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Endocrinology Vol. 146, No. 9 3843-3850
Copyright © 2005 by The Endocrine Society

17{alpha}-Estradiol: A Brain-Active Estrogen?

C. Dominique Toran-Allerand, Alexander A. Tinnikov, Ravinder J. Singh and Imam S. Nethrapalli

Departments of Anatomy and Cell Biology, Obstetrics and Gynecology, (C.D.T.-A., A.A.T., I.S.N.), and Neurology (C.D.T.-A.) and the Centers for Neurobiology and Behavior and Reproductive Sciences (C.D.T.-A., A.A.T., I.S.N.), Columbia University College of Physicians and Surgeons, New York, New York 10032; and Department of Laboratory Medicine and Pathology (R.J.S.), Mayo Clinic and Foundation, Rochester, Minnesota 55905

Address all correspondence and requests for reprints to: Dr. C. Dominique Toran-Allerand, Department of Anatomy and Cell Biology, Columbia University College of Physicians and Surgeons, 650 West 168th Street, Black Building, Room 1615, New York, New York 10032. E-mail: cdt2{at}columbia.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The estrogen 17ß-estradiol has profound effects on the brain throughout life, whereas 17{alpha}-estradiol, the natural optical isomer, is generally considered less active because it binds less avidly to estrogen receptors. On the contrary, recent studies in the brain document that 17{alpha}-estradiol elicits rapid and sustained activation of the MAPK/ERK and phosphatidylinositol 3-kinase-Akt signaling pathways; is neuroprotective, after an ischemic stroke and oxidative stress, and in transgenic mice with Alzheimer’s disease; and influences spatial memory and hippocampal-dependent synaptic plasticity. The present study measured the endogenous content of 17{alpha}-estradiol in the brain and further clarified its actions and kinetics. Here we report that: 1) endogenous levels of 17{alpha}-estradiol and its precursor estrone are significantly elevated in the postnatal and adult mouse brain and adrenal gland of both sexes, as determined by liquid chromatography/tandem mass spectrometry; 2) 17{alpha}-estradiol and 17ß-estradiol bind estrogen receptors with similar binding affinities; 3) 17{alpha}-estradiol transactivates an estrogen-responsive reporter gene; and 4) unlike 17ß-estradiol, 17{alpha}-estradiol does not bind {alpha}-fetoprotein or SHBG, the estrogen-binding plasma proteins of the developing rodent and primate, respectively. 17{alpha}-Estradiol was also found in the brains of gonadectomized or gonadectomized/adrenalectomized mice, supporting the hypothesis that 17{alpha}-estradiol is locally synthesized in the brain. These findings challenge the view that 17{alpha}-estradiol is without biological significance and suggest that 17{alpha}-estradiol and its selective receptor, ER-X, are not part of a classical hormone/receptor endocrine system but of a system with important autocrine/paracrine functions in the developing and adult brain. 17{alpha}-Estradiol may have enormous implications for hormone replacement strategies at the menopause and in the treatment of such neurodegenerative disorders as Alzheimer’s disease and ischemic stroke.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN THE EARLY 1970s, circulating estrogen levels in the postnatal rodent were initially reported to be very elevated (1). However, when RIA became sufficiently specific to distinguish 17{alpha}-estradiol from 17ß-estradiol, these apparently high estradiol (E2) levels were subsequently termed spurious estrogen because they were not immunoreactive 17ß-estradiol and disappeared after adrenalectomy (1). Far from being spurious, however, these elevated estrogen levels may have partly represented 17{alpha}-estradiol, whose precursors epitestosterone and estrone (E1), originate to a certain extent from the adrenal gland and ovary (2).

17{alpha}-estradiol was originally isolated from pregnant mare urine (3), but essentially nothing is known about its endogenous content in the blood and other tissues. The biosynthetic pathways of 17{alpha}-estradiol are complex and not entirely known (Fig. 1Go). 17{alpha}-Estradiol is synthesized from aromatization of epitestosterone (also known as 17{alpha}-testosterone), a natural optical isomer (enantiomer) of testosterone (also known as 17ß-testosterone), to 17{alpha}-estradiol by the enzyme cytochrome P450 aromatase (estrogen synthase) (4) in sites that have not been fully characterized but that include the brain (5). In addition, 17ß-estradiol is interconverted to the estrogen E1 by 17ß-hydroxysteroid dehydrogenase (6). However, neither the biosynthetic pathways for 17{alpha}-estradiol and its most direct precursor epitestosterone nor the site(s) of their formation have been unequivocally confirmed to date. Aromatase is conserved phylogenetically in the central nervous system (7). Extragonadal estrogen production by aromatization of androgens is a significant source of tissue estrogens, which is independent of circulating estrogen levels. Expression of aromatase activity is present in various regions of the developing and adult brain, including the basal forebrain, preoptic area, neocortex, hippocampus, and amygdala (5).



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  		FIG. 1. The biosynthetic pathways of 17{alpha}-estradiol are complex and not fully characterized. 17ß-HSD, 17ß-hydroxysteroid dehydrogenase.

 
17{alpha}-Estradiol has strong neuroprotective activity, as shown by its ability to reduce mortality dramatically in a rat model of middle cerebral artery occlusion (8); decrease the levels of the amyloid ß-precursor protein in transgenic mice with Alzheimer’s disease (9); and rescue PC12 cells from oxidative stress (10). Moreover, the ability of 17{alpha}-estradiol at 1 pM to elicit the rapid and sustained activation of MAPK/ERK1/2 and phosphatidylinositol 3-kinase-Akt (11, 12, 13) and ERK5 (Sétáló Jr., G., and D. Toran-Allerand, unpublished data), signaling mediators of the differentiative and neuroprotective effects of estrogen in the brain, demonstrates their exquisite sensitivity to this estrogen. In addition, that 17{alpha}-estradiol is able to induce short-latency effects on spatial memory in rats, presumably through influences on hippocampal synaptic plasticity (14), further emphasizes strongly the potential importance of this enantiomer for the brain.

