0013-7227/91/1296-2834$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society

Vol. 129, No. 6 Printed in U.S.A.

Immunocytochemical Distribution of Aromatase Cytochrome P450 in the Rat Brain Using PeptideGenerated Polyclonal Antibodies* MANJIT K. SANGHERA, EVAN R. SIMPSON, MICHAEL J. McPHAUL, GERALD KOZLOWSKI, ALAN J. CONLEY, AND EDWIN D. LEPHART Departments of Biochemistry (E.R.S., A.J.C., E.D.L.), Internal Medicine (M.J.M.), Physiology (G.K.), Obstetrics and Gynecology (E.R.S., A.J.C., E.D.L.), and Psychiatry (M.K.S.), and The Cecil H. and Ida Green Center for Reproductive Biology Sciences (E.R.S., A.J.C., E.D.L.), The University of Texas Southwestern Medical Center, Dallas, Texas 75235

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 P450 ARO M 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. (Endocrinology 129: 2834-2844, 1991)


HE FORMATION of estrogens from Ci9 steroids is catalyzed by a specific form of cytochrome P450, aromatase cytochrome P450 (P450AROM)- Aromatase activity has been detected in several tissue sites including the placenta (1), granulosa (2), Sertoli (3), and Leydig (4) cells, adipose tissue (5), several areas of the brain of both sexes (6), and the preimplantation blastocyst (7). The regulation of estrogen formation is coordinated in a tissue-specific fashion and involves multiple factors. Although aromatase has been reported in brain tissue of Received May 14,1991. Address all correspondence and requests for reprints to: Dr. Edwin D. Lephart, Green Center, Y6.316, The University of Texas Southwestern Medical Center, Dallas, Texas 75235-9051. * This work was supported in part by USPHS Grants AG-08174 (to E.R.S.), NS-24290 (to M.K.S.), and USPHS Training Grant T32HD07062 (to E.D.L.).

many species (6, 8-11), the physiological significance of brain aromatase has been studied most extensively in the rat (12-15). In the rat brain, local formation of estrogens from androgens is believed to mediate the sexual differentiation of neural structures perinatally (16-19) and to be involved in the regulation of reproductive behavior (20, 21). The distribution of aromatase has been evaluated by measuring enzymatic activity, and it has been observed that the rates are highest in hypothalamic and limbic areas of the rat brain (22, 23). However, in only a few cases have immunocytochemical techniques been used in an attempt to localize the enzyme to discrete anatomical structures (24-26). In these studies, staining was not detected in several rodent brain areas with known aromatase enzyme activity when a human placental


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ICC DISTRIBUTION OF BRAIN AROMATASE P450AROM antibody was used, suggesting that the antibody may not recognize the rodent P450AROM enzyme in

certain brain structures. Therefore, to determine the location of this enzyme in rat brain, we developed polyclonal antibodies to a synthetic peptide corresponding to a 20-amino acid segment common to the rat, human, and chicken cytochrome P450AROM proteins, as deduced from the nucleic acid sequence of the P450AROM CDNAS (26). Using light microscopy the distribution of P450AROM immunoreactivity in neural structures of Sprague-Dawley male and female rats (30 and 60 days of age) was studied using the avidin biotin peroxidase immunocytochemical technique. Our findings indicate that: 1) the distribution of P450AROM immunoreactive cells was similar among male and female rats; 2) immunoreactivity was located where aromatase enzyme activity has been reported; and 3) immunoreactivity was detected in areas of the brain where enzyme activity has not been examined previously, suggesting a role for local estrogen biosynthesis in neural function. Materials and Methods Antibody preparation A 20-amino acid peptide corresponding to residues 379-398 of rat P450AROM protein (27) was synthesized as shown in Fig. 1. This segment was chosen because the amino acid sequence is identical in the rat, human, and chicken P450AROM proteins, as deduced from the nucleic acid sequence of the rat, human, and chicken P450AROM complementary DNAs (cDNAs) (27) but has minimal homology to other P450s within this corresponding region. The synthesized peptide was coupled to the carrier protein keyhole limpet hemocyanin (KLH; Sigma Chemical Co., St. Louis, MO) through a cross-linking reagent (j8-maleimidopropionic acid iV-hydroxysuccinimide; Sigma Chemical Co.) at an additional N-terminal cysteine residue, by standard proHomology Domains

rat aromatase


Heme binding region

Rut 379 Hun 310

FlG. 1. Schematic representation of the rat aromatase protein and the corresponding 20-amino acid synthetic peptide (residues 379-398). The amino acid sequence (1-496) of the aromatase protein is based upon the rat cDNA characterized by Lephart et al. (27). The DNA and hormone binding domains were based on the human and chicken cDNAs and predicted amino acid sequences. Positions of identity across the rat, human (hum), and chicken (chk) amino acid sequences are shown as asterisks.


