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Am J Physiol Endocrinol Metab 293: E147-E158, 2007. First published March 20, 2007; doi:10.1152/ajpendo.00101.2007
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Ciliated epithelial-specific and regional-specific expression and regulation of the estrogen receptor-beta2 in the fallopian tubes of immature rats: a possible mechanism for estrogen-mediated transport process in vivo

Ruijin Shao,1 Birgitta Weijdegård,1,2 Julia Fernandez-Rodriguez,3 Emil Egecioglu,1 Changlian Zhu,4 Niklas Andersson,5 Ann Thurin-Kjellberg,2 Christina Bergh,2 and Håkan Billig1

1Department of Physiology/Endocrinology, Institute of Neuroscience and Physiology; 2Reproductive Medicine, Department of Obstetrics and Gynecology, Institute of Clinical Sciences, Sahlgrenska University Hospital; 3Centre for Cellular Imaging, Core facilities; 4Perinatal Center, Institute of Neuroscience and Physiology; and 5Division of Endocrinology, Department of Internal Medicine, The Sahlgrenska Academy at Gothenburg University, Gothenburg, Sweden

Submitted 14 February 2007 ; accepted in final form 14 March 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Several ERbeta isoforms have been identified in human and rodent tissues, but it is unclear whether each isoform has distinctly different cellular targeting characteristics and physiological functions. We have investigated the intracellular localization and regulatory patterns for ERbeta isoforms in rat fallopian tubes. Western blot analysis reveals that two ERbeta isoforms corresponding to ERbeta1 and ERbeta2 are expressed in rat fallopian tubes. However, ERbeta2 is the predominant form of ERbeta in this tissue. High-resolution confocal imaging and immunohistochemical analysis provide ample evidence that ERbeta expression is limited almost exclusively to the ciliated epithelial cells, in contrast to ER{alpha}, which is widely distributed. Furthermore, within the ciliated epithelial cells, ERbeta is colocalized with beta-tubulin IV at stem portion of the cilia. We show that ERbeta2 protein expression is tightly regulated by E2 or DPN in a time-dependent manner without changes in ERbeta1 expression. These estrogenic effects are inhibited by an ER antagonist, ICI 182,780. In addition, significant alteration of ERbeta immunoreactivity is detected only histologically in the ampullary region. Since the cilia are considered an essential determinant of tubal transport, we further demonstrate that E2- or DPN-induced ERbeta2 activation is associated with alterations in tubal protein expression crucial for the regulation of calcium-dependent ciliary beating. Given the coordinated regulation and interaction of ER and progesterone receptor in the cilia, we hypothesize that tubal ERbeta2 may facilitate the estrogen-mediated transport process by processing protein-protein interaction under physiological and/or pathological conditions. We show for the first time that a previously unrecognized localization of ERbeta isoform in rat fallopian tubes can combine with estrogen to individually control the expression of ER beta-isoforms in normal target tissues.

estrogen receptor-beta isoform; ciliated epithelial cells; ampulla

THE MAMMALIAN FALLOPIAN TUBE (oviduct) is one of the venues of reproductive events leading to the establishment and maintenance of pregnancy (38). In most animal species, including the rat, the fallopian tube is generally divided into four major regions: the infundibulum (fimbriae), the ampulla, the isthmus, and the uterotubal junction (5), which appear to be specialized to perform different functions (1, 26, 31). It is recognized that all regions are composed of heterogeneous cell types: ciliated and secretory epithelial cells as well as smooth muscle cells (31). These cells can be distinguished by the expression of cell type-specific markers (39, 47, 50, 68). In mammals it is likely that transport of the gametes, fertilization, initial embryonic development, and transport of the embryo to the uterus for implantation, a biological process, depend on the fallopian tube function (11, 20). Although the endogenous estrogens [principally 17beta-estradiol, (E2)] critical for the tubal epithelial cells undergoing morphological and biological changes in the reproductive cycle (26) and for normal tubal function (20) are described, the molecular mechanisms and cellular participants in the events that initially lead to E2-mediated tubal transport process remain largely unknown. Furthermore, fallopian tube malfunction may result in tubal ectopic pregnancy (when the embryo implants in the fallopian tube rather than in the uterus), which still remains a potentially life-threatening condition, without reliable diagnostics and prevention in women (38, 70). Of particular interest is the association between low levels of estrogen and the increased incidence of tubal ectopic pregnancy (4, 46), and there are indications that modulation of ovarian steroid levels may have clinical benefits in those patients (18). Therefore, elucidating the mechanisms of estrogens action involved in regulation of tubal functions in vivo is crucial to fully understanding this complex process.

It is well known that the key to the biological effects of estrogen is the activation of the estrogen receptor (ER), which is a ligand-dependent transcription factor belonging to the nuclear hormone receptor superfamily (72). At the cellular level, ligand-bound ERs homodimerize or tether to other nuclear transcription factors such as activator protein (AP) complex-1 (Jun/Fos) leading to gene transactivation, ultimately changing the phenotype of the target cell (21). There are two structurally related ER subtypes, ER{alpha} and ERbeta, derived from genes located on different chromosomes (chromosome 6 and chromosome 14, respectively) that appear to have overlapping but different tissue/cell expression and localization as well as ligand specificity (10, 21, 29, 69). Although recent studies have led to the identification of ER{gamma} specifically expressed in fish ovary and testis (23), the natural existence and possible functions of this subtype in mammals is undefined. Previously, sequence analysis has demonstrated a high degree of homology between ER{alpha} and ERbeta in the DNA-binding domain and the ligand-binding domain (21). Although hormone-activated ER{alpha} and ERbeta recognize the same estrogen-responsive element (ERE) in the promoter region of target genes, both receptors can display diverse transactivational properties in a ligand-dependent manner when they are coexpressed (54), and ERbeta also has the capacity to regulate ER{alpha} function in estrogen target cells (22, 57) by altered expression of c-Jun and c-Fos, which can form functional heterodimers for ER-mediated transcription (36). These observations strongly implicate that differential expression ER subtypes are crucial for the induction of tissue/cell-specific responses to various ligands. The importance of ER in female reproduction has been demonstrated using gene knockout models. Not surprisingly, ER subtype-specific knockout mice (15, 28, 30) or mice lacking both ER subtypes (9) show abnormal ovarian and uterine phenotypes. Additional evidence that the fallopian tube appears normal during development but lacks responsiveness to estrogen and is hypoplastic is found in the female ER{alpha}/beta knockout mice (9). However, one limitation to elucidating the physiological significance of specific ER subtype-mediated effects in the fallopian tubes has been the lack of availability of these knockout mice.

The relationship between hormone receptor-expressing cells and tubal function is a fundamental issue in fallopian tube biology, and earlier studies (43, 44, 60, 61) have demonstrated that specific binding sites for E2 can be observed using the ligand-binding assay in human fallopian tubes. This E2-binding activity is probably due to both ER subtypes. Furthermore, it has been demonstrated (2, 59, 62) that ERs display cyclic changes in human fallopian tubes during the menstrual cycle. Later studies have shown that ER mRNAs and proteins can be detected by RT-PCR (49, 50), in situ hybridization (41, 42, 56, 75), and immunohistochemical analyses (34, 49, 50, 56, 64, 75) in rat fallopian tubes and in particular that ER{alpha} expression is also regulated during development and the estrous cycle (41, 50, 75). All of these lines of observations suggest that intracellular ER levels are controlled by estrogen to favor the proper cellular functions in the fallopian tubes. However, the exact nature of expression and cellular localization of ERbeta in the fallopian tube remains controversial. In rat fallopian tubes, several studies (56, 75) have detected ER{alpha} mRNA and protein in the epithelial and muscle cells where ERbeta was absent. Others have reported ERbeta mRNA and protein in the epithelial cells (64) of the infundibulum and ampulla regions (50) and in the muscle cells (41, 42, 75). In contrast to ER{alpha} in the fallopian tubes, the cell-specific and regional-specific ERbeta localization in the fallopian tubes has not been clearly elucidated in most cases. Furthermore, several ERbeta isoforms have previously been identified in human and rodent tissues (13, 29, 40). However, the cellular distribution of ERbeta isoforms in the fallopian tubes has not been previously reported, and information on the steroid hormones controlling the ERbeta isoform expression in the fallopian tubes is lacking.

