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and ß with Potent Selective Ligands from Structure-Based Design
EnTec Gesellschaft für Endokrinologische Technologie GmbH (A.H., B.S., G.R., W.E.), and Jenapharm GmbH & Co. KG (O.P., D.K., G.M.), D-07745 Jena, Germany; and Schering AG (A.W., K.-H.F.), D-13342 Berlin, Germany
Address all correspondence and requests for reprints to: Dr. Karl-Heinrich Fritzemeier, Schering AG, Müllerstrasse 170–178, D-13342 Berlin, Germany. E-mail: karlheinrich.fritzemeier{at}schering.de.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and ERß, in mediating the physiological responses to estrogens are not completely understood. Although knockout animal experiments have been aiding to gain insight into estrogen signaling, additional information on the function of ER and ERß will be provided by the application of isotype-selective ER agonists. Based on the crystal structure of the ER ligand binding domain and a homology model of the ERß-ligand binding domain, we have designed steroidal ligands that exploit the differences in size and flexibility of the two ligand binding cavities. Compounds predicted to bind preferentially to either ER or ERß were synthesized and tested in vitro using radio-ligand competition and transactivation assays. This approach directly led to highly ER isotype-selective (
200-fold) and potent ligands. To unravel physiological roles of the two receptors, in vivo experiments with rats were conducted using the ER- and ERß-selective agonists in comparison to 17ß-estradiol. The ER agonist induced uterine growth, caused bone-protective effects, reduced LH and FSH plasma levels, and increased angiotensin I, whereas the ERß agonist did not at all or only at high doses lead to such effects, despite high plasma levels. It can thus be concluded that estrogen effects on the uterus, pituitary, bone, and liver are primarily mediated via ER. Simultaneous administration of the ER and ERß ligand did not lead to an attenuation of ER-mediated effects on the uterus, pituitary, and liver parameters. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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or ERß or both receptors. To answer these questions, mRNA and protein expression of the receptors in numerous tissues has been studied (2, 3, 4). Dominant expression of the respective ER isotypes in certain tissues suggests a distinct physiological role. ER has a broad expression pattern and is most abundant in uterus, vagina, liver, and pituitary. ERß is expressed in rat ovary, prostate, epididymis, lung, hypothalamus, and bladder (5). Low expression of ERß was observed in all uterine tissues (6).
In the human prostate, the majority of epithelial cells expresses ERß, whereas the stromal cells are negative (3). In the ovary, ER expression is observed in the stromal compartment and the theca cells. Expression of ERß is restricted to granulosa cells of developing follicles (7).
In addition to expression studies, the phenotypes of knockout mice in which each of the receptors or both were inactivated, have been analyzed (8, 9, 10, 11). The resulting knockout mouse phenotypes largely reflect the mRNA expression pattern of ER and ERß. The ERKO mice show a severe phenotype, are infertile (male and female), display elevated LH, estradiol, and testosterone levels (female), have decreased bone density and a disturbed breast development (8). In contrast, ERßKO mice develop normally and do not display a severely hampered reproductive function. Consistent with the high abundance of ERß in the ovary, the number of ovulated oocytes is reduced (9, 11). The prostate phenotype of these mice is still a matter of debate (3, 12).
Further insight into ER and ERß function has been provided by applying selective ligands to animals and studying the pharmacological effects (13, 14, 15). Compounds described for these purposes are the ER-selective agonist propyl pyrazole triol (PPT) (16), the ERß selective agonists diarylpropionitrile (DPN) (17), and the benzoxazole derivative ERB-041 (15).
In contrast to knockout animals, in which the entire receptors are inactivated throughout ontogeny and postnatal life, these experiments allow the ligand-dependent functions of both ERs and their interaction to be studied.
We have designed highly isotype-selective, steroidal ER agonists that, when applied to animals, directly stimulate either ER or ERß. In contrast to the above-mentioned nonsteroidal isotype-selective ER ligands, our compounds were designed on the basis of the available protein structure information and are close derivatives of the natural hormone estradiol. We suggest that the results obtained with these tool compounds mimic the physiological situation of estrogen action and provide additional insights into ER and ERß functions. Here we describe the structure-based design, the synthesis strategy, the in vitro compound profiling, and the in vivo pharmacological characterization of these tool compounds in female rats.
