Perinatal ovarian receptivity to estrogens.

<p>A–D: Quantitative RT-PCR for Esr1 (A), Esr2 (B), Gper (C) and Nr1i2 (D) performed on control ovaries at e18.5, day of birth (PND0) and PND12. Each point is constituted by three pools of at least four animals. Data points represent the mean ± SEM of the fold-change in target gene expression relative to a Snx17 reference gene and calibrator sample. Each point represents mRNA from 3 pools of ovaries from 3 animals. *p<0.05 (ANOVA, followed by PLSD test). <b>x</b> shows a statistically significant difference from e18.5 and <b>y</b> shows a statistically significant difference from PND0. E–P. <i>In situ</i> hybridizations for Esr1 (E, H), Esr2 (F, I, K), and Gper (G, J, L) in PND1 (E–G), PND6 (H-K) and PND2 (L-P) control ovaries show lower Esr1 expression in the ovary than in the oviduct epithelium (Ovd), higher expression of Esr2 in granulosa cells with follicle growth, and expression of Gper in the oocytes and granulosa cells. Inset in F shows a higher magnification of a group of follicles boxed in F. K shows a higher magnification of a primary (Iary and a secondary (IIary) follicles boxed in I. Inset in G shows another section of the ovary containing the oviduct. A comparison of <i>Gper</i> mRNA profile by <i>in situ</i> hybridization (L) with Esr2 (N, red) and Ybx2 (M, cytoplasmic, green) (and merged pictures O) by immunofluorescence revealed co-expression of both receptors in oocytes at the time of treatment. P shows hybridization with <i>Gper</i> sense probe. Scale bar: 100 µm except in insert in F and K (50 µm). Q-T. Quantitative RT-PCR for <i>Esr1</i> (Q), <i>Esr2</i> (R), <i>Gper</i> (S) and <i>Nr1i2</i> (T) using ovarian samples of controls (white bars) and animals treated with 10 µg E2 at PND0 (2 h after injection) and PND1 (black bars) shows E2 impairment of post-natal <i>Gper</i> up-regulation. Each bar represents mean ± SEM of the fold-change in target gene expression relative to a Snx17 reference gene and calibrator sample. Each point represents mRNAs from 3 to 6 pools of 6–16 and 10–26 ovaries, respectively. *p<0.05 (ANOVA, followed by PLSD test).</p

Plasma and hepatic reaction to acute estrogen exposure.

<p>A. Representative scheme of E2 biological activity and detoxification pathways. E2 can go through oxidative metabolism and be converted in E1 by 17bHsd2 or in hydroxyl-metabolites by enzymes of the Cyp family. Then it can be methylated by COMT, reduced by P450 reductase (resulting in DNA damage) or excreted after conjugative metabolism by GST. E2 can also be directly sulfo-conjugated by SULT or gluco-conjugated by UGT, and excreted. B–E. Plasma estradiol (B) and derivatives E2-sulfate (E2-S; C), estrone (D) and estrone-sulfate (E1-S; E) were measured by GC/MS from the day of birth (P0) to PND6 (P6). Each point represents the mean ± SEM of at least four pools of two animals each. A two-way ANOVA indicated that there was a significantly different profile for the 10 µg/d E2 treatment than for all other treated or control groups (p<0.001), E1 (p<0.001), E1-S (p = 0.002). Hormonal levels varied according to time, independent of treatment, for each metabolite tested: E1 (p<0.001), E2 (p<0.001), E1-S (p<0.001) and E2-S (p<0.001) (n = 4–5 pools of 3 to 4 animals at PND0, 4–5 pools of 2–4 animals at PND1, 4 pools of 2–3 animals at PND2, 4 pools of 2 animals at PND3 and 8 animals at PND6). F–J. Quantitative RT-PCR for <i>Hsd17b2</i> (F), <i>Ugt1a1</i> (G), <i>Cyp1b1</i> (H), <i>Cyp2b1/2</i> (I) and <i>Gsta2</i> (J) using liver samples of controls (white bars) and animals treated with 10 µg E2 at PND0 and PND1 (black bars) shows E2 impairment on post-natal <i>Hsd17b2</i>, <i>Cyp2b1/2</i> and <i>Gsta2</i> expression dynamics. Each bar represents mean ± SEM of the fold-change in target gene expression relative to a reference gene Snx17 and calibrator sample. Each point represents mRNA from 3 pools of ovaries from 3 animals. *p<0.05 (two-way ANOVA, followed by Tukey test). <b>a</b> shows an increase from PND0, <b>b</b> shows a decrease from PND1, <b>c</b> shows a decrease from PND3 and <b>*</b> shows a difference from the age-matched control group.</p