Therefore, the specific aims of this study were 1) to determine whether the different ERbeta protein isoforms are expressed in rat fallopian tubes with the use of a previously characterized antibody to ERbeta, 2) to investigate whether ERbeta expression is localized to specific regions and cell populations, and 3) to demonstrate how steroid hormones regulate this expression and localization. Furthermore, ciliary motility plays an essential role in fertilization (26), and intracellular calcium (Ca2+)-dependent ciliary beat frequency (CBF) (63) of ciliated epithelial cells has been suggested to play an important role in tubal transport in human fallopian tubes (31). The idea that estrogen-regulated ERbeta expression may provide this function was supported by analysis of Ca2+-binding proteins in rat fallopian tubes. In addition, the functional ER-mediated effect was evidenced by demonstration of progesterone (P4) receptor (PR), an estrogen-regulated protein, in the fallopian tubes by using the same experimental setting. Our results provided direct evidence of ERbeta protein expression in distinct cell populations and new sites for estrogen action in rat fallopian tubes.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Antibodies

The primary antibodies used for immunohistochemistry and Western blot analysis in the present study, their dilution, and sources are listed in Table 1. The antibody against ER{alpha} was a mouse monoclonal IgG raised against the full length of human ER{alpha} (50, 56). The antibody against ERbeta was a rabbit polyclonal IgG raised against a synthetic peptide consisting of amino acids 54–71 of rodent ERbeta. It recognizes both long form and splice variants of ERbeta but does not cross-react with ER{alpha} (27, 49). The specificity of these antibodies against the classical nuclear ER{alpha} and ERbeta have been validated by us in the uterine tissues from wild-type, ER{alpha}/beta, ER{alpha}, and ERbeta knockout mice by Western blotting analysis prior to use in this study (data not shown) and by others in previous studies (15, 55). A biotin-conjugated anti-rabbit antibody (711-066-152) streptavidin conjugated with fluorescein (DTAF; 016-010-084) and Cy3-conjugated anti-mouse antibody (715-166-150) was obtained from Jackson ImmunoResearch Laboratories (West Grove, PA) and used for fluorescence immunohistochemistry. Normal mouse IgG and rabbit IgG used as negative controls for immunohistochemistry were purchased from Santa Cruz Biotechnologies. Alkaline phosphatase conjugated goat anti-mouse IgG (A-1682; Sigma) and alkaline phosphatase-conjugated goat anti-rabbit IgG (AC31RL; Tropix, Bedford, MA) were used as secondary antibodies in the Western blot analysis.


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  		Table 1. Antibodies: species, clone/catalog number, method, dilution, and source

 
Animals

All experimental procedures and protocols used in the present study were approved by the local ethics committee, Gothenburg University, Gothenburg, Sweden. Prepuberal female Sprague-Dawley rats were obtained from B&K Universal (Sollentuna, Sweden) and maintained under defined conditions within a temperature-controlled (21 ± 2°C) and light-controlled (12:12-h light-dark cycles) room. They had access to standard pellet food and tap water ad libitum. All animals were allowed to adapt to their new environment for 5 days before studies were initiated.

Hormonal Treatment and Tissue Preparation

Experiment 1. To ascertain the expression of ER subtypes in female rat reproductive tissues, ovary, fallopian tube, and uterus from 21-day-old female rats (n = 3, 60 ± 5 g body wt) were collected and freed from fat and connective tissue immediately after being killed. Tissues were frozen in liquid nitrogen for Western blot analysis.

Experiment 2. It has been previously reported that the concentration of E2 is consistently low throughout neonatal development and starts to increase after day 28 of age in rats (45). Therefore, to examine whether acute exogenous estrogen regulated ERbeta expression in the fallopian tube, 21-day-old female rats, chosen at random, were given a single intraperitoneal injection of 50 ng/g body wt E2 (Sigma) in 200 µl of vehicle (sesame oil) or vehicle alone. The selected dose of E2 and the treatment regime has previously been shown to be effective in rat uterus in vivo (58).

Experiment 3. To further examine whether regulation of ERbeta expression by exogenous estrogen is mediated via ER{alpha} expression, 21-day-old female rats were given a single intraperitoneal injection of a selective ERbeta ligand (37), diarylpropionitrile [DPN; 2,3-bis(4-hydroxyphenyl) propionitrile; Tocris Cookson, Bristol, UK], 100 µg/g body wt, in 200 µl of vehicle (sesame oil) or vehicle alone. We evaluated data from previously published article (17) to establish an effective concentration for DPN in vivo.

Experiment 4. To determine the effect of steroidal ER antagonist ICI 182,780 on the regulation of ERbeta expression, 21-day-old female rats received either E2 (50 ng/g body wt ip) or DPN (100 µg/g body wt ip) and additionally either 8.3 mg/kg body wt of ICI 182,780 (Tocris Cookson) in 200 µl of vehicle (sesame oil) or vehicle alone. ICI 182,780 (faslodex, fulvestrant) has been used to ablate estrogen and ER signaling in several mammalian species (25).

Experiment 5. To examine the effect of progesterone (P4) on the expression of tubal ER subtypes, 21-day-old female rats received a single intraperitoneal injection of P4 (2.5 mg in 200 µl sesame oil/each animal) to achieve an effective dose as previously described (66).

At 0.5, 1, 3, 6, 12, and 24 h after injection in experiments 2–5, rat fallopian tubes (n = 3/time point in each experiment) were quickly collected, cleaned, and weighed. One fallopian tube in each animal was immediately frozen in liquid nitrogen and stored at –70°C for subsequent Western blot analysis. The other was fixed in 4% formaldehyde neutral buffered solution for 24 h at 4°C and embedded in paraffin for histochemical analysis. Protein was harvested from a matched portion of the organ in all instances to assure that results did not differ because of organ composition.

Protein Extraction and Western Blot Analysis

Whole tissue extracts for protein preparations were essentially carried out as described previously (66). Ten millimolars iodoacetamide were included in each buffer used for protein preparations to prevent nonspecific disulfide linkage. One micromolar sodium orthovanadate as a phosphatase inhibitor was also added in the buffer for protein preparation. The protein content was determined using the BCA protein assay (Pierce, Rockford, IL). Immunoblot analyses were performed using standard procedures to evaluate the abundance and distribution of ER{alpha}, ERbeta, beta-tubulin IV, pan-cytokeratin, {alpha}-smooth muscle actin, beta-actin, c-Jun, c-Fos, PR, calbindin-D9K, and calbindin-D28K (65, 66). Thirty micrograms of protein were directly electrophoresed on 4–12% sodium dodecyl sulfate (SDS)-polyacrylamide gels (Novex, San Diego, CA) using the MOPS-SDS running system. Blots generated with these extracts were probed with primary antibodies. The blots were also probed with an antibody to pan-cytokeratin to examine epithelial content (39) or {alpha}-smooth muscle actin to determine smooth muscle content (68). The immunosignal-CDP-Star substrate for alkaline phosphatase system (Tropix) was used to visualize protein bands. To reprobe the blot with another antibody, the blot was rehydrated in methanol, rinsed, and incubated with stripping buffer (65 mM Tris·HCl, 2% SDS, and 100 mM beta-mercaptoethanol, pH 6.8) at 50°C for 30 min. Immunoblotted signals were visualized using a LAS 1000-cooled charge-coupled device camera (Fujifilm) and ECL film (Amersham). Individual bands were quantified directly from membranes by densitometry using the Image Gauge software (Fujifilm). Signal intensities of the rat ER proteins were normalized to the gels stained with Coomassie blue as ratios to produce arbitrary densitometric units (ADU) of relative abundance. To standardize the assay for measurement of ER proteins, we examined different starting protein concentrations for each sample. This study demonstrated the linearity and validity of ADU for all immunoreactive bands in Western blot analysis (65, 66). All steps were carried out at room temperature unless otherwise stated.

Immunohistochemical Analyses and Microscope

Two different methods of immunohistochemistry were performed and confirmed in the same rat fallopian tube tissue. 3,3-Diaminobenzidine tetrahydrochloride (DAB) (65, 66) and dual-fluorescence (67) immunohistochemistry were based on the previously described methodology. After deparaffinization and rehydration, antigen retrieval was completed with 10 mM sodium citrate buffer (pH 6.0, 10 min in a 700-watt microwave). The endogenous peroxidase and nonspecific binding were removed by incubation with 3% H2O2 for 10 min and 10% normal goat serum for 1 h at room temperature. After incubation with primary antibody overnight at 4°C, sections were stained using the avidin-biotinylated-peroxidase complex detection system (ABC kit; Vector Laboratories, Burlingame, CA) according to the manufacturer's instructions, followed by a 2-min treatment with DAB. Sections were imaged on a Nikon E-1000 microscope under bright-field optics and photomicrographed using Easy Image 1 (Bergström Instruments).

