Division of Endocrinology, Metabolism, and Molecular Medicine, Northwestern University Medical School, Chicago, Illinois 60611
Address all correspondence and requests for reprints to: J. Larry Jameson, M.D., Ph.D., Department of Medicine, Northwestern University Medical School, 251 East Huron Street, Galter 3-150, Chicago, Illinois 60611. E-mail: ljameson{at}northwestern.edu.
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ABSTRACT |
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INTRODUCTION |
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In addition to this classical pathway of estrogen action, the ER can also alter gene transcription by a mechanism that does not require direct ER binding to an ERE. Instead, protein-protein interactions between the ER and other transcription factors, such as Sp1, Jun, or nuclear factor-kappa B, lead to changes in target gene transcription. Examples of genes regulated in this nonclassical pathway include heat shock protein Hsp27 (7), low density lipoprotein receptor (8), collagenase (9, 10, 11), IGF-I (12), choline acetyltransferase (13), lipoprotein lipase (14), IL-6 (15, 16), and others. In many of these cases, there is no evidence of ER binding to DNA, although sequences within the ER DNA binding domain (DBD) seem to be necessary (15, 17). In vitro studies suggest that non-ERE-dependent actions of the ER may be of physiological significance. It is also notable that many of the nonclassical pathway target genes are paradoxically stimulated by ER antagonists, raising the possibility that selective estrogen receptor modulators may act as agonists through the nonclassical pathway (9, 10).
The significance of nonclassical signaling for members of the nuclear receptor family has been elegantly demonstrated by studies of the glucocorticoid receptor (GR). Mice completely deficient in GR expression die shortly after birth due to lung atelectasis (18). Another model was created to identify GR actions that are mediated independent of DNA binding. A point mutation was introduced that abolished GR homodimerization and therefore GR binding to its response element (19). Animals homozygous for this mutation (GRdim/dim mice) survived, demonstrating that the GR activity through the classical pathway is not essential for survival. Further characterization of these animals allowed GR action to be classified as classical vs. nonclassical pathways. For example, proopiomelanocortin and PRL gene expression, as well as induction of thymocyte apoptosis, are dependent on DNA binding, whereas development and function of the adrenal medulla, and certain inflammatory responses, are independent of the classical pathway.
Knockout (KO) models of both the ER and ERß genes have demonstrated their important role in reproduction and development. Disruption of the ERß gene impairs ovulation but males are fertile (19). Mutagenesis of the ER
gene causes phenotypic effects in multiple reproductive tissues in males and females. ER
KO males are infertile and exhibit impaired fluid resorption and obstruction of the seminiferous tubules. ER
KO females are anovulatory and their ovaries contain hemorrhagic cysts. Their uteri are hypoplastic and unresponsive to estrogen, and the development of the mammary gland is arrested. Serum levels of estradiol (E2) and LH are elevated, and the levels of PRL are diminished (summarized in Ref. 20). Studies of mice deficient in both ER
and ß (ER
ßKO) provide additional evidence for the important role of the ERs in female reproductive development and function (21). Ovarian follicles of adult ER
ßKO females differentiate into structures that resemble seminiferous tubules of the testes, a phenotype that is unlike that seen in either ER
KO or ERßKO.
The goal of this study was to distinguish the classical and nonclassical ER actions in vivo. Previous in vitro work has demonstrated that it is possible to selectively eliminate classical ER signaling and preserve nonclassical signaling by introducing a mutation into the P box of the first zinc finger of the DBD (22). This mutation (E207A/G208A in mouse ER) abolishes ERE binding and activation of ERE-containing reporter genes, but preserves the nonclassical pathway, as demonstrated by ER regulation of reporter genes containing activator protein 1 (AP1) response elements. We hypothesized that targeted insertion (knock-in) of this mutation would contrast with the ER
KO model by preserving the physiological functions of ER that are independent of DNA binding (nonclassical pathway). Unexpectedly, heterozyous females are infertile due to anovulation and the occurrence of endometrial hyperplasia. The presence of such a strong phenotype in the heterozygous state confirms the role of nonclassical ER signaling in vivo and suggests that these features may be caused by dysregulation of classical and nonclassical signaling.
