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Fifty-two male Japanese quail (Coturnix japonica) obtained from the breeding colony established in our laboratory were castrated at the age of three weeks as previously described [49]. Animals were housed in isolation and allowed to recover for at least 3 weeks. The subjects were then randomly distributed into six experimental groups: sixteen subjects were implanted subcutaneously in the neck region with two 20 mm-long Silastic™ tubes (Silclear ™ Tubing, Degania Silicone, 1.57 mm i.d., 2.41 mm o.d.) that were empty (CX group, n = 7) or filled with crystalline testosterone (Sigma, T group, n = 9). These testosterone-filled implants restore in castrated male quail physiological levels of the steroid that are typical of sexually mature males and produce a full activation of male sexual behavior [50]. The remaining birds were injected daily in the pectoral muscle for 13 days with the general ER agonist diethylstilbestrol (Sigma; 250 µg in 50 µl vehicle, DES group, n = 9), the ERα specific agonist 4,4′,4″-(4-Propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol (Tocris, 250 µg in 50 µl vehicle, PPT group, n = 9), the ERβ specific agonist 2,3-bis(4-Hydroxyphenyl)-propionitrile (Tocris, 250 µg in 50 µl vehicle, DPN group, n = 9) or the vehicle propylene glycol (Sigma, 50 µl, PG group, n = 9). The cloacal gland, an androgen-dependent structure [51], [52], was measured with callipers (greatest width × greatest length in mm2) before and at the end of the experiment to confirm the effectiveness of the treatments. The body mass was also recorded at the same times. Throughout their life, birds were exposed to a photoperiod simulating long days (16 h light and 8 h dark per day) and had food and tap water available ad libitum. All experimental procedures were in agreement with the Belgian laws on the “Protection and Welfare of Animals” and on the “Protection of experimental animals” and were approved by the Ethics Committee for the Use of Animals at the University of Liège.
Behavioral testing and brain collection: The subjects were tested daily, alternatively for copulatory behavior (consummatory sexual behavior) or for rhythmic cloacal sphincter movements (appetitive sexual behavior) starting three days after the beginning of the treatments. The behavioral tests started approximately 2 h after the daily injection. To assess consummatory sexual behavior, the experimental bird was introduced into a test arena (60×40×50 cm) that contained a sexually mature female with which the male could freely interact. During these tests, the frequency of sexual behavior patterns including neck grabs (NG), mount attempts (MA), mounts (M) and cloacal contact movements (CCM) (see [53], [54] for a detailed description of these behaviors) was recorded by an observer blind to the treatment of the birds. Six tests were carried out during the thirteen experimentation days. To assess appetitive sexual behavior, the frequency of the rhythmic cloacal sphincter movements (RCSM) was quantified in a glass aquarium (40×20×25 cm) adjacent to another similar aquarium containing a female [55], [56]. A piece of opaque cardboard was attached to the exterior of the glass wall facing the experimenter to prevent the subject from being distracted by the presence of the experimenter. A mirror was placed at a 45° angle under the cage to allow the experimenter to view the cloacal area of the subject. A vertically sliding opaque panel was initially inserted between the two aquaria so that the experimental male could not see the female located in the second aquarium during 2.5 min. After this time, the sliding opaque panel was raised during the next 2.5 min. The male had then visual access to the female although he could not physically interact with her. The number of RCSM was recorded separately during the two 2.5 min periods when the male had or had not visual access to the female. The very low basal RCSM frequency observed in the absence of the female (usually less than 20) was then subtracted from the RCSM frequency observed in her presence to obtain a measure of the female-induced RCSM that is presented in the results. Four tests were carried out during the thirteen experimentation days. Twenty-four hours after the last behavioral test and the last injection, birds were killed by decapitation and checked for the completeness of castration and presence of Silastic™ implants (when relevant). All birds were found to exhibit complete castrations and all subjects in the CX and CX+T groups still possessed their hormone implants. Brains were dissected from the skull, fixed in 5% acroleine in phosphate buffered saline (PBS, 90 min), rinsed twice in buffer and cryoprotected in 30% sucrose for 48 h. They were then rapidly frozen on dry ice and kept at −80°C until used.
