Aromatase-immunoreactive Neurons Research Articles

Rapid testosterone-induced growth of the medial preoptic nucleus in male canaries

Testosterone activates singing within days in castrated male songbirds but full song quality only develops after a few weeks. Lesions of the medial preoptic nucleus (POM) inhibit while stereotaxic testosterone implants into this nucleus increase singing rate suggesting that this site plays a key role in the regulation of singing motivation. Testosterone action in the song control system works in parallel to control song quality. Accordingly, systemic testosterone increases POM volume within 1–2 days in female canaries, while the increase in volume of song control nuclei takes at least 2 weeks. The current study tested whether testosterone action is associated with similar differences in latencies in males. Photosensitive castrated male canaries were implanted with testosterone-filled Silastic™ implants and control castrates received empty implants, while simultaneously the photoperiod was switched from short- to long-days. Brains were collected from all subjects two days later. Plasma testosterone was elevated in testosterone-treated but not in controls. HVC volumes were not affected, but testosterone significantly increased the POM volume as identified by the dense group of aromatase-immunoreactive neurons, the number and somal area of these neurons and the fractional area they cover in POM. Testosterone-treated females from a previous experiment had a smaller POM volume in similar conditions suggesting the existence of a stable sex difference potentially affecting singing behavior. Thus testosterone induces male POM growth and aromatase expression in this nucleus within two days without affecting HVC size, further supporting the notion that testosterone increases singing motivation via its action in POM.

Testosterone-induced neuroendocrine changes in the medial preoptic area precede song activation and plasticity in song control nuclei of female canaries.

Testosterone plays a key role in the control of seasonal changes in singing behavior and its underlying neural circuitry. After administration of exogenous testosterone, song quality and song control nuclei volumes change over the course of weeks, but song rate increases within days. The medial preoptic nucleus (POM) controls sexual motivation and testosterone action in POM increases sexually motivated singing. In this study, we investigated the time course of testosterone action in the song control nuclei and POM, at the gross anatomical and cellular level. Photosensitive female canaries were injected with BrdU to label newborn neurons. One day later they were transferred to a long-day photoperiod and implanted with testosterone-filled or empty implants. Brains and blood were collected 1, 2, 9 or 21days later. Testosterone increased POM volume within 1day, whereas the volume of song control nuclei increased significantly only on day 21 even if a trend was already observed for HVC on day 9. The density of newborn neurons in HVC, labeled by Bromodeoxyuridine (BrdU) and doublecortin, was increased by testosterone on days 9 and 21 although a trend was already detectable on day 2. In POM, testosterone increased the number and size of aromatase-immunoreactive neurons already after 1day. This rapid action of testosterone in POM supports its proposed role in controlling singing motivation. Although testosterone increased the number of newborn neurons in HVC rapidly (9, possibly 2days), it is unlikely that these new neurons affect singing behavior before they mature and integrate into functional circuits.

Aromatase expression in the brain of the ruffed grouse (Bonasa umbellus) and comparisons with other galliform birds (Aves, Galliformes)

The enzyme aromatase is important for regulating sexual and aggressive behaviors during the reproductive season, including many aspects of courtship. In birds, aromatase is expressed at high levels in a number of different brain regions. Although this expression does vary among species, the extent to which the distribution of aromatase positive cells reflects species differences in courtship and other behaviors is not well established. Here, we examine the distribution of aromatase immunoreactive (ARO) neurons in the brain of a species with a unique courtship display, the ruffed grouse (Bonasa umbellus). Unlike most other galliforms, male ruffed grouse do not vocalize as part of their courtship and instead use their wings to create a non-vocal auditory signal to attract females. Because aromatase is involved in courtship behaviors in several bird species, including other galliforms, we hypothesized that aromatase distribution in the ruffed grouse would differ from that of other galliforms. We used an antibody raised against quail aromatase to examine aromatase immunoreactivity in the ruffed grouse, the closely related spruce grouse (Falcipennis canadensis) and the Japanese quail (Coturnix japonica). In all three species, ARO neurons were identified in the medial preoptic nucleus, the bed nucleus of the stria terminalis and the nucleus ventromedialis hypothalami. Both grouse species had ARO neurons in two regions of the telencephalon, the hyperpallium, and entopallium, and the ruffed grouse also in field L. ARO neurons were only found in one region in the telencephalon of the Japanese quail, the septum. In general, breeding male ruffed grouse had significantly more ARO neurons and those neurons were larger than that of both the non-breeding male and female ruffed grouse. Aromatase expression in the telencephalon of the ruffed grouse suggests that steroid hormones might modulate responses to visual and acoustic stimuli, but how this relates to species differences in courtship displays and co-expression with estrogenic receptors is yet to be determined.