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RESULTS |
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Knock-In Mutation of ER DBD
The aforementioned mutation (E207A/G208A) was introduced into the mER locus by targeted mutagenesis using positive-negative selection. A
-phage clone containing exon 3, which encodes the first zinc finger (23) (Fig. 2A
), was isolated from a mouse genomic library. The targeting construct contained a neomycin cassette flanked by lox P sites approximately 300 bp upstream of the mutated exon (Fig. 2B
). Transfection of the targeting vector into embryonic stem (ES) cells provided three correctly targeted clones, two of which were injected into blastocysts and implanted into foster mothers. Two of the male chimeras exhibited germline transmission and served as founders for the nonclassical ER knock-in (NERKI) colony. The modified allele designation is Er(tm2jlj). However, for the remainder of this manuscript, the knock-in mouse line will be referred to as NERKI. Initial screening was performed using the 5'-probe from the region of the ER
gene external to the targeting construct (Southern blot in Fig. 2C
; positions of the probe and restriction sites shown in Fig. 2B
). The neomycin cassette was excised by breeding the founder males to transgenic females expressing Cre recombinase under control of the cytomegalovirus promoter (Fig. 2B
). Genotyping was performed by PCR of sequences flanking the lox P site after excision of the neomycin cassette (Fig. 2D
). The presence of the mutated allele was confirmed by sequencing PCR products from genomic DNA. Expression of the mutated ER allele was confirmed by RT-PCR, followed by digestion of the PCR product at a mutation-specific site (SphI, Fig. 2E
). Expression of the mutant ER allele was comparable to the WT allele (data not shown). Western blot analyses indicated that total ER
protein levels were not altered (Fig. 2E
).
NERKI+/- Females Are Infertile
Heterozygous male NERKI+/- animals successfully produced progeny when bred to WT females. Unexpectedly, however, heterozygous female NERKI+/- mice were infertile when bred to either WT or NERKI+/- males. After 4 months, none of 14 NERKI+/- females became pregnant or gave birth. In parallel matings, their WT littermates reproduced successfully. Groups of six WT and six NERKI+/- females were superovulated and mated with WT or heterozygous males known to be fertile. All WT females had vaginal plugs and became pregnant. No plugs were seen in NERKI+/- females, indicating that they did not mate, and no pregnancies occurred. Examination of vaginal smears of NERKI+/- and WT littermates demonstrated that while WT animals cycled normally, NERKI+/- animals were in constant diestrus (data not shown).
NERKI+/- Females Have Decreased Serum Progesterone Levels
Serum gonadotropin and steroid hormone levels were determined for NERKI+/- animals and their WT littermates (Table 1A). LH, FSH, and E2 levels were not significantly different between the two groups. Progesterone levels were reduced in the NERKI+/- mice (P < 0.01), most likely because of abnormal follicular development and anovulation (see below).
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Differentiation of the Mammary Gland Is Inhibited in Untreated NERKI+/- Females
Mammary gland morphology was examined in young adult WT and NERKI+/- females. Ductal elongation at the borders of the mammary fat pad of NERKI+/- females was comparable to that of WT mice, indicating that mammary development was relatively normal at this stage of development (Fig. 3A). However, the subsequent side-branching and differentiation of ductal buds into lobuloalveolar structures, which occur after puberty, were decreased compared with WT littermates (Fig. 3B
). Quantitative comparison of mammary gland whole mounts demonstrated a decreased number of branching sites; the number of branching points per centimeter of duct was 91.2 ± 4.5 for WT females and 44.0 ± 1.9 for NERKI+/- females (P < 0.01). Analysis of the ducts at a higher magnification revealed that alveolar bud formation was impaired in NERKI+/- females (Fig. 3B
). We attempted to stimulate the development of the mammary glands by implanting progesterone pellets in WT and NERKI+/- females. Although some additional glandular development was observed in NERKI+/- animals, it was much less than that observed in WT animals.
