCG group and n = 6–9 for the group without gonadotrophin. Values are expressed as least squares means +/- SE. *p<0.05, **p<0.01, ***p<0.001 (two-way ANOVA, followed by a Holm-Sidak test or a Wilcoxon test to compare matched sample).</p