John B. Hutchison

Aromatase expression by astrocytes after brain injury: implications for local estrogen formation in brain repair.

Recent evidence indicates that 17beta-estradiol may have neuroprotective and neuroregenerative properties. Estradiol is formed locally in neural tissue from precursor androgens. The expression of aromatase, the enzyme that catalyses the conversion of androgens to estrogens, is restricted, under normal circumstances, to specific neuronal populations. These neurons are located in brain areas in which local estrogen formation may be involved in neuroendocrine control and in the modulation of reproductive or sex dimorphic behaviours. In this study the distribution of aromatase immunoreactivity has been assessed in the brain of mice and rats after a neurotoxic lesion induced by the systemic administration of kainic acid. This treatment resulted in the induction of aromatase expression by reactive glia in the hippocampus and in other brain areas that are affected by kainic acid. The reactive glia were identified as astrocytes by co-localization of aromatase with glial fibrillary acidic protein and by ultrastructural analysis. No immunoreactive astrocytes were detected in control animals. The same result, the de novo induction of aromatase expression in reactive astrocytes on the hippocampus, was observed after a penetrating brain injury. Furthermore, using a 3H2O assay, aromatase activity was found to increase significantly in the injured hippocampus. These findings indicate that although astrocytes do not normally express aromatase, the enzyme expression is induced in these glial cells by different forms of brain injury. The results suggest a role for local astroglial estrogen formation in brain repair.

Genital Sensory Field: Enlargement by Estrogen Treatment in Female Rats

Recordings of neuronal activity in the pudendal, genitofemoral, and pelvic nerves indicate that the sensory fields of these three nerves are the perineum, the caudal abdomen, and the vagino-cervical area and rectum, respectively. The sensory field of the pudendal nerve was significantly larger in estrogen-treated ovariectomized female rats than in uninjected controls. This effect of estrogen was not mediated by pudendal efferents.

Role of astroglia in estrogen regulation of synaptic plasticity and brain repair

Astroglia are targets for estrogen and testosterone and are apparently involved in the action of sex steroids on the brain. Sex hormones induce changes in the expression of glial fibrillary acidic protein, the growth of astrocytic processes, and the degree of apposition of astroglial processes to neuronal membranes in the rat hypothalamus. These changes are linked to modifications in the number of synaptic inputs to hypothalamic neurons. These findings suggest that astrocytes may participate in the genesis of androgen-induced sex differences in synaptic connectivity and in estrogen-induced synaptic plasticity in the adult brain. Astrocytes and tanycytes may also participate in the cellular effects of sex steroids by releasing neuroactive substances and by regulating the local accumulation of specific growth factors, such as insulin-like growth factor-I, that are involved in estrogen-induced synaptic plasticity and estrogen-mediated neuroendocrine control. Astroglia may also be involved in regenerative and neuroprotective effects of sex steroids, since astroglia formation after brain injury or after peripheral nerve axotomy is regulated by sex hormones. Furthermore, the expression of aromatase, the enzyme that produces estrogen, is induced de novo in astrocytes in lesioned brain areas of adult male and female rodents. Since astroglia do not express aromatase under normal circumstances, the induction of this enzyme may be part of the program of glial activation to cope with the new conditions of the neural tissue after injury. Given the neuroprotective and growth-promoting effects of estrogen after injury, the local production of this steroid may be a relevant component of the reparative process.

Brain aromatase expression after experimental stroke: Topography and time course

Brain aromatase has been shown to be increased in expression after neurotoxic damage and to exert neuroprotection via generation of local oestrogens. The present study investigates the topography and time course of brain aromatase expression after experimental stroke (middle cerebral artery occlusion (MCAO)). Ovariectomised stroke prone spontaneously hypertensive rats underwent distal MCAO by electrocoagulation. Immunohistochemistry revealed increased brain aromatase expression at 24h and 8 days in the cortical penumbra/peri-infarct zones with no increase evident at 2h or 30 days post-MCAO. Double label studies indicate that some of the increased aromatase expression is associated with astrocytic processes. Thus, this is the first evidence that aromatase protein is increased after MCAO and the location (peri-infarct), time course (within 24h) and cellular localisation (astrocytic) indicate the potential for aromatase to promote the survival of cells in the penumbra after experimental stroke by local synthesis of oestrogens.

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.

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.