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Treating animals with a super-physiological concentration of gonadotropins (superovulation) can sometimes bypass ovulatory defects (25). This technique was partially successful for inducing ovulation in NERKI+/- animals (data not shown). In WT animals, superovulation increased both the size of the ovary and the number of corpora lutea present. In NERKI+/- animals, superovulation also increased ovarian size, but not all mature follicles formed corpora lutea. Many of the follicles were arrested at the antral stage, and others formed hemorrhagic cysts, resembling those seen in untreated ERKO mice (26). Some follicles were successfully released, however, as demonstrated by the presence of corpora lutea in the ovaries and ova in the oviducts (data not shown).
The ovarian stroma in NERKI+/- animals was increased, and the stromal cells contained cytoplasmic vacuoles (Fig. 4A, NERKI). Staining of frozen sections with Oil Red O demonstrated increased lipid in these vesicles (Fig. 4B
). Similar lipid-filled cells have been observed in mice lacking steroidogenic acute regulatory protein (StAR) (27). Because of this similarity, levels of StAR expression were measured by RT-PCR and Western blot (Fig. 4C
). Levels of both StAR mRNA and protein were decreased in the ovaries of untreated NERKI+/- animals (Fig. 4C
). Quantitation of the results demonstrated that StAR expression was reduced by about 50% in the NERKI+/- ovary compared with WT. In the WT ovary, StAR is expressed at the highest levels in the corpus luteum, followed by the thecal cells (reviewed in Ref. 28), suggesting that the lack of ovulation may account for part of decreased StAR expression. No significant differences were seen in the expression of other genes involved in steroidogenesis, such as P450scc, P450c17, or aromatase (data not shown).
Uteri of NERKI+/- Mice Are Enlarged and Exhibit Cystic Endometrial Hyperplasia
NERKI+/- uteri are significantly elongated and heavier when compared with WT littermates (Fig. 5A, insets). The average wet uterine weight in an 8- to 12-wk-old animal was 104 ± 10 mg in WT compared with 265 ± 20 mg in NERKI+/- mice (P < 0.01). The difference in weight became even more pronounced in older animals (data not shown). Each of the structural compartments of the uterusthe myometrium, stroma, and endometriumare present in both WT and NERKI+/- uteri. However, NERKI+/- animals have grossly enlarged endometrial glands, filled with secretory material (Fig. 5A
), a phenotype that also progressed with age (data not shown).
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PR is one of the uterine estrogen-responsive genes that is regulated by the classical pathway (2). Its expression in intact animals was analyzed by immunohistochemistry and found to be similar in WT and NERKI+/- animals (Fig. 5C). When animals were ovariectomized and estrogen was replaced, PR expression increased comparably in WT and NERKI+/- animals (data not shown). These data indicate that one active ER
allele is sufficient for PR regulation in the uterus.
The Mutant ER Allele Functions in Vivo Similar to in Vitro
The activity of the classical and nonclassical pathways was examined in the uteri of NERKI+/- and WT animals. The classical pathway was assessed by measuring the expression of an ERE-regulated target gene PRL (1). Pituitary PRL levels were measured by RT-PCR using total RNA from females that were ovariectomized and treated either with placebo or estrogen (Fig. 6A). Basal levels of PRL were decreased in NERKI+/- females, but the degree of PRL induction by estrogen was comparable in both groups, indicating that the remaining allele of WT ER
was fully functional.
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DISCUSSION |
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A second possibility might involve an imbalance in the relative activities of the classical and nonclassical pathways. For example, the lack of ERE binding may selectively reduce the expression of genes in the classical pathway. In contrast, genes in the nonclassical pathway may not be altered. To the extent that some estrogen-responsive genes might control cell growth whereas others control differentiation, it is plausible that phenotypic effects might result from selective changes in the composition of expressed genes. This hypothesis can ultimately be tested by performing gene expression array studies that compare heterozygous ERKO and NERKI gene expression in specific tissues.