All animal procedures described here were run according to accepted standards of humane animal care, especially German animal welfare law with the permission of the District Government of Thuringia, Germany.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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in complex with 17ß-estradiol (E2) (18), an ERß homology model was generated. The sequence alignment of the ER and ERß LBDs shows 59% sequence identity with only two single amino acid deletions in loop regions in the case of ERß (Fig. 1
). The homology model was constructed by replacing the nonidentical amino acids according to this sequence alignment and altering the protein backbone in the region of deletions. The ERß model is shown in Fig. 2.
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and ERß-Selective Ligands L384 -> ERß M336 and ER M421 -> ERß I373, with ERß M336 being positioned above the B- and C-ring (near position 8ß) and I373 below the D-ring (near position 16 and 17) of E2 (Fig. 3A). Although the volume of the sulfur-containing methionine side chain (volume = 85.9 Å3) is slightly larger than the branched amino acid side chains of leucine and isoleucine (volume = 82.6 Å3 resp. 82.3 Å3), it was predicted that the increased flexibility of the linear methionine side chain would allow larger substituents to be accommodated. Thus, substitution of E2 with lipophilic groups containing one or two heavy atoms in 8ß position was predicted to lead to highly selective ERß agonists. These compounds would fit well into the ERß ligand binding pocket (Fig. 3C), although colliding with L384 in the case of ER. Similarly, a substitution at position 16 and/or 17 would lead to ER agonists. Again a larger substituent would replace the flexible methionine (ER-M421, Fig. 3B), although it interacts unfavorably with the rigid isoleucine (ERß I373) below the steroidal D-ring.
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- and ERß-Selective Ligands). To obtain ER-selective compounds, E2 was substituted with a five-membered ring bridging positions 16 and 17. This additional cyclic ether (compound 1) or lactone ring (compound 2: 3,17-dihydroxy-19-nor-17-pregna-1,3,5(10)-triene-21,16-lactone, named 16-LE2), respectively, provides bulkiness below the D-ring Figs. 3B
and 4).
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and ERß binding pockets (Figs. 3D and 4). The synthesis of the compounds is described in patent applications (19, 20) and will be published in detail elsewhere.
In Vitro Characterization of ER Isotype-Selective Ligands
The affinity of the synthetic ligands to rat (r) ER and human (h) ER and ß and the induction of reporter gene transcription via hER and ERß were determined using competitive radiometric and transactivation assays.
Compounds with larger substituents in positions 16 and 17 (compounds 1 and 2) show a high affinity for rER and hER, whereas they bind to rERß and hERß with an affinity about 2 orders of magnitude lower (Table 1). More polar and larger substituents increase the ER selectivity. Similar results are obtained with transactivation experiments, indicating a 250-fold selectivity in reporter gene activation via ER for 16-LE2 (Table 1 and Fig. 5).
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). The latter compound displays a 183-fold ERß selectivity in the transactivation assay (Table 1).
Neither 8ß-VE2 nor 16-LE2 exhibit antagonistic activity on E2-induced reporter gene activity in ER and ERß-dependent transactivation assays (data not shown). In these experiments, the pure antiestrogen ZM 182780 was used as a reference and caused half-maximal inhibition of the E2-induced reporter gene activity at concentrations of 1–3 nmol/liter.
In Vivo Characterization of ER Isotype-Selective Ligands
E2 and 16-LE2 caused a dose-dependent increase in uterine weight, dose-dependent decreases in serum rFSH and rLH (Fig. 6). 8ß-VE2 affected uterine weight and rLH only at the highest dose tested (100 µg/animal·d) and had no effect on rFSH in plasma (Fig. 6). Regarding hepatic estrogenicity, E2 caused a slight increase in angiotensinogen as measured by its enzymatic reaction product angiotensin I at a dose of 100 µg/animal·d. 16-LE2 exhibited considerable hepatic estrogenicity. A dose-dependent increase in angiotensin I (Fig. 6) and decreases in IGF-I, high-density lipoprotein (HDL), and total cholesterol were observed. 8ß-VE2 had no effect on any of these parameters. The bone mineral density (BMD) in the ovariectomized (OVX) model is fully recovered by treatment with E2 (1 µg/animal·d) or 16-LE2 at 10 µg/animal·d (Fig. 7). In case of the ERß agonist, significant effects were observed only at the highest dose (100 µg/animal·d). This dose restored 78% of BMD. In agreement with the effects on bone density, E2 and 16-LE2 caused significant decreases in deoxypyridinoline (DPD) and hydroxyproline levels (Fig. 7) at the 0.1 and 1 µg/animal·d doses, respectively, whereas the 8ß-VE2 caused a slight effect only at 100 µg/animal·d or was inactive.