Sex-Specific Aromatization of Testosterone in Mouse Hypothalamic Neurons

Conversion of androgens to oestrogens by neural aromatase during brain development appears to be a prerequisite for sexual differentiation of the mammalian central nervous system. In order to investigate the pre- and perinatal patterns of testosterone (T) aromatization in the male and female mouse brain, aromatase activity (AA) was measured in hypothalamic and cerebral homogenates of embryonic day (ED) 17 fetuses and neonates using an in vitro 3H2O product formation microassay. In addition, AA was examined in gender-specific neuronal cell cultures prepared from ED 15 mouse cerebral hemisphere and hypothalamus at 3 and 6 days in vitro (DIV), and this was compared with enzyme activities in homogenates. The aromatase has also been evaluated in glial-enriched cultures from ED 20 mouse hypothalamus and cortex as well as in ED 15 cultures treated with the neurotoxin kainic acid in order to localize AA to neurons and/or glial cells. Significant sex differences in AA were observed in hypothalamic tissue homogenates as early as ED 17, becoming even more distinct in neonates, AA being always higher in males compared to females. Similar AA was also found in cells from both sexes from cultured ED 15 hypothalamus after 3 DIV. However, significantly higher AA was observed after 6 DIV in ED 15 male hypothalamic cultures compared to female. ED 20 glial-enriched hypothalamic cultures (purity > 95%) from both brain regions exhibited very low AA after 6 DIV, and no sex differences were found.(ABSTRACT TRUNCATED AT 250 WORDS)

Steroid metabolising enzymes in the determination of brain gender

The neurotrophic effects of oestrogen formed in the brain are important in brain sexual differentiation of the central nervous system and behaviour. Aromatase, converting testosterone to oestradiol-17beta, is a key enzyme involved in brain development. In primary cell cultures of foetal hypothalamus, we have found that male neurones consistently have higher aromatase activity than in the female. Using a specific antibody to the mouse aromatase, immunoreactivity was localized in the neural soma and neurites in hypothalamic cultures. Additionally more male foetal hypothalamus neurones express aromatase than in the female. Testosterone increases aromatase activity in parallel with a greater number of aromatase-immunoreactive neurones. Testosterone also increases soma size, neurite length, and branching of cultured hypothalamic neurones. The neuronal aromatase activity appears to be sensitive to the inductive effects of androgen only during the later stages of foetal development. Endogenous inhibitors of the aromatase are also likely to have a regulatory role. This work suggests that regulation of a network of aromatase neurones, sensitive to the hormonal environment of the hypothalamus, may determine when oestrogens are available for neurotrophic effects underlying brain differentiation.

Differential Effects of Photoperiodic History on the Responses of Gonadotrophins and Prolactin to Intermediate Daylengths in the Male Syrian Hamster

The effect of photoperiodic history on the neuroendocrine response to intermediate daylengths (11-13.5 hr of light) was investigated in the male Syrian hamster. The duration of the nocturnal peak of pineal melatonin content was inversely proportional to photoperiod and independent of photoperiodic history. Serum levels of prolactin were lower in animals ex posed to shorter photoperiods. Photoperiodic history had little effect on the response of serum prolactin to intermediate daylengths. Serum luteinizing hormone (LH) concentrations were also lower in shorter photoperiods, but in addition were sensitive to the direction of photo periodic change, so that a single photoperiod could be interpreted as either stimulatory or inhibitory to LH secretion. This effect of photoperiodic history was expressed at intermediate photoperiods with 12-13.5 hr of light. The sensitivity of serum follicle-stimulating hormone (FSH) levels to photoperiodic history was masked by an early onset of photorefractoriness. Testicular size and serum testosterone levels revealed weaker effects of photoperiodic his tory; these were attributed to the dissociation between gonadotrophin and prolactin secretion induced by intermediate daylengths. The contrasting effects of photoperiodic history on the secretion of LH and prolactin may represent the expression of multiple photoperiodic time- measuring systems.

Neuroblastoma and Alzheimer’s Disease Brain Cells Contain Aromatase Activity

Human brain steroidogenic mechanisms, particularly aromatase, have been investigated in healthy and diseased conditions. Aromatase activity was measured in differentiated and undifferentiated neuroblastoma cell lines from mouse (TMN) and human (5H SY5Y) and in human post mortem brain samples. Neuroblastomas show much higher aromatase activity than human brain samples. Homogenates of adult human male and female cortex and frontal and temporal areas of both Alzheimers and control patients all show considerably lower activity. The temporal area has significantly higher aromatase activity than the frontal. Aromatisation activity in differentiated neuroblastoma cells of both species is lower than in undifferentiated cells. These results are consistent with an inverse relationship between brain estrogen formation and stage of neuronal differentiation and the hypothesis that aromatase may be involved in the early stages of neuronal growth. Significant but variable activities of other androgen-metabolising enzymes, such as 5 alpha-reductase, 3 alpha/beta-hydroxysteroid dehydrogenases, and 17 beta-hydroxysteroid dehydrogenase, which generate a spectrum of regulatory molecules, are also found.