Here we show by liquid chromatography/tandem mass spectrometry (LC-MS/MS) (15) that the endogenous content of 17{alpha}-estradiol and E1 in the postnatal and adult neocortex, hippocampus, hypothalamus, and cerebellum of both sexes is significantly elevated, compared with 17ß-estradiol. We further show that 17{alpha}-estradiol binds to estrogen receptors with binding affinities similar to 17ß-estradiol; is able to transactivate an estrogen-responsive reporter gene; unlike 17ß-estradiol, does not bind {alpha}-fetoprotein (AFP) or SHBG, the estrogen-binding plasma proteins of the developing rodent and primate, respectively, and brain levels are unaffected by gonadectomy and/or adrenalectomy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Tissue samples were obtained from postnatal day (P) 1, P7, and adult male and female C57BL/6J mice from a breeding colony under the direction of Dr. C. A. Mason (Columbia University, New York, NY). Adult (8 wk old) gonadectomized (ovariectomized or castrated) and gonadectomized-adrenalectomized male and female C57BL/6J mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Animals were kept under standard laboratory conditions, with tap water and mouse breeder chow ad libitum, under a 10-h light, 14-h dark cycle. All experiments conformed strictly to National Institutes of Health and international guidelines on the ethical use of animals in experiments. Experimental protocols were approved by the Institutional Animal Care and Use Committee of Columbia University. The gonadectomized and gonadectomized/adrenalectomized mice were used after 10 d of surgery. The gonadectomized/adrenalectomized mice were provided with the tap water containing 0.9% sodium chloride.

Reagents
[3H]17{alpha}-estradiol (17-{alpha}[6,9(n)-3H]estradiol, 34 Ci/mmol) was custom synthesized by Amersham Biosciences UK Ltd. (Buckinghamshire, UK). [3H]17ß-estradiol, ([2,4,6,7,16,17-3H(N)]estradiol, 110 Ci/mmol) was purchased from NEN Life Science Products, PerkinElmer (Boston, MA). Unlabeled 17{alpha}-estradiol and 17ß-estradiol were purchased from Sigma Chemicals (St. Louis, MO). The ERE-tk-luc expression plasmid, human estrogen receptor (ER)-ß, and mouse ER{alpha} expression vectors were a generous gift from Dr. Maria Sjoberg (Karolinska Institute, Stockholm, Sweden) (16). The pRL-CMV expression plasmid was from Promega (Madison, WI). All other reagents were from Sigma.

Tissue lysates
The tissues were homogenized in lysis buffer [50 mM Tris (pH 7.4), 150 mM NaCl, 10% glycerol, 1 mM EGTA, 1 mM Na3VO4, 5 mM ZnCl2, 100 mM NaF, containing antiprotease cocktail mini (Roche, Indianapolis, IN), and 60 mM n-octyl-ß-D-glucopyranoside]. The homogenates were centrifuged at 100,000 x g, and tissue lysate or serum samples were used for analysis by LC-MS/MS. The protein content in sample lysates was measured by the Bradford reagent (Bio-Rad Laboratories, Hercules, CA).

Analysis of estrogens by the LC-MS/MS method
LC-MS/MS assay of estrogens was carried out, as described previously (15), and was optimized for simultaneous analyses of 17{alpha}-estradiol, 17ß-estradiol, and E1, using deuterium-labeled internal standards. Samples were extracted with 6 ml methylene chloride, the upper aqueous layer removed, and the remaining solvent transferred to clean 13 x 100-mm glass tubes. The solvent was evaporated under nitrogen in a 45 C water bath, and the dried residue was redissolved in 50 µl sodium bicarbonate buffer [100 mmol/liter (pH 10.5)]. To derivatize the samples, we then added an equal volume of 1 mg/ml dansyl-chloride in acetone and vortexed each sample for 1 min, followed by incubation in a heating block at 60 C for 3 min. Immediately thereafter, the samples were transferred to autosampler glass vials, sealed, and assayed. After dansyl chloride derivatization, samples were separated by fast-gradient chromatography, injected into a tandem mass spectrometer, and ionized by atmospheric pressure chemical ionization.

The dansyl derivatives of 17{alpha}-estradiol, 17ß-estradiol, and E1 were monitored, using transitions of m/z 506–171 and m/z 504–171 and quantified using deuterium-labeled 17ß-estradiol and E1 internal standards (Fig. 2Go). The total run time per sample was 5 min. The multiple-reaction monitoring ion pairs were m/z 506/171 for 3-dansyl-estradiol and m/z 504/171 for 3-dansyl-estrone. The limits of detection of E1 and E2 by LC-MS/MS assay were 12.9 pmol/liter (3.5 pg/ml) and 23.2 pmol/liter (6.3 pg/ml), respectively. The limits of quantification (functional sensitivity) for E1 and E2 were 44.1 pmol/liter (11.9 pg/ml) and 10.3 pmol/liter (2.8 pg/ml), respectively. The assay was linear to 2200 pmol/liter (~600 pg/ml) for either analyte. Recoveries were 93–108% for E1 and 100–110% for E2. No cross-reactivity was observed. Method comparison with several different immunoassays revealed that the latter were inaccurate and prone to interferences at low E1 and E2 analyte levels. The E2 levels were measured, using the calibration curves for all three estrogens, and are presented as picogram per milligram protein in the tissue lysates (Tables 1Go and 2Go). To check for the specificity of 17{alpha}-estradiol, 17ß-estradiol, and E1, mouse serum and brain lysates were spiked with known amounts of the three estrogens, and the recovery of all three estrogens was linear (data not shown). To further confirm the specificity of the method, the brain lysates were diluted 1.5 times and 3.0 times with the lysis buffer, and these were measured for 17{alpha}-estradiol, 17ß-estradiol, and E1, as above. Estrogen extractions in these experiments was also linear, as exemplified in Fig. 3Go.