A third possibility could be that the phenotype in some of the tissues (e.g. ovary) is due to an inhibitory effect of the mutant ER on another signaling pathway. This hypothesis will ultimately be tested by generating a compound heterozygote (ER null/NERKI mice).
The NERKI+/- Phenotype Confirms Activity of the Nonclassical ER Pathway in Vivo
The ovaries of NERKI+/- animals contained cytoplasmic lipid deposits in the stromal cells; this feature is not seen in the ovaries of ERKO mice, suggesting that this phenotype is specific to the introduced mutation. Because of the similarity to the lipid deposits seen in StAR KO mice (27), the levels of StAR were examined and found to be decreased in untreated NERKI+/- ovaries. The StAR promoter is not known to contain an ERE, suggesting that the altered expression level is not the result of defective signaling through the classical pathway. Enhancer elements important for StAR expression include an SF1 binding site and a cAMP response element (31), the latter of which has been shown to participate in cross-talk with the ER (summarized in Ref. 32). Thus, it is possible that the lipid deposits are caused by inhibition through nonclassical ER activity, but this will require further studies of the StAR promoter. Interestingly, despite the decreased StAR levels, the serum levels of estrogen in NERKI+/- are normal, similar to the situation seen in StAR KO females (27).
The importance of ER for uterine growth is consistent with its high expression in the female reproductive tract during development, puberty, and adulthood (reviewed in Ref. 33). In the absence of ER
, the uterus is hypoplastic, resulting in a uterine weight that is only half that of WT littermates (26). The increased size and weight of the uterus in NERKI+/- females is strikingly different from the appearance of the uterus of ER
KO mice, suggesting a specific contribution of the nonclassical activity of ER
to uterine growth and development. Cross-talk between the ER and growth factors in the uterus is well established (34, 35, 36). It is known that estrogen can regulate levels of growth factors and their receptors in the uterus (37, 38, 39), possibly through the nonclassical pathway. For example, the epidermal growth factor promoter does not contain an ERE, but it has Sp1 sites that have been implicated in the regulation of epidermal growth factor expression (40). Given that impaired growth factor signaling causes uterine hypoplasia (41), and growth factor overexpression can increase uterine weight (42), altered growth factor expression could contribute to the increased uterine size in NERKI+/- animals.
Examination of IGF-I expression demonstrated that estrogen induces IGF-I in uteri of NERKI+/- animals to a similar or greater extent than in WT mice. More investigation is needed to establish the effects of the introduced mutation on growth factor expression in NERKI+/- animals. However, these initial data suggest that the mutation preserves the ER activity in the nonclassical pathway in vivo.
Although sporadic cystic endometrial hyperplasia has been observed in WT mice aged 1 yr or older (43), NERKI+/- animals exhibited this phenotype as young adults (812 wk of age). The extent of hyperplasia worsens with age, as observed in the 6-month-old NERKI+/- animals, whereas the WT littermates appear normal (data not shown). Endometrial hyperplasia can also be induced by treatment with certain ER ligands, such as TMX (44), or triarylalkenes similar to TMX (45). These agents act as agonists in the nonclassical pathway (9, 10), consistent with the possibility that activation of nonclassical signaling may contribute to the endometrial hyperplasia seen in NERKI mice. Mice lacking PR also exhibit uterine hyperplasia and inflammation (46). Although the PR levels in the uterus appear to be normal, it is possible that lower levels of progesterone, or defective PR signaling, may contribute to endometrial phenotype in NERKI+/- females.
ER is necessary for the uterine response to E2 or TMX after ovariectomy, as demonstrated by diminished hormonal responses in the ER
KO mouse (47, 48). On the other hand, a single functional ER
allele is sufficient for these responses (20). Similarly, the uteri of NERKI+/- mice are estrogen responsive, indicating that the mutation does not abolish the signaling pathways necessary for this response. This result provides additional evidence that the mutant does not exert a dominant-negative effect on estrogen signaling. Instead, it is more consistent with the provocative possibility that the phenotype results from an altered balance between the classical and the nonclassical pathways. For example, the WT allele may provide the necessary background for uterine development, as demonstrated by the active estrogen response. Subsequently, the nonclassical pathway can be fully engaged or even exaggerated due to reduced expression of genes in the classical pathway.
