Sexual differentiation of the central nervous system.

Sexual differentiation of reproductive and behavior patterns is largely effected by hormones produced by the gonads. In many higher vertebrates, an integral part of this process is the induction of permanent and essentially irreversible sex differences in central nervous function, in response to gonadal hormones secreted early in development.

Xenopus oocytes injected with rat uterine RNA express very slowly activating potassium currents.

Under the influence of estrogen, uterine smooth muscle becomes highly excitable, generating spontaneous and prolonged bursts of action potentials. In a study of the mechanisms by which this transition in excitability occurs, polyadenylated RNA from the uteri of estrogen-treated rats was injected into Xenopus oocytes. The injected oocytes expressed a novel voltage-dependent potassium current. This current was not observed in oocytes injected with RNA from several other excitable tissues, including rat brain and uterine smooth muscle from ovariectomized rats not treated with estrogen. The activation of this current on depolarization was exceptionally slow, particularly for depolarizations from relatively negative membrane potentials. Such a slowly activating channel may play an important role in the slow, repetitive bursts of action potentials in the myometrium.

Estrogen induction of a small, putative K+ channel mRNA in rat uterus.

Estrogen causes dramatic long-term changes in the activity of the uterus. Here we report the molecular cloning of a small (700 base) uterine mRNA species capable of inducing a slow K+ current in Xenopus oocytes. The 130 amino acid protein encoded by this mRNA species has a predicted structure that does not resemble that of previously described voltage-dependent channels from mammalian sources. It is, however, similar to structural motifs found in certain prokaryotic ion channels. The induction of this mRNA by estrogen is rapid; this uterine mRNA species is not detectable in uteri from estrogen-deprived rats, but is substantially induced after 3 hr of estrogen treatment. These results support a critical role for regulation of ion channel expression by estrogen in the uterus.

Chronic stress and neural function: accounting for sex and age.

Cognitive responses to stress follow the temporally dependent pattern originally established by Selye (1) wherein short-term stressors elicit adaptive responses whereas continued stress (chronic) results in maladaptive changes--deleterious effects on physiological systems and impaired cognition. However, this pattern for cognitive effects appears to apply to only half the population (males) and, more specifically, to young, adult males. Females show different cognitive responses to stress. In contrast to impaired cognition in males after chronic stress, female rodents show enhanced performance on the same memory tasks after the same stress. Not only cognition, but anxiety, shows sex-dependent changes following chronic stress--stress is anxiolytic in males and anxiogenic in females. Moreover, behavioral responses to chronic stress are different in developing as well as aging subjects (both sexes) as compared to adults. In aged rats, chronic stress enhances recognition memory in both sexes, does not alter spatial memory, and anxiety effects are opposite to young adults. When pregnant dams are exposed to chronic stress, at adulthood the offspring display yet different consequences of stress on anxiety and cognition, and, in contrast to adulthood when the behavioral effects of stress are reversible, prenatal stress effects appear enduring. Changing levels of estradiol in the sexes over the lifespan appear to contribute to the differences in response to stress. Thus, theories of stress dependent modulations in CNS function--developed solely in male models, focused on peripheral physiological processes and tested in adults--may require revision when applied to a more diverse population (age- and sex-wise) at least in relation to the neural functions of cognition and anxiety. Moreover, these results suggest that other stressors and neural functions should be investigated to determine whether age, sex and gonadal hormones also have an impact.

Androgen modulation of hippocampal synaptic plasticity.

This review briefly summarizes recent developments in our understanding of the role of androgens in maintaining normal hippocampal structure. Studies in rats and vervet monkeys have demonstrated that removal of the testes reduces the density of synaptic contacts on dendritic spines of cornu ammonis 1 (CA1) pyramidal neurons. This effect is rapidly reversed by treatment with either testosterone or the non-aromatizable androgen dihydrotestosterone, suggesting that maintenance of normal synaptic density is androgen-dependent, via a mechanism that does not require intermediate estrogen biosynthesis. Similar effects of these androgens are observed in ovariectomized female rats, except that in the female the actions of testosterone include a substantial contribution from estrogen formation. The ability to stimulate hippocampal spine synapse density is not directly related to systemic androgenic potency: thus, weak androgens such as dehydroepiandrosterone exert effects that are comparable to those of dihydrotestosterone; while partial agonist responses are observed after injection of the synthetic antiandrogen, flutamide. These data provide a morphological counterpart to observations that androgens enhance cognitive function and mood state, suggesting that these effects may result at least in part from hippocampal neurotrophic responses. The unusual specificity of these responses raises the possibility that effects of androgens on the brain may be mediated via different mechanisms than the masculinizing actions of these steroids in non-neural androgen target organs.