Sex differences in projections from preoptic area aromatase cells to the periaqueductal gray in Japanese quail

In many vertebrate species the medial preoptic area projects to a premotor nucleus, the periaqueductal central gray (PAG). This connection plays an important role in the control of reproductive behavior. In male Japanese quail (Coturnix japonica) specifically, the medial preoptic nucleus (POM), where various types of sensory inputs converge, is a critical site for the activational action of testosterone on male sexual behavior. To activate male copulatory behavior, testosterone must be aromatized to estradiol within the POM and aromatase-immunoreactive cells in the POM are the main source of projections to the PAG. The POM-PAG connection is thus an important functional circuit integrating the sensory with premotor components of sexual behavior. Contrary to what is observed in males, testosterone does not activate male-typical copulatory behavior in females and we investigated here via retrograde tracing methods whether this behavioral sexual difference is associated with a sex difference in connectivity between POM and PAG. Fluorescent microspheres were injected in the PAG of male and female quail and retrogradely labeled fluorescent cells counted in four fields of the POM in sections that had been immunolabeled for aromatase. Males had more aromatase-immunoreactive neurons projecting to the PAG than females and this difference was most prominent in the caudolateral part of the nucleus that has been specifically implicated in the control of male copulatory behavior. These data therefore support the hypothesis that sex differences in POM-PAG connectivity are causally linked to the sex difference in the behavioral response to testosterone.

Topography and function of androgen-metabolizing enzymes in the central nervous system

The present review describes concisely the topography and function of the three androgen-metabolizing enzymes, namely aromatase, 5alpha-reductase and 3alpha-hydroxysteroid dehydrogenase, in the central nervous system (CNS). Aromatase, estrogen synthetase, is the key enzyme for converting androgens to estrogens. Aromatase is indispensable for the sexual differentiation of the brain and the enzyme activity and expression of aromatase are high during the critical period of neural development, which extends from the late embryonal to the early neonatal period in rodents. Aromatase is expressed in neurons within specific hypothalamic and limbic regions. The locations of aromatase-immunoreactive neurons are divided into three groups according to the period of enzyme expression. Steroid 5alpha-reductase converts a number of steroids with a C3 ketone group and a C4-C5 double bond (delta4; androgens, progestins and glucocorticoids) to their 5alpha-reduced metabolites. Two isoforms of 5alpha-reductase are found and type 1 is predominant in neural tissues. The enzyme activity of 5alpha-reductase is found widely in the CNS and is high in white matter regions. The enzyme expression of 5alpha-reductase peaks during the late embryonic period. 3alpha-Hydroxysteroid dehydrogenase is the oxidoreductase that interconverts 3-ketosteroids to 3alpha-hydroxysteroids. Four isozymes have been found in humans and only one type has been found in rats. The enzyme converts 5alpha-reduced steroids (e.g. 5alpha-dihydroprogesterone) to tetrahydrosteroids (e.g. 3alpha,5alpha-tetrahydroprogesterone). The latter steroid is a potent stimulator of the GABA(A) receptor. The activity of 3alpha-hydroxysteroid dehydrogenase is high during the first 1-2 postnatal weeks, decreases with development and this enzyme is highly expressed in astrocytes.

