Proc. Natl. Acad. Sci. USA Vol. 83, pp. 513-516, January 1986 Neurobiology Estrogen formation and binding in the cerebral cortex of the developing rhesus monkey (sex determination/hormone receptors/brain differentiation) N. J. MACLUSKY*tt, F. NAFTOLIN*, AND P. S. GOLDMAN-RAKICt Departments of *Obstetrics and Gynecology and tNeuroanatomy, Yale University School of Medicine, New Haven, CT 06510-8063 Communicated by Clement L. Markert, September 12, 1985 ABSTRACT These studies were undertaken to determine present studies demonstrate that estrogen receptors and the whether estrogen receptors and the microsomal enzyme system microsomal enzyme system called the aromatase complex, called the aromatase complex, which is responsible for con- which is responsible for conversion of androgen to estrogen, version of androgen to estrogen, are present in the brain of the are present in the brain ofthe rhesus monkey during perinatal rhesus monkey during perinatal life. Four monkeys (three development. The regional distribution of aromatase com- females-one fetus removed on day 153 of gestation and two plex activity in the cerebral cortex is consistent with a infants, 5 and 6 days postnatal-and 1 male, 2 days postnatal) putative action oflocally formed estrogen in the development were studied. Cytosol estrogen receptors were detected in all of areas of the association cortex concerned with cognitive brain regions examined. The apparent equilibrium dissocia- processes. tions constants for reaction of these sites with [3H]moxestrol were similar to those for uterine and pituitary cytosol estrogen receptors (0.3-1.1 nM). Within the brain, highest levels of MATERIALS AND METHODS binding were observed in the hypothalamus-preoptic area, Four monkeys (three females-one fetus removed on day 153 with fairly even, lower concentrations throughout the cortical of gestation and two infants, 5 and 6 days postnatal-and 1 structures. Aromatase complex activity was detected in the male, 2 days postnatal) were used in these studies. The fetus majority of the tissue specimens. The highest levels of estrogen was removed by caesarian section: the pregnant monkey was formation were observed in the hypothalamus. However, the sedated with ketamine (3 mg/kg of body weight) and anes- amygdala, the hippocampus, and several of the cortex samples thetized by intratrachial administration of a halothane/oxy- also contained measurable aromatase complex activity. Among gen mixture. The brain was removed from the cranium the cortical samples, the highest levels of aromatase complex immediately after delivery of the fetus from the uterus. The activity were found in regions of the association cortex (the postnatal animals were anesthetized with sodium pento- dorsolateral-prefrontal, orbital-prefrontal, anterior cingu- barbital (35 mg/kg ip.). Cerebral circulation and respiration late, and parietal cortices). The lowest levels of aromatase were maintained while a craniectomy was performed. The activity were found in the somatosensory and motor cortices of brain was removed within 12 min after sedation. the postnatal animals. These results suggest that locally-formed In all cases the brain was placed in a bath of ice-cold saline estrogen may be involved in the effects ofcirculating androgens for an additional 2-5 min after removal and then dissected on on the developing primate neocortex. a bed of crushed ice. Hypothalamus-preoptic area, amyg- dala, hippocampus, dorsolateral-prefrontal cortex, orbital- In many mammals, sexual differentiation of the brain results prefrontal cortex, parietal cortex, motor cortex, somatosen- primarily from sex differences in gonadal steroid hormone sory cortex, and visual cortex were dissected as described secretion during early life (1). Virtually all of the information (14). The anterior cingulate cortex was taken as the anterior about effects of gonadal steroids on the developing nervous half of the cingulate gyrus between the level of the genu and system pertains to differentiation of diencephalic structures the splenium. that underlie gender differences in gonadotrophin release and Cytosol estrogen receptors were assayed by using the reproductive behavior. Evidence for direct effects of the synthetic estrogen [3H]moxestrol (11P-methoxy-17a- gonadal hormones on the maturation ofother brain structures ethynyl-estradiol) to label the receptor sites and Sephadex is much more limited (2) and has never been reported for LH-20 gel filtration to separate bound and free steroid as primates. There is, however, abundant behavioral evidence described (15). Tissues were weighed and homogenized in 10 for sex differences in cerebral function in both humans and mM Tris buffer containing 1.5 mM Na2EDTA, 1 mM dithio- nonhuman primates. In the rhesus monkey (Macaca threitol, and 10% glycerol adjusted to pH 7.4 with hydro- mulatta), sex differences have been reported in play behavior chloric acid (TEGD buffer). The homogenates were centri- and in