THE JOURNAL OF COMPARATIVE NEUROLOGY 322~360-376 (1992)

An Aromatase-Associated Cytoplasmic Inclusion, the "Stigmoid Body," in the Rat Brain: I. Distribution in the Forebrain KOH SHINODA, SHIRO MORI, TSUKASA OHTSUKI, AND YOSHIO OSAWA Department of Anatomy 11, Kinki University School of Medicine, Osakasayama, Osaka 589 (K.S., S.M.) and Department of Neuroanatomy, Biomedical Research Center, Osaka University Medical School, Suita, Osaka 565 (T.O.), Japan; Endocrine Biochemistry Department, Medical Foundation of Buffalo, Buffalo, New York 14203 (Y.O.)

ABSTRACT An aromatase-containing neural system was examined in the rat forebrain, using a

polyclonal antibody against aromatase-associated human placental antigen X-P2(hPAX-PZ). Numerous dot-like structures, which we have called stigmoid bodies, were immunostained in the preoptico-hypothalamic region, the bed nucleus of the stria terminalis, the medial amygdala, the arcuate nucleus, the subfornical organ, and the area extending from the hypothalamic area to the central gray through the medial forebrain bundle and the periventricular fiber system of the posterior diencephalon. The stigmoid bodies were always found as inclusions in the neuronal cytoplasm. Their diameter was usually 1-3 pm, but exceptionally large forms, over 3 pm, were found in some brain regions, including the area extending from the median preoptic nucleus surrounding the organosum vasculosum laminae terminalis to the anterior medial preoptic nucleus, the periventricular nucleus of the preoptic area, and some parts of the medial preoptic nucleus. Most of these nuclei show sexual dimorphism. The distribution pattern of the hPAX-P2 immunoreactive stigmoid bodies agreed well with that of aromatase activity previously reported in many biochemical studies. Brain regions where the stigmoid bodies were prominent largely coincide with steroid binding locations common to both androgen and estrogen, or regions where both sex steroid receptors are present. Although it still remains to be determined whether aromatase is localized within these stigmoid bodies, it appears likely that they are closely associated with the function of sex steroids at their target sites in the brain. (c) 1992 Wiley-Liss, Inc. Key words: human placental antigen X-Pz, aromatase cytochrome P-450, medial preoptic region, hypothalamus, immunohistochemistry

Sex steroids have critical effects on the organization and activation of certain brain regions involved in such reproductive functions as the development of sexual dimorphism, the induction of sexual feeling or behavior, and the control of the neuroendocrinergic milieu. With respect to controlling the sexual differentiation of the brain, androgen and estrogen appear to have opposite effects. It has been reported, however, that estrogen can successfully mimic the central actions of androgen (Baum and Vreeburg, '73; Davis and Barfield, '79; Doughty et al., '75; Westley and Salaman, '76). This is especially so in the case of brain differentiation in rats (Gorski, '63; Gorski and Barraclough, '63). Administration of excess estrogen during pregnancy or perinatal periods in female rats causes defeminization in adults. It blocks lordosis (Levine and Mullins, '64) and leads to postpuberal sterility, characterized by anovulation and persistent vaginal cornification (Docke and Dorner, '75;

o 1992 WILEY-LISS, INC.

Dorner et al., '71; Gorski, '63). Pretreatment with antiestrogens can block androgen-induced sexual behavior (Beyer and Vidal, '71; Doughty et al., '73; Lieberburg et al., '77; McDonald and Doughty, '72) and intermale aggression (Luttge, '79). The apparently paradoxical and overlapping actions of androgen and estrogen in the brain may be attributable, in part, to metabolic interconversion of androgen and estrogen at target sites. Androgen is directly converted into estrogen by aromatization of its A ring. Regulation of the converting enzyme aromatase affects the actions of androgens and thus can modify sexual dimorphism, sexual behavior, and sex-specificpatterns of gonadoAccepted April 23,1992. Address reprint requests to Koh Shinoda, Department of Anatomy 11, Kinki University School of Medicine, 377-2, Ohno-Higashi, Osakasayama, Osaka 589, Japan.