Electrophysiological and neurochemical characterization of neurons of the medial preoptic area in Japanese quail ( Coturnix japonica)

Intracellular recordings of medial preoptic neurons demonstrated that most neurons show a spontaneous firing, a linear I– V relationship and low-threshold-like events suppressed by the application of Ni 2+. Some neurons had a depolarizing sag of the membrane potential in response to hyperpolarizing current pulses. The majority of the cells exhibited a robust spontaneous synaptic activity suppressed by SR95531 (100 μM), a GABA A receptor antagonist, and/or by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 μM), an ( RS)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate (KA) glutamate receptor antagonist. Most neurons were affected by the application of AMPA (10 μM), kainate (30 μM), N-methyl- d-aspartic acid (NMDA, 10 μM), isoguvacine (a GABA A receptor agonist, 100 μM), dopamine (100 μM), and norepinephrine (100 μM). Biocytin injections coupled to aromatase immunocytochemistry identified 19 recorded neurons including 3 displaying a dense aromatase immunoreactivity. All of them responded to kainate, dopamine, and norepinephrine, while only one responded to isoguvacine and NMDA. Taken together, these results demonstrate a relative electrical and neurochemical homogeneity of the medial preoptic neurons, including a few aromatase-immunoreactive neurons that could be identified by immunocytochemistry after biocytin labeling of the recorded neurons.

Aromatase (Estrogen Synthase) Activity in the Dorsal Horn of the Spinal Cord: Functional Implications

The presence of aromatase (estrogen synthase) in neurons in the dorsal horn of the spinal cord in Japanese quail suggests that estrogens produced locally from androgens could control spinal sensory processes including nociception. We used the hot water nociceptive test (54 degrees C) to appraise the long-term effect of an inhibition of aromatization on the foot withdrawal latency in male quail. Four weeks after the ablation of their main source of testosterone (testes), castrated males displayed a significantly higher foot withdrawal latency than gonadally intact males. A prolonged treatment with subcutaneous capsules filled with testosterone or 17 beta-estradiol restored the baseline latency within 2 weeks. The effect of testosterone in castrated quail was almost completely blocked by systemic injections of Vorozole(TM), a nonsteroidal aromatase inhibitor or tamoxifen, an estrogen receptor antagonist (one injection per day for 10 days). Taken together, these data demonstrate for the first time to our knowledge an effect of estrogens formed by aromatization of androgens on nociception. Because aromatase-immunoreactive neurons and aromatase activity are present in the dorsal horns of the spinal cord, this control of pain thresholds is presumably mediated, at least in part, by estrogens produced at the spinal level that act locally via slow, presumably genomic, mechanisms mediated by the activation of spinal nuclear estrogen receptors.

Anatomical relationships between aromatase-immunoreactive neurons and nitric oxide synthase as evidenced by NOS immunohistochemistry or NADPH diaphorase histochemistry in the quail forebrain