Synaptic remodeling induced by gonadal hormones: neuronal plasticity as a mediator of neuroendocrine and behavioral responses to steroids.

During recent decades, it has become a generally accepted view that structural neuroplasticity is remarkably involved in the functional adaptation of the CNS. Thus, cellular morphology in the brain is in continuous transition throughout the life span, as a response to environmental stimuli. The effects of the environment on neuroplasticity are mediated by, to some extent, the changing levels of circulating gonadal steroid hormones. Today, it is clear that the function of gonadal steroids in the brain extends beyond simply regulating reproductive and/or neuroendocrine events. In addition, or even more importantly, gonadal steroids participate in the shaping of the developing brain, while their actions during adult life are implicated in higher brain functions such as cognition, mood and memory. A large body of evidence indicates that gonadal steroid-induced functional changes are accompanied by alterations in neuron and synapse numbers, as well as in dendritic and synaptic morphology. These structural modifications are believed to serve as a morphological basis for changes in behavior and cellular activity. Due to their growing functional and clinical significance, the specificity, timeframe, as well as the molecular and cellular mechanisms of hormone-induced neuroplasticity have become the focus of many studies. In this review, we briefly summarize current knowledge and the most significant recent discoveries from our laboratories on estrogen- and dehydroepiandrosterone-induced synaptic remodeling in the hypothalamus and hippocampus, two important brain areas heavily involved in autonomic and cognitive operations, respectively.

Aromatase in the central nervous system.

Central (central nervous system and pituitary) aromatization appears to be a fundamental process for endocrine control and development. Metabolism of androgens to estrogens and the subsequent metabolism of estrogens have been proven in many species, including humans, and linked to estrogen action. Thus, aromatization appears to initiate or to be involved in activities of importance to endocrine function at the central level and their effects peripherally. In the context of breast cancer, central aromatization relates to the control of gonadotrophins and other pituitary-brain hormones which may effect metabolism at the level of the breast. For example, follicle-stimulating hormone can increase aromatization and may be a factor in the control of such metabolism in breast tissue.

Antiproliferative actions of tamoxifen to human ovarian carcinomas in vitro.

The cytosolic estrogen receptor (ERc) and progestin receptor (PRc) levels were measured in 56 human epithelial ovarian tumors. The maximum ERc content in a tumor sample was 163 fmol/mg cytosolic protein. Forty-six of the tumor samples were evaluable for clonogenic growth in soft agar, and 19 samples produced 15 or more colonies per 10(5) cells plated. Four of the samples that grew had ERc levels of greater than 30 fmol/mg cytosolic protein. No correlation, however, between growth in soft agar and ERc or PRc content was observed. The antiproliferative properties of the antiestrogen, tamoxifen, were studied. Although no decrease in colony formation was observed after a 1-hr exposure to 0.2 or 2 microM tamoxifen, continuous exposure of cells to 2 microM tamoxifen reduced clonogenicity in 8 of 18 solid ovarian carcinomas examined. The maximum diminution in colony formation was approximately 50% of that of the control and was seen in 2 tumor samples. Both tumors that displayed the maximum response to continuous tamoxifen treatment had ERc and PRc levels greater than 30 fmol/mg cytosolic protein. None of the 14 tumors with ERc levels less than or equal to 30 fmol/mg cytosolic protein exhibited a decrease in colony formation of more than 50%. Exposure of cells for 1 hr to the combination of doxorubicin and tamoxifen produced a significant antagonism of the individual doxorubicin or tamoxifen antiproliferative effects in 7 of 9 samples examined. These data suggest that in a subset of human ovarian epithelial carcinomas tamoxifen alone can have some direct antiproliferative action on the clonogenic cells. The maximum antiproliferative effect of tamoxifen observed was related to ERc content in ovarian tumors.

The effect of seizures and kindling on reproductive hormones in the rat.