For dual-fluorescence immunohistochemistry, tubal sections were blocked in PBS containing 1% BSA-3% fat-free milk for 1 h at room temperature. Slides were incubated with the primary antibody in PBS supplemented with Triton X-100 (PBST) containing 1% BSA-3% fat-free milk overnight at 4°C. After being washed in PBST for 3–5 min, sections were incubated with secondary antibody at room temperature for 1 h. To detect the bound primary antibodies, the secondary antibody, either a biotin-conjugated anti-rabbit antibody together with streptavidin conjugated to DTAF or Cy3-conjugated anti-mouse antibody was employed for different ER subtype staining. Sections were washed and mounted with fluorescent Vectashield with 4,6 diamidino-2-phenylindole (DAPI). Slides were viewed on an Axiovert 200 confocal microscope (Carl Zeiss, Jena, Germany) equipped with a laser-scanning confocal imaging LSM 510 META system (Carl Zeiss) and photomicrographed. Background settings were adjusted from examination of negative control specimens. Images of positive staining were adjusted to make optimal use of the dynamic range of detection. Figures were composed in Adobe Photoshop with minimal alteration for presentation and layout. All final immunohistochemistry was carried out in parallel under identical conditions. To control for nonspecific staining, adjacent sections were stained as above, except the primary antibody was replaced with either PBST, normal mouse IgG, or rabbit IgG 1) in place of both primary antibodies to control for nonspecific staining and to obtain the background level of fluorescence and 2) to replace the second sequence primary antibody to ensure fsno cross-reactivity between the two staining sequences. Rat ovarian and uterine tissues served as a positive control and were stained as above for ER subtypes. The immunohistochemical findings illustrated are representative of those observed in randomly selected sections from multiple animals. The resulting stain was evaluated by two blind observers.

Statistical Analysis

Data are means ± SE of the number of independent experiments indicated in the figure legends. Multiple comparisons between data were performed using one-way ANOVA with correction of P values using the Bonferroni's multiple-range tests under the Analyze-It program (Analyze-It Software). Significance was accepted at the 0.05 level of probability.


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Expression of ERbeta Isoforms in the Reproductive Tissues in Prepuberal Rats

Known ERbeta protein isoforms result from alternative splicing of the ERbeta gene, and four ERbeta isoforms exist, including the ~59-, 56-, 54-, and 52-kDa ERbeta1, ERbeta2, ERbeta4, and ERbeta5 (29). To validate the molecular identity of ER subtypes, we performed Western blot analysis with protein extracts from rat ovary, fallopian tube, and uterus. All extracts exhibited a protein doublet of 51–64 kDa, which was specific for ERbeta (Fig. 1A), although the long-form band was substantially larger than the predicted size of ERbeta1 (~59 kDa). The antibody to ERbeta revealed that ERbeta1, considered to be the long form, is the most prominent in the ovary (10, 69). However, the short form of ERbeta appeared to be more pronounced in the fallopian tube and uterus than in the ovary (Fig. 1, A and B). In support of this notion, a mouse monoclonal anti-ERbeta antibody (EMR02; Novocastra Laboratories, Newcastle, UK) that did not show cross-reactivity to the other isoforms of ERbeta was used to confirm that rat fallopian tubes contained a minor amount of ERbeta1 (data not shown), in agreement with a previous study (10). When the membrane proteins from the fallopian tubes were run in the same gel and the blot was cut and probed with either the anti-ERbeta, the anti-ER{alpha}, or the anti-beta-tubulin IV antibody, the immunoreactive band for the ERbeta short form migrated slightly slower than beta-tubulin IV (55 kDa; Fig. 1C), suggesting that this short form is in close agreement with the molecular mass of ERbeta2 (56 kDa) (29). Furthermore, the average relative expression of ERbeta1 to ERbeta2 proteins in rat ovary (Fig. 1B) was similar to that of their mRNAs in human ovary, as previously reported (29), supporting the concept that the short form of ERbeta detected in the present study is ERbeta2. Although it remains possible that one additional smaller band in all tissues examined represents proteolytic products generated from the ERbeta bands, we do not favor this interpretation, since this smaller band was detected in the uterine tissues isolated from ER{alpha}/beta and ERbeta knockout mice (data not shown). In addition, the expression patterns of ER{alpha} and cell type-specific marker proteins are shown for comparison (Fig. 1A). Western blot analysis demonstrated the ER{alpha} was most abundant in the fallopian tube and uterus (Fig. 1, A and B). It is important to note that the fallopian tubes expressed both beta-tubulin IV [a specific marker for the ciliated epithelial cells in the neonatal and adult fallopian tubes (48, 50)] and pan-cytokeractin, which is widely used for identification of epithelial cells (39). The expression of {alpha}-smooth muscle actin protein (68) was included as a positive control for tubal and uterine protein content. Antibody to the ubiquitously expressed beta-actin was used to confirm equal loading. In conjunction with the levels of cell type-specific marker proteins detected by Western blot analyses, it can be concluded that ERbeta2 protein is dominant in rat fallopian tubes.


Figure 1
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  		Fig. 1. Expression of estrogen receptor (ER) subtypes and cell type-specific marker proteins in rat reproductive tissues. A: Western blot analysis of ovary, fallopian tube, and uterus from 21-day-old rats. Protein extracts from total lysates (30 µg/lane) were analyzed using antibodies to ERbeta, ER{alpha}, beta-tubulin IV, pan-cytokeratin, {alpha}-smooth muscle ({alpha}-SM) actin, and beta-actin as described in MATERIALS AND METHODS. The molecular weight of the protein is indicated. ERbeta1 and ERbeta2 (expected size ~59 and 56 kDa) were identified. B: quantification of ER subtype protein expression. The correct loading was evaluated by staining the gels with Coomassie blue. Relative levels of ER subtype proteins were expressed as a ratio of ER densitometric value to whole proteins in Coomassie blue staining. Graphed results are means ± SE of 3 rats/group. ***P < 0.001. C: representative Western blots of tubal protein for ER subtypes and beta-tubulin IV. Blot was probed with specific antibody against either ERbeta, ER{alpha}, or beta-tubulin IV as described in MATERIALS AND METHODS, demonstrating specific of the antibody for the ERbeta isoforms of appropriate molecular weight in rat fallopian tubes. The apparent molecular weights are shown along the ordinate for 2 standard proteins (ER{alpha} and beta-tubulin IV).

 
Region-Specific Localization of ERbeta in Rat Fallopian Tubes

Given that both ER{alpha} and ERbeta are expressed in the fallopian tubes, we next examined the precise cellular distribution of ER subtypes in each cell compartment of rat fallopian tubes by double-immunofluorescence labeling using a combination of ER{alpha} and ERbeta antibodies. In all regions examined, the cellular localization of ER{alpha} and ERbeta was distinguishable. Immunoreactivity of ER{alpha} was detected in ciliated and secretory epithelial cells as well as smooth muscle cells (Fig. 2, A–C, left, red), which is consistent with the previous reports on the predominance of ER{alpha} in rat fallopian tubes (34, 48–50, 56, 75). However, the positive staining of ERbeta was located mainly in the epithelium, placing it close to gametes and or early embryos found in the tubal lumen (Fig. 2, A–C, green). Furthermore, there was a progressive decrease in the proportion of ERbeta-positive cells from the infundibulum to the isthmus regions (Fig. 2, A–C), in agreement with the existence of epithelial cell number gradient in the fallopian tubes (1, 26, 31).


Figure 2
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  		Fig. 2. Localization of endogenous ER subtype proteins in the rat fallopian tubes. Tubal sections were immunolabeled for ER{alpha} and ERbeta visualized with the appropriate wavelength for ER{alpha} (red), ERbeta (green), and 4,6 diamidino-2-phenylindole (DAPI; blue) as described in MATERIALS AND METHODS. Antibodies used in Western blot analysis were used for immunohistochemical localization. Confocal microscopy revealed that the pattern of dual-fluorescent labeling of the 2 fluorochromes was almost the difference in the infundibulum (A), ampulla (B), and isthmus (C) regions. Whereas ER{alpha} was ubiquitously expressed in all cell types without regional difference in the fallopian tubes, ERbeta was detected mainly in the tubal epithelium, with the most intense signals in the infundibulum region (A), and detected less in the isthmus region (C). Sections were subsequently counterstained with DAPI to visualize cell nuclei (A–C, right). Using tubal tissues from different rats, the same results were obtained for replicate experiments. Scale bars are shown. epi, Epithelial cells; m, smooth muscle cells; lu, lumen.

 
Cell Type-Specific Localization of ERbeta in Rat Fallopian Tubes

To clearly visualize the exact localization of ERbeta in the epithelial cells, we subsequently examined the expression of ERbeta in the tubal epithelium using DAB immunostaining. The ciliated epithelial cells were intensely labeled by ERbeta antibody (Fig. 3, A and B), which is consistent with Fig. 2, whereas secretory epithelial cells were negative for ERbeta (Fig. 3B). Interestingly, intense ERbeta immunoreactivity was localized in the stem portion of cilia in the epithelial cells. There was no ERbeta immunoreactivity in the cytoplasm and nuclear bodies of ciliated epithelial cells (Fig. 3B). Preincubation of the antibody with the respective immunogen (data not shown) or the control rabbit IgG (Fig. 3C) resulted in no staining, whereas intensive nuclear ERbeta immunostaining was detected in rat ovarian granulosa cells when the same antibody was applied (Fig. 3, D and E), thus demonstrating the specificity of the DAB staining in the fallopian tube.