In Japanese quail ( Coturnix japonica), previous studies indicated that the distribution of reduced nicotinamide dinucleotide phosphate (NADPH) diaphorase overlaps with steroid-sensitive areas that contain dense populations of aromatase-immunoreactive (ARO-ir) cells. We investigated here the anatomical relationships between aromatase (ARO) and nitric oxide synthase (NOS)-containing cells that were visualized both by NOS-immunohistochemistry and NADPH-histochemistry. The distribution of ARO-ir and of NADPH-positive cells in the forebrain observed here matched exactly the distribution previously reported. The distribution of NOS-immunoreactive material in the vicinity of ARO-ir cell groups appeared similar to the distribution of NADPH-positive structures previously identified by histochemistry. The number of NOS-immunoreactive cells was similar to the number of NADPH-positive cells and they were found in the same brain regions. In contrast, few NOS-immunoreactive fibers were observed whereas numerous NADPH-positive fibers and punctuate structures were present in many areas. Major groups of NOS-immunoreactive/NADPH-positive neurons were adjacent to the main ARO-ir cell groups, such as the medial preoptic nucleus, the bed nucleus of the stria terminalis and the nucleus ventromedialis hypothalamic. However, examination of adjacent sections indicated that there is very little overlap between the NOS-immunoreactive and ARO-ir cell populations. This notion got further support by double-labeled sections where no double-labeled cells could be identified. In sections stained simultaneously by histochemistry for NADPH and immunohistochemistry for ARO, many NADPH-positive fibers and punctate structures were closely associated with ARO-ir perikarya. Taken together, the present data indicate that NOS is not or very rarely colocalized with ARO but that NOS inputs are closely associated with ARO-ir cells. Based on previous work in a variety of model systems, it can be hypothesized that these inputs modulate the expression or activity of ARO in the quail brain.

Preoptic Aromatase Cells Project to the Mesencephalic Central Gray in the Male Japanese Quail (Coturnix japonica)

Previous tract-tracing studies demonstrated the existence of projections from the medial preoptic nucleus (POM) to the mesencephalic central gray (GCt) in quail. GCt contains a significant number of aromatase-immunoreactive (ARO-ir) fibers and punctate structures, but no ARO-ir cells are present in this region. The origin of the ARO-ir fibers of the GCt was investigated here by retrograde tract-tracing combined with immunocytochemistry for aromatase. Following injection of fluorescent microspheres in GCt, retrogradely labeled cells were found in a large number of hypothalamic and mesencephalic areas and in particular within the three main groups of ARO-ir cells located in the POM, the ventromedial nucleus of the hypothalamus, and the bed nucleus striae terminalis. Labeling of these cells for aromatase by immunocytochemistry demonstrated, however, that aromatase-positive retrogradely labeled cells are observed almost exclusively within the POM. Double-labeled cells were abundant in both the rostral and caudal parts of the POM and their number was apparently not affected by the location of the injection site within GCt. At both rostro-caudal levels of the POM, ARO-ir retrogradely labeled cells were, however, more frequent in the lateral than in the medial POM. These data indicate that ARO-ir neurons located in the lateral part of the POM may control the premotor aspects of male copulatory behavior through their projection to GCt and suggest that GCt activity could be affected by estrogens released from the terminals of these ARO-ir neurons.

Effects of brain testosterone implants on appetitive and consummatory components of male sexual behavior in Japanese quail

Aromatization of testosterone (T) into an estrogen is necessary for the activation of consummatory and appetitive sexual behavior in male Japanese quail. T action within the medial preoptic nucleus (POM) is necessary and sufficient to activate consummatory behavior, and some evidence suggests that POM might be involved in the control of appetitive behavior, but other brain regions, such as the bed nucleus of the stria terminalis (BST), an area that contains a dense population of aromatase-immunoreactive neurons, are also likely to be involved. This study was performed to assess the effects of stereotaxic T implants targeting either the POM or the BST on the activation of both components of sexual behavior in castrated male quail. Appetitive sexual behavior was measured by an acquired social proximity response in which a male will approach a window providing visual access to a female after the window has been repeatedly paired with physical access to a female and the possibility to freely interact with her. Rhythmic cloacal sphincter movements that are produced by the male when given visual access to a female were used as another measure of appetitive sexual behavior that does not appear to depend on sexual learning. The experiments confirmed that copulation is necessary for males to develop the social proximity response that is used to measure the appetitive sexual behavior. T implants in the POM activated both components of sexual behavior, suggesting that these components cannot be completely dissociated. In contrast, T implants located within the BST did not affect either component, but because implants in the BST did not activate copulatory behavior, these results do not preclude a role for BST in the expression of a previously acquired appetitive sexual behavior.