Reproductive dysfunction and endocrine disorders are common among both women and men with epilepsy, and, in particular, with temporal lobe epilepsy. In clinical studies, it is hard to separate the effects of seizures from the effects of medication and life style. Studies in rodents, however, suggest that seizures per se can contribute to reproductive dysfunction. In female rats, generalized seizures disrupt normal ovarian cyclicity in adults, and repeated electroshock seizures delay the onset of puberty in juveniles. Right amygdala kindling in adult female rats causes acyclicity, the development of polycystic ovaries and premature aging of the hypothalamic-pituitary neuroendocrine axis, leading to chronic anovulation and continuous estrogen exposure. In adult male rats, repeated electroshock seizures result in transient hypogonadism, characterized by decreased serum testosterone levels and lowered gonadal tissue weight. In contrast, right amygdala kindling increases serum testosterone, estradiol levels and gonadal weight. These findings suggest that reproductive dysfunction in women and men with epilepsy may result from recurrent seizure activity, due to seizure-related interference with the normal functions of the hypothalamic-pituitary-gonadal axis.

Progestin receptors in rat brain: distribution and properties of cytoplasmic progestin-binding sites.

Putative progestin receptors have been characterized in brain and pituitary tissue from untreated and estrogen-primed ovariectomized-adrenalectomized rats. The properties of these sites appear indistinguishable from those of cytoplasmic progestin receptors from the uterus: 1) sedimentation coefficient of 7S, which is reduced by half in the presence of 0.3 M KCl; 2) specificity of binding which strongly favors synthetic and natural progestins as opposed to glucocorticoids, androgens, and estrogens; 3) a dissociation constant for binding the synthetic progestin [3H]R5020 (17 alpha, 21-dimethyl-19-norpregna-4, 9-diene-3,20-dione) of 0.3 nM; and 4) similar rates of formation and dissociation of the [3H]R5020-receptor complexes. In these respects, the estrogen-inducible and noninducible receptors of the brain also appear to be indistinguishable from each other. Estrogen induction of progestin receptors is apparent in uterus (6-fold), pituitary (8-fold), mediobasal hypothalamus (4-fold), and preoptic area (4-fold), all estrogen receptor-containing areas. The corticomedial amygdala does not show an estrogen effect on progestin receptor levels even though it contains estrogen receptor sites. The midbrain of the rat doeogen-insensitive receptors in the brain is relatively low and of the same order of magnitude as in nonstimulated hypothalamus, preoptic area, and pituitary, yet variations are seen among the estrogen-insensitive structures, with lowest levels occurring in cerebellum (6-7 fmol/mg protein) and highest levels occuring in cerebral cortex (approximately 25 fmol/mg protein). These findings are discussed in relation to the actions of progesterone which do and do not require estrogen priming and in relation to intracranial progesterone implantation studies.

Estrogen stimulation of 3-o-methyl-D-glucose uptake in isolated rat hepatocytes.

The effect of in vivo and in vitro estrogen treatment on 3-O-methyl-D-glucose (30MDG) uptake in isolated rat hepatocytes was examined. A 1-h preincubation of isolated hepatocytes with 17 beta-estradiol (E2) or 17 alpha-ethynyl estradiol stimulated uptake of 30MDG in a dose-dependent fashion. The response appeared to be sterospecific, in that 17 alpha-estradiol was approximately 100-fold less effective than its 17 beta isomer. By itself, the triarylethylene antiestrogen, nafoxidine, slightly increased hepatocyte 30MDG uptake, while in combination with estrogen, nafoxidine completely blocked the effects of both E2 and 17 alpha-ethynyl estradiol. The protein synthesis inhibitor, cycloheximide, inhibited basal 30MDG uptake and abolished the stimulatory effect of estrogen. Kinetic analysis indicated that the estrogen effect resulted from an increase in the Vmax of the 30MDG transport system, with no measurable change in its Km. In vivo estrogen treatment, using either estrogen injections or continuous release Silastic E2 capsules, produced an increase in hepatocyte 30MDG uptake of 52-86%. A similar (66%) increase was seen in pregnant animals between the 14th and 19th days of gestation. These findings demonstrate that estrogens exert a rapid stimulatory effect on hepatocyte hexose transport. This effect is qualitatively similar to the response to estrogen of the hexose transport systems in other estrogen target tissues, such as the uterus. In addition, the results provide further support for the concept that the effects of the triarylethylene antiestrogens are both tissue and end-point dependent. Although previous studies have shown that nafoxidine exerts estrogen-like effects on hepatic renin substrate production, the present data indicate that, with respect to hepatocyte 30MDG, nafoxidine is almost a pure estrogen antagonist.