Figure 3
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  		Fig. 3. Identification of ERbeta localization in the ciliated epithelical cells. Tubal sections were immunolabeled for ERbeta visualized with diaminobenzidine (DAB; brown) staining as described in MATERIALS AND METHODS. Antibodies used in Western blot analysis were used for immunohistochemical localization. A: strong ERbeta immunoreactivity was selectively restricted to ciliated epithelial cells (secretory epithelial cells lacked specific ERbeta immunoreactivity). A zoom of the epithelial cells positive for ERbeta in B is indicated by a red solid arrow. C: the same concentration of rabbit IgG instead of the primary antibody and secondary antibody was used as a negative control in an adjacent tubal section. D: in the same experiment, intensive nuclear ERbeta immunoreactivity was detected in rat ovarian granulosa cells. E: an enhanced magnification, noted by a black solid arrow in D, indicated an ovarian follicle. Using tubal tissues from different rats, the same results were obtained for replicate experiments. Scale bars are shown. CEC, ciliated epithelical cell; SEC, secretory epithelical cell; GC, granulosa cell; TC, theca cell. F: confocal microscopy of dual-fluorescent staining of ERbeta and beta-tubulin IV in epithelial cells of rat fallopian tubes. Tubal sections were immunolabeled for ERbeta and beta-tubulin IV visualized with the appropriate wavelength for ERbeta (green), beta-tubulin IV (red), and merged (yellow) as described in MATERIALS AND METHODS. Figure shows the high degree of overlap coefficiency of ERbeta and beta-tubulin IV.

 
With the goal of further identifying the subcellular compartment containing the majority of ERbeta in the ciliated epithelial cells, confocal microscopy was used to compare the distribution of ERbeta and beta-tubublin IV. Colocalization of ERbeta (green) and beta-tubulin IV (red) immunoexpression in the ciliated epithelial cells was clearly visible in the merged pictures (Fig. 3F, yellow). Taken together, these results definitively show that ERbeta is localized in the cilia of epithelial cells.

Estrogen Directly Regulates ERbeta2 Expression in Rat Fallopian Tubes

Next, we tested the hypothesis that the expression levels of ERbeta2 in the ciliated epithelial cells would be altered by acute E2 stimulation. Following treatment of prepuberal rats with E2 (0–24 h), ERbeta2 levels gradually increased, whereas additional treatment with ICI 182,780 slowly lowered ERbeta2 levels, consistent with its known ER antagonist activity (25). This inhibitory effect was shown to be time dependent (Fig. 4A). In contrast, ERbeta1 expression remained at almost background levels in all tubal extracts, and there were virtually no corresponding changes in ERbeta1 expression during the same time treatment period (data not shown). Therefore, most of the cell-specific immunoreactivity corresponding to ERbeta reflects ERbeta2 activity in the cilia.


Figure 4
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  		Fig. 4. Time-course effect of 17beta-estradiol (E2; A), diarylpropionitrile (DPN; B), and/or ICI 182,780 (A and B) on ERbeta2 protein levels in the rat fallopian tubes. Representative Western blots of tubal proteins for ER subtypes. All extracts were prepared during E2, DPN, ICI 182,780, or oil (C) at the time intervals indicated. Blot was probed with specific antibody against either ERbeta or ER{alpha} as described in MATERIALS AND METHODS. The correct loading was evaluated by staining the gels with Coomassie blue. Relative levels of ERbeta2 proteins were expressed as a ratio of ERbeta2 densitometric value to whole proteins in Coomassie blue staining. Graphed results are means ± SE of 3 rats/group. ***P < 0.001. Immunohistochemical analysis of estrogenic effects on ERbeta expression in the ampulla (D–F) and isthmus (G–I) regions. Tubal sections were immunolabeled for ERbeta visualized with DAB (brown) staining as described in MATERIALS AND METHODS. Antibody used in Western blot analysis was used for immunohistochemical localization. Higher intensity of ERbeta staining in the ampullary epithelial cells was seen after 24-h E2 treatment (E) and declined after treatment with ICI 182,780 (F), whereas the infundibulum (data not shown) and isthmus (G–I) regions did not show obvious changes in ERbeta immunoreactivity. Using tubal tissues from different rats, the same results were obtained for replicate experiments. Scale bars are shown.

 
To further test the specificity of the estrogenic effect on ERbeta2 expression, DPN, a selective ERbeta ligand (37), was used to mimic the action of E2 that directly regulates and activates ERbeta. DPN induced ERbeta2 expression in a similar fashion as E2, and expression was also inhibited by ICI 182,780 (Fig. 4B). There was no corresponding increase in ER{alpha} expression during DPN treatment, although a significant reduction in ER{alpha} levels was observed after addition of ICI 182,780 (Fig. 4B). In contrast, no changes in either ER subtype were observed in the fallopian tubes from rats treated with vehicle alone (sesame oil; Fig. 4C). In addition, the specificity of the E2 effect in the fallopian tubes was determined by testing 17{alpha}-estradiol (50 ng/g body wt in 200 µl of sesame oil) that does not activate the ER subtypes (data not shown). These results suggest that induction of ERbeta2 expression by E2 treatment is unlikely to be mediated via increases in ER{alpha} levels in rat fallopian tubes.

To examine whether the expression of ERbeta2 increases differentially in different tubal regions, we analyzed ERbeta2-positive epithelial cells in the tubal epithelium following E2 and/or ICI 182,780 treatment by immunohistochemistry. Consistent with the Western blot data (Fig. 4A), immunoreactive ERbeta increased in the ampullary region with E2 treatment (Fig. 4E) and was reduced after addition of ICI 182,780 (Fig. 4F). No change was observed in the infundibulum (data not shown) and isthmus (Fig. 4, G–I) regions during the same time treatment period. In addition, to evaluate whether treatment regime for ICI 182,780 used in the present study would activate cellular apoptotic pathways in the epithelial cells in rat fallopian tubes, the adjacent fallopian tube sections were used to analyze immunostaining of activated caspase-3, a key determinant of programmed cell death (24). However, immunoreactivity of activated caspase-3 is barely observed in tubal epithelial cells of rats treated with ICI 182,780 compared with that in rats treated with E2 or vehicle (data not shown). These localization studies indicate that downregulation of ERbeta2 expression by ICI 182,780 treatment is unlikely to be due to epithelical cell death. Taken together, the present results suggest that expression of ERbeta2 is enhanced after exposure to ER agonists and reduced by ER antagonists in a time- and a regional-dependent manner.

Effects of Estrogen on ERbeta-Associated Signaling Proteins in Rat Fallopian Tubes

The functional effects of estrogen occur through activation of ERs and modulation of gene expression, one of the events probably initiated by binding to AP-1, which is composed of Jun and Fos proteins (21, 36). Since most AP-1 transcription factors are present at low levels in cells but are rapidly induced and activated in response to specific stimuli, we measured the "activational" expression of c-Jun and c-Fos protein in the fallopian tubes from rats treated with E2, DPN, or ICI 182,780. c-Jun protein was abundantly increased after 0.5 and 1 h of E2 treatment. In addition, the level of c-Jun protein appeared to be increased by DPN in a time-dependent fashion. The amounts of c-Jun were not largely altered after ICI 182,780 treatment in E2-primed rats. In contrast, treatment with ICI 182,780 markedly decreased c-Jun protein levels in DPN-primed rats (Fig. 5, A and B), suggesting that the regulation of c-Jun may be an ERbeta-related manner. On the other hand, little or no change in the expression levels of c-Fos was observed after either E2, DPN, or ICI 182,780 exposure (Fig. 5, A and B). The results of this analysis demonstrate that changes in ER subtype protein levels by E2, DPN, or ICI 182,780 occurred independently of c-Fos status.


Figure 5
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  		Fig. 5. Western blot analysis of ER-associated signaling proteins in rat fallopian tubes. Representative Western blots of tubal proteins for c-Jun, c-Fos, progesterone (P4) receptor (PR), calbindin-D9K, and calbindin-D28K protein expression. All extracts were prepared at the time intervals indicated. Blot was probed with specific antibody against either c-Jun, c-Fos, PR, calbindin-D9K, or calbindin-D28K as described in MATERIALS AND METHODS. Antibody to the ubiquitously expressed beta-actin was used to confirm equal loading. The data are representative of 2 separate experiments.

