Hypothalamic Aromatase Research Articles

Relevance of c-Src and protein phosphatase 2A to aromatase activity: Evidence of an acute self-regulating oestrogenic signalling complex in rat central nervous system.

We previously reported that aromatase protein levels do not parallel aromatase enzyme activity. This suggests that oestrogenic signalling may be modulated via post-translational modification of aromatase protein. The tyrosine and serine phosphorylation state of aromatase are known to influence its activity. To investigate the possible relevance of aromatase phosphorylation to the incongruity observed between aromatase protein and its activity, we explored interactions between aromatase and the tyrosine kinase c-Src and the serine protein phosphatases 2A and 5 (PP2A and PP5), as well as the relationship between levels of tyrosine-phosphorylated aromatase and the extrapolated aromatase activity. We found that (a) hypothalamic aromatase was significantly more heavily tyrosine-phosphorylated than spinal aromatase; (b) aromatase was oligomerized with c-Src and PP2A/PP5, potentially activating aromatase via tyrosine-phosphorylation and serine-dephosphorylation; (c) the associations of c-Src and PP2A/PP5 with hypothalamic aromatase were substantially greater than with spinal aromatase; and (d) aromatase, oestrogen receptor α, PP2A, and c-Src were present in a common membrane oligomer. The existence of c-Src and PP2A in an oligomer that also contains aromatase and membrane oestrogen receptor α (and presumably other signalling molecules) indicates the presence in the CNS of a potentially self-regulating oestrogenic signalling unit. The degree to which such a complex operates autonomously and the regulatory factors thereof are likely to have substantial physiological implications and clinical relevance.

Programming of the reproductive axis by hormonal and genetic manipulation in mice

In mammals, the reproductive function is controlled by the hypothalamic–pituitary–gonadal axis. During development, mechanisms mediated by gonadal steroids exert an imprinting at the hypothalamic–pituitary level, by establishing sexual differences in the circuits that control male and female reproduction. In rodents, the testicular production of androgens increases drastically during the fetal/neonatal stage. This process is essential for the masculinization of the reproductive tract, genitals and brain. The conversion of androgens to estrogens in the brain is crucial for the male sexual differentiation and behavior. Conversely, feminization of the brain occurs in the absence of high levels of gonadal steroids during the perinatal period in females. Potential genetic contribution to the differentiation of brain cells through direct effects of genes located on sex chromosomes is also relevant. In this review, we will focus on the phenotypic alterations that occur on the hypothalamic–pituitary–gonadal axis of transgenic mice with persistently elevated expression of the human chorionic gonadotropin hormone (hCG). Excess of endogenously synthesized gonadal steroids due to a constant hCG stimulation is able to disrupt the developmental programming of the hypothalamic–pituitary axis in both transgenic males and females. Locally produced estrogens by the hypothalamic aromatase might play a key role in the phenotype of these mice. The ‘four core genotypes’ mouse model demonstrated a potential influence of sex chromosome genes in brain masculinization before critical periods of sex differentiation. Thus, hormonal and genetic factors interact to regulate the local production of the neurosteroids necessary for the programming of the male and female reproductive function.

Sex differences in behavioral and neurochemical effects of gonadectomy and aromatase inhibition in rats

