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Am J Physiol Endocrinol Metab 293: E1430-E1442, 2007. First published September 11, 2007; doi:10.1152/ajpendo.00384.2007
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Dynamic regulation of estrogen receptor-{alpha} isoform expression in the mouse fallopian tube: mechanistic insight into estrogen-dependent production and secretion of insulin-like growth factors

Ruijin Shao,1 Emil Egecioglu,1 Birgitta Weijdegård,1 John J. Kopchick,2 Julia Fernandez-Rodriguez,3 Niklas Andersson,4 and Håkan Billig1

1Department of Physiology/Endocrinology, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at Gothenburg University, Gothenburg, Sweden; 2Edison Biotechnology Institute, Konneker Research Laboratories, Ohio University, Athens, Ohio; and 3Centre for Cellular Imaging, Core facilities, and 4Division of Endocrinology, Department of Internal Medicine, The Sahlgrenska Academy at Gothenburg University, Gothenburg, Sweden

Submitted 19 June 2007 ; accepted in final form 3 September 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Estrogen receptors (ERs) are members of the nuclear receptor superfamily and are involved in regulation of fallopian tube functions (i.e., enhancement of protein secretion, formation of tubal fluid, and regulation of gamete transport). However, the ER subtype-mediated mechanisms underlying these processes have not been completely clarified. Recently, we identified ERbeta expression and localization in rat fallopian tubes, suggesting a potential biological function of ERbeta related to calcium-dependent ciliated beating. Here we provide for the first time insight into the less studied ER{alpha} isoforms, which mediate estrogen-dependent production and secretion of IGFs in vivo. First, Western blot studies revealed that three ER{alpha} isoforms were expressed in mouse fallopian tubes. Subsequent immunohistochemical analysis showed that ER{alpha} was detected in all cell types, whereas ERbeta was mainly localized in ciliated epithelial cells. Second, ER{alpha} isoform levels were dramatically downregulated in mouse fallopian tubes by treatment with E2 or PPT, an ER{alpha} agonist, in a time-dependent manner. Third, the presence of ICI 182,780, an ER antagonist, blocked the E2- or PPT-induced downregulation of tubal ER{alpha} isoform expression in mice. However, alteration of ER{alpha} immunoreactivity following ICI 182,780 treatment was only detected in epithelial cells of the ampullary region. Fourth, changes in ER{alpha} isoform expression were found to be coupled to multiple E2 effects on tubal growth, protein synthesis, and secretion in mouse fallopian tube tissues and fluid. In particular, E2 exhibited positive regulation of IGF-I and IGF-II protein levels. Finally, using growth hormone receptor (GHR) gene-disrupted mice, we showed that regulation by E2 of IGF production was independent of GH-induced GHR signaling in mouse fallopian tubes in vivo. These data, together with previous studies from our laboratory, suggest that the long-term effects of estrogen agonist promote IGF synthesis and secretion in mouse tubal epithelial cells and fallopian tube fluid via stimulation of ER{alpha}.

estrogen receptor-{alpha} isoforms; tubal epithelial cells

A CRITICAL FUNCTION of the mammalian fallopian tube (24) is to provide an optimal microenvironment for the necessary transport and maturation of gametes and the establishment of pregnancy. It excretes the intraluminal tubal fluid, containing a number of growth factors (1, 4, 29, 30, 34). Compelling evidence suggests that estrogen may be involved in the regulation of biological processes in the fallopian tube. Studies have shown that ovarian-derived estradiol (E2), the principal bioactive estrogen, is involved in the regulation of tubal protein secretion in human fallopian tubes in vivo (34) and in vitro (63). Insulin-like growth factors (IGFs) have been identified in human and rodent fallopian tube fluid (4). Accumulating data suggest that IGF-I may support embryonic development and survival (1). Moreover, E2 has been shown to increase endogenous IGF-I mRNA levels in rat fallopian tube and uterus (5, 46), and estrogen-responsive elements are located in the regulatory regions of the IGF-I gene (62). On the other hand, it has been known for a long time that tissue-specific IGF-I synthesis depends on growth hormone (GH) stimulation (15). Treatment with GH induces IGF-I expression in bovine tubal epithelial cells in vitro (38) and in human follicular fluid in vivo (65). Moreover, it has been demonstrated that rat fallopian tube expresses functional GH receptors (35). This raises the possibility of an indirect effect of E2 via GH and growth hormone receptor (GHR) on the production and secretion of IGFs in mouse fallopian tube. However, to date, the detailed mechanisms regulating the production and secretion of growth factors such as IGF into the tubal fluid are not fully understood.

