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

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

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

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

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

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

Plasma 11-deoxycorticosterone (DOC) and mineralocorticoid receptor testicular expression during rainbow trout spermiation: implication with 17alpha, 20beta-dihydroxyprogesterone on the milt fluidity?-0

(pg/ml) (B) and in testis mineralocorticoid receptor (rtMR) mRNA abundance (C) according to time of year and stage of sexual maturation. Means+ SD (n = 3–5). Mature males were only studied until December. with from a spring spawning strain. Kinetics of spermiation in a group of mature males, assessed by the spermiation index (D). Coincident changes in blood plasma DOC concentrations (pg/ml) (E) and in blood plasma MIS (ng/ml) (F) according to time of year. Means+ SD (n = 8). Different letters indicate significant differences between months. * Significant difference between immature and maturing/mature fish, p < 0.05.<p><b>Copyright information:</b></p><p>Taken from "Plasma 11-deoxycorticosterone (DOC) and mineralocorticoid receptor testicular expression during rainbow trout spermiation: implication with 17alpha, 20beta-dihydroxyprogesterone on the milt fluidity?"</p><p>http://www.rbej.com/content/6/1/19</p><p>Reproductive biology and endocrinology : RB&E 2008;6():19-19.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2430966.</p><p></p

Plasma 11-deoxycorticosterone (DOC) and mineralocorticoid receptor testicular expression during rainbow trout spermiation: implication with 17alpha, 20beta-dihydroxyprogesterone on the milt fluidity?-3

T different times post treatment. Means+ SD (n= 48–66). (B) Daily decomposition of this treatment effect. Means+ SD (n = 6–18). Different letters indicate significant differences between treatments, p < 0.05.<p><b>Copyright information:</b></p><p>Taken from "Plasma 11-deoxycorticosterone (DOC) and mineralocorticoid receptor testicular expression during rainbow trout spermiation: implication with 17alpha, 20beta-dihydroxyprogesterone on the milt fluidity?"</p><p>http://www.rbej.com/content/6/1/19</p><p>Reproductive biology and endocrinology : RB&E 2008;6():19-19.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2430966.</p><p></p

Plasma 11-deoxycorticosterone (DOC) and mineralocorticoid receptor testicular expression during rainbow trout spermiation: implication with 17alpha, 20beta-dihydroxyprogesterone on the milt fluidity?-2

Ndicate significant differences between treatments, p < 0.05.<p><b>Copyright information:</b></p><p>Taken from "Plasma 11-deoxycorticosterone (DOC) and mineralocorticoid receptor testicular expression during rainbow trout spermiation: implication with 17alpha, 20beta-dihydroxyprogesterone on the milt fluidity?"</p><p>http://www.rbej.com/content/6/1/19</p><p>Reproductive biology and endocrinology : RB&E 2008;6():19-19.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2430966.</p><p></p

Plasma 11-deoxycorticosterone (DOC) and mineralocorticoid receptor testicular expression during rainbow trout spermiation: implication with 17alpha, 20beta-dihydroxyprogesterone on the milt fluidity?-5

(pg/ml) (B) and in testis mineralocorticoid receptor (rtMR) mRNA abundance (C) according to time of year and stage of sexual maturation. Means+ SD (n = 3–5). Mature males were only studied until December. with from a spring spawning strain. Kinetics of spermiation in a group of mature males, assessed by the spermiation index (D). Coincident changes in blood plasma DOC concentrations (pg/ml) (E) and in blood plasma MIS (ng/ml) (F) according to time of year. Means+ SD (n = 8). Different letters indicate significant differences between months. * Significant difference between immature and maturing/mature fish, p < 0.05.<p><b>Copyright information:</b></p><p>Taken from "Plasma 11-deoxycorticosterone (DOC) and mineralocorticoid receptor testicular expression during rainbow trout spermiation: implication with 17alpha, 20beta-dihydroxyprogesterone on the milt fluidity?"</p><p>http://www.rbej.com/content/6/1/19</p><p>Reproductive biology and endocrinology : RB&E 2008;6():19-19.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2430966.</p><p></p