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tropin secretion (Hutchison and Steimer, '84; Kaplan and effects of testosterone on female rats and can feminize the McGinnis, '89; MacLusky and Naftolin, '81; McEwen, '81; sexual behavior of males (Christensen and Clemens, '75; McEwen et al., '77; McGinnis et al., '81; Naftolin et al., '75; Lieberburg et al.,'77). Knowledge of the cellular and subcellular localizations of Olsen, '85). A comparison of autoradiographic results obtained with tritiated testosterone labeled in the la,2a- aromatase is useful in understanding the action of sex positions (from which tritium is lost during aromatization) steroids on the brain. We have reported the distribution of and in the lp,2@-positions(at which tritium remains even immunoreactive neurons in rats (Shinoda et al., '89b) and after aromatization) has strongly suggested that testoster- monkeys (Shinoda et al., '89c), using a polyclonal antibody one is aromatized into estradiol in the medial preoptic against an aromatase-associated human placental antigen region (Sheridan and Melgosa, '83). Even in the brains of X-P2 (hPAX-PZ)(Shinoda et al., '89a), which biochemically testicular feminized mice which lack androgen receptors, inhibits aromatase activity (Osawa and Nakamura, '82). radioactive testosterone uptake can be observed by autora- Aromatase antibodies marked reticular or filamentous strucdiography in the medial preoptic nucleus, parts of the tures in the neuronal perikarya, which were distributed amygdala, and the bed nucleus of the stria terminalis, mainly in the ventral pallidum, but were absent from the where the radioactivity can be remarkably reduced by medial preoptico-hypothalamic region. Balthazart and his pretreatment with "cold" estrogens, but not with "cold" collaborators ('90a,b), using an aromatase antibody which dihydrotestosterone (Scheleicher et al., '86). It thus ap- was produced in a different way from ours (Harada, '881, pears likely that testosterone is taken up by neurons in the reported presence of the immunoreactive neurons in the above-mentioned regions and aromatized into estrogen, medial preoptic-hypothalamic region of the Japanese quail, which, in turn, acts on estrogen receptors. Aromatase the ring dove, and the zebra finch. Their results appeared to activity has been reported in the brains of a wide variety of agree with the biochemical data for aromatase activity in species (Borg et al., '87; Callard et al., '78, '79; Canick et al., the bird brains (Hutchison and Steimer, '84; Schumacher '86; Flores et al., '73; McEwen et al., '77; Naftolin et al., '71; and Balthazart, '87; Steimer and Hutchison, '80; Vockel et Ryan et al., '72; Schumacher and Balthazart, '86a,b, '87; al., '90a'b). Nevertheless, the antibody which Balthazart Steimer and Hutchinson, '80, '81; Timmers and Lambert, and his collaborators used failed to stain any neurons in the '87). Radioenzyme assays of microdissected brain tissues medial preoptico-hypothalamic regions of any mammalian have confirmed that it is high in the medial preoptic- brains tested, including those of monkeys and rodents. hypothalamic region and medial amygdala (George and Some neurons in the lateral hypothalamus of mice were Ojeda, '82; Kobayashi and Reed, '77; Naftolin et al., '75; reported to have been stained, but the distribution was Roselli, '91; Roselli and Resko, '87, '89; Roselli et al., '85; completely different from that of aromatase activity (BalSelmanoff et al., '77; Timmers et al., '87; Tobet et al., '85; thazart et al., '91). To clarify the discrepancy between bioWeisz and Gibbs, '74). Aromatase inhibitors such as an- chemical and neuroanatomical findings, we have underdrosta-1,4,6-triene-3,17-dione can block the defeminizing taken to re-examine the rat forebrain, using antibody to

Abbnwiations ac Acb

AH AHipm AMP Ar BSTm cc CeAm CG CP CP csc db DG

Dk DLG DM em1

f fi fr GP Hb Hip ic LA LH LM lo LO LP LSd LSi

anterior commissure nucleus accumbens anterior hypothalamic area posteromedial part of the amygdalo-hippocampalarea anterior medial preoptic nucleus arcuate nucleus medial division of bed nucleus of stria terminalis corpus callosum central amygdaloid nucleus central gray cerebral peduncule caudate-putamen commissure of superior colliculus diagonal band dentate gyrus nucleus of Darkschewitsch dorsal lateral geniculate nucleus dorsomedial hypothalamic nucleus external medullary lamina fornix fimbria fasciculus retroflexus globus pallidus habenula hippocampus internal capsule lateroanterior hypothalamic nucleus lateral hypothalamus lateral mammillary nucleus lateral olfactory tract nucleus of the lateral olfactory tract lateral preoptic area dorsal part of lateral septum intermediate part of lateral septum

LSV MAm MG ml MM MnP MP MPA mt oc ot OVLT PC Pe PH Pir PV PVS

sc

sco SF sm SN

so st SPF STh sx TM Tu VLG VM 21

ventral part of lateral septum medial amygdaloid nucleus medial geniculate body medial lemniscus medial mammillary nucleus median preoptic nucleus medial preoptic region medial preoptic area mammillothalamic tract optic chiasm optic tract organosum vasculosum laminae terminalis posterior commissure periventricular nucleus of hypothalamus nucleus posterior hypothalamic area piriform cortex paraventricular hypothalamic nucleus periventricular fiber system suprachiasmatic nucleus superior colliculus subfornical organ stria medullaris thalami substantia nigra supraoptic nucleus stria terminalis subparafascicular thalamic nucleus subthalamus supramammillary decussation tuberomammillary nucleus olfactory tubercle ventral lateral geniculate nucleus ventromedial hypothalamic nucleus zona incerta

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Fig. 1. Coronal tissue sections of rat anterior medial preoptic nucleus (a)incubated with antiserum against hPAXP2 and (b)stained with antiserum absorbed with purified antigen. The immunoreactive, dot-like structures (stigmoid bodies) seen in a are not stained in b (control). PAP method. Asterisks indicate blood vessels as landmarks. Insert: Enlargement of the area indicated by the arrow. AMP, anterior medial preoptic nucleus; oc, optic chiasm. a: bars = 50 Fm, 5 pm (insert). b: bar = 50 pm.

hPAX-P2,and now report a previously overlooked finding of intriguing immunoreactive dot-like structures, “stigmoid bodies.” In this paper, we focus on their cellular distribution in the forebrain and discuss their relation to reproductive functions. The subcellular ultrastructure of these “stigmoid bodies” will be discussed in a subsequent paper (Shinoda et al., ’92).

