Estrogen-induced activation of the mitogen-activated protein kinase cascade in the cerebral cortex of estrogen receptor-alpha knock-out mice.

We have shown previously in the developing cerebral cortex that estrogen elicits the rapid and sustained activation of multiple signaling proteins within the mitogen-activated protein (MAP) kinase cascade, including B-Raf and extracellular signal-regulated kinase (ERK). Using estrogen receptor (ER)-alpha gene-disrupted (ERKO) mice, we addressed the role of ER-alpha in mediating this action of estrogen in the brain. 17beta-Estradiol increased B-Raf activity and MEK (MAP kinase/ERK kinase)-dependent ERK phosphorylation in cerebral cortical explants derived from both ERKO and their wild-type littermates. The ERK response was stronger in ERKO-derived cultures but, unlike that of wild-type cultures, was not blocked by the estrogen receptor antagonist ICI 182,780. Surprisingly, both the ER-alpha selective ligand 16alpha-iodo-17beta-estradiol and the ER-beta selective ligand genistein failed to elicit ERK phosphorylation, suggesting that a different mechanism or receptor may mediate estrogen-induced ERK phosphorylation in the cerebral cortex. Interestingly, the transcriptionally inactive stereoisomer 17alpha-estradiol did elicit a strong induction of ERK phosphorylation, which, together with the inability of the ER-alpha- and ER-beta-selective ligands to elicit ERK phosphorylation, and of ICI 182,780 to block the actions of estradiol in ERKO cultures, supports the hypothesis that a novel, estradiol-sensitive and ICI-insensitive estrogen receptor may mediate 17beta-estradiol-induced activation of ERK in the brain.

Characterization of 11 beta-methoxy-16 alpha-[125I]iodoestradiol binding: neuronal localization of estrogen-binding sites in the developing rat brain.

The binding properties of the gamma-emitting 125I-labeled 11 beta-methoxy analog of 16 alpha-iodoestradiol, 11 beta-methoxy-16 alpha-iodoestradiol (MIE2), were characterized for its use in vivo as a ligand for the measurement and localization of estrogen-binding sites. In binding displacement studies, MIE2 bound to rat, rabbit, and human estrogen receptors with high affinity. Association of MIE2 with uterine cytosol estrogen receptors reached maximum values within 30 min at 25 C. At 0-4 C, association was much slower, with maximum binding values not achieved until 16-24 h after the start of the incubation. Once formed, the MIE2-estrogen receptor complex was quite stable at 0-4 C (t1/2 much greater than 24 h). At 25 C, dissociation of MIE2-estrogen receptor complexes occurred nearly 3 times more slowly than that of E2-estrogen receptor complexes (t1/2, 3.3 vs. 1.2 h). The iodinated estrogen was highly specific for the estrogen receptor and did not bind appreciably to androgen, progestin, or glucocorticoid receptors or to either human sex hormone-binding globulin or rat alpha-fetoprotein. MIE2 is also not a ligand for human sex hormone-binding globulin. Dose-dependent uptake of [125I]MIE2 into pituitary and brain cell nuclei was observed after its in vivo administration to 25-day-old female rats. In 10-micron brain sections from immature female rats treated with [125I]MIE2 (7.5 microCi/g BW), regional localization of estrogen-sensitive brain areas could be obtained by autoradiography using LKB Ultrofilm with an exposure time of only 16 h. In comparison, after an identical dose of 16 alpha-[125I]iodoestradiol, an exposure time of 72 h was required to achieve an image of similar density. Combined autoradiographic and immunocytochemical studies in 5- to 11-day-old female rats demonstrated nuclear binding of [125I]MIE2 in cells immunoreactive for neurofilament protein but not glial fibrillary acidic protein, indicating that estrogen receptors in the developing postnatal brain are restricted to neurons and are not present in astroglial cells. The biological characteristics of [125I]MIE2 combined with its high specific activity make it an estrogenic probe with a wide range of possible uses for the study of estrogen action in the developing brain as well as other estrogen target tissues.

Immunoreactive gonadotropin-releasing hormone (Gn-RH) during maturation in the rat: Ontogeny of regional hypothalamic differences.

