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Sexual behavior in vertebrates depends on the cyclic release of steroids and their binding to the brain receptors. Previously, we demonstrated the presence of specific binding of 3H-testosterone and staining with PG-21 in the brain of the adult male frog, Rana esculenta. Here, we report our further receptor characterization using an anti–androgen receptor antiserum, PG-21, and the androgen site of action in frog brain. Nuclei, which contained cells labeled for the androgen receptor (AR), were mainly identified in the olfactory bulbs, preoptic-septal region, infundibulum, amygdala, thalamus, tectum, torus semicircularis, and medulla. The neuroanatomical AR staining appears similar to that in other lower vertebrates.
Throughout vertebrate classes, androgens exert important functions in male reproduction.1 Autoradiography has been used successfully to describe androgen-concentrating neurons in mammals, birds, reptiles, amphibians, and fish.2 Thus, implants of testosterone in the rostral hypothalamus of castrated Rana pipiens restore male sexual behavior.3 The mechanism of action of androgens in the brain is related to aromatase activity,4 and binding with estrogen and androgen receptors is believed to be an initial event in the mechanism of central androgenic mediated action.5 In the present study, the neuroanatomical distribution of androgen receptor (AR) immunoreactivity (ir) has been investigated in the brain of the adult male frog, Rana esculenta, using an anti-AR antiserum.
Adult male frogs, Rana esculenta, collected in the breeding season were submitted to transcardial perfusion with saline and the brains were removed immediately following decapitation. For immunohistochemical procedures, the brains were dissected, fixed, cryoprotected, and stored in liquid nitrogen until sectioned. For biochemical procedures, the hypothalamic region was sectioned under a stereomicroscope using the following margins: anterior, septo-mesencephalic tract; posterior, where the third nerve enters the brain; dorsal, the level of the anterior commissure. Hypothalamic and extrahypothalamic areas were pooled into two different groups. Preparation of cytosolic and nuclear extracts of hypothalamic and extrahypothalamic areas was performed according to standard procedures reported in ref. 5. The anti-AR specificity of the PG-21 serum was tested, using immunohistochemical and biochemical studies, by omission of the primary antiserum, with a biotinylated antirabbit IgG, by competition studies6,7 and by Western blotting.8
Immunostaining only appeared in cell nuclei, whereas no detectable reaction product could be observed in the cytoplasm. FIGURE 1 shows that AR-ir is widely distributed throughout the brain, namely, in the olfactory bulbs (OB), striatum (ST), septum (SP), amygdala (AMY), thalamus (TH), preoptic area (POA), tectum (TEC), torus semicircularis (TOR), infundibulum (INF), pituitary (PIT), interpeduncular nucleus (IPN), cerebellum (CB), and motor nuclei of the medulla (MED). Highest density was observed in the hypothalamus. FIGURE 2 demonstrates immunoreactivity in the cytosolic and nuclear extracts of the hypothalamic and extrahypothalamic areas of the male frog, Rana esculenta, with PG-21 and the antirabbit IgG. PG-21 cross-reacted only in the nuclear extract of both brain areas, with a band corresponding to a molecular weight of about 110 kDa.
Our results show the distribution of AR-ir in the brain of a lower vertebrate: the anuran, Rana esculenta. The major areas of AR-ir in the male brain of the breeding period are the OB, SP, ST, AMY, POA, TEC, TOR, INF, and MED. A similar pattern is reported in the brain of other lower vertebrates: the urodele, Taricha granulosa;9 and the reptile, Sceloporus undulatus.6 Furthermore, as in urodela, ARs in anura occur in more brain regions than in mammals and fish.10–13 No autoradiographic evidence has been provided for the androgen site in TH, PIT, IPN, and CB of Rana esculenta.14 AR in vertebrates shows a large variability in molecular weight, but Western analysis in Rana esculenta reveals one immunoreactive band, with an apparent molecular weight of about 110 kDa. Our data reinforce our previous biochemical studies of androgen-binding molecules in the nuclear extract of frog brain with the characteristics of a true receptor15 and with the majority of the 3H-T binding activity attributable to the hypothalamic area. Further research may help to define the role of the AR in different brain areas and throughout the reproductive cycle.