Aromatase inhibitors, which are widely used for the treatment of estrogen-dependent cancers, have been associated with psychiatric side effects ranging from mania to depression. In the present study, we investigated sex differences in the behavioral and neurochemical effects of aromatase inhibition on male and female, sham-operated or gonadectomized adult rats. Three weeks after surgery, rats received chronic treatment with the aromatase inhibitor letrozole or vehicle and were then subjected to the open field test, which assesses general activity. Half of the subjects were subsequently exposed to the stressful procedure of the forced swim test (FST), which is also a test of antidepressant activity. Aromatase activity was analyzed in the hypothalamus and testosterone and corticosterone were assayed in the blood serum of all rats. The hippocampus and prefrontal cortex (PFC) were analyzed for monoamine (noradrenaline, dopamine and serotonin), as well as amino acid (GABA, glutamate, glycine, taurine, alanine and histidine) levels. The observed decrease in hypothalamic aromatase activity confirmed the efficacy of letrozole treatment in both sexes. Moreover, letrozole enhanced testosterone levels in sham-operated females. In the open field test, females were overall more active and explorative than males and gonadectomy eliminated this sex difference. In the FST, females exhibited overall higher immobility than males and gonadectomy further enhanced this passive behavior in both sexes. However, sustained aromatase inhibition had no effect on open field and FST behaviors. Head shakes during FST, which were fewer in females than in males, were reduced by castration in males and by letrozole treatment in ovariectomized females, suggesting a role of testosterone and extra-gonadal estrogens in the expression of this behavior. Sustained aromatase inhibition also decreased noradrenaline and the dopaminergic turnover rates [DOPAC/DA, HVA/DA] in the hippocampus and PFC of male and female rats, irrespectively of gonadectomy. Moreover, letrozole treatment enhanced the serotonergic turnover [5HIAA/5HT] rate in the hippocampus of males and females, irrespectively of gonadectomy. Amino acid levels were not influenced by letrozole, but sex differences were demonstrated with higher levels in the PFC of females vs. males. Present findings suggest that the neuropsychiatric effects of aromatase inhibition can be attributed to the inhibition of extragonadal estrogen synthesis, presumably in the brain, and could be further associated with serotonergic and catecholaminergic changes in brain regions involved in mood and cognition. Importantly, present data could be linked with the neurobiology of affective side-effects in post-menopausal women receiving aromatase inhibitors.

Glucocorticoid-induction of hypothalamic aromatase via its brain-specific promoter

In the brain, a 36-kb distal promoter (I.f) regulates the Cyp19a1 gene that encodes aromatase, the key enzyme for estrogen biosynthesis. Local estrogen production in the brain regulates critical functions such as gonadotropin secretion and sexual behavior. The mechanisms that control brain aromatase production are not well understood. Here we show that the glucocorticoid dexamethasone robustly increases aromatase mRNA and protein by up to 98-fold in mouse hypothalamic cell lines in a dose- and time-dependent fashion. Using deletion mutants of the brain-specific promoter I.f and chromatin immunoprecipitation-PCR, we isolated a distinct region (−500/−200bp) which becomes enriched in bound glucocorticoid receptor upon dexamethasone stimulation. A glucocorticoid antagonist or siRNA based knockdown of glucocorticoid receptor ablated dexamethasone stimulation of aromatase expression. Our findings demonstrate how glucocorticoids alter aromatase expression in the hypothalamus and might indicate a mechanism whereby glucocorticoid action modifies gonadotropin pulses and the menstrual cycle.

Seasonal changes in courtship behavior, plasma androgen levels and in hypothalamic aromatase immunoreactivity in male free-living European starlings ( Sturnus vulgaris)

In songbirds from temperate latitudes, singing during spring has an essential role in mate attraction, while during the non-breeding season it is connected to territorial aggression and/or maintaining dominance hierarchies or flock cohesion. Courtship behavior is regulated by plasma testosterone (T) levels. Other androgens, like dehydroepiandrosterone (DHEA) could be responsible for aggression. The aromatization of androgens in the brain is an essential step in mediating their effects on behavior. Our goal was to determine whether the seasonal changes in male courtship behavior (measured by average song bout length and wing-waving/flicking) are related to seasonal changes in androgen activity (measured by plasma T, DHEA levels) and aromatase (ARO) immunoreactivity in the preoptic area/medial preoptic nucleus (POA/POM) of free-living male starlings. DHEA increased during pair formation, decreased at nesting and remained at low levels. The number of ARO cells – in line with the T levels – increased during the courtship and nesting periods, but outside the breeding season it was low. Song bout length showed a similar pattern, namely the peak was reached during the courtship period, and after that males stopped singing when chicks started to hatch. Short and fast wing-flicking and wing-waving behavior was observed only during the breading season. Summarizing, we have found that song bout length of male starlings changes parallel with plasma T levels and ARO immunoreactivity in the POA/POM. Furthermore, DHEA levels were low during the sexually inactive period which suggests that other mechanisms could be involved in the aggressive non-courtship behavior/vocalization in these birds.