Ovarian reaction to acute estrogen exposure.

<p>A. A false-color heatmap shows cases of increasing, decreasing, detectable (but without differential expression) and undetectable transcript signal intensities across the replicates for different total ovary samples at the time points given (top). Each line corresponds to a probe set and each column to a sample replicate. A color scale is shown for signal intensity percentiles (bottom). Gene symbols and numbers of transcripts are shown at the right. B. Conventional RT-PCR screening of the expression of various enzymes involved in E2 metabolism in PND0, control (C) and E2-treated (E) PND1 and adult female ovaries, and PND0 livers reveals changes in expression of <i>Ugt1a1</i>, <i>Gsta2</i>, <i>Gstm5</i> between newborn and adult ovaries, stable expression of <i>Hsd17b2</i>, <i>Cyp1b1</i>, <i>Gstp1</i>, and the faint expression of <i>Sult1e1</i> and <i>Cyp2b1/2</i> by contrast to control <i>Snx17</i> RNAs. C–H. Quantitative RT-PCR for <i>Hsd17b2</i> (C), <i>Cyp1b1</i> (D), <i>Cyp2b1/2</i> (E), <i>Gsta2</i> (F), <i>Ugt1a1</i> (G) and the <i>Rbp4</i> E2 target gene (H) in ovaries of controls (white bars) and animals treated with 10 µg E2 (black bars) at PND0 (2 h after injection) and PND1 shows E2 impairment of post-natal <i>Gper</i> up-regulation. Each bar represents mean ± SEM of the fold-change in target gene expression relative to a Snx17 reference gene and calibrator sample. Each point represents mRNAs from 3 to 6 pools of 6–16 and 10–26 ovaries, respectively. *p<0.05 (two-way ANOVA, followed by Tukey test).</p

Primers used for qPCR and <i>in situ</i> hybridization (*).

<p>Primers used for qPCR and <i>in situ</i> hybridization (*).</p

Characterization of oocyte depletion.

<p>A. Time-course showing the decline in oocyte number per ovary in control (white points and dotted lines) and 10 µg E2-treated ovaries (black points and continuous lines) from PND0 to PND3. Data are expressed as mean ± SEM of 5–14 ovaries. *p<0.01 <i>vs.</i> age-matched controls (ANOVA followed by PLSD test). B. Time-course of apoptotic oocyte number per ovary in control (white points and dotted lines) and 10 µg E2-treated ovaries (black points and continuous lines) from PND0 to PND2. Data are expressed as mean ± SEM of 4–8 ovaries. C–D. Quantitative RT-PCR for <i>Bax</i> (C), <i>Bcl2</i> (D), <i>Nobox</i> (E), <i>Figla</i> (F), <i>Scp1</i> (G), <i>Hnrnpk</i> (H), <i>Foxo3a</i> (I) and <i>Eif4e</i> (J) in ovarian samples of controls (white bars) and animals treated with 10 µg E2 (black bars) at PND0 (2 h after injection) and PND1 shows transient down-regulation of <i>Bcl2</i> mRNA at PND0 and the absence of <i>Nobox</i> mRNA up-regulation between PND1 and PND2 in E2-treated ovaries. Each bar represents mean ± SEM of the fold-change in target gene expression relative to a Snx17 reference gene and calibrator sample). Data from 3–6 pools of 6–14 ovaries. *p<0.05 <i>vs.</i> age-matched control (two-way ANOVA, followed by t-test).</p

Estrogen dose-dependent depletion of oocytes.