MATERIALS AND METHODS Tissue preparation Animals were housed a t a constant temperature (20°C) with a 12 hour--12 hour light-dark cycle, and were given food and water ad libitum. Eight male and four female Wistar rats (30-300 g) were anesthetized with sodium pentobarbital (i.p. 65 mg/kg) and perfused through the ascending aorta with 20-50 cc of ice-cold saline, followed by 0.1 M sodium phosphate buffer (PB, pH 7.4) containing 4% paraformaldehyde and 0-0.1% glutaraldehyde (200-500 cc). The brains were removed and immersed in the same fixative for 24 hours, after which they were soaked in cold

0.1 M PB containing 30% sucrose (pH 7.4) until they sank. The brains were then frozen in dry ice and frontally sectioned in a cryostat at a thickness of 20-25 km. Frozen sections were collected in ice-cold 0.02 M sodium phosphatebuffered saline (PBS, pH 7.4) and rinsed several times with PBS to remove fixative. Free-floating sections were preincubated for 24 hours at 4°C in PBS containing 1%normal goat serum (NGS) and 0.3% Triton-X, bleached for 20 minutes with 40% methanol containing 1.5% H202,and rinsed three times, for 20 minutes each time, with PBS containing 1% NGS. Sections were then incubated for 3 days at 4°C in the first antibody (described below) diluted 1:3,000 with PBS containing 1%NGS and 0.3% Triton-X. After the primary antibody was washed out with PBS containing 1%NGS, sections were incubated for 24 hours at 4°C in anti-rabbit goat IgG (Behringer Institute) diluted 1:500 with PBS containing 1% NGS and 0.3% Triton X. They were then washed three times, for 20 minutes each time, with PBS and incubated for 24 hours at 4°C in peroxidase-antiperoxidase rabbit IgG complex (DAKO) diluted 1:500 with PBS. Sections were then washed four times (each time for 10

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Fig. 2. Higher magnification of a photomicrograph showing subcellular localization of hPAX-P2 immunoreactive dot-like structures. Neurons in the medial preoptic nucleus. Dot-like stigmoid bodies are located in neuronal perikarya as cytoplasmic inclusions (arrowheads). Some of these structures have clear spaces inside (double arrowheads). Bar = 10 km.

minutes) with 0.05 M Tris-HC1 buffer (pH 7.4). They were Terminology then treated with 0.05 M Tris-HC1 buffer containing 0.02% Most nomenclature is based on the atlas of Paxinos and diaminobenzidine and 0.6%nickel ammonium sulfate (DABnickel solution) in the presence of 0.005% hydrogen perox- Watson (’86). In addition, the studies of Simerly et al. (’84) ide (nickel-enhanced DAB reaction) for 10-15 minutes at and Bloch and Gorski (’88a) were consulted for the descriproom temperature. After three 10-minute rinses in Tris- tions of the medial preoptic region, while that of J u and HC1 buffer, the sections were mounted onto glass slides in Swanson (’89) was used for the descriptions of the bed 1%gelatinlsaline. After being air-dried, they were dehy- nucleus of the stria terminalis. The anterior medial preopdrated with a graded series of ethanol, immersed in xylene, tic nucleus in our text is homologous with the “medial and embedded in Enteran New. Neighboring sections were preoptic nucleus” of Bleier et al. (’79, ’82), with the stained with cresyl violet and their outlines were drawn “anteroventral periventricular nucleus” of Swanson (’76), under a camera lucida. After light microscopic observations Simerly et al. (’841, and Bloch and Gorski (’88a), and with were made of the specimens, the immunoreactive dot-like the “preoptic suprachiasmatic nucleus or area” of Konig structures found (stigmoid bodies) were plotted according and Klippel(’63)and Fahrbach et al. (’86). The term medial to an atlas. preoptic nucleus as used here is homologous with the “anterior hypothalamic nucleus” of Bleier et al. (’79, ’82). Antibody and control The subdivisions of the medial preoptic nucleus were based The antibody suppresses 73% of human placental mi- on the studies of Simerly et al. (’84) and Bloch and Gorski (’88a). For the posterodorsal nucleus, see the study of crosome aromatase activity, but does not suppress NADPHcytochrome P-450 reductase activity. It recognizes the Simerly et al. (’84). The lateroanterior hypothalamic nucytochrome P-450 portion of aromatase (cyt P-450arom) cleus in our description [following that of Paxinos and (Osawa and Nakamura, ’82). Immunohistochemically, the Watson (’8611is homologous with the “anterior part of the antibody stains placental chorionic villi and ovarian luted, anterior hypothalamic area” of Saper et al. (’79, ’78) and thecal, and interstitial cells of humans and rodents (Mat- Swanson (’761, with the “lateral anterior hypothalamic suda et al., ’84). It does not stain liver or adrenal cortex. The nucleus” of Bleier et al. (’79, ’821, and with the “anterior recognized antigen has been termed hPAX-P2,since the possi- hypothalamic nucleus” of J u and Swanson (’89).The oval bility cannot be excluded that the antibody recognizes not nucleus and theprincipal nucleus of the bed nucleus of the only aromatase, but also other aromatase-associated sub- stria terminalis were identified using the study of Ju and stances common to estrogen biosynthetic organs (Shinoda Swanson (’89). The former largely corresponds to the et al., ’89a). As controls, rat brains were stained with the dorsal part of the lateral division of de Olmos et al. (’85). antibody preincubated with an excess of purified antigen The latter is homologous with the “encapsulated nucleus (Osawa et al., ’78; Osawa and Higashiyama, ’80).Immunor- (or cell group)” of Young (’36) and Simerly et al. (’90; Simerly and Swanson, ’86, ’88) and largely corresponds to eaction was strongly inhibited in the control (Fig. 1).

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Figure 3

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Figs. 3 and 4. Schematic representation of the distribution of hPAX-P2 immunoreactive stigmoid bodies in the rat forebrain. The brain atlas was made on the basis of our own Nissl preparations of young male rats (30-90 g). Coronal sections (a-k) are arranged from rostra1 to caudal. Small dots marked on the right side of each plane indicate locations and relative density of the immunoreactive stigmoid bodies. For more details of their location. see Results in the text.

the posteromedial and posterointermediate part of the medial division of de Olmos et al. (’85).