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  		FIG. 2. LC-MS/MS profiles for adult mouse ovarian (A) and hippocampal (B) tissue lysates. LC-MS/MS extracted ion chromatograms of 17{alpha}-estradiol (17{alpha}-E2) and 17ß-estradiol (17ß-E2) and E1. The extracted samples were injected, using LC-MS/MS conditions described in Materials and Methods (15 ).

 

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  		TABLE 1. 17{alpha}-Estradiol, 17ß-estradiol, and estrone levels in mouse tissue lysates (pg/mg protein) and serum (pg/ml)

 

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  		TABLE 2. 17{alpha}-Estradiol, 17ß-estradiol, and estrone levels in gonadectomized and gonadectomized/adrenalectomized mouse tissue lysates (pg/mg protein)

 


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  		FIG. 3. Specificity test for 17{alpha}-estradiol. The P7 neocortical lysate was diluted 1.5 and 3.0 times, and the 17{alpha}-estradiol was measured by LC-MS/MS to check the linearity of estrogen extraction.

 
To determine cross reactivity and interferences, we added various amounts of E1, 17{alpha}-estradiol, 17ß-estradiol, estriol, ethynylestradiol, and E1-sulfate to a sample pool with previously measured E1 and E2 levels. More than 100 random samples from patients, as opposed to normal volunteers, were also assayed to detect potential drug or disease-related interferences (data not shown).

Binding assays
Recombinant ER{alpha} and ERß were obtained from Panvera-Invitrogen (Carlsbad, CA) and were diluted to 2 nM in the manufacturer’s recommended assay buffer [10 mM Tris (pH 7.4), containing 1 mM dithiothreitol, 10% glycerol, and 1 mg/ml BSA]. Adult rat or mouse uterus was homogenized in Tris buffer [10 mM Tris, 10% glycerol, 2 mM dithiothreitol (pH 7.4)], and cytosol was obtained by centrifugation of the homogenate at 100,000 x g at 4 C. Recombinant ER{alpha} or ERß or 50 µg of the uterine cytosol protein were incubated in a total volume of 0.1 ml for 16 h at +4 C with 0.25 nM-5 nM of [3H]17ß-estradiol or [3H]17{alpha}-estradiol with (for nonspecific binding) or without (for total binding) 500-fold molar excess of the cold ligand. Unbound E2 was removed by the addition of 0.1 ml of ice-cold 1% dextran-coated charcoal (DCC) suspension in the assay buffer for 5 min. The charcoal was removed by centrifugation, and the supernatant was taken for liquid scintillation counting. Specific binding was determined as the difference between total and nonspecific binding. The binding data were analyzed by Scatchard analysis. For displacement studies, the displacement of 2 nM of [3H]17ß-estradiol from ER{alpha}, ERß or uterine cytosol with different concentrations (10–12 to 10–6 M) of cold 17{alpha}-estradiol or 17ß-estradiol was determined. The relative binding affinity (RBA) of 17{alpha}-estradiol was calculated as the ratio of concentrations of the steroids required to reduce the specific binding by 50% (RBA of 17ß-estradiol = 100).

P7 mouse serum, rich in AFP (17), was used to study the binding of estrogens to AFP. The serum was treated with an equal volume of 1% DCC suspension for 15 min at 37 C to remove endogenous steroids and other low-molecular-weight substances and then diluted 2000 times in the assay buffer (as was done for the ERs). The binding assays for [3H]17{alpha}-estradiol and [3H]17ß-estradiol were performed, as described above for the ERs. Binding of [3H]17{alpha}-estradiol and [3H]17ß-estradiol to adult mouse and human serum was performed on serum pretreated with DCC and diluted 200 times. SHBG was diluted to 1 nM in the assay buffer. Binding of 2 nM of [3H]17{alpha}-estradiol or [3H]17ß-estradiol to mouse or human serum or to SHBG was measured, as described for the ERs. Human serum and SHBG were purchased from Sigma.

Cell culture and transfections
COS-7 cells were cultured in DMEM (American Type Culture Collection, Manassas, VA) with 10% fetal bovine serum (Summit Biotechnology, Fort Collins, CO) at 37 C in a humidified 5% CO2 atmosphere. For transient transfection assays, the cells were seeded in phenol red-free DMEM (Invitrogen, Carlsbad, CA) supplemented with 2% dextran-charcoal-stripped fetal bovine serum (Gemini, Woodland, CA). Cells were transfected with 100 ng of reporter plasmid, 50 ng of pRL plasmid, as an internal control, and 50 ng of ER{alpha} or ERß expression plasmids, or empty expression vector pSG5, to a total of 200 ng DNA per well. The transfection medium was changed after 24 h to phenol red-free DMEM, and 17{alpha}-estradiol or 17ß-estradiol (100 pM to 10 nM) with or without ICI 182,780 (10–7 M) was added, as described in the figure legends. Cells were harvested after 24 h, and a luciferase assay was performed, using the dual luciferase reporter assay system (Promega).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
LC-MS/MS was used to identify and compare the endogenous content of 17{alpha}-estradiol, 17ß-estradiol, and E1 in lysates of the brain (neocortex, hippocampus, hypothalamus, and cerebellum), adrenals, ovary, uterus, testis, and serum of P7 and adult male and female C57BL/6J mice (Fig. 2Go and Table 1Go). Whereas significantly elevated levels of 17ß-estradiol were found predictably in the adult ovary (Fig. 2AGo) and uterus, 17ß-estradiol levels in the P1, P7, and adult neocortex, hippocampus (Fig. 2BGo), hypothalamus, cerebellum, and adrenals of both sexes were below detectable limits (Table 1Go). However, in striking contrast, this pattern was completely reversed in the P1, P7, and adult brain of both sexes, in which elevated endogenous levels of 17{alpha}-estradiol and E1 were found (Fig. 2BGo and Table 1Go). Elevated endogenous levels of 17{alpha}-estradiol and E1 were also found in the P7 and adult ovary, uterus, and female adrenals (Table 1Go), whereas levels of these estrogens in P7 and adult serum were very low and frequently at or below the limit of sensitivity of the method (5 pg/ml). The gonadectomized (ovariectomized or castrated) and gonadectomized-adrenalectomized mice of both sexes had even higher levels of 17{alpha}-estradiol, whereas 17ß-estradiol was detected only in some samples and E1 was not detected at all (Table 2Go).