Increase in hypothalamic aromatase in macaque monkeys treated with anabolic-androgenic steroids

In an earlier study in rodents, we showed that the aromatase that converts androgens to estrogens in the preoptic area and bed nucleus of stria terminalis was significantly increased in concentration after exposure to anabolic-androgenic steroids. To confirm whether this occurs in primates, we conducted a positron emission tomographic study using macaque monkeys. Male rhesus monkeys were treated with nandrolone decanoate for 3 weeks. To measure aromatase concentrations, we performed positron emission tomographic imaging using a 11C-labeled specific aromatase inhibitor, [11C]vorozole. After treatment with nandrolone, significant increase in [11C]vorozole binding was observed in the hypothalamus but not other areas including the amygdala, which is also aromatase enriched. These findings in monkeys are consistent with those we obtained earlier in rats. These findings strongly suggest that aromatase in the hypothalamus may play a crucial role in the emotional instability of anabolic-androgenic steroids abusers.

Seasonal changes in courtship behavior, testicular development and in hypothalamic aromatase immunoreactivity in male free-living European starlings (Sturnus vulgaris)

Event Abstract Back to Event Seasonal changes in courtship behavior, testicular development and in hypothalamic aromatase immunoreactivity in male free-living European starlings (Sturnus vulgaris) Ottó Pintér1, 2*, Péter Péczely2 and Dóra Zelena1 1 Institute of Experimental Medicine of the Hungarian Acadamy of Sciences, Department of Behavioural Neurobiology, Hungary 2 Szent István University, Laboratory of Reproductive Biology, Faculty of Agricultural and Environmental Sciences, Hungary Male European starlings (Sturnus vulgaris) sing prior to copulation and together with wing-waving these behaviors play essential role in mate atraction. In birds the activity of testis - as a primary source of androgens production-gradually increases during the breeding season (spring). The aromatization of testosterone - the most important androgen - in brain plays an important role in regulating the behavior. Our goal was to determine wheather the seasonal changes in male courtship behavior (measured by song bout length and wing-waving) are related to seasonal changes in androgen activity (measured by testis volume and aromatase (ARO) activity in the preoptic area (POA)) of free-living male starlings. Song bout length was recorded with a presence of a female and a nesthole. The number of ARO cells - together with the testis volume - increased during the courtship and egg laying period but outside the breeding season (August) they were on minimum. Song bout length showed simmilar pattern, namely the peak was reached during the courtship and egg laying period and after that males stoped their singing, when chicks started to hatch. A second elevation of song bout length was observed during fledging of youngs. The short and fast wing-flicking behavior was observed in males which were singing in the near of the nestholes but the typical wing-waving display occured almost exclusively after introduction of a female. This behavior did not show seasonal changes. Summarizing, we found that song bout length of free-living male starlings went parallel with testicular growth and ARO activity in the brain, except at the hatching time, which suggest that although androgens are essential for inducing male sexual arousal, but they are not exclusive regulators of courtship behavior. Keywords: comparative endocrinology Conference: 25th Conference of the European Comparative Endocrinologists, Pécs, Hungary, 31 Aug - 4 Sep, 2010. Presentation Type: Conference Presentation Topic: Comparative endocrinology Citation: Pintér O, Péczely P and Zelena D (2010). Seasonal changes in courtship behavior, testicular development and in hypothalamic aromatase immunoreactivity in male free-living European starlings (Sturnus vulgaris). Front. Endocrinol. Conference Abstract: 25th Conference of the European Comparative Endocrinologists. doi: 10.3389/conf.fendo.2010.01.00042 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 26 Aug 2010; Published Online: 29 Aug 2010. * Correspondence: Mr. Ottó Pintér, Institute of Experimental Medicine of the Hungarian Acadamy of Sciences, Department of Behavioural Neurobiology, Budapest, Hungary, pinter.otto@koki.hu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Ottó Pintér Péter Péczely Dóra Zelena Google Ottó Pintér Péter Péczely Dóra Zelena Google Scholar Ottó Pintér Péter Péczely Dóra Zelena PubMed Ottó Pintér Péter Péczely Dóra Zelena Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Porcine Hypothalamic Aromatase Cytochrome P450: Isoform Characterization, Sex-Dependent Activity, Regional Expression, and Regulation by Enzyme Inhibition in Neonatal Boars1