Identification of catecholaminergic inputs to and outputs from aromatase-containing brain areas of the Japanese quail by tract tracing combined with tyrosine hydroxylase immunocytochemistry

In the quail brain, aromatase-immunoreactive (ARO-ir) neurons located in the medial preoptic nucleus (POM) and caudal paleostriatum ventrale/nucleus accumbens/nucleus striae terminalis complex (PVT/nAc/nST) receive catecholaminergic inputs identified by the presence of tyrosine hydroxylase-immunoreactive (TH-ir) fibers and punctate structures. The origin of these inputs was analyzed by retrograde tracing with cholera toxin B subunit (CTB) or red latex fluospheres (RLF) combined with TH immunocytochemistry. CTB and RLF injected in the POM or PVT/nAc/nST were found in cells located in anatomically discrete areas in the telencephalon (hippocampus, septum, archistriatum), hypothalamus (many areas in periventricular position), thalamus, mesencephalon, and pons. In these last two regions, many retrogradely labeled cells were located in dopaminergic areas such as the retroruberal field (RRF), substantia nigra (SN), and area ventralis of Tsai (AVT) but also in noradrenergic cell groups such as the locus ceruleus and subceruleus. CTB tracing showed that most of these connections are bidirectional. Many retrogradely labeled cells contained TH-ir material. As a mean, 10-20% and 40-60% of the RLF-containing cells in the dopaminergic areas were TH-ir when RLF had been injected in the POM or PVT/nAc/nST, respectively. TH-ir cells projecting to the POM appeared to be mostly located in the periventricular hypothalamus and in AVT, whereas projections to the PVT/nAc/nST originated mainly in the SN (with significant contributions from the RRF and AVT). These data support the existence of functional relationships between aromatase and catecholamines.

Androgens stimulate the morphological maturation of embryonic hypothalamic aromatase-immunoreactive neurons in the mouse

Gonadal steroids play an important role as developmental factors for the rodent brain and are implicated in the sexual differentiation of neural structures. Estrogens have been linked to survival and plasticity of central neurons, thereby regulating the development of hypothalamic and limbic structures associated with reproductive functions. Besides estrogens, androgens also contribute actively to CNS maturation. We have shown recently that androgens stimulate the receptor-mediated functional differentiation of cultured hypothalamic aromatase-immunoreactive (Arom-IR) neurons by stimulating the expression of Arom, the key enzyme in estrogen formation. In the present study, we investigated whether androgens are capable of influencing morphological differentiation of hypothalamic Arom-IR neurons. Androgen treatment, unlike estrogen, stimulated the morphological differentiation of cultured embryonic hypothalamic Arom-IR cells by increasing neurite outgrowth and branching, soma size, and the number of stem processes. This effect was brain region- and transmitter phenotype-specific; neither cortical Arom-IR neurons nor hypothalamic GABAergic neurons responded to androgens. Moreover, morphogenetic effects depended on androgen receptor (AR) activation, since morphological changes were completely inhibited by flutamide. Double-labeling of hypothalamic Arom-IR neurons revealed a considerable number of cells coexpressing AR, whereas cortical Arom-IR cells did not label for AR. Our data demonstrate that androgens function as morphogenetic signals for developing hypothalamic Arom-IR cells, thus being potentially effective in influencing plasticity and synaptic connectivity of hypothalamic Arom-systems.

Gender-specific steroid metabolism in neural differentiation.