cedures. Adult female New Zealand White rabbits (Myrtle's Rabbitry, Thompson Station, TN) were immunized using the following standard protocol. Animals were injected sc with 50 Hg peptide-coupled KLH in 0.5 ml Freunds' adjuvant at various dorsal sites at 14-day intervals on three occasions. The animals received a booster injection 28 days later using the same peptide concentration and subsequently were bled 7 days later and at 14-day intervals. The sera was tested for titer in an enzyme-linked immunosorbent assay. The immunoglobulin G fractions were prepared using diethylaminoethyl-Affigel Blue chromatography, and the immunoreactive titer was determined by enzyme-linked immunosorbent assay. The chromatographed peak fractions were pooled and lyophilized in 0.02 M Tris-HCl, pH 8.0 and stored at -20 C with 0.02 (wt/vol) sodium azide, NaN3. Animals Immature female (20-24 days old) and male and female (3060 or 90 days old) Sprague-Dawley rats were purchased from Dominion Labs (Omaha, NE). For Western analysis, the tissues were pooled from 10-15 adult (60 or 90 days old) male and female rats for preparation of homogenate and microsomal samples. In the immunocytochemical studies male and female rats (30 or 60 days of age; n = 4-6 animals of each sex) were studied, while immature anestrous rats (24 days old; n = 2-4 animals per treatment) served as positive controls. Western analysis Tissues were homogenized in 1% sodium cholate, 0.1% sodium dodecyl sulfate (SDS) and sonicated. Protein content of each tissue homogenate was determined by the method of Lowry et al. (28) using BSA as standard. The tissue homogenates (100 ng protein) with loading buffer (0.1 M Tris-HCl, pH 6.8, 0.4 SDS, 2% glycerol, 0.4 mM EDTA, 0.4 mM 0-mercaptoethanol, 0.01% bromophenol blue; final concentrations) were boiled for 2 min, then the samples were loaded into wells of a 3% acrylamide stacking gel. The proteins were resolved by electrophoresis on an 8% polyacrylamide gel in a buffer containing 50 mM Tris, 383 mM glycine, 0.1% SDS, and 2 mM EDTA, and transferred to polyvinyl membranes (Immobilon, Millipore Corp., Bedford, MA) at 100 V for 75 min in buffer containing 20 mM Tris, 150 mM glycine and 20% methanol. All immunoblotting procedures were carried out at room temperature in buffer containing 10 mM Tris-HCl, pH 7.4,0.15 M NaCl, and 0.2% Nonidet P-40 (Sigma Chemical Co.). The membranes were preincubated for 30 min in buffer, then incubated with the aromatase antibody (dilution 1:1000) for 2 h. After the incubation, the membranes were washed for 15 min then incubated with [125I]protein A (ICN Biomedicals Inc., Costa Mesa, CA; 106 dpm/ml) for 30 min and finally washed for 60 min. Autoradiography was performed using Kodak X-Omat AR-2 flim (Eastman Kodak Co., Rochester, NY) at -70 C with two intensifying screens. Exposure time varied between 3-12 h. Immunocytochemistry Sprague-Dawley male and female rats (30 and 60 days of age) were deeply anesthetized with sodium pentobarbital (50