5alpha-Dihydrotestosterone (DHT) receptors in rat brain and pituitary cell nuclei.

Gonadectomized-adrenalectomized (GX-ADX) adult male rats were injected iv with 2-4 mug/kg [1,2-3H]DHT and sacrificed 2 h later. Whole tissue homogenates and purified cell nuclear fractions were prepared from various brain regions and the pituitary and analyzed for radioactivity. Cell nuclei from pituitary and most limbic-hypothalamic regions (but not cerebral cortex) concentrated radioactivity (per unit protein) over whole tissue levels. The highest levels of nuclear-associated radioactivity were present in pituitary, hypothalamic and septal tissues. Analysis of radioactivity by double isotope dilution, chromatography, and recrystallization revealed that unmetabolized DHT represented 96 and 95% of the nuclear-associated radioactivity in pituitary and pooled limbic-hypothalamic structures, respectively. Simultaneously administered 100-fold molar excesses of unlabeled 5betaDHT, progesterone or corticosterone did not reduce nuclear-retained [3H]DHT, while unlabeled 5alphaDHT competed strongly. The anti-androgen, cyproterone acetate, competed at doses capable of of blocking androgen-mediated neuroendocrine effects. Levels of nuclear-retained [3H]DHT were negligible in intact males, but rose markedly 24 h after castration-adrenalectomy, remaining stable for 2 weeks post-operatively. Density gradient centrifugation of pituitary or brain cell nuclear salt extracts (0.4M KCl) revealed the [3H]DHT bound to a macromolecule sedimenting at 3--4S. The characteristics of DHT brain and pituitary cell nuclear binding are compared with the respective characteristics of neural cytosol and ventral prostate nuclear androgen binding components. These preliminary data suggest the existence of the functional, steroid-specific, stereospecific cell nuclear androgen receptor in the adult rat brain and pituitary.

Insulin specific binding sites in the myometrium of pregnant rats.

Insulin (INS) specific binding sites were detected in the myometrium of pregnant rats. The apparent equilibrium dissociation constants for the higher and lower affinity INS binding components were 0.01 X 10(-9) M and 2.5 X 10(-9) M, respectively. During the latter half of pregnancy INS binding increased gradually and peaked on the day of parturition (159.9 +/- 19.8, 196.3 +/- 24.6, 254.7 +/- 36.8, and 272.6 +/- 29.1 and femtomolars per mg protein, mean +/- SEM on days 15, 20, 21, and 22 of gestation, respectively). The pattern of increased INS binding paralleled the prepartum increase in myometrial cell nuclear estrogen receptors as well as myometrial glycogen levels. In contrast, low INS binding was associated with elevated cell nuclear progestin receptor levels, on day 15 and immediately postpartum. These results suggest that, during the latter half of pregnancy, myometrial INS binding capacity and glycogen concentration gradually increase. Since INS receptor levels begin to increase in parallel with increasing nuclear estrogen receptors, INS receptor synthesis may be under estrogen control.

Androgen binding and metabolism in the cerebral cortex of the developing rhesus monkey.

The influence of gonadal steroids on the maturation of the cerebral cortex and their possible influence on cortical function are not well understood. The present study examines androgen binding and metabolism in regions of the cerebral cortex and several subcortical structures at different time points during the development of the rhesus monkey. Androgen binding and metabolism were assessed in selected cortical and subcortical regions from neonatal, juvenile, and adult rhesus monkeys. Specific high affinity androgen-binding sites (apparent equilibrium dissociation constants for [3H]R1881, 0.04-0.3 nM) were observed in many areas of the rhesus monkey brain. These areas included (but were not limited to) the hypothalamus, amygdala, dorsolateral prefrontal cortex, orbital prefrontal cortex, visual and somatosensory cortex, and corpus callosum. Although the highest level of androgen binding was observed in the hypothalamus (8-20 fmol/mg protein), cortical samples also had measurable levels of binding (1-5 fmol/mg protein). No apparent regional or developmental differences in the number or affinity of androgen-binding sites were detected in animals ranging from 1 week to 8 yr in age. Androgen metabolism via aromatization and 5 alpha-reduction was observed in all regions of the neocortex examined, although at lower levels than in the hypothalamus and amygdala. Overall production of estrogens and 5 alpha-reduced androgens declined approximately 10-fold from prenatal to early postnatal life in both cortical and subcortical structures. The presence of androgen binding and metabolism in the monkey cerebral cortex indicates that steroid hormones may have considerable impact on cortical function in primates at postnatal as well as prenatal ages.