1. Both the neuroendocrine system and the brain mechanisms underlying gender-specific behavior are known to be organized by steroid sex hormones, androgen and estrogen, during specific sensitive phases of early fetal and perinatal development. The factors that control these phasic effects of the hormones on brain development are still not understood. Processes of masculinization and defeminization are thought to be involved in the sex differentiation of mammalian reproductive behavior. 2. The P450 aromatase, converting androgen to estrogen, is a key enzyme in the development of neural systems, and the activity of this enzyme is likely to be one of the factors determining brain sex differentiation. 3. We have examined the localization and regulation of brain aromatase using the mouse as a model. Measurement of testosterone conversion to estradiol-17 beta, using a sensitive radiometric 3H2O assay, indicates that estrogens are formed more actively in the male mouse brain than in the female during both the prenatal and the neonatal periods. In primary cell cultures of embryonic mouse hypothalamus there are sex differences in aromatase activity during early and late embryogenesis, with a higher capacity for estrogen formation in the male than the female. These sex differences are regionally specific in the brain, since on gender differences in aromatase activity are detectable in cortical cells. 4. Aromatase activity in the mouse brain is neuronal rather than glial. Using a specific antibody to the mouse aromatase, immunoreactivity is restricted to neuronal soma and neurites in hypothalamic cultures. There are more neurons containing expressed aromatase in the male hypothalamus than in the female. Therefore, gender-specific differences in embryonic aromatase activity are neuronal. 5. Testosterone increases aromatase activity specifically in hypothalamic neurons, but has no effect on cortical cells. The neuronal aromatase activity appears to be sensitive to the inductive effects of androgen only in the later stages of embryonic development. Androgen also increases the numbers of aromatase-immunoreactive neurons in the hypothalamus. 6. This work suggests that the embryonic male hypothalamus and other androgen target areas contain a network of neurons which has the capacity to provide estrogen for the sexual differentiation of brain mechanisms of behavior. The phasic activity of the key enzyme, aromatase, during development is influenced by androgen. What determines the developmental action of androgen and the other factors involved in the regulation and expression of this neuronal enzyme still have to be established.

Immunohistochemical localization of estrogen receptors within aromatase-immunoreactive neurons in the fetal and neonatal rat brain.

We elucidated the anatomical relationship between estrogen receptors and aromatase, the enzyme converting androgens to estrogens, in the fetal and neonatal rat brain by means of double immunohistochemical labeling, using antibodies against rat estrogen receptors and human placental aromatase cytochrome P450. Numerous aromatase-immunoreactive neurons were found in the medial preoptic area, the bed nucleus of the stria terminalis, the medial amygdaloid nucleus and the ventromedial nucleus. Estrogen receptors were also abundant in these areas. Most of the aromatase-immunoreactive neurons showed immunoreactivity for estrogen receptors in the medial subdivision of the bed nucleus of the stria terminalis and in the posterodorsal, division of the medial amygdaloid nucleus. There were also many double-labeled cells in the ventromedial nucleus. However, in the medial preoptic area the localization of aromatase-immunoreactive neurons was distinct from that of neurons containing estrogen receptors. These results suggested that estrogens, which are converted from androgens in aromatase-containing neurons, are involved in the sexual differentiation of the brain through estrogen receptors within aromatase-immunoreactive neurons in the bed nucleus of the stria terminalis, the medial amygdaloid nucleus and the ventromedial nucleus, but through estrogen receptors in aromatase-immunonegative neurons in the medial preoptic area.

Presence of estrogen receptors in aromatase-immunoreactive neurons in the mouse brain

The present study was undertaken to demonstrate estrogen receptors in aromatase-immunoreactive neurons in the fetal and neonatal mouse brain by using the double immunohistochemical technique. The majority of aromatase-immunoreactive neurons showed the immunoreactivity for estrogen receptors in the cell nuclei in the medial subdivision of the bed nucleus of the stria terminalis and the posterodorsal division of the medial amygdaloid nucleus, but a few did in the medial preoptic area. These results suggest that aromatase-immunoreactive neurons in the former two areas are regulated by steroids through estrogen receptors in their cell nuclei.

Sexual differentiation of brain and behavior in quail and zebra finches: studies with a new aromatase inhibitor, R76713.