The developmental appearance of immunoreactive gonandotropin releasing hormone (Gn-RH) was measured by radioimmunoassay in male and female rats from the 12th day of gestation to adulthood. Gn-RH content of the whole hypothalamus of both sexes increased from birth to 21 days of age. By the 28th day, Gn-RH content in females had approximately reached adult levels, while that in males continued to increase. Adult male rats exhibited significantly higher levels than adult females. The ontogeny of the regional differences previously observed in cycling female rats (4) was also studied. Regional analysis of Gn-RH development was characterized in the anterior hypothalamus by the rapid attainment of adult levels by day 7. In striking contrast, in the mid-hypothalamic region, Gn-RH content increased gradually, rising dramatically just prior to vaginal opening. The developmental pattern of Gn-RH content is consistent with the maturation of those hypothalamic structures concerned with synthesis, transport and storage of Gn-RH. The regional patterns observed may thus represent sequential maturation of the neural pathways involved.

Estradiol (E2) elicits SRC phosphorylation in the mouse neocortex: the initial event in E2 activation of the MAPK cascade?

In neocortical explants, E2 activates various signaling components of the MAPK cascade, including B-Raf and MAPK kinase-dependent ERK, suggesting a possible role in the differentiative actions of E2 in the brain. To further characterize the signaling pathways activated by E2, we determined whether c-Src, a member of the Src family of nonreceptor tyrosine kinases and an important modulator of both the MAPK cascade and neuronal differentiation, may play a role in E2 signaling. The present studies show for the first time in the brain that E2 elicits phosphorylation of c-Src on three functionally critical tyrosine residues (Y220, Y423, and Y534), and that this phosphorylation occurs despite disruption of ER alpha (in ER knockout mice). PP2, a Src family kinase inhibitor, suppressed not only E2-induced phosphorylation of c-Src, but ERK phosphorylation as well, suggesting that c-Src may be an upstream regulator of E2 signaling. E2-induced phosphorylation of c-Src is associated with increased tyrosine phosphorylation of Shc, increased association of Shc with Grb2, and induction of Ras, but not Rap1, activation. Together, these data provide evidence that E2 activates a novel c-Src-dependent signal transduction pathway in the developing brain.

Estrogen and the brain: beyond ER-alpha and ER-beta.

17beta-Estradiol is a greatly under-appreciated neural growth and trophic factor for the mammalian brain of all ages. Like other growth factors, such as the neurotrophins, 17beta-estradiol influences neurogenesis, neuronal differentiation, and neuronal survival of its targets throughout life. Estrogen elicits developmentally regulated differentiative effects, which are not normally seen in the adult brain. However, re-expression of this developmental response occurs in the adult, following loss of trophic support, whether induced by estrogen deprivation or brain injury. In addition to the classical intranuclear estrogen receptors (ER) ER-alpha and ER-beta, we have recently identified a novel, plasma membrane-associated, putative ER that is neither ER-alpha nor ER-beta, which we have designated 'ER-X'. ER-X is a developmentally regulated estrogen-binding protein, present in wild-type, ER-alpha gene-disrupted (alphaERKO) and ER-alpha null mice, which is re-expressed following ischemic brain injury. The preferred ligand of ER-X is 17alpha-estradiol. Although ER-X shares some homology with the C-terminus of ER-alpha, it is not an alternative splicing variant and may be a new gene. While ER-X appears to mediate 17alpha- and 17beta-estradiol activation of the MAPK cascade, ER-alpha, in contrast, is inhibitory to its activation. Estradiol activation of MAPK/ERK may be particularly relevant for neuroprotection during aging and Alzheimer's disease.

Novel sites and mechanisms of oestrogen action in the brain.

