Percentage of birds that displayed at least one mount attempt (A) or one cloacal contact movement (B) and cumulative frequencies of these two behaviors in the 5 experimental groups (C–D).

<p>Data were analyzed by appropriate analyses of variance or χ2 tests that were followed by post-hoc tests specifically comparing the 4 experimental groups to the controls (see text). Results of these post-hoc comparisons are shown at the top of the bars as follows: * =  p<0.05, ** =  p<0.01 and *** =  p<0.001.</p

Labelling indices and deduced BrdU concentrations after incubation of HEK293T cells with serum from BrdU-injected animals.

<p>(A). Percentage (means ± SD) of the total numbers of cells visible in culture that were immunoreactive for BrdU after incubation with serum collected from adult canaries at various times after BrdU injection. All individual male (black spots) and female (open circles) data points are represented in the figure. (B) Average concentrations (means ± SD) of BrdU in these canary samples based on the calibration curve illustrated in the inset of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063692#pone-0063692-g001" target="_blank">Figure 1G</a>. (C–D) Concentrations of BrdU in blood samples collected in two female Japanese quail (C) or one male mouse (D) at various times after a single BrdU injection.</p

Aromatase activity (mean±SEM) measured in different regions of gonadally intact male and female Japanese quail.

<p><b>A</b>. A mixed-design ANOVA with all regions considered individually revealed a significant effect of sex (F_1,17 = 9.284; p = 0.007) and brain region (repeated factor; F_5,85 = 20.640; p<0.001) as well as an interaction between these two factors (F_5,85 = 2.473; p = 0.038). <b>B</b>. A mixed-design ANOVA of results after pooling data for POM and mBST as well as for VMN and tuber (MBH) revealed significant effects of the sex (F_1,17 = 9.407; p = 0.007) and regions (F_3,51 = 43.392; p<0.001) as well as a significant interaction (F_3,51 = 4.776; p = 0.005). Individual means were compared by the Fisher Protected Least Significance Difference test. Comparisons between males and females within a given region are reported at the top of the columns as follows: (*) 0.05 </p

Plasma corticosterone concentrations measured in the 4 experimental groups before the experiment and after 4 and 21 days of exposure to the different social conditions.

<p>* = p<0.05.</p

Dose-response curve of labelling index after incubation of HEK293T cells with BrdU.

<p>(A–F) Cultured cells stained for BrdU, after incubation with BrdU at different concentrations (as indicated at the bottom of each image). Images were consistently modified to enhance contrast and remove colour, and are all shown to the same scale. (A) At very low concentrations, very few cells were labelled even weakly. (B–F) As BrdU concentration increased, the proportion of visible cells that were weakly-labelled (w), moderately-labelled (m), or strongly-labelled (s) increased. Every culture plate also contained some unlabelled cells (arrowhead). (G) Total numbers of labelled cells (weakly+moderately+strongly) reached a plateau with BrdU concentrations above 50 µg/mL; note the break in the x-axis scale<b>.</b> At lower concentrations, however, (inset) there was a strong linear relationship between BrdU concentration and the total number of labelled cells (Y = 1.651 X, line forced through [0,0]; confidence interval for slope = 1.403 to 1.898, r = 0.9606, p<0.0001). Dashed lines show 95% confidence interval around the (solid) line of best fit.</p

Linear regression of number of EdU-positive cells versus number of BrdU-positive cells in the VZ of the combined sections of birds injected with EdU only 4 and 24 hours before brain collection.

<p>Linear regression of number of EdU-positive cells versus number of BrdU-positive cells in the VZ of the combined sections of birds injected with EdU only 4 and 24 hours before brain collection.</p

Effect of the embryonic treatments with estradiol benzoate (EB) or Vorozole (VOR) on the cloacal gland area and on different aspects of male sexual behavior of gonadectomized male (black bars) and female (white bars) quail that were treated as adults with exogenous testosterone.

<p>All data are means ± SEM. The number of subjects per group is indicated in bars of panel A. <b>A</b>. The cloacal gland area was significantly affected by the sex of the birds (F_1,54 = 26.068; p<0.001) and their treatments (F_2,54 = 27.798; p<0.001) but there was no interaction between these two factors (F_2,54 = 1.836; p = 0.169). <b>B</b>. The frequency of female-elicited rhythmic contractions of sphincter muscles (RCSM) did not differ between sexes (F_1,53 = 0.208; p = 0.650) or treatments (F_2,53 = 0.493; p = 0.613) and was not influenced by the interaction of these two factors (F_2,53 = 0.817; p = 0.447). <b>C</b>. The total frequency of mount attempts (MA) displayed during the 3 copulatory tests significantly differed between sexes (F_1,54 = 5.879, p = 0.0187) and treatments (F_2,54 = 7.989, p = 0.009) and an interaction between the 2 factors was detected (F_2,54 = 6.808, p = 0.002). <b>D</b>. The mount attempt (MA) latency on the last copulatory test was also different between sexes (F_1,54 = 12.104, p = 0.001) and treatments (F_2,54 = 32.564, p<0.001) and a significant interaction was found (F_2,54 = 8.464, p<0.001). <b>E</b>. The total frequency of cloacal contact movements (CCM) displayed during the 3 copulatory tests significantly differed between sexes (F_1,54 = 5.106, p = 0.0279) and treatments (F_2,54 = 10.975, p<0.001) and an interaction between the 2 factors was detected (F_2,54 = 1.134, p = 0.329). <b>F</b>. The cloacal contact movements latency on the last copulatory test was also different between sexes (F_1,54 = 1.895, p = 0.174) and treatments (F_2,54 = 8.497, p<0.001) and a significant interaction was found (F_2,54 = 0.449, p = 0.640). When appropriate, individual means were compared by Fisher Protected Least Significance Difference test. Results are reported at the top individual bars as follows: * p<0.05 compared to vehicle injection (VEH) same sex, Δ p<0.05 compared to Vorozole injection (VOR) same sex, # p<0.05 compared to males same treatment, and ◊ p<0.05 compared to other treatment in both sexes (no interaction in this analysis).</p