Domestic pigs have three CYP19 genes encoding functional paralogues of the enzyme aromatase cytochrome P450 (P450arom) that are expressed in the gonads, placenta, and preimplantation blastocyst. All catalyze estrogen synthesis, but the gonadal-type enzyme is unique in also synthesizing a nonaromatizable biopotent testosterone metabolite, 1OH-testosterone (1OH-T). P450arom is expressed in the vertebrate brain, is higher in males than females, but has not been investigated in pigs, to our knowledge. Therefore, these studies defined which of the porcine CYP19 genes was expressed, and at what level, in adult male and female hypothalamus. Regional expression was examined in mature boars, and regulation of P450arom expression in neonatal boars was investigated by inhibition of P450arom with letrozole, which is known to reprogram testicular expression. Pig hypothalami expressed the gonadal form of P450arom (redesignated the "gonadal/hypothalamic" porcine CYP19 gene and paralogue) based on functional analysis confirmed by cloning and sequencing transcripts. Hypothalamic tissue synthesized 1OH-T and was sensitive to the selective P450arom inhibitor etomidate. Levels were 4-fold higher in male than female hypothalami, with expression in the medial preoptic area and lateral borders of the ventromedial hypothalamus of boars. In vivo, letrozole-treated neonates had increased aromatase activity in hypothalami but decreased activity in testes. Therefore, although the same CYP19 gene is expressed in both tissues, expression is regulated differently in the hypothalamus than testis. These investigations, the first such studies in pig brain to our knowledge, demonstrate unusual aspects of P450arom expression and regulation in the hypothalamus, offering promise of gaining better insight into roles of P450arom in reproductive function.

Prenatal Aroclor 1254 exposure and brain sexual differentiation: Effect on the expression of testosterone metabolizing enzymes and androgen receptors in the hypothalamus of male and female rats

Polychlorinated biphenyls (PCBs) are industrial pollutants detected in human milk, serum and tissues. They readily cross the placenta to accumulate in fetal tissues, particularly the brain. These compounds affect normal brain sexual differentiation by mechanisms that are incompletely understood. The aim of this study was to verify whether a technical mixture of PCBs (Aroclor 1254) would interfere with the normal pattern of expression of hypothalamic aromatase and 5-alpha reductase(s), the two main enzymatic pathways involved in testosterone activation and of androgen receptor (AR). Aroclor 1254 was administered to pregnant rats at a daily dose of 25 mg/kg by gavage from days 15 to 19 of gestation (GD15–19). At GD20 the expression of aromatase, 5-alpha reductase types 1 and 2 and androgen receptor (AR) and aromatase activity were evaluated in the hypothalamus of male and female embryos. The direct effect of Aroclor was also evaluated on aromatase activity adding the PCB mixture to hypothalamic homogenates or to primary hypothalamic neuronal cultures. The data indicate that aromatase expression and activity is not altered by prenatal PCB treatment; 5-alpha reductase type 1 is similarly unaffected while 5-alpha reductase type 2 is markedly stimulated by the PCB exposure in females. Aroclor also decreases the expression of the AR in females. The observed in vivo effects are indicative of a possible adverse effect of PCBs on the important metabolic pathways by which testosterone produces its brain effects. In particular the changes of 5-alpha reductase type 2 and AR in females might be one of the mechanisms by which Aroclor exposure during fetal development affects adult sexual behavior in female rats.

Decreased hypothalamic aromatization in female rats of true precocious puberty

The true precocious puberty animal model induced by the single dose of danazol was used for investigating the expressions of hypothalamic aromatase in the advanced onset of puberty in rats. The day of vaginal opening and first estrus showed significant advancement in the model rats compared with the normal and vehicle rats ( P < 0.01, respectively). The hypothalamic gonadotropin-releasing hormone (GnRH) mRNA expression increased significantly in the model rats compared with that in the normal and vehicle ones ( P < 0.01). The levels of aromatase mRNA and protein expressions detected by RT-PCR and Western blot both decreased in the model rats compared with those in the normal and vehicle groups ( P < 0.05). The results suggested that the hypothalamic aromatization might diminish in the onset of true precocious puberty of female rats.

Androgenic regulation of hypothalamic aromatase activity in prepubertal and postpubertal male golden hamsters.