As shown in Fig. 4Go, [3H]17{alpha}-estradiol bound to human recombinant ER{alpha} and ERß with affinities similar to those of 17ß-estradiol [affinity constant (Kd) = 0.4 nM and Kd = 0.5 nM, respectively, a finding confirmed by binding assays performed on adult rat uterine cytosol (Kd = 0.4 nM)]. We studied 17{alpha}-estradiol displacement of [3H]17ß-estradiol from recombinant human ER{alpha} and ERß and uterine cytosol receptors. In these assays, the RBA of 17{alpha}-estradiol for ER{alpha} and ERß was 51 and 64%, respectively, of that of 17ß-estradiol. However, displacement of [3H]17ß-estradiol by 17{alpha}-estradiol from uterine cytosol gave an RBA of 15% of 17ß-estradiol. Pretreatment of the cytosol with DCC suspension, which removed endogenous steroids and low-molecular-weight substances, resulted in a rise of the RBA for 17{alpha}-estradiol to 65%.



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  		FIG. 4. [3H]17{alpha}-Estradiol binds to recombinant ERs with an affinity similar to that of [3H]17ß-estradiol. Scatchard analyses of the data on specific binding of 17{alpha}-estradiol and 17ß-estradiol to recombinant human ER{alpha}, ERß, and rat uterine cytosol. Each point represents the mean of a duplicate measurement. Specific binding of 0.25, 0.5, 1.0, 1.3, 2.5, and 5 nM of [3H]17{alpha}-estradiol or 17ß-estradiol was measured in three independent experiments for ER{alpha} and in two independent experiments for ERß or rat uterine cytosol.

 
Although estrogen binding studies were attempted with brain tissue, the counts for specific activity were too low, compared with the total binding, and increasing the amount of protein per assay resulted only in higher nonspecific binding activity. The specific activity of the commercially available 17ß-estradiol is higher than 100 Ci/mmol, but a specific activity of only 34 Ci/mmol was the best we could obtain for our custom tritium-labeled 17{alpha}-estradiol from the manufacturer. In the absence of a 17{alpha}-estradiol of higher specific activity, we used recombinant ER{alpha} and ERß proteins for our studies and confirmed our results, using uterine ERs. We extrapolated our results from the recombinant ERs and the uterine lysates to the brain because the brain and uterine ERs have the same protein sequence (18).

To complement the binding studies, we tested whether 17{alpha}-estradiol could activate transfected ER{alpha} and ERß (Fig. 5Go). Both 17{alpha}-estradiol and 17ß-estradiol activated a luciferase reporter gene, containing an ERE in COS-7 cells transfected with ER{alpha} or ERß. Activation of the transfected ERs was blocked by the ER antagonist ICI 182,780. No response to estrogens was observed in the cells transfected with empty vectors.



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  		FIG. 5. Both 17{alpha}-estradiol and 17ß-estradiol activated a luciferase reporter gene, containing an ERE in COS-7 cells transfected with ER{alpha} or ERß. The ERE-luciferase activity was normalized against Renilla luciferase (Promega) activity. Each column represents mean ± SEM of three independent experiments done in duplicates.

 
Because 17ß-estradiol is known to bind AFP, the principal estrogen-binding plasma protein of the developing rodent, with high affinity (17), we characterized the extent to which 17{alpha}-estradiol bound AFP. It had been reported earlier that 17{alpha}-estradiol has a high affinity for AFP as well (19), and whereas we also obtained some displacement of [3H]17ß-estradiol from serum AFP by 500-fold molar excess of 17{alpha}-estradiol, the displacement was minimal. For a total binding of 20–25 x 1000 cpm/sample, 500-fold molar excess of 17{alpha}-estradiol resulted in 17–20 x 1000 cpm, some 20% displacement. This suggests that 17{alpha}-estradiol has very low affinity for AFP. In fact, the concentration of 17{alpha}-estradiol used in the displacement studies was in the micromolar range, which could result in some nonspecific suppression of 17ß-estradiol binding. However, when [3H]17{alpha}-estradiol was used to determine its affinity for AFP, AFP did not bind 17{alpha}-estradiol at all (Fig. 6Go). For example, 17{alpha}-estradiol may interact very weakly with the AFP ligand binding pocket and may compete with 17ß-estradiol at very high concentrations. But no specific binding could be detected, when we used the labeled 17{alpha}-estradiol at a nanomolar concentration. Similarly, SHBG, the estrogen-binding plasma protein of the developing primate, did not bind 17{alpha}-estradiol to any extent (data not shown) and was not further evaluated. On the other hand, no marked differences in specific binding to adult mouse or human serum were found between the two estradiols (Fig. 6Go).