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transactivational activity (21), we tested various combinations of both tool compounds in vivo. No signs of inhibition of ER effects by the ERß ligand were observed on uterine weight (Fig. 8), rFSH, rLH, angiotensin I, IGF-I, HDL, and total cholesterol (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-LBD (24). Interestingly, the predicted flexibility of the methionine ERß-M336 was observed experimentally in the ERß LBD-genistein complex x-ray structure. This side chain adopts two unequally (70:30) occupied alternative positions, indicating high flexibility. In another homology model of ERß (25), it was predicted that the ERß ligand binding pocket at the ß-face of E2 is smaller due to the bulkier side chain of methionine (ERß-M336) in comparison to leucine (ER-L384). Indeed, it was confirmed in the ERß x-ray structure that the ligand binding cavity is smaller than in the case of ER. However, the prediction that substitution at the ß-face of E2 (positions 8ß and 15ß) should result in ER-selective compounds and substitution at the -face (positions 16 and 17) should yield ERß-selective compounds (25) is not supported by our studies. Although methionine is slightly larger than leucine and isoleucine, we considered it to be more flexible, leading to the prediction that larger substituents can be accommodated in the vicinity of methionine residues. The in vitro test results of the isotype-selective ligands support this prediction and demonstrate the importance of considering protein (side chain) flexibility in view of designing ligands. Studies involving the systematic derivatization of hexestrol have resulted in DPN, another highly ERß selective agonist (17). Site-directed mutagenesis and transactivation studies also suggest that the difference in size of the ER/ß binding pockets due to the difference ER-L384/ERß-M336 is responsible for the high isotype selectivity of DPN (26). It has recently been shown that single point mutations of those amino acids that differ between ER and ERß alone cannot account for the ER selectivity of PPT and that long-range interactions have to be taken into consideration (27). This may be explained by the completely different structure of PPT in comparison to estradiol. Certain conformational rearrangements within the ligand binding pocket must occur to accommodate PPT (16). This is in contrast to our ligands, which are designed to be structurally close to 17ß-estradiol and effectively exploit the few differences within the binding pockets with only relatively small substituents. It is thus supposed that our ligands do not induce larger conformational rearrangements within the binding pockets with respect to 17ß-estradiol and that ligand selectivity is not dependent on long-range interactions.
In Vitro Studies
Because ER is the predominat ER isotype in the uterus, and ERß shows high expression levels in prostate, estrogen isotype-selective compounds could be identified using preparations of these tissues for receptor binding studies. The results obtained with the rat cytosolic preparations correlate with the relative binding affinities determined with in vitro-expressed hERs (Table 1). The binding affinities also agree with the transactivation data obtained with cell-based reporter gene assays (Table 1). Especially in the case of the ERß-selective agonists (compound 3 and 4), a correlation between ligand binding and reporter gene transcription was obtained. 16-LE2 and 8ß-VE2 are thus estrogens that bind to and act on ER and ERß selectively but with a similar potency as E2.