 
To answer the question of whether estrogen exposure could affect the sensitivity of estrogen-regulated protein expression, rats were treated at different times in the presence of E2 and/or ICI 182,780 and PR, and calbindin-D9K and calbindin-D28K were examined. Western blot analysis revealed that E2 was able to induce PR(A/B) and calbindin-D9K expression in a time-dependent manner. This E2 effect was reversed by ICI 182,780, suggesting that both PR(A/B) and calbindin-D9K proteins are involved in E2-mediated ER activation (Fig. 5A). A slight decrease in calbindin-D28K protein could be detected at 3 h after E2 treatment. The expression of this protein increased thereafter, becoming highly expressed at a time when high levels of ERbeta2 were detected in rat fallopian tubes (Fig. 4A). However, there was no significant change in calbindin-D28K expression during ICI 182,780 treatment (Fig. 5A).

Confirmation of the role of ERbeta in the regulation of PR, calbindin-D9K, and calbindin-D28k expression was obtained by DPN treatment. Similarly to E2, DPN was also able to increase PR(A/B), but with a smaller magnitude compared with E2 treatment. DPN also increased calbindin-D9K protein levels in a time-dependent manner. Blockage of E2 effects on PR(A/B) expression was observed by ICI 182,780 treatment (Fig. 5B). Interestingly, when we examined calbindin-D28K protein levels in the same tissue extracts, a decrease in the expression of this protein with time of DPN treatment was observed. The minimum expression of calbindin-D28K was detected after 12-h E2 treatment, and the level of this protein increased thereafter in a manner similar to E2 treatment. Furthermore, ICI 182,780 exhibited similar effects to DPN on calbindin-D28K expression (Fig. 5B). There results support the hypothesis linking the regulation of ERbeta expression to the functions of Ca2+-regulated processes.

P4 Fails to Regulate ERbeta2 Expression in Rat Fallopian Tubes

P4 is often considered as an antagonist in modulating the effects of estrogen in the fallopian tubes (26, 66). It is possible that the response to estrogen is not specific, and P4 could also regulate ERbeta2 expression. To address this possibility, a similar time course experiment was performed to evaluate whether P4 participates in the regulation of ERbeta2 protein expression in rat fallopian tubes. Western blot analysis showed that levels of ERbeta2 remained unchanged, whereas ER{alpha} protein tended to decrease during P4 treatment (Fig. 6). Moreover, there were no significant differences in serum P4 concentrations among rats given oil, E2, DPN, and/or ICI 182,780 during the treatment period (data not shown), suggesting that regulation of ERbeta2 expression is unlikely to be related to P4 action.


Figure 6
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  		Fig. 6. Time course effect of P4 on ERbeta2 protein levels. Representative Western blots of tubal protein for ER subtypes. All extracts were prepared at the time intervals indicated. Blot was probed with specific antibody against either ERbeta or ER{alpha} as described in MATERIALS AND METHODS. The proper loading was evaluated by staining the gels with Coomassie blue. Relative levels of ERbeta2 proteins were expressed as a ratio of ERbeta2 densitometric value to whole proteins in Coomassie blue staining. Graphed results are means ± SE of 3 rats/group.

 

   DISCUSSION
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Interest in the functions of ERbeta has increased due to not only the distinct tissue/cell distribution and opposite transcriptional directions compared with ER{alpha} (10, 29, 54, 69) but also due to the discovery of multiple isoforms of this receptor in a variety of tissues/cell types in humans and rodents (13, 29, 40). Furthermore, most experiments on the involvement of ERbeta in various cellular (22, 36, 57) and physiological (15, 28) functions have dealt only with the full-length ERbeta1, leaving the functional significance of other ERbeta isoforms elusive. Using several molecular approaches, the present study offers a detailed description of the expression, cellular localization, and regulation of ERbeta isoforms within the rat fallopian tubes in response to estrogen stimulation.

Using analysis of protein expression patterns and cell type-specific markers, we observe that ERbeta2 is the predominant ERbeta isoform and that ERbeta1 expression is very low in rat fallopian tubes. The ratio of the two ERbeta isoforms has been known to vary depending on the tissue (29, 40), but such an exclusive expression of either isoform as seen in the fallopian tubes in vivo has not been shown to date. Strikingly, our study clearly shows that the expression of ERbeta is intense in ciliated but not secretory epithelial cells by demonstrating the colocalization of ERbeta and beta-tubulin IV, a cellular marker for the cilia in the fallopian tubes (48, 50). Similarly, immunoreactivity for ciliated ERbeta is also found in human and mouse (Shao R, Weijdegård B, Fernandez-Rodriguez J, and Billig H, unpublished data) fallopian tube epithelium. However, previous studies investigating ER expression in rat fallopian tubes have found either no ERbeta protein expression (48, 56) or selective localization in the smooth muscle cells (64). The reasons for divergent results are likely to relate to differences in ERbeta antibodies (the differential epitope and antigen recognition), experimental techniques (no discrimination between ERbeta isoforms), and/or the phase of the cycle of the animals used in the studies. Also, the failure to observe ERbeta in the past is probably due to the extremely low levels of ERbeta1, originally described as ERbeta (Fig. 1A) (10, 64), in rat fallopian tubes.

To our knowledge, this is the first study to demonstrate that ERbeta2 protein but not ERbeta1 protein in rat fallopian tubes responds dynamically to estrogen stimulation by undergoing a time-dependent regulation in vivo. As a complementary approach, DPN, an ERbeta agonist, was used to test whether estrogen regulates ERbeta2 expression directly, since ER{alpha} is also present in the same tissues, suggesting that ERbeta2 itself may play a role in the modulation of estrogenic action in the fallopian tubes. Moreover, biochemical studies indicate that ERbeta2 encodes most functional domains related to ERbeta1 (13). Studies in vitro (40) have shown that ERbeta2 protein retains DNA binding activity, recognizing the ERE in its regulatory region as opposite to ERbeta1 protein. Therefore, it is conceivable that regulation and activation of ERbeta2 protein may be involved in estrogen-induced biological effects in vivo. The existence of ERbeta with undetectable ER{alpha} in the cilia of epithelial cells indicates that cilia may represent a target for estrogen action mediated exclusively by ERbeta. Our results are of high importance since the main task of ciliated epithelial cells is to transport the female and male gametes, as well as the embryos through the fallopian tubes, in particular cilia perform essential motile and sensory functions in the mucociliary transport system (5, 11, 20, 31). It is generally accepted that changing the mammalian CBF, a Ca2+-regulated process, is necessary for an effective transport process during physiological conditions (63). A previous study (74) has demonstrated a rise in intracellular free Ca2+ concentration, and rapid fluxes in Ca2+ levels increase CBF in the ciliated epithelial cells of rabbit fallopian tubes in vitro. Due to the difficulties in demonstrating an endogenous estrogen effect on Ca2+ level and activity in fallopian tubal cells, we used an indirect approach to address the contribution of ERbeta protein in mediating the overall effect of estrogen on the regulation of Ca2+ concentration and activity. There is evidence that Ca2+-binding proteins are responsible for intracellular Ca2+ storage and that the functions of Ca2+-regulated processes are reflected in the variety of Ca2+-binding proteins (32). Experimental studies performed in vivo have shown that calbindin-D9K is localized in the ciliated epithelial cells (35) and that it is regulated by estrogen in rat fallopian tubes (7). In addition, the dependency of calbindin-D28K expression on estrogen has been reported in mouse fallopian tubes (19). The time course studies show that the upregulation of ERbeta2 expression by both E2 and DPN coincides with an increase in the amounts of calbindin-D9K, whereas calbindin-D28K is transiently decreased in rat fallopian tubes. These correlative findings support the notion that estrogen-induced Ca2+-binding protein changes may be in part responsible for ERbeta2 expression and activation, which implicates ERbeta2 in coordinating Ca2+-related cilia functions such as ciliary beat. The ability of steroid hormones to regulate CBF has been demonstrated in vitro. For instance, treatment with E2 prevents P4-induced inhibition of CBF in human fallopian tubes, although E2 itself has no effect on CBF (33), suggesting that estrogen may promote cilia to change CBF indirectly. Although enhancement of ovum transport by exogenous E2 is observed in cyclic and pregnant rats (52), any mechanistic explanation of how estrogen regulates CBF in the ciliated epithelial cells and influence tubal transport process (20) is necessarily speculative. We and others have previously shown that in the fallopian tubes gonadotropins increase the level of PRs, which are required for endogenous E2 stimulation of steroidogenesis in vivo (66, 71) and in vitro (53). In this study, we demonstrate that treatment with E2 or DPN results in the induction of PR proteins in rat fallopian tubes, further strengthening the notion that estrogen is a positive regulator of PR in rodent fallopian tubes. Both ER and PR are members of the steroid/nuclear receptor superfamily with similar functional domains for ligand binding, nuclear location, transcriptional activation, and DNA binding (72). A previous study has demonstrated that ER is able to interact with the PR at the AP-1 response element (73), and data suggest that the potential exists for cross-talk between ER and PR respond to the integration of the steroid hormone signals (3). More recently, it has been reported that PR can be detected in the cilia of ciliated epithelial cells in human and mouse fallopian tubes (71), which is consistent with the fact that ERbeta is localized in the cilia (Fig. 3). In line with these observations, it is likely that an ERbeta and PR interaction is also present in the cilia in rat fallopian tubes. Since P4 is not able to regulate ERbeta2 expression in vivo, we hypothesize that ERbeta2 acts on cilia to regulate CBF via PR, which may explain the indirect effect of estrogen on the regulation of CBF during transport process in human fallopian tubes. Obviously, our data together with a previous study showing that local exposure to ICI 182,780 in the fallopian tubes blocks E2-induced ovum transport in cyclic rats (51) lend strength to the physiological relevance of ERbeta in this tissue in vivo.