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  		FIG. 6. Specific binding of [3H]17ß-estradiol (open triangle) and [3H]17{alpha}-estradiol (solid triangle) to P7 mouse serum, which is rich in AFP (diluted 2000 times). The binding isotherm for [3H]17ß-estradiol represents one assay done in duplicates. No specific binding to AFP was detectable for [3H]17{alpha}-estradiol in the range of 1.25–10 nM. This was repeated at least five times with 10 nM of [3H]17ß-estradiol as a positive control, in which high specific binding was seen each time (data not shown). Inset, Specific binding of 2 nM of [3H]17ß-estradiol (open columns) and [3H]17{alpha}-estradiol (closed columns) to adult mouse and human serum (diluted 200 times) and SHBG (1 nM).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We report here the identification of significantly elevated endogenous levels of 17{alpha}-estradiol and E1 in the brains of postnatal and adult mice of both sexes. The presence of such elevated levels was unexpected because the stereo-specificity of the ER for 17ß-estradiol has been presented for decades as a classical example of the preference of living organisms for only one of two enantiomers. Nonetheless, previous studies (19) have reported that 17{alpha}-estradiol elicits genomic (classical) estrogenic responses in a human breast cancer cell line, with an estimated Kd of 0.7 nM, which is comparable with that of 17ß-estradiol (0.2 nM) and with what we found for uterine cytosol (Kd = 0.4 nM).

The presence of significantly elevated endogenous levels of 17{alpha}-estradiol in various brain regions, in association with barely detectable serum levels of this estrogen, strongly suggest that the endogenous levels of 17{alpha}-estradiol in the brain must be derived from local synthesis. To investigate this possibility, we measured the levels of 17{alpha}-estradiol, 17ß-estradiol, and E1 in gonadectomized and gonadectomized-adrenalectomized mice, in which we found even more elevated levels of 17{alpha}-estradiol in all the samples. Moreover, whereas 17ß-estradiol levels were elevated in a few samples, E1 levels were below the limits of detectability, strongly suggesting that 17{alpha}-estradiol is indeed synthesized locally in the brain (Table 2Go) and that E1 is of ovarian origin.

Although a recent report documented the presence of 17ß-estradiol in the neonatal rat brain by RIA (20), identification of steroids by LC-MS/MS is considered much more specific. LC-MS/MS identifies the different estrogens after they have been separated chromatographically, whereas RIA, which uses antibodies to 17ß-estradiol, is unable to distinguish the various estrogens because of cross-reactivity. Moreover, it has been reported that although there were large differences between the concentration of estradiol measured in the same samples by the different commercially available RIA kits, the apparent changes in concentration that might be regarded as clinically significant were nothing more than the imprecision of the assay (15, 21).

Although the prevailing view is that 17{alpha}-estradiol is inactive and does not interact with the classical ERs, ER{alpha} and ERß (22), we found that [3H]17{alpha}-estradiol bound to and transactivated human recombinant ER{alpha} and ERß with affinities similar to those of 17ß-estradiol (Kd = 0.4 nM and Kd = 0.5 nM, respectively), a finding confirmed by binding assays performed on adult rat uterine cytosol (Kd = 0.4 nM). 17{alpha}-Estradiol activation of transfected ER{alpha} and ERß has been reported previously (23, 24), and our data are in good agreement with those studies. Whereas our binding results contradict the generally held belief, that 17{alpha}-estradiol is much less efficient than 17ß-estradiol in displacing [3H]17ß-estradiol from ERs, Korenman (25), Edwards and McGuire (26), and Kuiper et al. (27) have all reported that 17{alpha}-estradiol can bind to ERs with an affinity of approximately 50% of the RBA of 17ß-estradiol. An RBA of 58% of 17ß-estradiol has also been reported for 17{alpha}-estradiol binding to human recombinant ER{alpha} (27). This is consistent with our findings of RBAs of 51 and 64% of 17{alpha}-estradiol binding to ER{alpha} and ERß, respectively. The discrepancy with numerous data concerning the lower displacing activity of 17{alpha}-estradiol may be explained by the presence of other proteins, endogenous substances, or reagents in the assay buffers, which may influence binding. Some DCC-removable low-molecular-weight substances in the uterine cytosol may prevent 17{alpha}-estradiol from binding to the ERs. For example, free fatty acids are known to influence steroid-protein interactions dramatically (28). Proteins have also been shown to affect the RBA. For example, whereas diethylstilbestrol had an RBA higher than that of 17ß-estradiol (RBA = 100) in the rat uterine nuclear fraction, the RBA of DES dropped from 245 to 43 in the presence of rat serum and 10-fold in the presence of human serum albumin, with no effect on the RBA of 17ß-estradiol (29).

It is unclear why other workers have failed to detect the high binding affinity of 17{alpha}-estradiol. We attribute our findings of high affinity to the use of DCC, which removed the endogenous steroids and low-molecular-weight agents, such as free fatty acids, from the cytosol, thereby increasing the affinity of 17{alpha}-estradiol for the ERs. However, removal of low-molecular-weight substances to achieve binding of 17{alpha}-estradiol to ER{alpha} and ERß may not be physiological. There may be regional brain differences with respect to the presence of proteins, fatty acids, and other molecules that would normally prevent binding of 17{alpha}-estradiol to these ERs.