We are investigating novel, non-transcriptionally mediated mechanisms that may contribute to the differentiative effects of oestrogen in developing forebrain neurons. Recent findings in the cerebral cortex document that 17 alpha- and 17 beta-oestradiol elicit rapid and sustained activation of the Ras-Raf-MAP kinase cascade, a major growth factor signalling pathway. Using oestrogen receptor (ER) alpha knockout (ERKO) mice, we addressed the identity of the receptor mediating activation of the MAP kinase cascade. 17 beta-oestradiol increased B-Raf activity and MEK-dependent ERK phosphorylation in explants of wild-type and ERKO cerebral cortex. Although neither the ER alpha-selective ligand, 16 alpha-iodo-17 beta-oestradiol (16 alpha-IE2) nor the ER beta-selective ligand, genistein, elicited ERK phosphorylation, as little as 0.1 nM 17 beta-oestradiol did so. Moreover, 16 alpha-IE2 acted as an inhibitory modulator of ERK activation, and the ER antagonist ICI 182 780 blocked oestradiol action only in wild-type cultures. These data suggest that neither ER alpha nor ER beta mediate activation of the MAP kinase cascade. A putative, novel, oestradiol-sensitive and ICI 182 780-insensitive receptor, designated ER-X may, rather, be involved. Association of ER-X with flotillin, the neuronal homologue of the caveolar protein, caveolin, places ER-X within plasma membrane caveolae and supports the hypothesis that a membrane-associated ER may mediate rapid oestrogen activation of the MAP kinase cascade.

Organotypic culture of the developing cerebral cortex and hypothalamus: relevance to sexual differentiation.

Addition of estradiol to organotypic cultures of the rodent hypothalamus, preoptic area and cerebral cortex elicits a striking enhancement of neurite growth which is restricted to estrogen receptor-containing regions of the explants. The morphogenetic consequences of enhanced neurite growth help explain the origin of structural sexual dimorphisms in the CNS. Neurite enhancement may result from both neuronotrophic and growth-promoting actions of steroids. Steroid effects may be mediated by interactions with endogenous growth factors or their receptors. Growth-promoting effects of steroids on the developing cerebral cortex, in particular, may have relevance for sexual differentiation of cognitive functions.

Mechanisms of estrogen action during neural development: mediation by interactions with the neurotrophins and their receptors?

Estrogen enhances the growth and differentiation of neurites within the developing forebrain. A critical issue is whether these developmental actions of estrogen are mediated directly or indirectly by means of autocrine responses or local paracrine mechanisms, through interactions with growth factors, such as the neurotrophins, and their receptors. Support for the latter hypothesis comes from our recent observations of co-expression of estrogen receptor mRNA with the mRNAs for the neurotrophins and their receptors; differential and reciprocal up-regulation of estrogen and NGF receptor mRNA and protein expression by estrogen in adult female rat sensory neurons, PC12 cells; and cerebral cortical cultures; and putative estrogen response elements in the NGF, BDNF, trkA and p75 genes. Estrogen and the neurotrophins may influence each other's actions by regulating receptor and ligand availability or by reciprocal regulation at the level of signal transduction or gene transcription. The neurotrophins may serve as regulatory "switches" for the apparent developmentally-regulated, differential pattern of estrogen receptor regulation by its ligand, whereby their ability to increase estrogen receptor levels significantly may be sufficient to override the intrinsic suppressive action of estrogen on its receptor. Estrogen and the neurotrophins, acting in concert and reciprocally, may stimulate the synthesis of proteins required for neuronal differentiation, survival and maintenance of function.

Neurite-like outgrowth from CNS explants may not always be of neuronal origin.

In living explants of the developing CNS 'true' neurites (neuronal processes) may appear virtually indistinguishable from the processes of radial and Bergmann glia. This inability to clearly distinguish the phenotype of responsive 'neurites' in the living state has profound implications for the interpretation of responses to 'neurite-promoting' substances. Labeling techniques, specific for intermediate filament proteins that are characteristic of neurons and neuroglia, should always be used to supplement morphologic observations on neurite-like outgrowth from living CNS explants.

Regional differences in intraneuronal localization of alpha-fetoprotein in developing mouse brain.

Estrogen receptor-containing regions of limbic, hypothalamic and amygdaloid areas of the developing mouse brain exhibited little or no intraneuronal immunofluorescence reactivity for AFP in comparison with the bright fluorescence of adjacent regions. These regional differences in the topographic distribution of intraneuronal AFP may represent intrinsic differences in the uptake, turnover or immunoreactivity of the internalized protein, perhaps related to estrogen metabolism by such neurons.

Sex steroids and the development of the newborn mouse hypothalamus and preoptic area in vitro. II. Morphological correlates and hormonal specificity.

