Immunocytochemical Distribution of Aromatase Cytochrome P450 in the Rat Brain Using Peptide-Generated Polyclonal Antibodies*

Abstract

Estrogen formation is catalyzed by the aromatase cytochrome P450 (P450AROM) enzyme. Aromatase activity has been detected in several regions in the rat brain. In the present study, we used peptide-generated polyclonal antibodies raised against a 20-amino acid synthetic fragment of the rat P450AROM protein (as deduced from the nucleic acid sequence of the rat P450AROM complementary DNA), to determine the location of this enzyme in rat brain sections. Immunoreactive antisera were titered by means of an enzyme-linked immunosorbent assay and purified by diethylaminoethyl-Affigel Blue chromatography. Specific immunoreactivity was confirmed by Western blot analysis using known aromatase-containing tissue (rat ovary homogenates and microsomal fractions). Evaluation of the distribution of P450AROM immunoreactivity in brain sections of male and female rats (30 and 60 days of age) was performed using the avidin biotin peroxidase immunocytochemical technique and light microscopy. P450AROM immunoreactivity appeared to be localized to neurons, and was present in brain regions and nuclei where enzymatic activity has been reported. For example, intense immunoreactivity was observed in the amygdaloid structures and supraoptic nucleus, whereas moderate to light immunoreactivity was evident in the paraventricular and arcuate nuclei and hippocampus. Surprisingly, neurons in the bed nucleus stria terminalis, medial basal hypothalamic, and preoptic areas displayed little aromatase immunoreactivity. However, P450AROM immunoreactivity was detected in specific brain regions not previously recognized to contain the enzyme (i.e. intense staining was seen in the reticular thalamic nucleus, olfactory tract and piriform cortex, as well as other brain structures). The pattern, distribution, and intensity of P450AROM immunoreactivity was similar regardless of sex or age. In this study, microsomal preparations derived from a new brain area (i.e. the reticular thalamic nucleus; Rt) displaying P450AROM immunoreactivity were observed to contain detectable levels of aromatase enzymatic activity, as determined by the 3H2O-release assay. The activity in the Rt was inhibited by a known aromatase inhibitor, 4-hydroxyandrostenedione. These results confirm histologically the localization of P450AROM to brain regions where aromatase enzymatic activity has been detected and extend the knowledge of its location to areas previously unknown as sites of aromatase activity, which may be involved in the modulation of neuroendocrine function and reproductive behavior.

Effect of Fasting on c-Fos Expression in the Hypothalamic Nuclei and Nucleus of the Solitary Tract in Male Rats: Time Course Study and the Role of Testosterone.

“…Testosterone could be converted to estrogen and then act through the estrogen receptors (ER). The immunocytochemical distribution of c-Fos expression found in the brain of testosteronetreated, fasted castrates is consistent with that of aromatase activity or immunoreactivity [34][35][36], or ER mRNA [37] previously reported. Increased expression of ER mRNA has been shown in the PVN, SON, AMYG and NTS [37] where FLI was also found after 6-h fasting in testosterone-treated castrates in the present study.…”

Hippocampal Synapses Depend on Hippocampal Estrogen Synthesis

“…Results from several laboratories over the past decade have established unequivocally that the enzymes found in classic steroidogenic tissues are present in the CNS (Compagnone and Mellon, 2000). Aromatase, which transforms testosterone to estradiol, has been detected in rat hippocampal neurons both at the mRNA level (Abdelgadir et al, 1994;Wehrenberg et al, 2001) and protein level (Sanghera et al, 1991;Garcia-Segura et al, 1999, 2001). Recently, it was shown that adult hippocampal neurons indeed synthesize estrogens (Prange-Kiel et al, 2003).…”

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

“…Since it appears that aromatase is present in or near the PVN (Lund et al 2006, Sanghera et al, 1991 this presents a potential interpretive problem for the HPA of both males (as a result of testosterone aromatization to estradiol) and females. One possibility lies in the ratio of ERalpha to ERbeta that exists within neurons in and around of the PVN.…”