<p>A–C. Immunolabeling of TRA98 in oocytes of control animals at PND3 (A) and females treated daily with 10 µg/d E2 between PND0 and PND2 (B). Higher magnification of TRA98 labeling shows oocyte-specific expression (C). Scale bar = 100 µm. D–E. Stereological estimation of ovarian volume (D) and oocyte numbers per ovary (E) at PND3 in controls, and as a function of E2 dose, show a dose-dependent decrease of both parameters. Each bar represents means ± SEM. Number of ovaries are indicated in brackets on the abscissa. *p<0.01; **p<0.001 (ANOVA followed by Fisher test).</p

Adult CYP20-exposed offspring displayed maladaptive behavior in response to highly stressful conditions.

<p><b>A</b>: The openfield was used as a weakly challenging task. Total distance travelled in the apparatus (1–2), time spent and the mean speed in different parts of the openfield were used to assess emotional reactivity (3). <b>B</b>: Emotional reactivity in response to highly challenging environments was evaluated by confronting mice to a novel cage with no sawdust. Total distance travelled was monitored over the total duration of the test (1). Data were also analyzed on a minute-by minute basis (2–3). <b>C-D</b>: Emotional reactivity in response to highly challenging environments was evaluated in the forced swimming task and the tail suspension task. In each condition, total time spent immobile was scored over the total duration of the test (1) and on a minute-by-minute basis (2). <b>E</b>: Habituation to a second exposure to stressful environments was assessed by confronting mice to the forced swimming task 24h later the first trial. Performance on day 2 is depicted on (1) and (2), and habituation between day 1 and day 2 is depicted on (3) and (4). Data are mean +/- sem; n = 20–21 /group. *** p < 0.001, ** p < 0.01, *p < 0.05 and # p < 0.09.</p

Genes commonly dysregulated in the brain of CYP20 and CYP5-exposed offspring.

<p>Fold change ≥ 1.2.</p

General toxicity induced by perinatal exposure to cypermethrin.

<p><b>A</b>: Cypermethrin did not modify maternal weight gain during pre and postnatal exposure. Maternal weight gain was evaluated every two days, during the pre and postnatal period. <b>B</b>: Each treatment session was associated with a clinical and behavioral assessment of the mothers (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184475#sec002" target="_blank">Materials and methods</a> for details). <b>C</b>: On the day of delivery, sex ratio within each group and the number of pups by litter were determined. Data are mean +/- sem; Experiment 1: n = 20-21/group; Experiment 2: n = 13-14/group. ** p < 0.01 and *** p < 0.001 compared to CTL.</p

Adult CYP5-exposed offspring displayed maladaptive behavior in response to highly stressful conditions.

<p><b>A</b>: The elevated plus maze (EPM) was used as a weakly challenging task. Total distance travelled in the apparatus (1–2), time spent and mean speed in different parts of the EPM were used to assess emotional reactivity (3). Time spent in stretched attend postures was also measured to collect data on risk assessment (4). <b>B</b>: Emotional reactivity in response to weakly challenging environments was also evaluated by confronting mice to a novel slightly lighted environment during 5 min: total distance travelled was monitored over the total duration of the test (1). Data were also analyzed on a minute-by minute basis (2). <b>C</b>: Emotional reactivity in response to highly challenging environments was evaluated in the forced swimming task by measuring total time spent immobile over the total duration of the test (1) and on a minute-by-minute basis (2). <b>D</b>: Habituation to a second exposure to stressful environments was assessed by confronting mice to the EPM 24h later the first trial. Distance travelled on day 2 is depicted on (1) and (2), and habituation between day 1 and day 2 is depicted on (3) and (4). (5) and (6) show how offspring habituate between day 1 and day 2 in terms of spatial exploration of the apparatus. Data are mean +/- sem; n = 13–14 / group. *p < 0.05, ** p < 0.01, *** p < 0.001.</p