In studies designed to elucidate morphogenetic mechanisms involved in the neurogenesis of sexual differentiation of the brain, estradiol or testosterone was added to organotypic cultures of the newborn mouse hypothalamus and preoptic area. Both gonadal hormones selectively accelerated and enhanced neuritic proliferation in specific regions of the preoptic area and infundibular/premamillary levels. This regional localization suggests specific induction of neuritic branching perhaps only in those neurons shown by autoradiography to contain the steroid receptor. The significance of estradiol per se is emphasized by the reduction and retardation of neuritic outgrowth in those same regions following exposure to steroid-deficient medium or blockade of the nuclear receptor (CI-628) and by the failure of testosterone alone to induce a significant response. The importance of aromatization of androgen to estradiol is supported by the failure of non-aromatizable 5 alpha-dihydrotestosterone to elicit an effect even in the presence of estradiol. This apparent hormonal specificity suggests that the neuritic response may be a component of sexual differentitation and that the trophic effects of estradiol may influence significantly the ontogeny of target neural circuits in the brain of both genders.

Androgen increases the number of cells in fetal mouse spinal cord cultures: implications for motoneuron survival.

Androgen effects were studied in organotypic cultures of the E12 fetal mouse lumbosacral spinal cord labeled in utero with [3H]thymidine on E10. Following continuous exposure to androgens for one month in vitro, the number of labeled cells was significantly increased in whole explants, and in hemisected segments in the presence or absence of co-cultured fetal thigh muscle. Because lumbosacral motoneurons undergo their final mitosis predominantly on E10 and thus remain permanently labeled, the results suggest that androgens increase neuronal numbers by directly modulating motoneuron survival rather than stimulating mitosis. These findings demonstrate for the first time that in addition to the well documented role of the muscle target in motoneuron survival, the direct neuronotrophic effects of androgen at the level of the spinal cord must also be considered.

Intraneuronal alpha-fetoprotein and albumin are not synthesized locally in developing brain.

Alpha-Fetoprotein and albumin are found intraneuronally in developing mammalian brains. To investigate whether this results from local biosynthesis, the existence of the corresponding brain messenger RNAs and the ability of brain cultures to synthesize these proteins were studied in the mouse. The findings show that neither the proteins nor the mRNAs coding for them are made in sufficient quantities to account for the material detected immunologically, supporting an extracellular origin.

Sex steroids and the development of the newborn mouse hypothalamus and preoptic area in vitro: III. Effects of estrogen on dendritic differentiation.

Gonadal steroids have previously been shown to enhance neuritic outgrowth from mouse preoptic area explants in vitro. To examine effects within the explant proper, dendritic fields of Golgi-stained neurons of estradiol-treated and untreated cultures were compared quantitatively. Neurons of gonadal steroid-treated cultures had significantly more dendrites arising from the cell body. Since bifurcation probability did not differ between the treatments, the primary effect of estradiol treatment appears to be induction of dendritic branches from the soma. This indicates that intra-explant dendrites, like extra-explant neurites, proliferate in response to estradiol treatment and suggests the involvement of hormone-stimulated dendrogenesis in sexual differentiation of the brain.

Estrogen and insulin synergism in neurite growth enhancement in vitro: mediation of steroid effects by interactions with growth factors?

Addition of estradiol to organotypic cultures of the fetal murine hypothalamus, preoptic area and cerebral cortex has been shown to elicit a striking enhancement of neurite growth which appears restricted to estrogen receptor-containing explant regions. The mechanisms underlying this response are unknown. An important question is whether the neurite enhancement which follows exposure to estradiol is due directly to the interaction of estrogen with the cell that was stimulated (the receptor-containing cell) or whether intermediate steps involving the possible interaction of estrogen and the endogenous polypeptide neurite-promoting growth factors or their receptors may play an important role. Recent findings in the cultures suggest that the effect of estrogen on neurite growth may involve synergistic interactions between estradiol and insulin-related peptides and may be important in regulating estrogen-responsive neurite growth in the central nervous system. Concurrent addition of estradiol and high levels of insulin (10 micrograms/ml or 50 micrograms/ml) to cultures of the olfactory bulb, hypothalamus, preoptic area and cerebral cortex of the fetal rat and mouse results in a dramatic acceleration and increase of neurite outgrowth which appears localized to estrogen receptor-containing explant regions. The supraphysiological concentrations of insulin required to elicit this response suggest that the factor(s) involved is unlikely to be insulin per se. Insulin may activate the receptor of different but closely related molecules such as the insulin-like growth factors (IGF)-I or -II to which it exhibits a relatively low affinity. Interactions between hormones and endogenous growth factors have been implicated in the modulation or mediation of an increasing number of endocrine-dependent, differentiative processes in vivo and in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Androgen action in fetal mouse spinal cord cultures: metabolic and morphologic aspects.