RESULTS Numerous hPAX-P2 immunoreactive “dot-like structures” were distributed in forebrainregions distinct from those where

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hPAX-P2 immunoreactive, reticulo-filamentous structures were distributed (Shinoda et al., ’89b). These structures were spherical or sometimes ovoid, the majority being 1-3 pm in diameter. They resembled nucleoli in shape and size and were present in neuronal perikarya or in the basal portion of large dendrites (Fig. 2). They were detected in neurons, but not in deep glial cells. Occasionally ependymal cells of the third ventricle had such structures. Their anatomic distribution throughout the rostrocaudal extent of the forebrain is shown in drawings (Figs. 3 , 4 ) . The major regions included the preoptico-hypothalamic region and medial amygdala. In some brain regions, including the preoptic nuclei, there were larger dot-like structures with diameters of 3 pm up to 4 pm (Figs. 6, 8).Immunoreactive intensity was directly proportional to size. In the neurons containing them, the inclusions were usually single, although occasionally more than two were present (Figs. 5 , 81, in which case they were mostly smaller (less than 1 pm in diameter). Dot-like structures larger than 2 pm appeared to be hollow, with one or two internal clear spaces (Fig. 2), the average diameter of which was about 0.5 pm, sometimes reaching 1 pm. We have provisionally termed these inclusions stigmoid (dot-like or stigma-like) bodies. Their ultrastructural details will be reported in detail elsewhere (Shinoda et al., ’92). In brief, these bodies consisted of aggregates of fine granulo-fibrillar materials with moderate to low affinity for electron-dense metallic stains and with no clearly defined limiting membranes. Their distribution was similar in males and females and basically similar in rats of different weights. However, the stigmoid bodies were more immunoreactive in younger rats (30-50 g) than in adults (200-300 g), in general, decreasing in size and becoming dispersed as the animals grew older. In the telencephalon, immunoreactive stigmoid bodies were rarely seen in the neocortex or allocortex (including the piriform, cingulate, retrosplenial, and entorhinal cortices and Ammon’s horn of the hippocampus). Exceptionally weakly stained, very small stigmoid bodies were observed in the granular cells of the dentate gyrus of the hippocampus. In the amygdaloid area, numerous medium-sized stigmoid bodies were localized in the medial nucleus, being especially prominent in its posterior divisions (Fig. 5a,b). Less prominent medium-sized stigmoid bodies were seen in the central nucleus, especially in the lateral division near the entrance of the stria terminalis. Small stigmoid bodies were disseminated in the anterior cortical amygdaloid nucleus, some extending to the anterior amygdaloid area, the bed nucleus of the accessory olfactory tract, and the basomedial nucleus. Medium densities of small stigmoid bodies were distributed in the posteromedial part of the amygdalo-hippocampal area. In the septum, basal forebrain regions, and basal ganglia, small-sized stigmoid bodies were sparsely distributed in the area extending from the ventral division of the lateral septa1 nucleus to the angle between the horizontal and vertical limbs of the diagonal band. Compared to the prominent neuronal groups containing immunoreactive, reticulofilamentous structures in the ventral pallidum and diagonal band, no remarkable populations of stigmoid bodies were detected in other parts of the septum, the basal forebrain (including the Calleja islands, the ventral pallidum, the nucleus accumbens, and the olfactory tubercle), or the basal ganglia (including the dorsal striatum and pallidum). In the bed nucleus of the stria terminalis, many immunoreactive stigmoid bodies were distributed in certain regions,

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Fig. 5. hPAX-P2immunoreactive stigmoid bodies in the posterodorsal part of the medial amygdaloid nucleus (MAm) (a,b) and the principal nucleus of the bed nucleus of the stria terminalis (BSTpr) (c,d).b and d are enlargements of a and c, respectively. Asterisks indicate blood vessels as landmarks, a: A large number of stigmoid bodies are concentrated in the medial amygdaloid nucleus, whereas very few are seen in the intercalated amygdaloid nucleus (Ic) (arrow-

head in a) or in the intraamygdaloid division of the bed nucleus of the stria terminalis (BSTi). c: Arrow indicates medial direction. b and d Some neurons contain more than two stigmoid bodies, which are mostly small in size (less than 1 pm). ot, optic tract; sm, stria medullaris thalami; st, stria terminalis. a: bar = 100 pm. b: bar = 25 pm. c: har = 50 pm. d: bar = 25 km.

most prominently in the posteromedial or posterointermediate part of the medial division extending from the junction of the septum and thalamus to the area dorsal to the preoptic region (the principal or encapsulated nucleus; see Terminology section above) (Fig. 5c,d). Rostrally, the neuronal group merged into a scattered neuronal group containing stigmoid bodies in the ventral division of the lateral septal nucleus, whereas caudally it extended into the stria terminalis. In the posteromedial part of the medial division of the bed nucleus of the stria terminalis, there was another prominent neuronal group containing stigmoid bodies in an area largely corresponding to the magnocellular nucleus in the anteroventral area of the anterior division (Ju and Swanson, ’89) Immunoreactive stigmoid bodies were also prominent in the dorsal part of the lateral division (the oval nucleus; see Terminology section above). In contrast, only a few neurons containing stigmoid bodies were scattered in other parts or the intraamygdaloid division of the bed nucleus. The distribution pattern of these bodies in the principal nucleus of the bed nucleus of the stria terminalis resembled that in the medial amygdaloid nucleus, while that in the oval nucleus of this region resembled that in the central amygdaloid nucleus. These amygdala/bed nucleus