ER Loss-of-Function May Contribute to the NERKI+/- Phenotype
Analysis of the ovarian phenotype reveals similarities between NERKI+/- and homozygous ERKO females. Females of both models are anovulatory. However, ER
KO ovaries contain hemorrhagic cysts as a result of elevated gonadotropins (49). The cysts in NERKI+/- ovaries were not hemorrhagic, unless animals were treated with superphysiological levels of gonadotropins. This finding is consistent with the role of gonadotropins in the establishment of the hemorrhagic ovarian phenotype in both models.
Treatment with gonadotropins induced ovulation in NERKI+/- females. However, none of the superovulated NERKI+/- animals became pregnant when mated to fertile males (data not shown). It is likely that the endometrial hyperplasia, which has been implicated in infertility in several species (50, 51, 52), contributes to infertility in the NERKI+/- model, independent of the ovarian phenotype.
Mouse mammary glands develop through two major stages: ductal elongation, occurring during puberty, and postpubertal side-branching and alveolar development (53). Mammary glands of ERKO mice are growth inhibited at the prepubertal stage (54). Interestingly, mammary glands of NERKI+/- animals undergo ductal elongation normally, but subsequent branching and lobuloalveolar development are arrested. As a similar phenotype was seen in animal models that had defective progesterone or PRL signaling (55, 56, 57, 58), it is possible that there is a related defect in NERKI+/- animals. The regulation of both progesterone and PRL signaling pathways is largely dependent on classical ER
activity, and an ERE has been identified in both the PR and PRL promoters (1, 2). However, PR levels are comparable in the mammary glands of WT and NERKI+/- females, and treatment of NERKI+/- females with progesterone did not induce mammary gland development. It is still possible that the mammary phenotype in NERKI+/- animals is caused by decreased activity of the classical pathway (59), but the phenotype is not a direct effect of decreased levels of progesterone.
The NERKI Model Provides an Opportunity to Examine the Molecular Basis of ER Signaling in Vivo
The phenotype of NERKI+/- females illustrates the complexity of estrogen signaling in whole-animal physiology. It is notable that the effect of the NERKI mutant is distinctly different in various estrogen target tissues, i.e. it causes hyperplasia in the uterus and hypoplasia in the mammary gland. This apparent discrepancy resembles the effects of the selective estrogen receptor modulators, which are used for clinical therapy. For example, TMX acts as an antagonist in the breast and results in decreased growth, but it acts as an agonist in the uterus, where it induces hyperplasia (reviewed in Ref. 60). Such differences could arise from varied contributions of the classical vs. the nonclassical pathways in each estrogen target tissue. Understanding the molecular mechanisms by which the ER mediates its effects opens new possibilities for tissue-specific therapies. It will be especially interesting to study the NERKI mutation in the homozygous state. Efforts are currently underway to create homozygous NERKI animals using in vitro fertilization techniques and foster mothers to bypass the ovulatory and uterine defects.
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MATERIALS AND METHODS |
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Southern Blots
A 444-bp probe from the 5'-region outside of the targeting construct was prepared by PCR using a cloned genomic fragment of mER as a template. The same probe was used for screening ES cells and for screening of the progeny of chimeric mice. Total genomic DNA purified from tail fragments was restricted with EcoRV, separated on an 0.8% agarose gel at 20 V in 0.5x Tris-borate-EDTA and DNA and transferred onto a Nytran membrane using the Turbo Blotter rapid downward transfer system (Schleicher \|[amp ]\| Schuell, Inc., Dassel, Germany) according to the manufacturers protocol. DNA was attached to the membrane by cross-linking. The membrane was blocked in hybridization buffer [1 M NaCl, 50 mM Tris-HCl (pH 7.5), 40% formamide, 10% dextran sulfate, 1% sodium dodecyl sulfate (SDS)] in the presence of 200 µg/ml denatured herring testes DNA at 42 C overnight. The denatured probe was added at 106 cpm/ml hybridization buffer and incubated overnight at 42 C. After incubation with the probe, the blots were washed at room temperature twice with 2x standard sodium citrate, 0.1% SDS prewarmed to 37 C, followed by one wash with 2x standard sodium citrate, 0.1% SDS prewarmed to approximately 65 C. Blots were then exposed to film.