In many species of vertebrates, major sex differences affect reproductive behavior and endocrinology. Most of these differences do not result from a direct genomic action but develop following early exposure to a sexually differentiated endocrine milieu. In rodents, the female reproductive phenotype mostly develops in the absence of early steroid influence and male differentiation is imposed by the early action of testosterone, acting at least in part through its central conversion into estrogens or aromatization. This pattern of differentiation does not seem to be applicable to avian species. In Japanese quail (Coturnix japonica), injection of estrogens into male embryos causes a permanent loss of the capacity to display male-type copulatory behavior when exposed to testosterone in adulthood. Based on this experimental result, it was proposed that the male reproductive phenotype is "neutral" in birds (i.e. develops in the absence of endocrine influence) and that endogenous estradiol secreted by the ovary of the female embryo is responsible for the physiological demasculinization of females. This model could be recently confirmed. Females indeed display a higher level of circulating estrogens that males during the second part of their embyronic life. In addition, treatment of female embryos with the potent aromatase inhibitor, R76713 or racemic vorozole which suppresses the endogenous secretion of estrogens maintains in females the capacity to display the full range of male copulatory behaviors. The brain mechanisms that control this sexually differentiated behavior have not been identified so far but recent data suggest that they should primarily concern a sub-population of aromatase-immunoreactive neurons located in the lateral parts of the sexually dimorphic preoptic nucleus. The zebra finch (Taeniopygia guttata) exhibits a more complex, still partly unexplained, differentiation pattern. In this species, early treatment with exogenous estrogens produces a masculinization of singing behavior in females and a demasculinization of copulatory behavior in males.(ABSTRACT TRUNCATED AT 400 WORDS)

Aromatase immunoreactive neurons and neuronal processes in the hypothalamus of human (IR-ARO) fetuses and adult subjects

Aromatase immunoreactive neurons and neuronal processes in the hypothalamus of human (IR-ARO) fetuses and adult subjects

Aromatase- (estrogen synthetase) immunoreactive neurons in the rat septal area. A light and electron microscopic study

The aromatase enzyme (estrogen synthetase) catalyzes the conversion of testosterone to estrogen in peripheral and central nervous tissue. Light and electron microscopic immunocytochemistry was used to study the localization of this enzyme in the septal area of adult male and female albino rats. Aromatase-immunoreactivity was found restricted to neuronal somata and dendritic arbors, and no sex differences were detected in its distribution or intensity. Most aromatase-immunoreactive neurons formed two oblique bands in the lateral and the medial zones of the lateral septum; in addition, labeled cells were present in the septohippocampal nucleus and the laterodorsal portion of the bed nucleus of the stria terminalis. Electron microscopy revealed that the majority of aromatase-positive neurons in the lateral septum exhibit somatic spines, a characteristic marker of a neuron population that is known to contribute to local and extraseptal projections. The presence of aromatase in lateral septal somatospiny neurons suggests that estrogen formed by these neurons may be critically involved in the septal control of steroid-dependent behaviors.

Androgens influence sexual differentiation of embryonic mouse hypothalamic aromatase neurons in vitro.