Doses of testosterone that fully activate male reproductive behavior in castrated adult male hamsters fail to elicit mounting and intromissions in prepubertal castrates, even when circulating levels of testosterone are equivalent in the two age groups. We hypothesize that this differential responsiveness to testosterone is mediated at least in part by the efficacy with which testosterone in the hypothalamus is aromatized to estradiol, an important hormone mediating male sexual behavior. Therefore, hypothalamic aromatase activity, as measured by the conversion of [3H]testosterone to [3H]estradiol in tissue homogenates, was assessed in four separate experiments: 1) intact prepubertal and adult male golden hamsters, 2 and 3) castrated adult or prepubertal males that received either a 0- or 2.5-mg dose of testosterone, and 4) castrated adult and prepubertal males treated with the 2.5-mg dose oftestosterone. These studies demonstrate that hypothalamic aromatase activity is significantly higher in adult males compared with prepubertal males, and that hypothalamic aromatase activity is increased by testosterone to the same extent in both the adult and prepubertal male hamster. Therefore, the failure of testosterone-treated castrated prepubertal male hamsters to engage in the full suite of male reproductive behaviors is not due to the inability of testosterone to be converted into estradiol in the hypothalamus. Differences in the ability of testosterone to increase aromatase activity in other brain regions, or differences in the action of testosterone and/or estradiol on other cellular processes must account for the inability of testosterone to facilitate male reproductive behavior in juvenile males.

Androgenic Regulation of Hypothalamic Aromatase Activity in Prepubertal and Postpubertal Male Golden Hamsters

Androgenic Regulation of Hypothalamic Aromatase Activity in Prepubertal and Postpubertal Male Golden Hamsters

Developmental expression and regulation of aromatase- and 5alpha-reductase type I mRNA in the male and female mouse hypothalamus.

Androgen metabolites synthesized by neural aromatase and 5alpha-reductase are implicated in many aspects of mammalian brain development and, in particular, in the masculinization of distinct central nervous system structures and brain functions. The present study was designed to determine (1) the developmental profile of aromatase- and 5alpha-reductase type I mRNA expression in the mouse hypothalamus and (2) to relate ontogenetic sex differences in aromatase activity which have been described in the past to sex-specific aromatase gene expression. In addition, we analysed the effect of androgens on the perinatal regulation of hypothalamic aromatase and 5alpha-reductase type I mRNA expression. By applying semiquantitative reverse transcription-polymerase chain reaction analysis, we found hypothalamic aromatase mRNA expression to be developmentally regulated and to display sex differences at birth and on postnatal day 15 with higher mRNA levels in males. Newborn males and females, which were treated in utero with the androgen receptor antagonist cyproterone actetate, exhibited significantly reduced aromatase mRNA levels compared with untreated controls. In contrast to aromatase, expression levels of hypothalamic 5alpha-reductase mRNA did not reveal a clear-cut developmental profile or sex differences, and no regulatory role for androgens in controlling 5alpha-reductase mRNA expression was found. In conclusion, these results demonstrate perinatal sex differences in hypothalamic aromatase- but not 5alpha-reductase gene expression and suggest that sex differences in perinatal aromatase activity are reflected by corresponding differences in mRNA levels. Androgens are found to control brain estrogen formation pretranslationally at the level of aromatase gene expression. Our findings imply that sex differences in androgen availability and responsiveness are important regulatory factors for aromatase expression in the developing male hypothalamus.

Hypothalamic aromatase cytochrome P450 and 5α-reductase enzyme activities in pregnant and female rats