The potential importance of the elevated endogenous levels of 17{alpha}-estradiol in the brain is significantly heightened by our observations that 17{alpha}-estradiol is the preferred ligand of ER-X (30), the novel, plasma membrane-associated, putative ER we recently identified in the developing mouse (13) and baboon (Guan, X., and D. Toran-Allerand, unpublished data) brain. ER-X is considered the preferred putative receptor for 17{alpha}-estradiol because 17{alpha}-estradiol activates MAPK/ERK at a dose level of 1 pM, whereas 17ß-estradiol requires a level 100-fold higher to achieve the same degree of activation (13), presumably because 17ß-estradiol has a greater affinity for ER{alpha} and ERß than for ER-X and would activate those receptors before ER-X. Moreover, whereas activation of ER-X by 17{alpha}-estradiol elicits activation of MAPK/ERK, activation of ER{alpha} by ER{alpha}-selective ligands inhibits MAPK/ERK dramatically (13, 31) and exposure of ERß to ERß-selective ligands is without effect on MAPK activation (31). Thus, the high levels of 17{alpha}-estradiol may be more relevant for neural functions mediated by ER-X than ER{alpha} and ERß. The 17{alpha}-estradiol/ER-X association may be particularly relevant because, although ER-X is developmentally regulated, it is reexpressed in the adult brain after an ischemic brain injury (13) and in a transgenic mouse model (J20) of Alzheimer’s disease (Nethrapalli, I. S., and D. Toran-Allerand, unpublished data). Thus, the likely target of 17{alpha}-estradiol in the brain is ER-X and not the classical intranuclear ERs, ER{alpha} and ERß.

The presence of significantly elevated endogenous levels of 17{alpha}-estradiol and E1 in the brains of postnatal and adult mice raises questions regarding their possible biological roles. 17{alpha}-Estradiol and/or 17ß-estradiol have been shown to have differentiative effects in the developing brain (17) and neuroprotective effects in the adult with respect to damage from age-associated, neurodegenerative states such as Alzheimer’s (9, 32) and Parkinson’s (33) diseases as well as multiple sclerosis (34), schizophrenia (35), and ischemic stroke (8, 36, 37). 17{alpha}-Estradiol and ER-X thus form an estrogen/receptor system that would be readily available for neuroprotection during development and in the adult. The importance of 17{alpha}-estradiol for the adult brain in particular is further emphasized by noting that, although endogenous levels of 17ß-estradiol were elevated in the adult uterus, the classical estrogen (17ß-estradiol) target tissue, 17{alpha}-estradiol, but not 17ß-estradiol, levels were significantly elevated in the adult brain of both sexes.

The uterotrophic activity of 17 {alpha}-estradiol has also been reported (38). However, it was not possible in these studies to determine whether the uterotrophic stimulation was due to the direct effects of 17{alpha}-estradiol because this enantiomer was partially metabolized to 17ß-estradiol, and both enantiomers were found in uterine nuclei after an implant of 17{alpha}-estradiol (38).

The importance of age-specific functions of 17{alpha}-estradiol is suggested by our findings of its lack of binding to plasma AFP, the major estrogen binding plasma protein of the developing rodent, whose circulating postnatal levels are very high (2 mg/ml) (17). AFP has generally been considered the principal barrier that protects tissues of the perinatal rodent, particularly the brain, from excessive exposure to circulating estrogens (17). The dramatic difference in the bioavailability of the two E2 enantiomers for the ERs in the postnatal brain suggests that, during the developmental period, 17{alpha}-estradiol may have important age-specific functions that are distinct from sexual differentiation of the brain (39) as, for example, influences on neurogenesis and neuronal survival. Although immunoreactivity for AFP and other plasma proteins has been found within neurons of the neonatal rodent brain (17), the binding of 17{alpha}-estradiol to its intraneuronal ERs (ER-X) would not be modulated or regulated by AFP, as 17ß-estradiol binding to ER{alpha} and ERß would be, thus enabling 17{alpha}-estradiol to exert its actions freely.

Treatment with exogenous 17{alpha}-estradiol may have unpredictable consequences. For example, Hajek et al. (40) reported that exogenous 17{alpha}-estradiol was carcinogenic in the reproductive tract of neonatal BALB/c mice but not in the adult. They proposed that the biological effects of 17{alpha}-estradiol may be age dependent, which may explain why this estrogen has been considered inactive in the adult. Moreover, because 17{alpha}-estradiol does not bind to AFP, its bioavailability, as compared with 17ß-estradiol, is much higher in perinatal animals, which may explain its reported carcinogenic effects at this age (40).

Here we show, for the first time to our knowledge, that high endogenous levels of 17{alpha}-estradiol are present in the postnatal and adult male and female brain. Our findings suggest that because 17{alpha}-estradiol is not present in the circulation and is unaffected by ovariectomy, castration, and/or adrenalectomy, it is likely to be made in the brain. Moreover, that the endogenous levels of 17{alpha}-estradiol are significantly elevated in the brain, after gonadectomy and/or adrenalectomy, suggests that the gonads and adrenals may normally exert some sort of regulatory influence on the endogenous brain content of 17{alpha}-estradiol, which disappears after their removal. Because 17{alpha}-estradiol elicits both MAPK/ERK and phosphatidylinositol 3-kinase and Akt phosphorylation (11, 12, 13), its presence in the brain makes it uniquely positioned to activate the entire subsequent range of differentiative and neuroprotective signaling pathways. Moreover, because 17{alpha}-estradiol is capable of both binding and activating ER-X, these findings challenge the view that 17{alpha}-estradiol is without biological significance.

17{alpha}-Estradiol and its specific receptor ER-X do not constitute a classical endocrine hormone/receptor system but, rather, a novel system with important local autocrine and paracrine functions in both the developing and adult brain of both sexes. Far from being a biologically inactive estrogen, as is generally believed, 17{alpha}-estradiol may have very important neural functions throughout life, with enormous implications for hormone replacement strategies at the menopause and in the treatment of various neurodegenerative disorders, including Alzheimer’s disease and ischemic stroke.


