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 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 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 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 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

In human fetal gonads, exposure to MEHP significantly increases the mRNA expression of LXRα downstream genes involved in the cholesterol and lipid pathways.

<p>Ovaries and testes from 7 to 12 week/old human fetuses were cultured with or without 10⁻⁴ M MEHP for three days. mRNAs were isolated from whole gonads and the transcriptional level of LXRα downstream genes was analyzed by real-time qPCR in testes (A) and ovaries (B). Results were normalized to <i>Actin β</i> expression and are shown as fold changes relative to control values. Histograms represent the mean ± SEM of 4 to 8 different ovaries/testes from different fetuses (as indicated in the respective column). *p<0.05, **p<0.01, ***p<0.001 in paired t-tests recommended when comparing few samples.</p

MEHP exposure affects the expression of nuclear receptors in human fetal testes and ovaries.

<p>Testes and ovaries from 7 to 12 week-old human fetuses were cultured with or without 10⁻⁴ M MEHP for 3 days and then mRNAs were isolated from whole gonad. (A), NRs superfamily TLDA plates were run with whole testis samples. Results were normalized to <i>actin β</i> or <i>HMBS</i> expression and the 3 differentially expressed NRs are shown as fold changes relative to the control values. Histograms represent the mean ± standard error of the results using 4 independent fetal testis cultures. (B), Transcriptional level of <i>PPARγ</i>, <i>LXRα</i> and <i>NR4A1</i> was analyzed by real-time qPCR in fetal ovaries. Results were normalized to <i>Actin β</i> expression and are shown as fold changes relative to control values. Histograms represent the mean ± SEM of 4 different ovaries from different fetuses (as indicated in the respective column). *p<0.05, **p<0.01 in paired t-tests recommended when comparing few samples.</p

<i>In vitro</i> exposure to MEHP does not affect the number or apoptosis rate of germ cells in human fetal ovaries.

<p>Ovaries from 7 to 12 week/old human embryos were cultured with or without 10⁻⁴ M MEHP for three days. At the end of the culture period, they were fixed with Bouin’s solution and stained with hematoxylin and eosin. Oogonia density (number of germ cells per mm² tissue) was measured based on the morphological analysis (A) and germ cell apoptosis rate (B) based on the expression of cleaved Caspase-3 (brown) (C). Histograms represent the mean ± SEM of four different ovaries from different fetuses (as indicated in the columns). Arrowheads indicate oogonia and arrows cleaved Caspase- 3 positive oogonia. Bar, 15 µm.</p

mRNA expression of <i>PPARγ</i>, <i>LXRα</i> and <i>NR4A1</i> as well as of LXRα downstream genes in sorted germinal and somatic cells from fetal testes.

<p>Human fetal testes were treated, or not, with 10⁻⁴ M MEHP for 3 days in organotypic cultures. Testes were then dissociated and the M2A-positive and -negative cell fractions were sorted. The relative mRNA expression of <i>PPARγ</i>, <i>LXRα</i> and <i>NR4A1</i> (A) as well as of LXRα downstream genes involved in cholesterol and lipid synthesis (B) was quantified in the M2A-positive and -negative cell populations by real-time qPCR. Results were normalized to <i>actin β</i> expression and are shown as fold changes relative to controls. The black columns represent M2A-positive cells and the grey columns the M2A-negative cells. The histograms are the mean ± SEM of 4 to 8 different testis from different fetuses (as indicated in the respective column). *p<0.05, **p<0.01 in paired t-tests as recommended when comparing few samples.</p