Estrogen formed perinatally in the brain from testicular androgen by aromatase is involved in the irreversible determination of male brain development. Perinatal sex differences in aromatase activity have been observed in the hypothalamus. Testosterone (T) is a major modulator for aromatase in the adult rat hypothalamus. However, it is not known whether circulating T influences aromatase neurons during fetal brain development. To study the influence of androgen exposure on embryonic neuronal aromatase, gender-specific primary cell cultures were prepared from embryonic day 15 mouse hypothalamus and cortex. Estrogen formation by cultured neurons was measured using an in vitro 3H2O product formation assay, and aromatase neurons were identified by immunocytochemistry using a highly specific antiserum. Aromatase activity (AA) per well and numbers of aromatase-immunoreactive (IR) neurons per microtubulus associated protein II-IR neurons x 10(5) were significantly higher in male hypothalamic cultures compared with female when grown in the absence of sex steroids. When AA was calculated per aromatase-IR neuron, no differences in enzyme activity were found between male and female. Therefore, the level of AA in individual male hypothalamic neurons is similar to the female, but a higher proportion of male neurons express aromatase. After T treatment, AA per well (P < or = 0.001) and AA/aromatase-IR cell (P < or = 0.005) in male and female hypothalamic cultures was significantly increased vs. controls. In addition, numbers of aromatase-IR neurons/microtubulus associated protein II-IR neurons x 10(5) were significantly higher after T exposure compared with controls (P < 0.001). Androgenic effects on hypothalamic AA and aromatase-IR cell numbers were dose-dependent and mediated via androgen receptor stimulation, since the observed effects were inhibited by the androgen-receptor antagonist flutamide. There was no effect of T on cortical AA or aromatase-IR cell numbers, indicating area-specific regulation of brain aromatase. We conclude that 1) sex differences in hypothalamic AA are due to a higher percentage of neurons expressing aromatase in males rather than to higher AA in individual male hypothalamic aromatase-IR cells, and 2) androgens influence the development of the fetal hypothalamic aromatase system. Because T influenced both the embryonic male and female hypothalamic neurons in culture, the developing mouse brain aromatase appears to be bipotential in response to androgen. The data suggest that environmental and genetic factors affecting androgen level and/or androgen receptor function in the developing brain could interfere with the sexual differentiation of estrogen forming neurons.

Androgens influence sexual differentiation of embryonic mouse hypothalamic aromatase neurons in vitro

Estrogen formed perinatally in the brain from testicular androgen by aromatase is involved in the irreversible determination of male brain development. Perinatal sex differences in aromatase activity have been observed in the hypothalamus. Testosterone (T) is a major modulator for aromatase in the adult rat hypothalamus. However, it is not known whether circulating T influences aromatase neurons during fetal brain development. To study the influence of androgen exposure on embryonic neuronal aromatase, gender-specific primary cell cultures were prepared from embryonic day 15 mouse hypothalamus and cortex. Estrogen formation by cultured neurons was measured using an in vitro 3H2O product formation assay, and aromatase neurons were identified by immunocytochemistry using a highly specific antiserum. Aromatase activity (AA) per well and numbers of aromatase-immunoreactive (IR) neurons per microtubulus associated protein II-IR neurons x 10(5) were significantly higher in male hypothalamic cultures compared with female when grown in the absence of sex steroids. When AA was calculated per aromatase-IR neuron, no differences in enzyme activity were found between male and female. Therefore, the level of AA in individual male hypothalamic neurons is similar to the female, but a higher proportion of male neurons express aromatase. After T treatment, AA per well (P < or = 0.001) and AA/aromatase-IR cell (P < or = 0.005) in male and female hypothalamic cultures was significantly increased vs. controls. In addition, numbers of aromatase-IR neurons/microtubulus associated protein II-IR neurons x 10(5) were significantly higher after T exposure compared with controls (P < 0.001). Androgenic effects on hypothalamic AA and aromatase-IR cell numbers were dose-dependent and mediated via androgen receptor stimulation, since the observed effects were inhibited by the androgen-receptor antagonist flutamide. There was no effect of T on cortical AA or aromatase-IR cell numbers, indicating area-specific regulation of brain aromatase. We conclude that 1) sex differences in hypothalamic AA are due to a higher percentage of neurons expressing aromatase in males rather than to higher AA in individual male hypothalamic aromatase-IR cells, and 2) androgens influence the development of the fetal hypothalamic aromatase system. Because T influenced both the embryonic male and female hypothalamic neurons in culture, the developing mouse brain aromatase appears to be bipotential in response to androgen. The data suggest that environmental and genetic factors affecting androgen level and/or androgen receptor function in the developing brain could interfere with the sexual differentiation of estrogen forming neurons.