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mg/kg ip) then perfused transcardially, using a speed-controlled minipump (Cole-Parmer Instruments Co., Chicago, IL), with 50 ml 0.15 M sodium chloride followed by 100 ml Zamboni's fixative (pH 7.4) at room temperature. The brains and adrenal glands were removed. Immature anestrous Sprague-Dawley female rats (24 days of age) were treated with 5 or 30 IU PMSG to stimulate follicular development and estrogen biosynthesis. Eighteen to 24 h later, the animals were anesthetized with sodium pentobarbital and perfused as described above, and the ovaries were removed. The brains, adrenal glands, and ovaries were placed in Zamboni's fixative for 1-3 h and then soaked in 20% phosphate sucrose solution for 1 h. Sections of brain (through the entire hypothalamic region), adrenals, and ovaries were cut on a freezing microtome (50 microns), collected in cold PBS (0.1 M; pH 7.4). Free-floating serial sections were immunostained for P450AROM using the ABC immunocytochemical technique. The sections were immersed sequentially in PBS containing the following: 1) 1% H2O2 in 0.1 M phosphate buffer, pH 7.4, for 30 min; 2) 2 normal goat serum (NGS) for 45 min; 3) 0.5% triton X-100, 2% NGS for 10 min; 4) rabbit antirat aromatase (1:1,000 in 0.5% Triton X-100, 2% NGS) at room temperature for 36-48 h; 5) biotinylated goat antirabbit immunoglobulin G (1:200) for 1 h; 6) avidin-biotin complex (ABC kit, Vector Labs, Burlingane, CA) for 1 h; and 7) 0.035% 3-3'-diaminobenzidene HCl 4 (Sigma Chemical Co.) and 3% H2O2 for 1 min. Between each step, the sections received three 10-min washes in PBS containing 2% NGS. The sections were mounted on clean slides, air dried, and coverslipped. As controls, some sections in each experiment were processed 1) without primary antibody; and 2) with the primary antibody which had been preincubated with an excess of the peptide (5x or 10x) that was used as the antigen. Sections were viewed under a light microscope, and the positions of P450AROM-positive perikarya were plotted onto sections taken from the rat brain atlas of Paxinos and Watson (29). Subcelluar fractionation of rat brain and human placental aromatase activity Rat amygdaloid and reticular thalamic (Rt) regions were dissected (in blocked tissue fragments) from 30- to 60-day-old male rats. The rat brain tissues were pooled from 12 animals. Human placental tissue (obtained at cesarean sections) and rat samples were homogenized in ice-cold buffer (10 mM potassium phosphate/100 mM postassium chloride/1 mM EDTA), pH 7.35, containing 10 mM dithiothreitol and 320 mM sucrose. Subcellular fractions were prepared by centrifugation as previously described (15). The pellets were resuspended in 2 ml homogenization buffer, and the protein content was determined by the method of Lowry et al. (28). Aromatase activity was measured in the subcellular fractions using the 3H2O-release assay. 3

H2O-Release assay and in vitro inhibition of aromatase enzyme activity by 4-hydroxyandrostenedione Preparation of the substrate, aromatase inhibitor (4-hydroxyandrostenedione), and the methodology of the 3H2O-release assay have been described in detail elsewhere (15, 30, 31).

Endo • 1991 Voll29«No6

However, in brief, microsomal samples (100 /A homogenization buffer containing 50 fig for human placental and 0.4 to 0.7 mg for brain tissues) were preincubated in the presence of 5.0 fiM 4-hydroxyandrostenedione for 20 min at 35 C. The incubations were initiated by the addition of 100 fd homogenization buffer containing 2 mM NADPH, 2 mg/ml BSA, and 2.0 fiM [1|8-3H] androstenedione or [l/3-3H]testosterone as substrate. The reaction tubes were incubated for 1 h at 35 C in air. At the end of the incubations, trichloroacetic acid was added (10% final concentration) to precipitate the proteins, and 1 ml chloroform was added to each tube for steroid extraction. Isolation and purification of the 3H2O was performed as previously described (31), and an aliquot of the 3H2O was quantified by scintillation counting. Blank incubations (using BSA protein equivalents) were included in each experiment to determine background radioactivity. The results were expressed as picomoles of aromatase activity per h/mg protein for human placental samples and femtomoles of aromatase activity per h/mg protein for rat brain samples.

Results Immunoblotting To determine the immunoreactivity of the purified antisera, homogenates of various tissues were electrophoresed and examined by immunoblotting using the P450AROM antibody. The antibody recognized a single protein band with an apparent mol wt of 56,000 in rat ovary homogenate and microsomal samples (Fig. 2). The estimated mol wt of the band from rat ovarian samples is similar to the predicted mol wt of the proteins as deduced from the respective cDNAs (27) and previous size determination by SDS-poly aery lamide gel electro2 S S ? - X & w

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Immunocytochemical distribution of aromatase cytochrome P450 in the rat brain using peptide-generated polyclonal antibodies.

Estrogen formation is catalyzed by the aromatase cytochrome P450 (P450AROM) enzyme. Aromatase activity has been detected in several regions in the rat...
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