The metabolism of steroid hormones in the medial basal hypothalamus (MBH) is known to play a critical role in neural development, the modulation of neuroendocrine function and regulating sexual behavior. While the important biological functions of the aromatase enzyme are well established, the importance of brain 5α-reductase has been revealed and elucidated only in the last few years. The distribution and regulation of brain aromatase and 5α-reductase enzyme activities have been investigated for the most part in male rats. Therefore, in the present study, MBH aromatase cytochrome P450 and 5α-reductase activities were characterized in pregnant and female rats during postnatal development under various hormonal conditions. MBH aromatase activity was determined in each tissue sample using the ‘tritiated water release’ assay, whereas, the 5α-reductase rates were determined by thin layer chromatography and scintillation counting of the isolated 5α-metabolites. Both activities were highest in infantile animals, then declined with increasing postnatal age; whereas, in aged non-cycling or ovariectomized/adrenalectomized (Ovx/Adx) rats high rates of androgen metabolism were seen in MBH tissue. No significant alterations in MBH aromatase were observed when the 5α-reductase pathway was blocked in pregnant animals during late gestation with a known 5α-reductase inhibitor (Proscar). However, plasma estradiol levels were significantly increased in the Proscar-treated animals. These results indicate that: 1) the decreasing MBH aromatase and 5α-reductase profile (in infantile to adult cycling animals) is developmentally regulated, 2) evidently, there is a divergent regulatory mechanism controlling MBH aromatase versus 5α-reductase in aged animals where the aromatase activity increased in aged non-cycling and Ovx/Adx rats while 5α-reductase rates remained at moderate levels and, 3) apparently, the 5α-reductase pathway is not involved in regulating MBH aromatase activity during late pregnancy.

Developmental profile and regulation of brain estrogen synthesis by aromatase

Aromatase cytochrome P450 enzyme catalyzes the formation of estrogen from androgen in distinct regions of the vertebrate brain. During neural development, local estrogen synthesis is required for the sexual differentiation of male brain characteristics and the differentiation of neural circuits Involved in sex-specific behaviors and neuroendocrine functions. A common phenomenon in all species studied so far is that the male brain displays much higher and brain region-specific aromatase expression during distinct periods of brain development than the female one. An important question arising from these findings is which factor(s) play a role in regulating estrogen formation in a sex- and brain region-specific fashion. Among the factors investigated, androgens have been found to be the most powerful regulators of brain aromatase. In vitro experiments using primary cell cultures of embryonic mouse brains showed that sex differences in aromatase activity in hypothalamic cells develop, at first, independently of gonadal steroids. Later during embryonic development, aromatase expression is regulated in a region- and sex-specific way by circulating androgens. Hypothalamic aromatase neurons are most sensitive to androgen exposure whereas cortical and midbrain ones are insensitive. Moreover, androgens affect morphological maturation of aromatase-immunoreactive cells by stimulating neurite outgrowth and dendritic arborization. These findings suggest that androgens function as major morphogenetic factors during the differentiation of the mammalian hypothalamic aromatase system. During late embryonic development and perinatally, androgen levels differ between sexes, being significantly higher in males. This time period corresponds exactly to the developmental stage when aromatase activities are highest in the male hypothalamus. It seems plausible therefore that higher androgen concentrations in the male circulation during ontogenesis are causally connected with the observed sex differences in hypothalamic aromatase activity. Biomedical Reviews 1997; 7: 41-50.

Sex differences in the regulation of embryonic brain aromatase

Oestrogen formed from androgen by aromatization plays a critical role in the sexual differentiation of the male brain and behaviour. A question which has still to be answered is what regulates the gender-specific changes in aromatase activity forming oestrogen during sensitive periods of brain growth. Using a primary cell culture technique and sexed embryos, we have shown that in the fetal mouse brain, oestrogen formation in the male is neuronal rather than glial and aromatase activity is regionally localized, being higher in the hypothalamus than in the cortex. The aromatase activity measured from cells in culture has the same enzyme binding affinity (apparent Km ∼ 40 nM) as intact brain samples. Neurones developing in the embryonic male brain (embryonic day (ED) 15) contain higher aromatase activity (Vmax, 895 fmol/h/mg protein) than the female (Vmax, 604). Although a sex difference exists at early stages of embryonic development (ED 13), the embryonic aromatase system is regulated by steroids later in fetal development. The developing aromatase-containing neuroblasts probably form processes which connect to other aromatase neurones. Immunoreactive staining with an aromatase polyclonal antibody identifies an increase in numbers of aromatase-immunoreactive hypothalamic neuronal cell bodies following testosterone treatment. Testosterone treatment also causes both stimulation of neurite growth and branching as well as functional maturation of aromatase neurones. In particular, there is an increase in aromatase activity per neurone as well as a dramatic increase in the number of neurones expressing the enzyme. Both the functional and morphological changes depend on androgen receptor stimulation for several days in vitro. This conclusion is supported by colocalization studies which reveal a high number of fetal hypothalamic aromatase neurones co-expressing androgen receptor. We conclude that testosterone influences the growth of male hypothalamic neurones containing aromatase at a sensitive period of brain development. Endogenous steroid inhibitors of aromatase, probably formed within the neuroglia, also play a role in the control of oestrogen production. An endogenous 5α-reduced metabolite of testosterone, 5α-androstanedione, is almost as potent in inhibiting neuronal hypothalamic aromatase activity (Ki = 23 nM) as the synthetic non-steroidal inhibitors such as the imidazole, fadrozole, and the triazoles, arimidex and letrozole. It is clear that the oestrogen-forming capacity of the male hypothalamus has the special characteristics and plasticity of regulation which could affect brain differentiation at specific steroid-sensitive stages in ontogeny.