“pairs” have been shown to be homologous in fiber connection patterns and in neuropeptide-, neuronal amine-, neuronal metal-, and enzyme activity-histochemistry and have recently been organized as the medial extended amygdaloid group and central extended amygdaloid group, respectively (Alheid and Heimer, ’88; de Olmos, ’90; de Olmos et al., ’85). The largest numbers of immunoreactive stigmoid bodies in the forebrain were concentrated in the preoptic region, especially in the median preoptic, the anterior preoptic, the medial preoptic, and the periventricular nuclei. Very large stigmoid bodies (diameter over 3 km) with strong immunoreactivity frequently occurred in these nuclei, especially in the region extending from the anterior medial preaptic nucleus to the rostroventral tip of the median preoptic nucleus, where they surrounded the organosum vasculosum laminae terminalis (Figs. 1 , 6 ) . The subfornical organ, which is associated with the dorsal tip of the median preoptic nucleus, also contained many medium to large and, occasionally very large, stigmoid bodies (Fig. 9). In the medial preoptic nucleus, numerous small to medium stigmoid bodies were widely distributed (Fig. 7); however, as this nucleus is heterogenous, neurons in some parts often

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Fig. 6. hPAX-Pt immunoreactive stigmoid bodies in the rostroventral tip of the median preoptic nucleus (MnP) adjacent to the organosum vasculosum laminae terminalis (OVLT). b is an enlargement of a.Very large stigmoid bodies are frequent (arrowheads). Asterisk indicates blood vessel as a landmark. a: bar = 50 km. b: bar = 20 km.

contained very large stigmoid bodies (Fig. 8c). Compared to the small, round neurons of the central division, these neurons were larger in size and were elongated or ovoid in shape. Rostrally, the large inclusions were situated in the medial division surrounding the lateral side of the central division, but more caudally, they were progressively partitioned into the lateral division (Figs. 7, 8). Most caudally, they were situated in the lateral division and extended dorsolaterally to the posterodorsal preoptic or magnocellular nuclei of the bed nucleus of the stria terminalis. Neurons containing stigmoid bodies were present throughout the rostrocaudal extent of the periventricular hypothalamic nucleus (Figs. 7, lo), large stigmoid bodies occurring more frequently in the preoptic part of this nucleus than in the posterior hypothalamic part. In addition to the abovementioned preoptic nuclei, some smaller stigmoid bodies were localized in the anterodorsal preoptic, the anteroventral preoptic, the parastrial, and the posterodorsal preoptic

nuclei. Other neurons containing stigmoid bodies were diffusely distributed in the medial preoptic area. Some were present in the lateral preoptic area. In this area, neurons containing medium-sized stigmoid bodies were frequent in the medial forebrain bundle where it begins to diverge and to mingle with the inferior thalamic radiation. More caudally, there were fewer, but still many immunoreactive stigmoid bodies in the anterior hypothalamus which is continuous with the preoptic area. Some immunoreactive stigmoid bodies were widely scattered throughout the rostrocaudal extent in the anterior hypothalamic area. A neural group containing medium- to large-sized stigmoid bodies was localized in the lateroanterior hypothalamic nucleus. Many neurons containing stigmoid bodies were scattered in the periventricular nucleus of the anterior hypothalamic region, although their density was less than that in the preoptic region. A few stigmoid bodies were observed in the dorsal periphery of the suprachiasmatic

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Fig. 7. hPAXP2 immunoreactive stigmoid bodies in the medial preoptic nucleus at the posterior level. The medial preoptic nucleus (MP) is further subdivided roughly into the medial (m) central ( c ) and lateral (I) parts by broken lines. b is an enlargement of the central part and of the periventricular nucleus (Pe) in a. Areas indicated by asterisks in a and b are greatly enlarged in Figure 8. a: bar = 100 km. b: bar = 50 +m.

nucleus and in the anterodorsal aspect of the supraoptic nucleus. In the paraventricular nucleus of the hypothalamus, the density of stigmoid bodies was low and they were largely localized to the parvicellular subdivision. In the posterior hypothalamus, the most prominent area containing immunoreactive stigmoid bodies was the arcuate nucleus. A few medium to large stigmoid bodies were observed at the rostral levels of this nucleus, while more were found at the levels of the median eminence and mammillary region (Fig. 10);stigmoid bodies were more densely distributed at the more caudal levels of the nucleus.

In the mammillary region, especially, almost all the neurons in the arcuate nucleus appeared to contain wellstained medium-sized stigmoid bodies (Fig. 10d). Large stigmoid bodies were occasionally present in this nucleus, but only at the level of the median eminence (Fig. lob). In the dorsomedial hypothalamic nucleus, small- to mediumsized stigmoid bodies were distributed in both the compact and diffuse parts, being more prominent in the former (Fig. lld). In the ventromedial hypothalamic nucleus, weakly stained stigmoid bodies were sparse and did not show regional concentrations. Neurons of the tuberomagnocellu-

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Fig. 8. High magnification of the stigmoid bodies in four parts of the medial preoptic regions indicated by corresponding asterisks in Figure 7: the medial (a),central (b),and lateral ( c ) parts of the medial preoptic nucleus and the periventricular nucleus (d).Note that the average size of the stigmoid bodies in the medial (a) and central parts (b) of the medial preoptic nucleus is relatively small compared to that in the

lateral part (c) and in the periventricular nucleus (d), very large structures being frequent in the lateral part. Diameters of some stigmoid bodies (arrowheads in c) are approximately 4 pm. Double arrowheads in d indicate neurons which contain more than three very small stigmoid bodies (less than 0.5 pm in diameter). a-d: bars = 10 pm.