The elucidation of the cellular functions of ERbeta proteins in rat-ciliated epithelial cells will allow us to reveal the connection between cell type-specific ERbeta contribution and disease-related tubal dysfunction. In vivo and in vitro observations (8, 14) have previously shown that the number of ciliated epithelial cells is increased by E2 stimulation in human follopian tubes. Notably, human tubal ectopic pregnancy shows a reduction in the number of ciliated epithelial cells (6) and low levels of circulating estrogen (4, 46). Beneficial effects of E2 supplementation have been found in women with tubal ecopic pregnancy (18). These observations indicate that estrogen may determine the number of ciliated cells and coordinate ciliary motility in the fallopian tubes. Results from previous studies (11, 20) also suggest that tubal cilia in the ampullary region rather than other regions are crucial for gamete transport function. Although ciliated-ERbeta protein is ubiquitously expressed in all tubal regions in rats, our results show that the significant alteration of ERbeta2 protein expression is observed mostly in the ampullary region. Treatment with ICI 182,780 reduces ERbeta2 expression in ampullary region cilia but not in other regions, which is in agreement with the finding that the ampulla is the main region affected by reduced number of ciliated epithelial cells when estrogen is abolished (1). Our data here point to a possible clinical relationship. Since fertilization occurs in ampullary region (26, 31), which is also the most common site for tubal ectopic pregnancies in humans (38, 70), it can be assumed that a decrease in ERbeta2 protein level may be, at least in part, a direct consequence of cilia loss (i.e., deciliation) (8, 14, 26) rather than ciliated epithelial cell death (in this study). Alternatively, lack of estrogen-mediated ERbeta2 function may result in the occurrence of tubal ectopic pregnancies and reduced fertility. Further studies will be needed to verify this concept and extend it to human fallopian tube abnormality.

In the mammalian fallopian tube, the process of successful fertilization is regulated by a complex interplay of endocrine and paracrine signals, including estrogens (26) that are mediated mostly by nuclear ER-dependent mechanisms via at least two distinct receptors, ER{alpha} and ERbeta (21). The identification of an ER subtype with restricted expression in specific cell populations yields insights into the physiological functions of the fallopian tubes and provides the basis for potential functional manipulations. Our results demonstrate that ER{alpha} is located in the epithelial and smooth muscles cells, wheras expression of ERbeta is detected only in the epithelial cells. The differential localization of ER{alpha} and ERbeta among populations of tubal cells may explain why estrogen affects target cell and respective cellular components differently. For example, E2 participates in the regulation of smooth muscle contraction and relaxation in rat fallopian tubes (51), suggesting that ER{alpha}-positive smooth muscle cells respond directly to E2 action. On the other hand, emerging evidence indicates that ER antagonist ICI 182,780 stimulates degradation of ERs and prevents receptor dimerization, inhibiting estrogen-dependent gene transcription (12, 16). In accordance with this, we observe that E2-induced ER{alpha} and ERbeta2 expression is inhibited efficiently by ICI 182,780. However, ERbeta-positive epithelial cells also contain ER{alpha}; therefore, it is not possible to dissect the individual role of ER subtypes in the fallopian tubes in our studies since ICI 182,780 usually results in an attenuated cellular response of E2 and transcriptional repression of both ER{alpha} and ERbeta regardless of the presence or absence of ER agonist (25). An interesting area for future investigations would be to enlarge our approach to select ER subtypes on the basis of previously established ER knockout mouse models (9, 15, 28, 30). Further studies to confirm the absence of individual ER subtype in the fallopian tubes will be informative in evaluating their specific function and comparing them with those observed in rat fallopian tubes in vivo.

In summary, we demonstrate for the first time a specific localization and possible function of ERbeta isoforms in rat fallopian tubes. We show that ERbeta2 represents a relatively high proportion of total ERbeta protein. Considering the cellular distribution and regulatory patterns of ERbeta2 in ciliated epithelial cells, we speculate that this isoform may function in female reproductive events within the fallopian tube (transport of the female and male gametes as well as embryos) and also when these events are dysfunctional (i.e., induction of tubal ectopic pregnancy). Furthermore, ERbeta2 apparently contributes to the action of E2 by changing ciliated epithelial cell function. In this context, the present work not only enhances our knowledge on tissue/cell-specific expression of ERbeta isoforms but also provides a physiological link between ERbeta2 and tubal transport processes affected by E2 signaling. Future challenges include establishing exactly how ERbeta2 is targeted to cilia, what other proteins it interacts with, and what the downstream target of ERbeta2 may be. These are under investigation in our laboratory.


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 ABSTRACT
 MATERIALS AND METHODS
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This work was supported by Grant 10380 from the Swedish Medical Research Council (to H. Billig) and (in part) by the Assar Gabrielssons Forsknings Foundation, the Scientific Foundation of Eva och Oscar Ahrens, Iwan och Eleonore Ljunggrens, Wilhelm och Martina Lundgrens, Hjalmar Svensson, and Emil och Maria Palms, as well as Adlerbertska Research Foundation (to R. Shao).


   ACKNOWLEDGMENTS
 
We thank Drs. Fu-Ping Zhang (Department of Physiology, Institute of Biomedicine, University of Helsinki, Helsinki, Finland) and Elisabet Stener-Victorin for their helpful comments on the manuscript as well as Dr. Joakim Larsson for useful suggestions during the course of experimentation.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. Shao, Instiute of Neuroscience and Physiology, The Sahlgrenska Academy at Gothenburg University, SE-41390 Gothenburg, Sweden (e-mail: ruijin.shao{at}fysiologi.gu.se)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