Sexual dimorphism in the developmental regulation of brain aromatase.

Steroid sex hormones have an organizational role in gender-specific brain development. Aromatase (cytochrome P450AR), converting testosterone (T) to estradiol-17 beta (E2) is a key enzyme in brain development and the regulation of aromatase determines the availability of E2 effective for neural differentiation. Gender differences in brain development and behaviour are likely to be influenced by E2 acting during sensitive periods. This differentiating action has been demonstrated in rodent and avian species, but also probably occurs in primates including humans. In rodents, E2 is formed in various hypothalamic areas of the brain during fetal and postnatal development. The question considered here is whether hypothalamic aromatase activity is gender-specific during sensitive phases of behavioural and brain development, and when these sensitive phases occur. In vitro preoptic and limbic aromatase activity has been measured in two strains of wild mice, genetically selected for behavioural aggression based on attack latency, and in the BALB/c mouse. Short attack latency males show a different developmental pattern of aromatase activity in hypothalamus and amygdala to long attack latency males. Using primary brain cell cultures of the BALB/c mouse, sex differences in hypothalamic aromatase activity during both early embryonic and later perinatal development can be demonstrated, with higher E2 formation in males. The sex dimorphism are brain region specific, since no differences between male and female are detectable in cultured cortical cells. Immunoreactive staining with a polyclonal aromatase antibody identifies a neuronal rather than an astroglial localization of the enzyme. T increases fetal brain aromatase activity and numbers of aromatase-immunoreactive hypothalamic neuronal cell bodies. T appears to influence the growth of hypothalamic neurons containing aromatase. Differentiation of sexually dimorphic brain mechanisms may involve maturation of a gender-specific network of estrogen-forming neurons which are steroid-sensitive in early development.

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.

Gender-specific brain formation of oestrogen in behavioural development

Steroid sex hormones have an organisational role in the development of brain mechanisms underlying gender-specific behaviour. Although peaks in gonadal androgen occur at developmental stages that coincide with sensitive periods for the differentiation of both structural sex differences in the brain and sexual behaviour, the factors that control the phasic effects of steroids are still not understood. Aromatase, converting androgen to oestrogen, is a key enzyme in development, and regulation of the activity of this enzyme is likely to be one of the factors determining availability of oestrogen effective for brain differentiation. Measurement of testosterone metabolism in vitro shows that in the mouse oestrogens are formed actively in the neonatal brain during male development. In cultured cells of the embryonic mouse hypothalamus there are sex differences in hypothalamic aromatase activity both during early embryonic and later perinatal development, with a higher capacity for oestrogen formation in the male than in the female. The sex differences are regionally specific, since no differences in aromatase activity are detectable in cultured cortical cells between male and female. Aromatase activity is neuronal rather than astroglial. Using a specific antibody to the mouse aromatase, immunoreactivity is also restricted to neuronal soma and neurites in hypothalamic cultures. Therefore, gender-specific differences in aromatase regulation are probably restricted to neurons. Testosterone increases oestrogen formation specifically in cultured hypothalamic neurones, but has no effect on cortical cells. Although there is a sex difference in early embryonic neuronal aromatase, aromatase activity appears to be sensitive to androgen only in later embryonic development. What determines the phasic sensitivity of the developing brain aromatase system to androgen has still to be determined.