Morphologic and metabolic aspects of androgen effects on the developing spinal cord were studied in organotypic cultures of the E13 (embryonic day 13) fetal mouse lumbosacral spinal cord, maintained as either hemisected, homologous explant pairs co-cultured with bulbocavernosus muscle (morphologic studies), or as whole cross-sectional segments without muscle in which the nutrient medium was supplemented with muscle extract (metabolic studies). Metabolic studies demonstrated the total absence of aromatase activity. 5 alpha-Reductase activity, on the other hand, increased differentially in a segment-dependent manner in spinal cord explants from 0 to 35 days in culture, suggesting regional differences in the utilization of testosterone and its 5 alpha-reduced metabolites. In all studies, spinal cord explants showed androgen-dependent increases in neurite outgrowth, although this was most pronounced in spinal cord-muscle co-cultures. These results indicate that androgens per se affect very early development throughout the entire lumbosacral spinal cord, and that this influence is not restricted to those segments reported to be sexually dimorphic in the adult.

Perinatal development of hypothalamic and cortical estrogen receptors in mouse brain: methodological aspects.

Binding of the estrogen, [3H]moxestrol, to fetal and neonatal mouse brain cytosol receptors was examined to determine the ontogeny of estrogen receptors in the hypothalamus and cerebral cortex from embryonic (E) day 15 to postnatal (P) day 18. Cytosol receptor assays were performed under exchange conditions at 25 degrees C for 4 h in order to measure receptors which had become occupied by estradiol during tissue homogenization. Scatchard analysis revealed high affinity (Kd = 0.4 nM) sites and was in good agreement with single point assays at 1 nM, which measured 70% of binding capacity. Binding was initially examined in the whole forebrain. Total binding of [3H]moxestrol in the forebrain increases between E15 and E18 and reaches adult levels at P9. The increase in binding relative to protein content peaks at P9 and then decreases, whereas the amount of binding relative to DNA content reaches a maximum between P12 and P15. The developmental time-course of the estrogen receptors was studied in the hypothalamus and 3 cortical regions. In the hypothalamus binding of [3H]moxestrol increases from P5 to P18 of the cortical areas. The cingulate cortex shows the highest amount of binding, increasing until P9 and then declining. In the other two cortical areas studied, the lateral and posterior cortex, binding expressed per mg DNA, is somewhat higher between P7 and P15 than in adults. When the binding is expressed per mg protein there is a sharp decline after P7, the magnitude of which is probably a result of a large increase in protein content relative to amount of receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Perinatal development of estrogen receptors in mouse brain assessed by radioautography, nuclear isolation and receptor assay.

The development of estrogen receptors was investigated in vivo in the brains of fetal and neonatal mice 2 h after administering [3H]moxestrol to the pregnant mothers or neonates. Moxestrol bypasses the alpha-fetoprotein 'protective barrier' and gains access to estrogen receptors. Analysis of [3H]moxestrol uptake by radioautography and by cell nuclear isolation and counting of radioactivity revealed a marked increase in the number of estrogen receptors and estrophilic cells in the brain during late fetal and early postnatal development. Assays of cytosol estrogen receptors were conducted in parallel and revealed a comparable pattern of development. The increase in estrogen receptors and labeling was especially great from embryonic day (E) 15 to E18. Cytosol assays revealed a low level of receptors in the whole brain on E13. Radioautography revealed that clearly labeled cells in the hypothalamus and preoptic area were virtually absent on E13 but were evident on E15, with marked increases occurring between E15 and E18, both in number of labeled cells and in intensity of labeling per cell. Within the cerebral cortex the dorsal cingulate cortex was the most extensively labeled area; however, clearcut labeling was not evident on E13 or E15. Thus, the development of cortical estrogen receptors occurs somewhat later than that in the hypothalamus and preoptic area. The perinatal increase in estrogen receptors usually begins several days after the birthdates of neurons in these estrophilic regions of the brain, and corresponds to the early responsiveness of these neurons to the organizational and activational influences of estrogen.