  1. Abe H. The mammalian oviductal epithelium: regional variations in cytological and functional aspects of the oviductal secretory cells. Histol Histopathol 11: 743–768, 1996.[Web of Science][Medline]
  2. Amso NN, Crow J, Shaw RW. Comparative immunohistochemical study of oestrogen and progesterone receptors in the fallopian tube and uterus at different stages of the menstrual cycle and the menopause. Hum Reprod 9: 1027–1037, 1994.[Abstract/Free Full Text]
  3. Ballare C, Uhrig M, Bechtold T, Sancho E, Di Domenico M, Migliaccio A, Auricchio F, Beato M. Two domains of the progesterone receptor interact with the estrogen receptor and are required for progesterone activation of the c-Src/Erk pathway in mammalian cells. Mol Cell Biol 23: 1994–2008, 2003.[Abstract/Free Full Text]
  4. Barnea ER, Oelsner G, Benveniste R, Romero R, DeCherney AH. Progesterone, estradiol, and alpha-human chorionic gonadotropin secretion in patients with ectopic pregnancy. J Clin Endocrinol Metab 62: 529–531, 1986.[Abstract/Free Full Text]
  5. Blandau RJ. Comparative aspects of tubal anatomy and physiology as they relate to reconstructive procedures. J Reprod Med 21: 7–15, 1978.[Web of Science][Medline]
  6. Brosens IA, Degraef R. Microbiopsy of the fallopian tube as a method for clinical investigation of tubal function in infertility. Int J Fertil 20: 55–60, 1975.[Web of Science][Medline]
  7. Choi KC, Leung PC, Jeung EB. Biology and physiology of Calbindin-D9k in female reproductive tissues: involvement of steroids and endocrine disruptors. Reprod Biol Endocrinol 3: 66, 2005.[CrossRef][Medline]
  8. Comer MT, Leese HJ, Southgate J. Induction of a differentiated ciliated cell phenotype in primary cultures of Fallopian tube epithelium. Hum Reprod 13: 3114–3120, 1998.[Abstract/Free Full Text]
  9. Couse JF, Hewitt SC, Bunch DO, Sar M, Walker VR, Davis BJ, Korach KS. Postnatal sex reversal of the ovaries in mice lacking estrogen receptors alpha and beta. Science 286: 2328–2331, 1999.[Abstract/Free Full Text]
  10. Couse JF, Lindzey J, Grandien K, Gustafsson JA, Korach KS. Tissue distribution and quantitative analysis of estrogen receptor-alpha (ERalpha) and estrogen receptor-beta (ERbeta) messenger ribonucleic acid in the wild-type and ERalpha-knockout mouse. Endocrinology 138: 4613–4621, 1997.[Abstract/Free Full Text]
  11. Croxatto HB. Physiology of gamete and embryo transport through the fallopian tube. Reprod Biomed Online 4: 160–169, 2002.[Medline]
  12. Dauvois S, Danielian PS, White R, Parker MG. Antiestrogen ICI 164,384 reduces cellular estrogen receptor content by increasing its turnover. Proc Natl Acad Sci USA 89: 4037–4041, 1992.[Abstract/Free Full Text]
  13. Deroo BJ, Korach KS. Estrogen receptors and human disease. J Clin Invest 116: 561–570, 2006.[CrossRef][Web of Science][Medline]
  14. Donnez J, Casanas-Roux F, Ferin J, Thomas K. Changes in ciliation and cell height in human tubal epithelium in the fertile and post-fertile years. Maturitas 5: 39–45, 1983.[CrossRef][Web of Science][Medline]
  15. Dupont S, Krust A, Gansmuller A, Dierich A, Chambon P, Mark M. Effect of single and compound knockouts of estrogen receptors alpha (ERalpha) and beta (ERbeta) on mouse reproductive phenotypes. Development 127: 4277–4291, 2000.[Abstract]
  16. Fawell SE, White R, Hoare S, Sydenham M, Page M, Parker MG. Inhibition of estrogen receptor-DNA binding by the "pure" antiestrogen ICI 164,384 appears to be mediated by impaired receptor dimerization. Proc Natl Acad Sci USA 87: 6883–6887, 1990.[Abstract/Free Full Text]
  17. Frasor J, Barnett DH, Danes JM, Hess R, Parlow AF, Katzenellenbogen BS. Response-specific and ligand dose-dependent modulation of estrogen receptor (ER) alpha activity by ERbeta in the uterus. Endocrinology 144: 3159–3166, 2003.[Abstract/Free Full Text]
  18. Gaddum-Rosse P, Rumery RE, Blandau RJ, Thiersch JB. Studies on the mucosa of postmenopausal oviducts: surface appearance, ciliary activity, and the effect of estrogen treatment. Fertil Steril 26: 951–969, 1975.[Web of Science][Medline]
  19. Gill RK, Christakos S. Regulation by estrogen through the 5'-flanking region of the mouse calbindin-D28k gene. Mol Endocrinol 9: 319–326, 1995.[Abstract/Free Full Text]
  20. Halbert SA, Tam PY, Blandau RJ. Egg transport in the rabbit oviduct: the roles of cilia and muscle. Science 191: 1052–1053, 1976.[Abstract/Free Full Text]
  21. Hall JM, Couse JF, Korach KS. The multifaceted mechanisms of estradiol and estrogen receptor signaling. J Biol Chem 276: 36869–36872, 2001.[Free Full Text]
  22. Hall JM, McDonnell DP. The estrogen receptor beta-isoform (ERbeta) of the human estrogen receptor modulates ERalpha transcriptional activity and is a key regulator of the cellular response to estrogens and antiestrogens. Endocrinology 140: 5566–5578, 1999.[Abstract/Free Full Text]
  23. Hawkins MB, Thornton JW, Crews D, Skipper JK, Dotte A, Thomas P. Identification of a third distinct estrogen receptor and reclassification of estrogen receptors in teleosts. Proc Natl Acad Sci USA 97: 10751–10756, 2000.[Abstract/Free Full Text]
  24. Hengartner MO. The biochemistry of apoptosis. Nature 407: 770–776, 2000.[CrossRef][Medline]
  25. Howell A, Osborne CK, Morris C, Wakeling AE. ICI 182,780 (Faslodex): development of a novel, "pure" antiestrogen. Cancer 89: 817–825, 2000.[CrossRef][Web of Science][Medline]
  26. Jansen RP. Endocrine response in the fallopian tube. Endocr Rev 5: 525–551, 1984.[Abstract/Free Full Text]
  27. Jensen EV, Cheng G, Palmieri C, Saji S, Makela S, Van Noorden S, Wahlstrom T, Warner M, Coombes RC, Gustafsson JA. Estrogen receptors and proliferation markers in primary and recurrent breast cancer. Proc Natl Acad Sci USA 98: 15197–15202, 2001.[Abstract/Free Full Text]
  28. Krege JH, Hodgin JB, Couse JF, Enmark E, Warner M, Mahler JF, Sar M, Korach KS, Gustafsson JA, Smithies O. Generation and reproductive phenotypes of mice lacking estrogen receptor beta. Proc Natl Acad Sci USA 95: 15677–15682, 1998.[Abstract/Free Full Text]
  29. Leung YK, Mak P, Hassan S, Ho SM. Estrogen receptor (ER)-beta isoforms: a key to understanding ER-beta signaling. Proc Natl Acad Sci USA 103: 13162–13167, 2006.[Abstract/Free Full Text]
  30. Lubahn DB, Moyer JS, Golding TS, Couse JF, Korach KS, Smithies O. Alteration of reproductive function but not prenatal sexual development after insertional disruption of the mouse estrogen receptor gene. Proc Natl Acad Sci USA 90: 11162–11166, 1993.[Abstract/Free Full Text]
  31. Lyons RA, Saridogan E, Djahanbakhch O. The reproductive significance of human Fallopian tube cilia. Hum Reprod Update 12: 363–372, 2006.[Abstract/Free Full Text]
  32. Lytton J, Nigam SK. Intracellular calcium: molecules and pools. Curr Opin Cell Biol 4: 220–226, 1992.[CrossRef][Medline]
  33. Mahmood T, Saridogan E, Smutna S, Habib AM, Djahanbakhch O. The effect of ovarian steroids on epithelial ciliary beat frequency in the human Fallopian tube. Hum Reprod 13: 2991–2994, 1998.[Abstract/Free Full Text]
  34. Mariani ML, Ciocca DR, Gonzalez Jatuff AS, Souto M. Effect of neonatal chronic stress on expression of Hsp70 and oestrogen receptor alpha in the rat oviduct during development and the oestrous cycle. Reproduction 126: 801–808, 2003.[Abstract]
  35. Mathieu CL, Mills SE, Burnett SH, Cloney DL, Bruns DE, Bruns ME. The presence and estrogen control of immunoreactive calbindin-D9k in the fallopian tube of the rat. Endocrinology 125: 2745–2750, 1989.[Abstract/Free Full Text]
  36. Matthews J, Wihlen B, Tujague M, Wan J, Strom A, Gustafsson JA. Estrogen receptor (ER) beta modulates ERalpha-mediated transcriptional activation by altering the recruitment of c-Fos and c-Jun to estrogen-responsive promoters. Mol Endocrinol 20: 534–543, 2006.[Abstract/Free Full Text]
  37. Meyers MJ, Sun J, Carlson KE, Marriner GA, Katzenellenbogen BS, Katzenellenbogen JA. Estrogen receptor-beta potency-selective ligands: structure-activity relationship studies of diarylpropionitriles and their acetylene and polar analogues. J Med Chem 44: 4230–4251, 2001.[CrossRef][Web of Science][Medline]
  38. Miller BT, Leondires M. The fallopian tube in health and disease. In: The Physiologic Basis of Gynecology and Obstetrics, edited by Seifer DB, Samuels P, and Kniss DA. Baltimore, MD: Lippincott Williams & Wilkins, 2003, chapt. 16, p. 222–244.