In Vivo Studies: Selective Activation of ER or ERß
The compounds 16-LE2 and 8ß-VE2 appear excellently suited for the study of ER/ß-mediated functions because they act on either ER or ERß with a comparable selectivity and are nearly equally potent with respect to E2. In addition, both compounds are close steroidal analogs of E2 (only two to four additional carbon/oxygen atoms) and thus reduce the probability of completely different biological activities at other receptors or enzymes with respect to 17ß-estradiol. Genistein for example, also described as weakly selective ERß-ligand (28, 29), is characterized by inhibition of tyrosine kinases (30), 17ß-hydroxysteroid dehydrogenases, and 5-reductase type II (31). It is as such not very well suited for studying ER isotype-mediated effects in in vivo models. A notable difference between the ER and ERß agonists is the substitution of position 17 in case of 16-LE2, which protects this compound against oxidation of the 17ß-hydroxy group through 17ß-hydroxysteroid dehydrogenases. 8ß-VE2 is not protected and was supposed to be metabolically less stable. The compounds were thus administered sc with osmotic pumps, leading to high plasma levels (see supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org).
The results of the in vivo studies with the steroidal ER and ERß ligands are in agreement with expectations based on the tissue distribution of ER and ERß, respectively, and ER knockout phenotypes. Organs expressing predominantly ER such as uterus, pituitary, and liver could be affected with E2 and 16-LE2, but not (or only at the highest dose) with 8ß-VE2. At this high dose, the residual ER activity of 8ß-VE2 (Table 1) is presumed to be responsible for the observed effects. The uterotropic activity of E2 as well as the feedback via the hypothalamic-pituitary axis is thus mediated predominantly via ER (Fig. 6). Similar results were obtained with the ER-selective agonist PPT (13). ERß plays an inferior role in these organs. Interestingly, the effects on hepatic parameters such as angiotensin I, IGF-I, HDL, and total cholesterol are stronger for 16-LE2 than for E2. The ERß agonist did not show effects on these parameters up to 100 µg/animal·d (Fig. 6). Liver effects are thus mediated via ER. It is well known that 17-alkyl-substituted steroids (like 16-LE2 or ethinylestradiol) are protected against oxidation of the 17ß-hydroxy group, a keystep in metabolic inactivation of steroidal estrogens. The metabolic stabilization through the substitution would impair metabolic inactivation and contribute to the substantial hepatic estrogenicity of the ER agonist (32). Protection against OVX-induced bone loss by 16-LE2 confirms that ER is the primary mediator of antiresorptive effects of estrogens on bone and are consistent with observations made with the ER agonist PPT (13). In trabecular bone, ER and ERß are coexpressed, although ER is about 10-fold more abundant than ERß at the mRNA level (33). 16-LE2 was 10 times less potent than E2 in preventing the OVX-induced loss of BMD, whereas 8ß-VE2 was 1000-fold less potent than E2. Bone-protective effects of 8ß-VE2 at very high doses appear to be mediated via ER. Several surrogate parameters for bone effects such as DPD and hydroxyproline (Fig. 7) are in agreement with the BMD measurements. DPD and hydroxyproline, markers that reflect the amount of degraded collagen, are significantly decreased with E2 and 16-LE2 treatment but not with 8ß-VE2.
Interestingly, throughout all in vivo experiments, the effects of 16-LE2 on uterus, pituitary, bone, and liver occur at doses that are slightly more than 2 orders of magnitude, but significantly less than 3 orders of magnitude lower than in the case of the ERß agonist. This ratio (ER/ERß) of more than 100-fold in the in vivo experiments reflects the selectivity of 16-LE2 determined in vitro (70- to 250-fold; Table 1). Also a comparison between the in vitro and in vivo potency of the ER agonist and E2 reveals a good agreement between the two methods.
It has been suggested that ERß reduces ER-mediated gene transcription, also referred to as a "Yin Yang" relationship (21). The availability of the two ER isotype-selective agonists permitted us to test this hypothesis. The simultaneous administration of 16-LE2 and 8ß-VE2 did not result in inhibition of ER-mediated effects on uterine weight (Fig. 8), pituitary (rFSH, rLH, data not shown), and liver (angiotensin I, IGF-I, HDL, and total cholesterol, data not shown). At least in these organs, the ERß agonist was unable to antagonize ER effects.
In all the in vivo experiments discussed, low serum levels of 8ß-VE2 cannot explain weak or undetectable effects of 8ß-VE2 on estrogen-sensitive parameters. At nonuterotropic doses (e.g. 10 µg/animal·d), plasma levels as high as 0.8 ng/ml (see supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org) were found. In comparison, E2 exerts its effects at plasma levels of 5–40 pg/ml (34). Indeed, we and others (15, 35) recently revealed distinct physiological roles that are solely mediated via ERß using ER isotype selective compounds.