Antiestrogen inhibits the induction of progestin receptors by estradiol in the hypothalamus-preoptic area and pituitary.

Estradiol treatment of ovariectomized-adrenalectomized rats produced an increase in cytosol progestin binding in the hypothalamus-preoptic area (HPOA), pituitary, and uterus. In the HPOA and pituitary, this induction of progestin receptors by estradiol was inhibited by the antiestrogen CI-628 under a variety of dose and time conditions. In the uterus, inhibition of the full effect of estradiol on progestin binding was observed after 3 days of injection of antiestrogen and estradiol. In the absence of estradiol, the antiestrogen produced a slight induction of progestin receptors in the HPOA and pituitary and a substantial induction of progestin receptors in the uterus. The results suggest that the induction of progestin receptors could be a key intermediate step in some behavioral and neuroendocrine actions of estradiol.

The catechol estrogen, 2-hydroxyestradiol-17 alpha, is formed from estradiol-17 alpha by hypothalamic tissue in vitro and inhibits tyrosine hydroxylase.

The ability of estradiol-17 alpha to serve as a substrate for estrogen-2/4-hydroxylase in rabbit hypothalamic tissue was determined and compared to that of estradiol-17 beta. Both 2- and 4-hydroxy metabolites of estradiol-17 alpha were formed by the hypothalamic tissue in vitro. The rates of formation of 2-hydroxyestradiol (2-OHE2)-17 alpha and -17 beta were similar as were their kinetic constants (Km and Vmax). In addition, 2-OHE2-17 alpha was shown to inhibit purified rat adrenal tyrosine hydroxylase with a potency comparable to that of 2-OHE2-17 beta, a finding similar to that reported by others with respect to catechol-o-methyltransferase. Since estradiol-17 alpha has a markedly reduced affinity for estrogen receptors compared with extradiol-17 beta, this steroid could be useful in studies designed to distinguish between receptor mediated effects of estrogens and effects that locally formed catechol estrogens may have through their direct interaction with catecholaminergic system in neural tissue.

Comparison of age- and sex-related changes in cell nuclear estrogen-binding capacity and progestin receptor induction in the rat brain.

Previous studies have suggested parallels between the mechanisms underlying sexual differentiation and age-related loss of reproductive cyclicity in the female rat. Both appear to involve the actions of estrogen on the brain and are associated with a reduction in hypothalamic estrogen sensitivity. In this study the effects of sex and aging on cell nuclear estrogen receptor-binding capacity and cytosol progestin receptor induction in the pituitary gland, periventricular preoptic area (PVP), medial preoptic area (mPO), bed nucleus of the stria terminalis, arcuate median eminence region (ARC), ventromedial nucleus (VMN), and corticomedial amygdala were directly compared. Young (2.5 months old) and middle-aged (8-10 months old) male and female and old (19 months old) female rats were gonadectomized 14 days before adrenalectomy (ADX). For comparison of cell nuclear estrogen receptor-binding capacity, animals received a saturating dose of estradiol (36.0 micrograms/kg BW) 3 days after ADX and 1 h before death. Cell nuclear estrogen binding was measured by an in vitro exchange assay. For comparison of estrogen-induced progestin receptors, animals received a sc placed Silastic capsule containing 10% 17 beta-estradiol at the time of ADX and were killed 3 days later. Cytosol progestin binding was measured by an in vitro binding assay. Cell nuclear estrogen-binding capacities and cytosol progestin receptor induction were lower in the PVP, mPO, and VMN of the young male than in the young female. In the female, the level of progestin receptor induction in the pituitary and brain was unaffected by age; however, cell nuclear estrogen-binding capacity in the mPO, VMN, ARC, and pituitary gland was lower in old than in middle-aged females. These results demonstrate that the effects of sexual differentiation and aging on the hypothalamus involve similar, but not identical, region-specific reductions in cell nuclear estrogen receptor-binding capacity. The consequences of these reduced estrogen receptor binding levels in terms of the induction of progestin receptor in response to estrogen exposure are, however, very different in the male compared to those in old female rats.