  39. Moll R, Levy R, Czernobilsky B, Hohlweg-Majert P, Dallenbach-Hellweg G, Franke WW. Cytokeratins of normal epithelia and some neoplasms of the female genital tract. Lab Invest 49: 599–610, 1983.[Web of Science][Medline]
  40. Moore JT, McKee DD, Slentz-Kesler K, Moore LB, Jones SA, Horne EL, Su JL, Kliewer SA, Lehmann JM, Willson TM. Cloning and characterization of human estrogen receptor beta isoforms. Biochem Biophys Res Commun 247: 75–78, 1998.[CrossRef][Web of Science][Medline]
  41. Mowa CN, Iwanaga T. Developmental changes of the oestrogen receptor-alpha and -beta mRNAs in the female reproductive organ of the rat—an analysis by in situ hybridization. J Endocrinol 167: 363–369, 2000.[Abstract]
  42. Mowa CN, Iwanaga T. Differential distribution of oestrogen receptor-alpha and -beta mRNAs in the female reproductive organ of rats as revealed by in situ hybridization. J Endocrinol 165: 59–66, 2000.[Abstract]
  43. Muechler EK, Cary D. Further characterization of estrogen receptors in the human oviduct. Fertil Steril 39: 819–823, 1983.[Web of Science][Medline]
  44. Muechler EK, Kohler D. Properties of the estrogen receptor in the human oviduct and its interaction with ethinylestradiol and mestranol in vitro. J Clin Endocrinol Metab 51: 962–967, 1980.[Abstract/Free Full Text]
  45. Noda S, Sawaki M, Shiraishi K, Yamasaki K, Yamaguchi R. Age-related changes of genital systems in the female Crj:CD (SD) IGS rats during sexual maturation. J Vet Med Sci 64: 315–319, 2002.[CrossRef][Web of Science][Medline]
  46. Norman RJ, Buck RH, Kemp MA, Joubert SM. Impaired corpus luteum function in ectopic pregnancy cannot be explained by altered human chorionic gonadotropin. J Clin Endocrinol Metab 66: 1166–1170, 1988.[Abstract/Free Full Text]
  47. Okada A, Ohta Y, Brody SL, Iguchi T. Epithelial c-jun and c-fos are temporally and spatially regulated by estradiol during neonatal rat oviduct differentiation. J Endocrinol 182: 219–227, 2004.[Abstract]
  48. Okada A, Ohta Y, Brody SL, Watanabe H, Krust A, Chambon P, Iguchi T. Role of foxj1 and estrogen receptor alpha in ciliated epithelial cell differentiation of the neonatal oviduct. J Mol Endocrinol 32: 615–625, 2004.[Abstract]
  49. Okada A, Ohta Y, Buchanan DL, Sato T, Inoue S, Hiroi H, Muramatsu M, Iguchi T. Changes in ontogenetic expression of estrogen receptor alpha and not of estrogen receptor beta in the female rat reproductive tract. J Mol Endocrinol 28: 87–97, 2002.[Abstract]
  50. Okada A, Ohta Y, Inoue S, Hiroi H, Muramatsu M, Iguchi T. Expression of estrogen, progesterone and androgen receptors in the oviduct of developing, cycling and pre-implantation rats. J Mol Endocrinol 30: 301–315, 2003.[Abstract]
  51. Orihuela PA, Parada-Bustamante A, Cortes PP, Gatica C, Croxatto HB. Estrogen receptor, cyclic adenosine monophosphate, and protein kinase A are involved in the nongenomic pathway by which estradiol accelerates oviductal oocyte transport in cyclic rats. Biol Reprod 68: 1225–1231, 2003.[Abstract/Free Full Text]
  52. Ortiz ME, Villalon M, Croxatto HB. Ovum transport and fertility following postovulatory treatment with estradiol in rats. Biol Reprod 21: 1163–1167, 1979.[Abstract]
  53. Ouhibi N, Menezo Y, Benet G, Nicollet B. Culture of epithelial cells derived from the oviduct of different species. Hum Reprod 4: 229–235, 1989.[Abstract/Free Full Text]
  54. Paech K, Webb P, Kuiper GG, Nilsson S, Gustafsson J, Kushner PJ, Scanlan TS. Differential ligand activation of estrogen receptors ERalpha and ERbeta at AP1 sites. Science 277: 1508–1510, 1997.[Abstract/Free Full Text]
  55. Pasqualini C, Guivarc'h D, Boxberg YV, Nothias F, Vincent JD, Vernier P. Stage- and region-specific expression of estrogen receptor alpha isoforms during ontogeny of the pituitary gland. Endocrinology 140: 2781–2789, 1999.[Abstract/Free Full Text]
  56. Pelletier G, Labrie C, Labrie F. Localization of oestrogen receptor alpha, oestrogen receptor beta and androgen receptors in the rat reproductive organs. J Endocrinol 165: 359–370, 2000.[Abstract]
  57. Pettersson K, Delaunay F, Gustafsson JA. Estrogen receptor beta acts as a dominant regulator of estrogen signaling. Oncogene 19: 4970–4978, 2000.[CrossRef][Web of Science][Medline]
  58. Pillai SB, Jones JM, Koos RD. Treatment of rats with 17beta-estradiol or relaxin rapidly inhibits uterine estrogen receptor beta1 and beta2 messenger ribonucleic acid levels. Biol Reprod 67: 1919–1926, 2002.[Abstract/Free Full Text]
  59. Pino AM, Devoto L, Soto E, Castro O, Sierralta W. Changes in cytosolic and nuclear estradiol receptors of normal Fallopian tube throughout the menstrual cycle. J Steroid Biochem 16: 193–197, 1982.[CrossRef][Web of Science][Medline]
  60. Pollow K, Inthraphuvasak J, Grill HJ, Manz B. Estradiol and progesterone binding components in the cytosol of normal human fallopian tubes. J Steroid Biochem 16: 429–435, 1982.[CrossRef][Web of Science][Medline]
  61. Pollow K, Inthraphuvasak J, Manz B, Grill HJ, Pollow B. A comparison of cytoplasmic and nuclear estradiol and progesterone receptors in human fallopian tube and endometrial tissue. Fertil Steril 36: 615–622, 1981.[Web of Science][Medline]
  62. Press MF, Nousek-Goebl NA, Bur M, Greene GL. Estrogen receptor localization in the female genital tract. Am J Pathol 123: 280–292, 1986.[Abstract]
  63. Salathe M. Regulation of mammalian ciliary beating. Annu Rev Physiol 69: 401–422, 2007.[CrossRef][Web of Science][Medline]
  64. Saunders PT, Maguire SM, Gaughan J, Millar MR. Expression of oestrogen receptor beta (ER beta) in multiple rat tissues visualised by immunohistochemistry. J Endocrinol 154: R13–R16, 1997.[Abstract/Free Full Text]
  65. Shao R, Ljungstrom K, Weijdegard B, Egecioglu E, Fernandez-Rodriguez J, Zhang FP, Thurin-Kjellberg A, Bergh C, Billig H. Estrogen-induced upregulation of AR expression and enhancement of AR nuclear translocation in mouse fallopian tubes in vivo. Am J Physiol Endocrinol Metab 292: E604–E614, 2007.[Abstract/Free Full Text]
  66. Shao R, Weijdegard B, Ljungstrom K, Friberg A, Zhu C, Wang X, Zhu Y, Fernandez-Rodriguez J, Egecioglu E, Rung E, Billig H. Nuclear progesterone receptor A and B isoforms in mouse fallopian tube and uterus: implications for expression, regulation, and cellular function. Am J Physiol Endocrinol Metab 291: E59–E72, 2006.[Abstract/Free Full Text]
  67. Shao R, Zhang FP, Tian F, Anders Friberg P, Wang X, Sjoland H, Billig H. Increase of SUMO-1 expression in response to hypoxia: direct interaction with HIF-1alpha in adult mouse brain and heart in vivo. FEBS Lett 569: 293–300, 2004.[CrossRef][Web of Science][Medline]
  68. Skalli O, Ropraz P, Trzeciak A, Benzonana G, Gillessen D, Gabbiani G. A monoclonal antibody against alpha-smooth muscle actin: a new probe for smooth muscle differentiation. J Cell Biol 103: 2787–2796, 1986.[Abstract/Free Full Text]
  69. Takeyama J, Suzuki T, Inoue S, Kaneko C, Nagura H, Harada N, Sasano H. Expression and cellular localization of estrogen receptors alpha and beta in the human fetus. J Clin Endocrinol Metab 86: 2258–2262, 2001.[Abstract/Free Full Text]
  70. Tay JI, Moore J, Walker JJ. Ectopic pregnancy. West J Med 173: 131–134, 2000.[CrossRef][Web of Science][Medline]
  71. Teilmann SC, Clement CA, Thorup J, Byskov AG, Christensen ST. Expression and localization of the progesterone receptor in mouse and human reproductive organs. J Endocrinol 191: 525–535, 2006.[Abstract/Free Full Text]
  72. Tsai MJ, O'Malley BW. Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annu Rev Biochem 63: 451–486, 1994.[CrossRef][Web of Science][Medline]
  73. Uht RM, Anderson CM, Webb P, Kushner PJ. Transcriptional activities of estrogen and glucocorticoid receptors are functionally integrated at the AP-1 response element. Endocrinology 138: 2900–2908, 1997.[Abstract/Free Full Text]
  74. Villalon M, Hinds TR, Verdugo P. Stimulus-response coupling in mammalian ciliated cells. Demonstration of two mechanisms of control for cytosolic [Ca2+]. Biophys J 56: 1255–1258, 1989.[Web of Science][Medline]
  75. Wang H, Eriksson H, Sahlin L. Estrogen receptors alpha and beta in the female reproductive tract of the rat during the estrous cycle. Biol Reprod 63: 1331–1340, 2000.[Abstract/Free Full Text]



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