The brains were cut with a cryostat in the coronal plane from the level of the tractus septopallio-mesencephalicus to the caudal end of the tuberal hypothalamus. 30 µm-thick free-floating sections were collected in four series. Two series were stained by immunohistochemistry respectively for aromatase (ARO) and vasotocin (VT) as previously described and validated for quail (ARO: [11], [57]; VT: [16], [58]) with slight modifications. Briefly, sections were incubated for 15 min in 0.1% sodium borohydride in PBS and washed in PBS (pH 7.3–7.4). Endogenous peroxidase activity was blocked by incubating the sections for 20 min in 0.6% hydrogen peroxide, the non-specific antibody binding sites were blocked with 5% goat normal serum, and the sections were incubated with the primary antibody at 4°C (rabbit anti-quail recombinant ARO antibody, QR2/05 (gift from Prof. N. Harada, Fujita Health University, Toyoake, Japan) 1∶3000, overnight incubation) or rabbit anti-VT antibody (gift from Dr. D.G. Gray, Max Plank Institute of Bad Nauheim, Germany) 1∶5000, 48 hours incubation). Sections were then left for 2 h in secondary biotinylated goat anti-rabbit antibody (1∶400, Dako A/S, Gosltrup, Denmark) and finally incubated in ABC Vectastain elite Kit PK-6100 (Vector Laboratories) for 90 min. All reagents were in Phosphate-buffered saline 0.05 M containing 0.1% triton X-100 (PBST) and several rinses were performed between each step. The peroxidase enzymatic activity was then visualized with 3, 3′ diaminobenzidine tetrahydrochloride (DAB, 2%), 0.012% hydrogen peroxide in PBS (aromatase, ARO) or with DAB (2%), Nickel sulphate (25 mg/ml), 0.012% hydrogen peroxide in Sodium Acetate (0.175 M) (vasotocin, VT). Reaction was terminated by several rinses in PBS and the sections were mounted in an aqueous gelatin medium (aromatase) or in Eukitt® quick-hardening mounting medium (Sigma, vasotocin) and coverslipped.
Image acquisitions were performed by an observer blind to the treatment groups using a CCD camera (Model CFW-1612C, Scion Corporation, MD, USA) attached to an Olympus microscope and connected to a MacIntosh computer (Software: ImageJ, Wayne Rasband, NIH, Bethesda, MD, USA). We first calculated the volume of the medial preoptic nucleus (POM), defined by aromatase-immunoreactive-cells (See figure 1A.). The area of the nucleus was measured in all sections containing the nucleus throughout its rostro-caudal extent (objective 10X). Areas were then summed and multiplied by the sampling interval (120 µm; every 4th 30 µm section was stained) to derive an estimate of volume. In addition, we analyzed the vasotocinergic innervation of the POM by calculating the relative optical density of the immunoreactive signal observed a with a 20 × objective. The quantification field (460×0.350 µm = 0.161 mm2 at 20X) was placed in the corner formed by the ventral edge of the anterior commissure and the lateral edge of the third ventricle at the level where the anterior commissure reaches its largest extension. The field was then moved one field ventrally (350 µm) and the optical density of the entire computer field at this location was quantified (see figure 1B–C). The relative optical density (ROD) was defined as the difference between of the optical density (gray levels) measured (after calibration) within the POM and in an equivalent area located in a vasotocin-free location in the telencephalon on the same section (background).
Figure data removed from full text. Figure identifier and caption: 10.1371/journal.pone.0018627.g001 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.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.
Preliminary analyses indicated that there was, as expected, no significant difference between the two control groups (CX and PG) for all variables considered. Results from these two groups were thus pooled (CX/PG) in all statistical analyses presented here to increase their power. The effects of treatments on the proportion of subjects displaying a specific behavior at least once during the six copulatory tests (NG, MA, M, CCM) or one the four RCSM tests was assessed by Chi square tests. One-tailed Fisher's exact probability tests were then used to identify a potential effect of specific compounds compared to the control group since increases only could possibly be observed (these behaviors are absent in castrated birds). The mean cumulative behavioral frequencies summed over all tests for each subject (active or inactive) were analyzed by non-parametric Kruskall-Wallis analyses of variance that were followed when appropriate by the post-hoc comparison of all experimental groups with the control group with the Dunn's test. All statistical results for NG and MA, on the one hand, and for M and CCM, on another hand, were nearly identical and to avoid redundancy we shall only present results relative to MA and CCM. The size of the cloacal gland, the volume of the POM defined by aromatase-staining, and the density of vasotocinergic innervation in the POM were analyzed by parametric one-way analyses of variance (ANOVA) followed when appropriate by post-hoc Dunnett' tests comparing all groups with the controls. All statistical analyzes were performed with GraphPad Prism 5.0 for MacOS X (GraphPad Software Inc, La Jolla CA) and all data are expressed as mean ± SEM. Differences were considered significant for p<0.05.
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