Intracellular partitioning of androgen receptor immunoreactivity in the brain of the male syrian hamster: Effects of castration and steroid replacement

Abstract

The effect of castration and steroid replacement on the intracellular partitioning of the androgen receptor in the brain of the male Syrian hamster was determined using immunocytochemistry. Androgen receptors were visualized using the PG-21 antibody (G. S. Prins) on 40-microns coronal brain sections from hamsters perfused with 4% paraformaldehyde with or without 0.4% glutaraldehyde. Control studies confirmed antibody specificity in gonad-intact and castrate males. In the normal adult male, androgen receptor immunocytochemistry reveals intense staining confined to the cell nucleus. Castration caused a gradual increase in cytoplasmic labelling within 2 weeks, accompanied by a reduction in nuclear staining intensity in androgen receptor-containing neurons throughout the brain. Cytoplasmic androgen receptor staining was eliminated after treatment of orchidectomized males for only 8 h with exogenous testosterone. Likewise, long-term exposure to testosterone and dihydrotestosterone, a nonaromatizable androgen, maintained nuclear androgen receptor immunoreactivity. However, exposure to low physiologic concentrations of estrogen was not effective in this regard. In addition, we determined that nuclear androgen receptor immunoreactivity decreases in response to inhibitory short-day photoperiod, but without an increase in cytoplasmic immunostaining. This appears to be due to the decrease in androgen production by the testis, rather than a direct photoperiodic effect, because testosterone supplementation to short-day males restored the intensity of nuclear androgen receptor immunoreactivity to levels comparable to those in the intact male. These findings are compatible with a new model for the intracellular localization of androgen receptors, in which a subset of unoccupied receptors is located in the cell cytoplasm in the absence of ligand. They further demonstrate the repartitioning of such cytoplasmic receptors, thereby confirming and extending previous observations using biochemical techniques on the regulation of neuronal androgen receptors.

Use of PG-21 immunocytochemistry to detect androgen receptors in the songbird brain.

“…Androgen binding increases nuclear localization of AR protein in transfected COS cells (Kemppainen et al, 1992;Simental et al, 1991). In both the paraventricular nucleus of hamsters and in ventral and dorsal lobes of rat prostate, castration decreased nuclear and increased cytoplasmic staining of ARs, and T treatment rapidly reversed this effect (Prins and Birch, 1993;Wood and Newman, 1992). Cytoplasmic ARs may be difficult to detect by ICC, possibly because they are distributed more diffusely in the cytoplasm than in the nucleus (Wood and Newman, 1992).…”

“…In both the paraventricular nucleus of hamsters and in ventral and dorsal lobes of rat prostate, castration decreased nuclear and increased cytoplasmic staining of ARs, and T treatment rapidly reversed this effect (Prins and Birch, 1993;Wood and Newman, 1992). Cytoplasmic ARs may be difficult to detect by ICC, possibly because they are distributed more diffusely in the cytoplasm than in the nucleus (Wood and Newman, 1992). Finally, it is also possible that the PG-21 antibody has a higher affinity for occupied than for unoccupied receptors.…”

“…It is possible that modifications of our protocol may enable the use of this antibody to label ARs in other systems. For example, our protocol might be modified for use on nonmammalian tissue fixed by perfusion or processed free-floating, as has been done with mammalian brain tissue (Iqbal et al, 1995;Wood and Newman, 1992).…”

“…Given these results, we explored the use of a polyclonal rabbit antibody (PG-21) that is raised against amino acids 1-21 of the rat AR (Prins et al, 1991). This antibody has been successfully used to label ARs in a variety of tissues in mammals and fish (Freeman et al, 1995;Iqbal et al, 1995;Ciofi et al, 1994;Gustavson et al, 1994;Wood and Newman, 1992;Prim et al, 1991). Using the PG-21 antibody, we selectively labeled AR+ cells in the song nuclei and hypothalamic regions of songbirds.…”

An alternate pathway for androgen regulation of brain function: Activation of estrogen receptor beta by the metabolite of dihydrotestosterone, 5α-androstane-3β,17β-diol

“…Similarly, a number of approaches have been taken to localize AR in neural tissues, including in vivo autoradiography (Sar and Stumpf, 1980), in vitro binding in microdissected brain tissues (Handa et al 1987), immunohistochemistry (Bingaman et al 1994a, b;Wood andNewman, 1993 andZhou et al, 1994), and in situ hybridization (Kerr et al 1995, Simerly et al, 1991. These studies have all shown that the AR is expressed in brain areas now known to mediate reproductive functions.…”