   Acknowledgments
 
We thank Drs. Carol A. Mason (Columbia University) for the generous gift of the C57BL/6J mice; Maria Sjoberg (Karolinska Institute) for providing expression plasmids for ER-{alpha}, ER-ß, and ERE-luciferase; Meharvan Singh (University of North Texas) for constructive criticisms of the manuscript; and Ms. Hranush Melikyan for expert technical assistance in the care of the mice. We also thank Dr. Michel Ferin (Columbia University) for allowing us to use his nitrogen evaporation facility.


   Footnotes
 
This work was supported in part by grants from National Institutes of Health (National Institute on Aging) and National Institute of Mental Health/Office of Research in Women’s Health and the Maffei Gift for Brain Research (all to C.D.T.-A.).

First Published Online June 9, 2005

1 A.A.T. and I.S.N. contributed equally to this work. Back

Abbreviations: AFP, {alpha}-Fetoprotein; DCC, dextran-coated charcoal; E1, estrone; E2, estradiol; ER, estrogen receptor; ERE, estrogen response element; Kd, affinity constant; LC-MS/MS, liquid chromatography/tandem mass spectrometry; P, postnatal day; RBA, relative binding affinity.

Received December 15, 2004.

Accepted for publication May 27, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Weisz J, Gunsalus P 1973 Estrogen levels in immature female rats: true or spurious-ovarian or adrenal? Endocrinology 93:1057–1065[Medline]
  2. Blaquier J, Dorfman RI, Forchielli E 1967 Formation of epitestosterone by human blood and adrenal tissue. Acta Endocrinol (Copenh) 54:208–214[Medline]
  3. Budavari S, ed 1996 The Merck index. 12th ed. Whitehouse Station, NJ: Merck Research Laboratories
  4. Finkelstein M, Weidenfeld J, Ne’eman Y, Samuni A, Mizrachi Y, Ben-Uzilio R 1981 Comparative studies of the aromatization of testosterone and epitestosterone by human placental aromatase. Endocrinology 108:943–947[Abstract]
  5. MacLusky NJ, Walters MJ, Clark AS, Toran-Allerand CD 1994 Aromatase in the cerebral cortex, hippocampus, and mid-brain: ontogeny and developmental implications. Mol Cell Neurosci 5:691–698[CrossRef][Medline]
  6. Steckelbroeck S, Watzka M, Reissinger A, Wegener-Toper P, Bidlingmaier F, Bliesener N, Hans VH, Clusmann H, Ludwig, M, Siekmann L, Klingmuller DJ 2003 Characterization of estrogenic 17ß-hydroxysteroid dehydrogenase (17ß-HSD) activity in the human brain. Steroid Biochem Mol Biol 86:79–92
  7. Callard GV, Petro Z, Ryan KJ 1978 Phylogenetic distribution of aromatase and other androgen-converting enzymes in the central nervous system. Endocrinology 103:2283–2290[Abstract]
  8. Simpkins JW, Rajakumar G, Zhang YQ, Simpkins CE, Greenwald D, Yu CJ, Bodor N, Day AL 1997 Estrogens may reduce mortality and ischemic damage caused by middle cerebral artery occlusion in the female rat. J Neurosurg 87:724–730[Medline]
  9. Levin-Allerhand JA, Lominska CE, Wang J, Smith JD 2002 17{alpha}-estradiol and 17ß-estradiol treatments are effective in lowering cerebral amyloid-ß levels in AßPPSWE transgenic mice. J Alzheimers Dis 4:449–457[Medline]
  10. Gelinas S, Bureau G, Valsatro B, Massicotte G, Cicchetti F, Chiasson K, Gagne B, Blanchet J, Matinoli MG 2004 {alpha}- And ß-estradiol protect neuronal but not native PC12 cells from paraquat-induced oxidative stress. Neurotox Res 6:141–148[Medline]
  11. Singh M, Sétáló Jr G, Guan X, Warren M, Toran-Allerand CD 1999 Estrogen-induced activation of mitogen-activated protein kinase in cerebral cortical explants: convergence of estrogen and neurotrophin signaling pathways. J Neurosci 19:1179–1188[Abstract/Free Full Text]
  12. Singh M 2001 Ovarian hormones elicit phosphorylation of Akt and extracellular-signal regulated kinase in explants of the cerebral cortex. Endocrine 14:407–415[CrossRef][Medline]
  13. Toran-Allerand CD, Guan X, MacLusky NJ, Horvath TL, Diano S, Singh M, Connolly Jr ES, Nethrapalli IS, Tinnikov AA 2002 "ER-X": a novel, plasma membrane-associated, putative estrogen receptor that is regulated during development and after ischemic brain injury. J Neurosci 22:8391–8401[Abstract/Free Full Text]
  14. MacLusky NJ, Luine VN, Hajszan T, Leranth C 2004 The 17{alpha} and 17ß isomers of estradiol both induce rapid spine synapse formation in the CA1 hippocampal subfield of ovariectomized female rats. Endocrinology 146:287–293
  15. Nelson RE, Grebe SK, OKane DJ, Singh RJ 2004 Liquid chromatography-tandem mass spectrometry assay for simultaneous measurement of estradiol and estrone in human plasma. Clin Chem 50:373–384[Abstract/Free Full Text]
  16. Bjornstrom L, Sjoberg M 2002 Mutations in the estrogen receptor DNA-binding domain discriminate between the classical mechanism of action and cross-talk with Stat5b and activating protein 1 (AP-1). J Biol Chem 277:48479–48483[Abstract/Free Full Text]
  17. Toran-Allerand CD 1984 On the genesis of sexual differentiation of the general nervous system: morphogenetic consequences of steroidal exposure and possible role of {alpha}-fetoprotein. Prog Brain Res 61:63–98[Medline]
