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

Sex differences in the percentage of DAPI cells that were immunoreactive for parvalbumin (%PV-ir cells) or were surrounded by perineuronal nets (%PNN cells), in the percentage of PV-ir cells surrounded by PNN (%PV with PNN) and in the percentage of PNN surrounding PV-ir cells (%PNN with PV) in 4 song control nuclei, HVC (used as a proper name), RA (robust nucleus of the arcopallium), Area X of the basal ganglia and LMAN (lateral magnocellular nucleus of the anterior nidopallium).

<p>The figure shows the mean ± SEM of data in males (black bars) and females (open bars). Numbers of data in each case are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123199#pone.0123199.t001" target="_blank">Table 1</a>. ** = p<0.01, * = p<0.05 and (*) = 0.10</p

Representative photomicrographs illustrating the distribution of chrondroitin sulfate immunoreactivity (Green signal) and of parvalbumin-immunoreactive cells (red signal) as observed in in parasagittal sections of male (C, C) and female (B, D) zebra finch brains.

<p>Panels A_B illustrate differences in HVC, panels C-D concern RA. The magnification bar in D (20 μm) also applies to all other panels.</p

Sex differences in the percentage of DAPI cells that were immunoreactive for parvalbumin (%PV-ir cells) or were surrounded by perineuronal nets (%PNN cells), in the percentage of PV-ir cells surrounded by PNN (%PV with PNN) and in the percentage of PNN surrounding PV-ir cells (%PNN with PV) in 3 auditory areas (Field L; the dorsal lateral mesencephalic nucleus, MLd; the medial nucleus of the dorsolateral thalamus, DLM), and 3 visual areas (the nucleus rotondus, ROT; the nucleus spiriformis lateralis, SpL; the nucleus subpretectalis, SP).

<p>The figure shows the mean ± SEM of data in males (black bars) and females (open bars). Numbers of data in each case are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123199#pone.0123199.t001" target="_blank">Table 1</a>. * = p<0.05 and (*) = 0.10</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

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

Sex differences in the numbers of DAPI positive cells, in numbers of parvalbumin-immunoreactive (PV-ir) cells, of perineuronal nets (PNN) and in PV-ir cells surrounded by PN (PV+PNN), and in the intensity and percentage of surface covered by chondroitin sulfate-immunoreactive material (PNN intensity and PNN fraction area) in 4 song control nuclei and 3 auditory and 3 visual areas of the zebra finch brain.

<p>In each case the table lists the mean + SEM (in parentheses) of values in males and females, followed by the associated F and p obtained in the corresponding ANOVA (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123199#sec002" target="_blank">methods</a> for detail of statistical analyses). The sample size in each case is indicated in a last column separately for all cell counts first and then for PNN intensity and fractional areas.</p><p>*** = p<0.001</p><p>** = p<0.01, = p<0.05 and</p><p>(*) = 0.10</p><p>Sex differences in the numbers of DAPI positive cells, in numbers of parvalbumin-immunoreactive (PV-ir) cells, of perineuronal nets (PNN) and in PV-ir cells surrounded by PN (PV+PNN), and in the intensity and percentage of surface covered by chondroitin sulfate-immunoreactive material (PNN intensity and PNN fraction area) in 4 song control nuclei and 3 auditory and 3 visual areas of the zebra finch brain.</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