RT-PCR
RNA was purified from tissues using TRIzol (Life Technologies, Inc.) according to the manufacturers protocol. RNA was subsequently treated with DNase I (Promega Corp., Madison, WI) for 15 min at 37 C followed by a 15-min incubation at 75 C. cDNA was prepared using 30 U of reverse transcriptase (Promega Corp.) per reaction in the presence of reverse transcriptase buffer (Promega Corp.), 0.8 mM deoxynucleotide triphosphates, 80 U of RNasin (Promega Corp.) and 1 µg random hexamers per reaction. The reaction mixture was incubated at 25 C for 10 min, followed by 42 C for 30 min and 99 C for 10 min. The primers used for ER PCR were sense: 5'-AGAACGAGCCCAGCGCCTACG and antisense: 5'-GACGCTTGTGCTTCAACATTC. The PCR reaction was performed according to the previous protocol for 3033 cycles. The PCR product was approximately 400 bp in size. The PCR product was purified using QIAGEN (Chatsworth, CA) PCR Purification kit and restricted using SphI restriction enzyme (Promega Corp.), generating two bands of approximately 200 bp. PCR primers used for semiquantitive PCR reactions were: tubulin sense, 5'-CCGGTGTCTGCTTCTATC; antisense, 5'-GAGCCGCTCCATCAGCAG; GAPDH sense, 5'-CCCTTCATTGAGACCTCAACCTA; antisense, 5'-CCAAAGTTGTCATGGATGAC; ß-actin sense, 5'-GAAGCTGTGCTATGTTGCTC; antisense, 5'-CCAGAGCAGTAATCTCCTTC; PRL sense, 5'-CTGCCAATCTGTTCCGCTGGTGAC; antisense, 5'-CCTACTGCAGTTATTAGTTGAAAC (63); IGF-I sense, 5'-CACATCTCTTCTACCTGGCG; antisense, 5'-TGAGTCTTGGGGCATGTCAGT. 32P-
-dCTP was added to the reaction, products were separated on a 6% polyacrylamide gel, and results were quantitated using a phosphor imager.
Animal Housing
Animals were housed in a barrier facility with a 14-h light,10-h dark cycle. Unless stated for the purpose of the experiments, animals of the same sex were housed five per cage. For continuous matings, one male and one female were placed in a cage. Superovulation was accomplish by ip injection of pregnant mares serum gonadotropin (10 IU/mouse; Sigma, St. Louis, MO) at the end of the light phase followed by ip injection of 5 IU/mouse of human chorionic gonadotropin (hCG; Sigma) 48 h later (25).
Animal Procedures
All procedures were performed according to the Master Protocol approved by the Northwestern University Animal Care and Use Committee. For tissue collection, animals were anesthetized by injection with nembutal, sodium solution (Abbott Laboratories, Abbott Park, IL; 200 µl/mouse), and blood was collected by retroorbital bleed. All results presented in this publication were obtained using females between 8 and 12 wk of age, except animals used for measuring the expression of IGF-I. For this experiment, 4-wk-old sexually immature females were used. Three animals were used for each group. At time 0, animals from the treatment group were injected with 5 µg 17ß-E2 (Sigma) per mouse. Animals were euthanized after 6 h. Ovariectomy was performed under anesthesia induced by an ip injection of 50 µl per mouse of cocktail containing 25 mg/ml ketamine (80 mg/kg; Ketaset; American Home Product Corp., Madison, NJ), 1.27 mg/ml xylazine (10 mg/kg; Rompun; Bayer Corp., Pittsburgh, PA), and 0.25 mg/ml acetopromazine (American Home Product Corp.). For ovariectomy, eight animals of each genotype were used. Animals were allowed to recover for 2 wk before implantation of pellets above the scapulae. For the implantation, animals were anesthetized using Metofane (Schering-Plough Corp., Union, NJ). Pellets contained 0.25 mg/pellet 17ß-E2 (21-d release), 0.5 mg/pellet TMX, free base (21-d release), or placebo (all pellets from Innovative Research of America, Sarasota, FL). There were four animals of each genotype in the placebo group, and two animals of each genotype in the E2 and TMX groups. Animals were euthanized 3 d after pellet implantation.