  18. Bettini E, Pollio G, Santagati S, Maggi A 1992 Estrogen receptor in rat brain: presence in the hippocampal formation. Neuroendocrinology 56:502–508[Medline]
  19. Laurant C, de Lauzon SD, Cittanova N, Nunez E, Jayle MF 1975 The comparative specificity of 3 estradiol-binding proteins. Rat {alpha}-foetoprotein, rat liver 17ß-hydroxy steroid dehydrogenase and anti-(estradiol-6-carboxymethyloxime-bovine serum albumin) antiserum. Biochem J 151:513–518[Medline]
  20. Amateau SK, Alt JJ, Stamps CL, McCarthy MM 2004 Brain estradiol content in newborn rats: sex differences, regional heterogeneity, and possible de novo synthesis by the female telencephalon. Endocrinology 145:2906–2917[Abstract/Free Full Text]
  21. Lee CS, Smith NM, Kahn SN 1991 Analytic variability and clinical significance of different assays for serum estradiol. J Reprod Med. 36:156–160
  22. Anstead GM, Carlson KE, Katzenellenbogen JA 1997 The estradiol pharmacophore: ligand structure-estrogen receptor binding affinity relationships and a model for the receptor binding site. Steroids 62:268–303[CrossRef][Medline]
  23. Preisler-Mashek MT, Solodin N, Stark BL, Tyriver MK, Alarid ET 2002 Ligand-specific regulation of proteasome-mediated proteolysis of estrogen receptor-{alpha}. Am J Physiol Endocrinol Metab 282:E891–E898
  24. Wade CB, Robinson S, Shapiro RA, Dorsa DM 2001 Estrogen receptor (ER){alpha} and ERß exhibit unique pharmacologic properties when coupled to activation of the mitogen-activated protein kinase pathway. Endocrinology 142:2336–2342[Abstract/Free Full Text]
  25. Korenman SG 1969 Comparative binding affinity of estrogens and its relation to estrogenic potency. Steroids 13:163–177[CrossRef][Medline]
  26. Edwards DP, McGuire WL 1980 17{alpha}-Estradiol is a biologically active estrogen in human breast cancer cells in tissue culture. Endocrinology 107:884–891[Abstract]
  27. Kuiper GG, Carlsson B, Grandien K, Enmark E, Haggblad J, Nilsson S, Gustafsson JA 1997 Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors {alpha} and ß. Endocrinology 138:863–870[Abstract/Free Full Text]
  28. Boonstra R, Tinnikov AA 1998 Increased corticosteroid binding capacity of plasma albumin but not of corticosteroid-binding globulin caused by ACTH-induced changes in free fatty acid concentrations in snowshoe hares and rabbits. J Endocrinol 156:205–212[Abstract]
  29. Okulicz WC, Johnson LD 1987 The relative binding affinity of diethylstilbestrol to uterine nuclear estrogen receptor: effect of serum and serum albumin. Proc Soc Exp Biol Med 185:478–483[Abstract]
  30. Toran-Allerand CD 2004 Novel mechanisms of estrogen action in the developing brain: beyond ER-{alpha} and ER-ß. Exp Gerontol 39:1579–1586[CrossRef][Medline]
  31. Singh M, Sétáló Jr G, Guan X, Frail DF, Toran-Allerand CD 2000 Estrogen-induced activation of the MAP kinase cascade in the cerebral cortex of estrogen receptor-{alpha} knock-out mice. J Neurosci 20:1694–1700[Abstract/Free Full Text]
  32. Green PS, Simpkins JW 2000 Estrogens and estrogen-like non-feminizing compounds. Their role in the prevention and treatment of Alzheimer’s disease. Ann NY Acad Sci 924:93–98[Abstract/Free Full Text]
  33. Currie LJ, Harrison MB, Trugman JM, Bennett JP, Wooten GF 2004 Postmenopausal estrogen use affects risk for Parkinson disease. Arch Neurol 61:886–888[Abstract/Free Full Text]
  34. Voskuhl RR 2003 Hormone-based therapies in MS. Int MS J 10:60–66[Medline]
  35. Akhondzadeh S, Nejatisafa AA, Amini H, Mohammadi MR, Larijani B, Kashani L, Raisi F, Kamalipour A 2003 Adjunctive estrogen treatment in women with chronic schizophrenia: a double-blind, randomized, and placebo-controlled trial. Prog Neuropsychopharmacol Biol Psychiatry 27:1007–1012[CrossRef][Medline]
  36. Yang SH, Liu R, Wu SS, Simpkins JW 2003 The use of estrogens and related compounds in the treatment of damage from cerebral ischemia. Ann NY Acad Sci 1007:101–107[Abstract/Free Full Text]
  37. Rau SW, Dubal DB, Bottner M, Gerhold LM, Wise PM 2003 Estradiol attenuates programmed cell death after stroke-like injury. J Neurosci 23:11420–11426[Abstract/Free Full Text]
  38. Clark JH, Williams M, Upchurch S, Eriksson H, Helton E, Markaverich BM 1982 Effects of estradiol-17{alpha} on nuclear occupancy of the estrogen receptor, stimulation of nuclear type II sites and uterine growth. J Steroid Biochem 16:323–328[CrossRef][Medline]
  39. McEwen BS 1999 Clinical review 108: the molecular and neuroanatomical basis for estrogen effects in the central nervous system. J Clin Endocrinol Metab 84:1790–1797[Free Full Text]
  40. Hajek RA, Robertson AD, Johnston DA, Van NT, Tcholakian RK, Wagner LA, Conti CJ, Meistrich ML, Contreras N, Edwards CL, Jones LA 1997 During development, 17{alpha}-estradiol is a potent estrogen and carcinogen. Environ Health Perspect 105(Suppl 3):577–581



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