Cellular variations in estrogen receptor mRNA translation in the developing brain: evidence from combined [125I]estrogen autoradiography and non-isotopic in situ hybridization histochemistry.

The spatial distribution of cells in the adult rodent forebrain which express estrogen receptor mRNA, as shown by in situ hybridization histochemistry with isotopically-labeled probes, has been reported to overlap with regions that are known targets of estrogen and which bind estrogen. The extent to which detection of estrogen receptor mRNA within developing forebrain neurons of the postnatal day 10-12 female rat is accompanied by translation into estrogen binding sites was investigated by combining [125I]estrogen autoradiography with non-isotopic (digoxigenin) in situ hybridization, using a 48-base oligodeoxyribonucleotide probe encoding a sequence of the estrogen-binding domain of rat uterine estrogen receptor cDNA. Estrogen receptor mRNA and estrogen binding sites appeared to be restricted to neurons. No mRNA or binding was seen in ependymal cells. Cells expressing estrogen receptor mRNA were widely distributed in the developing rat forebrain and were found in brain regions generally corresponding to those previously shown in the adult, with the addition of some regions not previously described, such as the medial habenula and dorsal endopiriform nucleus. Although there was widespread overlapping of estrogen receptor mRNA expression with known estrogen binding sites, there were regional and cellular variations in the extent of receptor mRNA translation. This pattern was true for developing forebrain regions previously defined as estrogen receptor-containing (hypothalamus, preoptic area, medial and lateral septum, vertical and horizontal nuclei of the diagonal band, cerebral cortex, hippocampus and amygdala) as well as for regions heretofore not considered estrogen targets (the thalamus, dorsal endopiriform nucleus, claustrum, ventral pallidum/substantia innominata and the basal nucleus of Meynert) or characterized as estrogen-responsive in the adult without previously documented estrogen binding [caudate-putamen (striatum)]. While estrogen binding and receptor mRNA expression always co-localized, neurons expressing estrogen receptor mRNA did not always exhibit ligand binding and there was no clear-cut relationship between the intensity of the hybridization signal and estrogen binding. Little, however, is known about translational control of estrogen receptor expression in the brain. Localization of estrogen binding sites to regions not generally considered targets of estrogen would appear to reflect the greater sensitivity of the iodinated ligand than the tritiated estrogens more commonly used for autoradiography. Non-isotopic in situ hybridization histochemistry combined with [125I]estrogen autoradiography represents a very powerful tool with which to study regulation of estrogen receptor gene expression at the single cell level with an exceptional degree of cellular and anatomical resolution.(ABSTRACT TRUNCATED AT 400 WORDS)

Insulin influences astroglial morphology and glial fibrillary acidic protein (GFAP) expression in organotypic cultures.

Variations in the levels and timing of exposure to insulin-related peptides influence the phenotypic appearance of astroglia present in organotypic cultures of the E17 mouse cerebellum as well as the expression of glial fibrillary acidic protein (GFAP) mRNA and its encoded protein. The morphology of GFAP-immunoreactive cells was influenced by the levels of insulin added in an age-specific manner. Fetal radial glia were selectively and significantly (P less than 0.001) increased by high (10 micrograms/ml) insulin levels, comprising the majority of the GFAP-positive cells seen. In contrast, there was an almost complete reversal of this pattern elicited by low (10 pg/ml) insulin levels, where GFAP-positive cells appeared undifferentiated and epithelioid (P less than 0.001). In newborn cultures, on the other hand, the morphological responses to both high and low levels of insulin were considerably attenuated and involved radial glia primarily, whose numbers were significantly increased by the high insulin levels. Exposure to high levels of insulin was accompanied by an increase in GFAP mRNA expression, as determined by non-isotopic (biotin) in situ hybridization histochemistry, and intense GFAP immunoreactivity, while low insulin levels elicited minimal expression of both message and protein product. In view of the critical interdependence of developing neurons and radial glia with respect to neuronal migration and the differentiation of neurons and astroglia, the responses observed suggest developmentally regulated mechanisms by which insulin-related peptides themselves may influence directly and indirectly both neuronal and astroglial differentiation.