Serum Hormone Measurements
Serum E2 RIA was performed using a double antibody RIA kit according to manufacturers protocol (ICN Biomedicals, Inc., Costa Mesa, CA). LH and FSH RIAs were performed using antibodies and reference preparations from the National Hormone Pituitary Program (provided by Dr. A. F. Parlow). Serum progesterone was measured using an immunochemical coated tube kit (ICN Pharmaceuticals, Inc., Costa Mesa, CA). E2 levels were measured in a group of 10 NERKI+/- and 10 WT mice. Progesterone was measured in groups of 11 mice. LH and FSH were measured in groups of 19 NERKI+/- and WT mice.
Histology
Excised tissues were fixed in 4% paraformaldehyde at 4 C overnight and subsequently stored in 70% ethanol at 4 C. Tissues were embedded in paraffin at the Pathology Core Facility (Northwestern University, Chicago, IL) and sectioned at thickness 35 µm. Standard hematoxylin-eosin (H&E) stain was performed and cover slips mounted using ACCU.MOUNT 60 (Baxter Scientific Products, Riverdale, NJ). For PR immunohistochemistry, primary antibody sc-538 was used (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Frozen sections were stained with Oil red O according to Ref. 64 and counterstained with hematoxylin (Vector Laboratories, Inc., Burlingame, CA). Uteri and ovaries were collected from 30 animals from each group (NERKI+/- and WT). Whenever possible, comparisons were made between littermates.
Whole Mounts of Mammary Glands
Whole mounts of mammary glands were prepared as described previously (59). Glands from 15 animals from each group (NERKI+/- and WT) were collected. Glands were excised and fixed overnight on a slide at room temperature in 25% glacial acetic acid, 75% ethanol. The next morning, slides were washed with 70% ethanol for 15 min, followed by a 5-min wash in water. Slides were incubated in carmine alum solution overnight at room temperature. The following morning the tissue was rinsed with water and washed with 70% ethanol for 15 min, 95% ethanol for 15 min, 100% ethanol for 15 min, and xylene for 15 min. Coverslips were mounted using ACCU.MOUNT 60 (Baxter Scientific Products).
Cell Culture
TSA cells were cultured and transfected as described previously (22).
Western Blots
Whole protein extracts were prepared by sonicating tissues in water containing protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN), 1 mM EDTA, and 1 mM EGTA. The extracts were fractionated using 10% SDS-PAGE gels and transferred onto Hybond-P transfer membranes (Amersham Pharmacia Biotech, Piscataway, NJ). Antibody D-12 was used to detect ER (Santa Cruz Biotechnology, Inc.). StAR antibody was a gift from Dr. J. F. Strauss III (University of Pennsylvania Medical Center, Philadelphia, PA). Proteins were visualized using an ECL+Plus kit (Amersham Pharmacia Biotech) according to the manufacturers protocols.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: AP1, Activator protein 1; DBD, DNA binding domain; E2, Estradiol; ER, estrogen receptor; ERE, estrogen response element; ES, embryonic stem; GR, glucocorticoid receptor; H&E, hematoxylin-eosin; KO, knockout; mER, mouse ER
; NERKI, nonclassical ER knock-in; PR, progesterone receptor; PRL, prolactin; SDS, sodium dodecyl sulfate; StAR, steroidogenic acute regulatory protein; TMX, tamoxifen; WT, wild-type.
Received for publication August 8, 2001. Accepted for publication July 11, 2002.
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REFERENCES |
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