Actions of an estradiol-17-fatty acid ester in estrogen target tissues of the rat: comparison with other C-17 metabolites and a pharmacological C-17 ester.

The C-17 fatty acid esters of estradiol (E2) are a unique family of nonpolar estradiol metabolites. They are potent long-acting estrogens that represent the natural analog of the synthetic esters used for estrogen therapy. We measured the uterotropic response and the formation of uterine nuclear estrogen receptors (ERn) produced by iv administration of a representative ester, E2-17-stearate, in comparison to E2, other natural C-17 conjugates of E2, E2-17-glucuronide, and E2-17-sulfate, and the pharmacological ester E2-17-cyclopentylpropionate. While E2-17-stearate produced a sustained and greater uterotropic response compared to E2, the maximal induction of ERn by the ester was only about one third of that induced by a similar dose of E2. However, the induction of ERn by E2-17-stearate was markedly sustained compared to that by E2. Furthermore, the initiation of the ERn response to E2-17-stearate was delayed. Since E2-17-esters do not bind to the ER, this delay is consistent with the requirement for hydrolysis of the esters before interaction with the ER. Neither of the ionic conjugates of E2 (sulfate and glucuronide) produced an increase in ERn concentrations or a uterotropic response. The synthetic ester cyclopentylpropionate, like E2, produced a rapid ERn response and a significantly shorter uterotropic response than the stearate ester. When the induction of ERn by E2-17-stearate was investigated in other target tissues there were no marked differences in the brain, pituitary, and liver. No blood-brain barrier was apparent for the formation of ERn, despite the fact that this steroidal ester circulates in the blood bound to lipoproteins. These findings suggest that this unusual family of steroidal esters has biological properties that differentiate them from other known estrogens, natural and synthetic, in terms of their ability to produce a slow-onset sustained estrogenic stimulus in a variety of different estrogen target tissues.

Distribution of occupied and unoccupied estrogen receptors in the rat brain: effects of physiological gonadal steroid exposure.

In vitro autoradiographic methods have been developed for selective measurement of occupied and unoccupied estrogen receptors (ERs) in brain tissue sections. Addition of protamine sulfate traps unoccupied ERs in the tissue sections, allowing them to be detected after a short period of incubation with labeled estrogen. Occupied ERs are assessed, after washing in buffer without protamine to eliminate unoccupied receptor, by incubating the sections for 2 h at 37 C to exchange isotopically labeled steroid for the endogenous unlabeled ligand. Total ER binding capacity is estimated by summing the values for occupied and unoccupied ER. In all brain regions of normal females, ER occupation is low at estrus, reflecting the very low levels of circulating estradiol present at this stage of the estrous cycle, rising to approximately 50% of binding capacity at proestrus. By contrast, in intact males ER occupation varies considerably between brain regions, from a high of 55% of binding capacity in the bed nucleus of the stria terminalis to a low of 21% in the hypothalamic arcuate nucleus. Gonadectomy or treatment of intact males with the aromatase inhibitor 4-hydroxy androstenedione greatly reduces or eliminates ER occupation, depending on the brain region. In both sexes, changes in levels of endogenous gonadal steroids have little effect on total (occupied plus unoccupied) ER concentrations, with the exception of the hypothalamic ventromedial nucleus of the female, in which total ER concentration declines at estrus. These results are consistent with the hypothesis that local aromatization may be the primary determinant of regional ER occupation in the brain of the male rat, in contrast to the female, in which high levels of ER occupation are found only during the preovulatory estrogen surge. Although physiological changes in circulating estradiol and aromatizable androgen concentrations induce large changes in ER occupation, they have little effect on total ER content in most regions of the brain, suggesting that previous reports of changes in ER messenger RNA levels under different conditions of gonadal steroid exposure may not be directly reflected in steady state levels of the cognate receptor site.

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.