lar nucleus occasionally contained stigmoid bodies. The stigmoid bodies were sparsely distributed in other posterior hypothalamus, including the tuber cinerium, the dorsal hypothalamic area, and the posterior hypothalamic area. In the lateral hypothalamus, scattered stigmoid bodies were found in the medial forebrain bundle, especially in the area just medial to the internal capsule (Fig. llb). A few of these bodies were dispersed in the supramammillary area and a limited part of the premammillary nucleus; they were rarely seen in the medial and lateral mammillary nuclei. Neurons of the tuberomammillary nucleus often contained stigmoid bodies (Fig. llc). In the zona incerta (Fig. l l a ) numerous stigmoid bodies were distributed in the anterior part rostra1 to the external medullary lamina. The neural group containing the stigmoid bodies extended widely from the area along the external medullary lamina rostrally to the caudal end of the medial division of the bed nucleus of the stria terminalis, medially to the area dorsal to the paraventricular and dorso- medial hypothalamic nuclei, and ventrocaudally to the lateral hypothalamic area adjacent to the internal capsule.

In the thalamus, stigmoid bodies were rarely seen at anterior levels. Some were found in limited areas of the posterior levels, including the subparafascicular thalamic nucleus and the posterior portion of the dorsal lateral geniculate body along the acoustic radiation and contiguous to the ventral lateral geniculate body. Some stigmoid bodies were present in the periventricular fiber system of the posterior diencephalon (Fig. lle). The neural group in this fiber system appeared to form a band along the third ventricle of the posterior diencephalon; it extended from the precommissural area or posterior part of the paraventricular thalamic nucleus ventrally to the posterior hypothalamic area, ventrocaudally to the medial forebrain bundle, ventrorostrally to the zona incerta, and dorsocaudally to the central gray. A few stigmoid bodies were seen in the area between the medial lemniscus and the ventral part of the anterior pretectal nucleus; these structures may be a lateral extension of the subparafascicular thalamic nucleus. A few stigmoid bodies were scattered in the lateral habenular nucleus at the middle levels of its rostrocaudal extent; a little more caudally these bodies were confined to

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Fig. 9. hPAXP2immunoreactive stigmoid bodies in the subfornical organ (SFO).b is an enlargement of a. Asterisks indicate blood vessels as landmarks. vhc, ventral hippocampal commissure. a: bar = 50 krn. b: bar = 25 um.

the medial part of this nucleus, and they were rare in other parts of the habenula, including the medial habenular nucleus and the rostral or posterior extremities of the lateral habenular nucleus. Some small stigmoid bodies were localized in the rostrolateral and dorsal parts of the midbrain central gray (Fig. 110.A few stigmoid bodies were scattered in other parts of the central gray, including the nucleus of Darkschewitsch and the rostral interstitial nucleus of the medial longitudinal fasciculus. In the circumventricular system, many immunoreactive stigmoid bodies were present in the subfornical organ (Fig. 9). A few of these bodies were present in the organosum vasculosum laminae terminalis (OVLT); larger bodies in the region appeared to belong to the median preoptic nucleus adjacent to the OVLT (Fig. 6). Stigmoid bodies were not clearly detected in the hypophysis, the pineal gland, or the subcommissural organ.

DISCUSSION Possible diversity of aromatase-associated structures Our first major finding was that, in addition to the reticulo-filamentous structures demonstrated in previous studies (Shinoda et al., '89b), stigmoid structures immunostained for hPAX-PZ were present in the neuronal cytoplasm of distinct brain regions. The second was that the main regions where such neurons were concentrated included the preoptico-hypothalamic region (especially the medial preoptic region), the medial amygdala, and the bed nucleus of the stria terminalis. The small size of these stigmoid structures probably accounts for the fact that they have previously been overlooked. They were not demonstrated with antibody absorbed with a purified antigen and thus were confirmed to be specifically immunostained for hPAX-P2associated with aromatase. It is important to note

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Fig. 10. hPAX-PZ immunoreactive stigmoid bodies in the arcuate nucleus (Arc) at levels of the median eminence (ME) (a) and of the mammillary region ( c ) .b is an enlargement of the framed area in a. b' and b" are enlargements of the dorsal and ventral parts in b, respectively. The dorsal part of the arcuate nucleus is indicated as a transitional area (T)between the arcuate nucleus and the periventricu-

lar nucleus at this level. d is an enlargement of c. Asterisks indicate blood vessels as landmarks. IIIV, third ventricle; MM, medial mammillary nucleus; MRe, mammillary recess of third ventricle. a: bar = 100 pm. b: bar = 50 pm. b': bar = 20 pm. b": bar = 20 pm. c: bar = 100 pm. d: bar = 25 pm.