Photomicrographs illustrating the aromatase-immunoreactive perikarya (A) and the vasotocin-immunoreactive fibers (B, C) present within the medial preoptic nucleus (POM) that were quantified in the present study.

<p>Panel A illustrates the dense group of aromatase-immunoreactive neurons that outline the entire POM. The dotted line marks the limits of the POM as they were defined for quantification. Panel B shows the accumulation of vasotocin-immunoreactive fibers in the POM at the level of the anterior commissure. The rectangle drawn with a solid line indicates the area where quantification was performed that is illustrated at higher magnification in panel C. The dotted rectangle indicates how the camera field was originally placed before being moved to its final location (see text). Note that quantification of fibers concerned the steroid-sensitive network located in the POM, not the denser network located more ventrally that originates from the magnocellular neurons. CA: commissural anterior, LFB: latera forebrain bundle. Magnification bar =  500 µm in A–B, 100 µm in C.</p

Effect of embryonic treatments with estradiol benzoate (EB) or Vorozole (VOR) on aromatase activity (AA; mean ± SEM) in the brain of adult gonadectomized male (black bars) and female (white bars) quail that were treated as adults with exogenous testosterone.

<p>Figures in the bars represent the numbers of final data points available for each group. Results for each nucleus were analyzed by a two-way ANOVA with the sex of the birds (Sex) and their embryonic treatment (TRT) as factors. Interactions (INT) between these factors were also evaluated. Data are illustrated for <b>A</b>) the medial preoptic nucleus (POM; Sex: F_1,54 = 0.382, p = 0.539, TRT: F_2,54 = 5.093, p = 0.009, Int: F_2,54 = 0.355, p = 0.703), <b>B</b>) the medial bed nucleus of the stria terminalis (mBST; Sex: F_1,52 = 0.810, p = 0.372, TRT: F_2,52 = 7.192, p = 0.002, Int: F_2,52 = 1.870, p = 0.164), <b>C</b>) the nucleus taeniae of the amygdala (TnA; Sex: F_1,53 = 1.408, p = 0.241, TRT: F_2,53 = 0.791, p = 0.459, Int: F_2,53 = 0.107, p = 0.899), <b>D</b>) the ventromedial nucleus of the hypothalamus (VMN; Sex: F_1,53 = 4.252, p = 0.044, TRT: F_2,53 = 0.170, p = 0.844, Int: F_2,53 = 0.276, p = 0.760); <b>E</b>) the tuber (Sex: F_1,54 = 0.230, p = 0.636, TRT: F_2,54 = 1.640, p = 0.204, Int: F_2,54 = 0.030, p = 0.975), <b>F</b>) the periacqueductal gray (PAG; Sex: F_1,52 = 0.039, p = 0.844, TRT: F_2,52 = 1.758, p = 0.183, Int: F_2,52 = 1.360, p = 0.266), <b>G</b>) the POM and mBST together (Sex: F_1,52 = 0.475, p = 0.494, TRT: F_2,52 = 7.901, p = 0.001, Int: F_2,52 = 1.162, p = 0.321) and <b>H</b>) the mediobasal hypothalamus (MBH = VMN+Tuber; Sex: F_1,53 = 1.428; p = 0.237, TRT: F_2,53 = 0.419; p = 0.660, Int: F_2,53 = 0.271; p = 0.764). Origins of the main effects of TRT in the POM, mBST and POM+mBST were analyzed by post-hoc Fisher Protected Least Significance Difference tests. Results are reported at the top of the two columns as follows: * p<0.05 compared to different treatment in both sexes.</p

Schematic drawings of coronal sections through the quail brain from anterior to posterior illustrating the areas that were punched to quantify aromatase activity.

<p>The circle diameter reflects the diameter of the micropunch needle (o.d. 0.8 or 1.2 mm) used to collect the tissue. Abbreviations: mBST, medial portion of the bed nucleus of the stria terminalis; CA, commissura anterior; Cb, cerebellum; CO, optic chiasma; CP, commissura posterior; DSD, decussatio supraoptica dorsalis; FPL, Fasciculus prosencephalii lateralis (lateral forebrain bundle); Tub, Tuber; PAG, periacqueductal gray; POM, medial preoptic nucleus; TSM, tractus septopallio-mesencephalicus; VIII, third ventricle; VL, ventriculus lateralis; VMN, ventromedial nucleus of the hypothalamus; TnA, nucleus taeniae of the amygdala.</p