The work presented here demonstrates that it is possible to use protein structure information, either from an x-ray structure or a homology model, to design receptor isotype-selective ligands and to apply such compounds as pharmacological tools in animal models for studying the physiological roles of the respective receptors. The results of the in vivo studies with the ER isotype-selective compounds described here illustrate that reproductive and metabolic estrogen effects are mediated via ER. The described tool compounds are suitable for studying estrogen effects in variety of other tissues, organs, and species.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and ß, androgen, glucocorticoid, mineralocorticoid, and PR) were aligned using the program ClustalW (36). Gonnet series matrices and gap opening/gap extension penalties of 10/0.2 were used. The homology model of ERß was built based on the crystal structure of ER (18). Because H12 of the ER-LBD forms contacts with a neighboring protein molecule in this crystal structure, a conformation of this helix similar to H12 in the PR-LBD was modeled. This model of ER was used as a template for the construction of the ERß model, using the SYBYL program (Tripos Inc., St. Louis, MO). Energy minimization with the AMBER 4.1 force field led to the final model of ERß. A detailed description of the homology modeling procedure is given in (37). Volume calculations of amino acids were performed with MOLCAD in SYBYL. All four ER isotype-selective agonists were manually placed into the binding pockets by superimposition with estradiol and subsequently docked by energy minimization using the MMFF94 force field (38).
ER Binding Studies
The binding affinity of the compounds to rER, rERß, hER, and hERß were determined by in vitro competition experiments using [3H]E2 (5 nmol/liter) as a ligand and unlabeled E2 as a reference (39). hER and hERß were produced in Sf9 insect cells using baculovirus expression vectors (an expression plasmid for the hER was obtained from P. Chambon, Strasbourg, France). Ligand binding to rER and rERß was determined using cytosol preparations (40) of rat prostate (41) and rat uterus (42). The binding affinity of the test compounds is given as relative binding affinity, defined as (IC50 E2/IC50 test compound) x 100 (IC50 of E2 is 5 nmol/liter), whereby the IC50 of E2 at hER equals 1.25 ± 0.7 x 10–8 (n = 4) and the IC50 of E2 at hERß equals 1.32 ± 0.7 x 10–8 (n = 4).
Transactivation Assays
The estrogenic potency of the ER ligands in vitro was determined by transactivation assays (39). U2-OS human osteosarcoma cells (ATCC, Manassas, VA) were transiently cotransfected with hER or hERß expression vector (hER: HEGO, P. Chambon), respectively, and an estrogen response element (ERE)2-luciferase reporter gene. Estrogen-induced reporter gene activity was determined 24 h after treatment of the cells with test compounds. The relative transcriptional potency is defined as (EC50 E2/EC50 test compound) x 100 (EC50 of E2 = 4.5 ± 1.6 x 10–12 M (n = 8) in the ER transactivation assay and EC50 of E2 = 3.3 ± 1.4 x 10–11 M (n = 8) in the ERß transactivation assay).
Animal Studies
Effect of 16-LE2 and 8ß-VE2 on uterus, pituitary, and liver.
Adult OVX Wistar rats (body weight 220–250 g) were treated for 8 d with E2, 16-LE2, 8ß-VE2, or vehicle (propylene glycol) starting 21 d after surgery. Osmotic pumps (Alzet, model 2001D) for sc treatment. Parameters determined were as follows: uterine weight; rFSH, rLH, angiotensin I, IGF-I, HDL, total cholesterol, and plasma levels of 8ß-VE2.
Effect of 16-LE2 and 8ß-VE2 on bone.
Six-month-old Wistar rats (body weight 250–270 g) were OVX and immediately treated for 29 d with E2, 16-LE2, 8ß-VE2, or vehicle (propylene glycol). The compounds were administered sc by osmotic pumps (Alzet, model 2004). Urine was collected on d 1, 8, and 29; blood on d 1, 8, 15, and 29. IGF-I, rGH, angiotensin I, HDL, total cholesterol, hydroxyproline, and DPD cross-links were determined. On d 29, the animals were killed, and uteri and tibiae were prepared. BMD was measured using quantitative x-ray computer tomography.