that the distribution pattern of stigmoid bodies coincides with that of the aromatase activity previously reported in several biochemical studies (Canick et al., '86; George and Ojeda, '82; Kobayashi and Reed, '77; Roselli et al., '85, '91; Roseli and Resko, '87, '89; Ryan et al., '72; Selmanoff et al., '77; Tobet et al., '85; Weisz and Gibbs, '74). These findings, taken together with the immunological characteristics of the antibody (Matsuda et al., '84; Osawa and Nakamura, '821, indicate that the stigmoid bodies are associated with the localization of aromatase itself or with aromatase activity in the rat brain. There is a discrepancy between our present findings and the immunohistochemical results recently obtained in bird brains by Balthazart and his collaborators ('90a,b). Although the results of both their and our studies show a similar distribution of aromatase-likeimmunoreactivestructures in homologous brain regions in birds and rats (e.g., the preoptic area contained an abundance of immunoreactive neurons with both antibodies), the subcellular localization appeared to be different. We found subcellular localization in a distinct cytoplasmic inclusion, the "stigmoid body" (Shinoda et al., '92), whereas the Balthazart group appears to have observed diffused staining in the cytosol. The cause of the discrepancy may be attributable to different characteristics of the two antibodies or to species differences in aromatase morphology. The hPAX-P2 is considered to be an aromatase-associated antigen complex and is found within estrogen-synthesizing tissues. Al-

though it contains the cytochrome P-450 of aromatase, it also possibly contains other aromatase-associated substances (Shinoda et al., '89a). In the present and previous immunohistochemical studies of the rat brain, at least two types of morphological structures have been immunostained. The first was a reticulo-filamentous structure, which was found predominantly in the primary olfactory receptors of the necklace olfactory system (Shinoda et al., '89a) and in the olfactory bulb (Shinoda et al., '90), and was widely distributed in the forebrain, including the ventral pallidum, the diagonal band, parts of the amygdala, and the cerebral neocortex (Shinoda et al., '89b). The second was the dot-like structure demonstrated here. The presence of two morphologicallydistinct types of immunoreactive structures may reflect antigen association with more than two kinds of heterogeneous components relevant to aromatase. Alternatively, this indicates that the antigen is localized in different subcellular structures in different brain regions. Aromatase isozymes have been reported in goldfish brains (Kitawaki et al., '87) and there is a possibility that there are more than two kinds of aromatase isozymes, which are differently localized in subcellular structures, depending on the brain region and the species. In the zebra finch, it has been suggested that aromatase in the hypothalamus and telencephalon is distinctive in structure and in hormonal regulation (Balthazart et al., '90b; Vockel et al., '90a,b). The immunological diversity of such isozymes also appears likely, in view of the finding that the antibody which

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Fig. 11 hPAX-Pp immunoreactive stigmoid bodies (arrowheads) in the anterior part of the zona incerta (a),the lateral hypothalamus nucleus (b),the tuberomammillary nucleus (c),the dorsomedial hypothalamic nucleus (d),the area along the periventricular fiber system of the posterior diencephalon (el,and the midbrain central gray (0. CGD,

dorsal part of the midbrain central gray; csc, commissure of the superior colliculus; DMHc, compact part of the dorsomedial hypothalamic nucleus; PVS, periventricular fiber system; IIIV, third ventricle. a: bar = 30 pm.b: bar = 30 pm. c: bar = 30 pin. d: bar = 30 pm, e: bar = 50 pm. f: bar = 50 pm.

Balthazart and his collaborators used cannot stain aromatase neurons in some mammalian brains, including those of the monkey and rat (Balthazart et al., '911, although it does recognize aromatase in bird brains (Balthazart et al., '90a,b). In mice, the antibody has been reported to stain some neurons in the lateral hypothalamus, but it does not stain neurons in the preopticohypothalamic region, and its distribution pattern is completely different from that of biochemical aromatase activity.

Overlapping with sex steroid-sensitive regions Brain regions rich in stigmoid bodies are largely involved in reproductive or maternal functions and are interrelated, being joined by anatomical fiber connections. The medial amygdaloid nucleus and the principal nucleus of the bed nucleus of the stria terminalis provide strong inputs to the medial preoptico-hypothalamic nuclei (especially a major projection to the medial preoptic nucleus), which appear to

STIGMOID BODY IN RAT FOREBRAIN be involved in mediating the neuroendocrine, autonomic, and somatomotor responses that are necessary for normal reproductive function (Cowan et al., '65; Heimer and Nauta, '69; Leonard and Scott, '71; Millhouse, '69; Simerly and Swanson, '88). The medial preoptic nucleus shares bidirectional connections with the anterior medial preoptic and the arcuate nuclei, as well as with the above-mentioned medial amygdaloid nucleus and bed nucleus of the stria terminalis (Simerly and Swanson, '86, '88). The amygdalohippocampal area supplies strong afferents to many of the medial preoptico-hypothalamic regions that contain a high density of neurons which express androgen and estrogen receptors (Canteras et al., '89; Krettek and Price, '78; Simerly and Swanson, '86). The ventral division of the lateral septal nucleus projects to the medial and periventricular zones of the preoptico-hypothalamic region (Price et al., '87; Swanson, '87; Swanson et al., '87). There is connectivity from the medial preoptic region to the central periaqueductal gray (Conrad and Pfaff, '76; Swanson et al., '78). Estrogen-concentrating neurons in the medial preoptic region have been shown to project to the medial midbrain (Fahrbach et al., '86). We found that the stigmoid bodies were not only localized in the prominent nuclei, but that they were also disseminated in the area along the stria terminalis, the medial forebrain bundle, and the periventricular fiber system. These fiber systems are intermingled, at the lateral preoptico-hypothalamus, with anterior or posterior parts of the medial forebrain bundle, and connect the preoptico-hypothalamic regions with the lateral septum, the medial and cortical amygdaloid nuclei, the intrahypothalamic nuclei, the neuronal groups in the periventricular zone of the posterior diencephalon, and the midbrain central gray (Chi, '70; Cowan et al., '64; Geeradets et al., '90a,b; Hamilton and Skultety, '70; Millhouse, '69; Nauta, '58; Nieuwenhuys et al., '82; Sutin, '66; Veening et al., '82; Wolf and Sutin, '66). Thus, the stigmoid bodies appear to occur in a functional system that links the medial preopticoanterior hypothalamic region with the other regions of the brain. The distribution patterns resemble the previously proposed "hormone-sensitive pathways" which correlate with the sites of the functional effects of androgen and estrogen (Pfaff and Keiner, '73). Indeed, the brain regions containing the stigmoid bodies largely coincide with steroid binding locations common to both androgen and estrogen (Sar and Stumpf, '75; Sheridan, '78; Sheridan et al., '75; Stumpf and Sar, '76). Such regions include parts of the neocortex, the ventral pallidum (especially in the vicinity of islands of Calleja), the medial preoptic region, the subfornical organ, the ventral division of the lateral septal nucleus, the bed nucleus of the stria terminalis, the medial and cortical amygdaloid nuclei, the hypothalamic periventricular zone, the arcuate nucleus, the ventral premammillary nucleus, the anterior part of the zona incerta, the area along the periventricular fiber system, and the midbrain central gray (Pfaff and Keiner, '73; Sar and Stumpf, '75; Sheridan et al., '75; Stumpf and Sar, '76). Our results demonstrate that the majority of these regions contained stigmoid bodies, exceptionsbeing parts of the neocortex,the ventral pallidum, the ventral premammillary nucleus, and the ventromedial hypothalamic nucleus. Interestingly, the neocortex and ventral pallidum do contain large numbers of hPAX-P2 immunoreactive, reticulofilamentous structures (Shinoda et al., '89b); the prominent group of reticulo-filamentous neurons around the islands of