the effects of neocortical lesions during the first 3 fuged at 1°C for 1 hr at 105,000 x g. The supernatant cytosol months of postnatal life (3-6). In humans, sex differences fractions were incubated with 0.1-2 nM [11f3-methoxy- have been reported in cognitive abilities, cerebral lateraliza- 3H]moxestrol (New England Nuclear; specific activity, 79 tion, play behavior, and incidence of a variety of childhood Ci/mmol; 1 Ci = 37 GBq) overnight at 0-4°C. Parallel learning disorders (7-11). Circulating androgen levels during incubations, used in correcting for nonspecific binding, early life have been implicated as a possible causative factor contained a 100-fold molar excess of unlabeled moxestrol in for at least some of these sexual dimorphisms (11, 12). addition to the labeled steroid. Macromolecular bound ra- In many subprimate mammalian species, local estrogen dioactivity in duplicate 200-,ul samples of each incubate was biosynthesis is believed to be critically important for andro- separated by Sephadex LH-20 gel filtration (15). Protein gen-induced sexual differentiation of the brain (1, 13). The content of the cytosols was measured by the method of Bradford (16). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: E1, estrone; E2, estradiol. in accordance with 18 U.S.C. §1734 solely to indicate this fact. fTo whom reprint requests should be addressed. 513 Downloaded by guest on September 27, 2021 514 Neurobiology: MacLusky et al. Proc. Natl. Acad Sci. USA 83 (1986) The specificity of the binding reactions was studied by incubating the cytosol fractions with a single concentration of [3H]moxestrol (2nM) in the presence of unlabeled estradiol, 0. progesterone, cortisol, testosterone, or the synthetic estro- gen diethylstilbestrol-all added to a final concentration of 100 nM. 00 Aromatase complex activity was measured by a procedure mc based on the product isolation methods as described by Callard et al. (17) and Milewich et al. (18) with 4- 0.1 [3H]androstene-3,17-dione as substrate. Tissues were ho- o . mogenized with a motor-driven Teflon-glass homogenizer in 4 8 12 200 400 buffer (1 ml per 100 mg of tissue) containing 250 mM sucrose Bound, fmol/mg of protein and 50 mM sodium phosphate (pH 7.2). Aliquots ofthe tissue homogenates (1.8 ml; in duplicate in the case of the larger FIG. 1. High-affinity binding for 13H]moxestrol in cytosol frac- cerebral cortex samples) were incubated for 2 hr at 370C with tions prepared from the tissues ofa 6-day-old female rhesus monkey. 0.2 ml ofphosphate buffer containing an NADPH-generating The graph depicts Scatchard (21) plots of [3H]moxestrol binding in system (4 mM ATP/4 mM NADP/10 mM glucose 6- cytosol fractions prepared from the pooled hypothalamus and phosphate/4 units of dehydrogenase) preoptic area (A) and orbital cortex (A) (Left) and from the uterus (o) glucose-6-phosphate and pituitary (e) (Right). Equilibrium dissociation constants [Kd and 50 pmol of [1,2,6,7- H(N)]andros-4-ene-3,17-dione (New (nM)] and saturation binding capacities [NS (fmol/mg of cytosol England Nuclear; specific activity, 87 Ci/mmol). Ten micro- protein)] for all of the tissues examined were as follows: uterus, Kd grams each of unlabeled estradiol (E2) and estrone (E1) were = 1.14, NS = 392; pituitary, Kd = 0.66, NS = 97; hypothalamus- added to the incubates as an estrogen "trap" (13). At the end preoptic area, Kd = 0.41, NS = 12.4; hippocampus, Kd = 0.47, NS ofthe incubation, the reaction was stopped by adding 5 ml of = 3.0; orbital-prefrontal cortex, Kd = 0.48, NS = 3.15; anterior toluene containing 0.25% Triton X-100. Unlabeled "carrier" cingulate cortex, Kd = 0.62, NS = 4.2; dorsolateral-prefrontal steroids (200 ,ug each ofE1 and E2) and 14C-labeled E1 and E2 cortex, Kd = 0.48, NS = 3.2; somatosensory cortex, Kd = 0.61, ND (2000 dpm each) were added. The incubates were extracted = 4.1; motor cortex, Kd = 0.71, NS = 3.5. with 3 x 5 ml oftoluene. The combined toluene extracts were evaporated to dryness and subjected to phenolic partition to motor cortices from the postnatal animals, sufficient [3H]E1 separate the androgen and estrogen metabolites present. The was obtained for analysis by reverse isotopic dilution and estrogens were purified by TLC on silica gel GF254 plates crystallization to constant specific activity. The 3H:14C ratios (Brinkmann) in the system described by Ruh (19). The in the purified E1 fractions changed by less than 5% over separated E1 and E2 fractions were eluted from the TLC three successive crystallizations, confirming the identity of plates and then acetylated by overnight incubation at room the major estrogen product. The results of the aromatase temperature with 0.2 ml of pyridine and 0.2 ml of acetic complex measurements are summarized in Fig. 3. As expect- anhydride. The acetate fractions were chromatographed ed, the highest levels of estrogen formation were observed in once more on silica gel plates in methylene chloride/ethyl the hypothalamus-preoptic area.