Coadministration of 16-LE2 and 8ß-VE2.
On d 14 after OVX, Wistar rats (body weight 180–200 g) were treated sc for 3 d with 16-LE2, 8ß-VE2, combinations of 8ß-VE2 plus 16-LE2 or vehicle alone (benzyl benzoate + castor oil: 1 + 4 vol/vol). Uterine weight, rFSH, rLH, IGF-I, HDL, total cholesterol, and angiotensin I were determined.
Statistical Analyses
One-way ANOVA was used to demonstrate statistical differences between animal groups. Calculations of statistical significance (P values < 0.05) between treatment groups and control group were performed using Dunnett’s t test.
Biochemical Methods
Plasma levels of 8ß-VE2 were measured by RIA (EnTec GmbH, Hamburg/Jena, Germany). The assay exploits the cross reactivity of the antiserum contained in the 125I-estradiol MAIA kit of ADALTIS, with 8ß-VE2 (coefficients of variation of intra- and interassay precision: 8.5%, limit of quantification 15.13pg/ml).
Parameters determined were as follows: rFSH, GH, and LH in serum were determined with RIAs from Amersham Biosciences (Amersham, Buckinghamshire, UK); cholesterol and HDL cholesterol in plasma by enzymatic colorimetric assays (Dr. Bruno Lange GmbH, Düsseldorf, Germany); IGF-I in serum with the 125I-RIA IGF-R20 (Mediagnost, Reutlingen, Germany); hydroxyprolin in serum using a colorimetric assay (EnTec GmbH); coefficients of variation of the intra- and interassay precision: less than 2.83% and less than] 5.25%); and free DPD cross-links in urine with the 125I-RIA 
-BCT DPD AA-60F1 (IDS Ltd., Boldon, UK).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: BMD, Bone mineral density; DPD, deoxypyridinoline; DPN, diarylpropionitrile; E2, 17ß-estradiol; ER, estrogen receptor; H12, helix 12; HDL, high-density lipoprotein; 16-LE2, 3,17-dihydroxy-19-nor-17-pregna-1,3,5(10)-triene-21,16-lactone; h, human; LBD, ligand binding domain: OVX, ovariectomized; PPT, propyl pyrazole triol; PR, progesterone receptor; r, rat; 8ß-VE2, 8-vinylestra-1,3,5(10)-triene-3,17ß-diol.
Received for publication February 5, 2004. Accepted for publication April 16, 2004.
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M. T. Follettie, M. Pinard, J. C. Keith Jr., L. Wang, D. Chelsky, C. Hayward, P. Kearney, P. Thibault, E. Paramithiotis, A. J. Dorner, et al. Organ Messenger Ribonucleic Acid and Plasma Proteome Changes in the Adjuvant-Induced Arthritis Model: Responses to Disease Induction and Therapy with the Estrogen Receptor-{beta} Selective Agonist ERB-041 Endocrinology, February 1, 2006; 147(2): 714 - 723. [Abstract] [Full Text] [PDF]
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O. Imamov, G.-J. Shim, M. Warner, and J.-A. Gustafsson Estrogen Receptor beta in Health and Disease Biol Reprod, November 1, 2005; 73(5): 866 - 871. [Abstract] [Full Text] [PDF]
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T. Pelzer, V. Jazbutyte, K. Hu, S. Segerer, M. Nahrendorf, P. Nordbeck, A. W. Bonz, J. Muck, K.-H. Fritzemeier, C. Hegele-Hartung, et al. The estrogen receptor-{alpha} agonist 16{alpha}-LE2 inhibits cardiac hypertrophy and improves hemodynamic function in estrogen-deficient spontaneously hypertensive rats Cardiovasc Res, September 1, 2005; 67(4): 604 - 612. [Abstract] [Full Text] [PDF]
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K. F. Koehler, L. A. Helguero, L.-A. Haldosen, M. Warner, and J.-A. Gustafsson Reflections on the Discovery and Significance of Estrogen Receptor {beta} Endocr. Rev., May 1, 2005; 26(3): 465 - 478. [Abstract] [Full Text] [PDF]
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