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Calleja which correspond to the rostra1 extension of the ventral pallidum, are present in the same regions as the densely estrogen-concentrating neurons demonstrated in autoradiographic studies of rat brains (Pfaff and Keiner, '73; Sar and Stumpf, '75; Sheridan et al., '75; Stumpf and Sar, '76). The combined distribution of hPAX-P2immunoreactive dot-likeand reticulo-filamentous structures largely matches the distribution of steroid-concentrating regions for androgen and estrogen in the rodent forebrain. The two forms of immunoreactive structures appear to be complementary. Recently, an in situ hybridization study (Simerly et al., '90) clearly showed that mRNA expression of androgen and estrogen receptors overlapped in the majority of their common binding regions. Thus, the distribution of stigmoid bodies appears to overlap not only with those brain regions where aromatase activity is high, but also with those in which androgen and estrogen receptors are present. It is likely that regions where immunoreactive stigmoid bodies are frequent develop sexual dimorphism as a result of exposure to high or low levels of circulating androgen during the perinatal period, e.g., most nuclei in the medial preoptic regions, the medial amygdaloid nucleus, and the principal nucleus of the bed nucleus of the stria terminalis (Bleier et al., '82; Bloch and Gorski, '88a; Guillamdn et al., '88; Simerly et al., '84). It is of interest to note that the size and immunoreactivity of the stigmoid bodies differed in different nuclei and at different ages. Large stigmoid bodies with diameters exceeding 3 pm were most frequent in the area extending from the median preoptic nucleus surrounding the OVLT to the anterior medial preoptic nucleus, in the periventricular nucleus of the preoptic region, and in the lateral part of the medial preoptic nucleus at posterior levels (Fig. 8). It has been demonstrated that the anterior medial preoptic nucleus is approximately 1.6 times larger in the female rat than in the male (Bleier et al., '82; Bloch and Gorski, '88b; Simerly et al., '84) and that, in female rats, it is involved in the regulation of the phasic release of luteinizing hormone (LH) in female rats (Wiegand and Terasawa, '82). The lateral division of the medial preoptic nucleus has also been reported to be proportionately larger in this nucleus of the female (Blochand Gorski, '88a; Simerly et al., '84). Furthermore, the periventricular hypothalamic nucleus has been shown to be larger, more densely cellular, and less clearly demarcated from the adjacent medial preoptic nucleus in the female than in the male (Bleier et al., '82; Simerly et al., '84). These nuclei which are more prominent in the female contain multiple stigmoid bodies. By contrast, small- t o medium-sized stigmoid bodies are predominant in the central division (Fig. 8) of the medial preoptic nucleus, which is prominently larger in the male (Bloch and Gorski, '88a; Simerly et al., '84). It thus is possible that large stigmoid bodies are associated with feminization of sexually dimorphic areas or with protection from neuronal masculinization induced by androgen in the target sites. Although quantitative analysis of the stigmoid bodies has yet to be done, it is already evident that these structures become smaller and more dispersed throughout most brain regions as rats grow older. This may be the morphologic counterpart of the biochemical decrease of aromatase activity reported in the preoptico-anterior hypothalamic region of older rats (George and Ojeda, '82; Tobet et al., '85). Taken together, these lines of evidence indicate that, in the rat, stigmoid bodies in gonadal hormone-sensitive

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forebrain regions involved in reproductive functions may play an important role in some mechanism that mediates aromatization from androgen to estrogen and the subsequent modulation of their receptor binding.

ACKNOWLEDGMENTS This research was supported by a Grant-in-Aid for Encouragement of Young Scientists from the Japanese Ministry of Education, Science, and Culture (04857009). The authors thank Dr. John Pearson for critical reading of the manuscript. We are also grateful to Mrs. Junko Shinoda for her encouragement throughout this work.

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An aromatase-associated cytoplasmic inclusion, the "stigmoid body," in the rat brain: I. Distribution in the forebrain.

An aromatase-containing neural system was examined in the rat forebrain, using a polyclonal antibody against aromatase-associated human placental anti...
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