It is well established that cellular responsiveness or susceptibility to E2 is mostly mediated by the intracellular estrogen receptor (ER), a member of the nuclear receptor superfamily of ligand-dependent transcription factors that regulate the expression of many genes to control physiological processes (20). Generally, ligand binding to ERs results in the dissociation of heat shock proteins, thereby allowing receptors to form dimers (ER{alpha}/ER{alpha}, ERbeta/ERbeta, or ER{alpha}/ERbeta) and either bind specific nucleotide sequences, known as estrogen response elements (ERE), in the promoter regions of ER-regulated genes or tether to DNA through interaction with other nuclear transcription factors such as activator protein complex-1 in the regulatory regions of target genes. ER interaction with ERE can result in either an increase or decrease in target gene transcription (48). Two major subtypes of ER have been identified: ER{alpha} and ERbeta (48). Although both receptors consist of similar DNA-binding and ligand-binding domains (16), they display overlapping but distinct tissue/cell distribution, different pharmacological properties with respect to ligands, and opposite transcriptional directions depending on the ligand concentration in estrogen target tissues/cells (9, 16, 32, 51, 61). Genetic ablation of the ER{alpha} and/or ERbeta genes in mice illustrates the vital role of the ER subtypes in maintaining normal female reproductive functions (19). Manipulation of the endogenous ER subtypes does not result in equivalent physiological effects in mouse ovary and uterus (8, 11, 28, 37). For example, mice in which ER{alpha} or ERbeta is absent display varying degrees of ovarian infertility, whereas ER{alpha} is an essential subtype mediating response to different ER agonists (i.e., E2 and diethylstilbestrol) in the uterus, further supporting the concept that the diversity of tissue/cell distribution of the ER subtypes may dictate different biological functions.

It is generally accepted that target tissue responsiveness to E2 is principally controlled by the amount and type of ERs. Numerous studies have demonstrated ER subtype expression in mouse (9, 47) and rat (39, 44, 45, 49, 50, 54, 66) fallopian tubes, and it has been shown that the expression of ER{alpha} is also regulated during both development and the estrous cycle (39, 44, 45, 50, 66). Furthermore, truncated forms of both ER{alpha} and ERbeta have been identified in many estrogen target tissues and cell types (2, 3, 32, 42), suggesting that the tissue/cell-specific responses to estrogen resulting in the divergence of biological function may at least in part be due to differences in the differential expression and regulation of ER{alpha}/beta isoforms. Recently, we have demonstrated the presence of ERbeta isoforms specifically in the ciliated epithelial cells of rat fallopian tube (56). Although the molecular mechanisms of ERbeta function in the fallopian tube still need to be clearly elucidated, our data suggest that ERbeta is involved in the estrogen-mediated regulation of calcium-dependent ciliated beating (56). In contrast to ERbeta, it has been postulated that ER{alpha} gene common polymorphisms in humans have been associated with female infertility (7), and ER{alpha} serves as a dominant regulator in fallopian tube development (44). However, the relative and respective contribution of ER{alpha} isoforms and functional significance of ER{alpha} in the action of estrogen related to tubal protein production and secretion have not been established.

In the present study, we have used histological, molecular, and functional approaches 1) to investigate the regulation of ER{alpha} isoform expression and localization in mouse fallopian tubes, 2) to demonstrate the specific biological roles of ER{alpha} in estrogen-mediated tubal function, and 3) to dissect out the estrogen-dependent molecular mechanism regulating IGF synthesis and secretion in tubal tissues and fluid. Our studies provide new evidence that tubal ER{alpha} is the critical link that mediates estrogen-dependent regulation of protein synthesis and secretion of IGF-I and IGF-II in mouse tubal epithelial cells and fallopian tube fluid.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Antibodies and reagents. The primary antibodies used for immunohistochemistry and Western blot analysis in the present study, their dilution, and sources are listed in Table 1. The rabbit polyclonal ER{alpha} antibody (MC-20) was raised against a short peptide sequence corresponding to 580-599 amino acids in the mouse ER{alpha} COOH-terminal region, which does not cross-react with ERbeta or other steroid receptors (11, 52). The mouse monoclonal ER{alpha} antibody (6F11) was raised against the full length of human ER{alpha} (50, 54). Whereas MC-20 recognizes different ER{alpha} isoforms, 6F11 only recognizes ER{alpha}1 (the full-length ER{alpha}). The antibody against ERbeta was a rabbit polyclonal IgG raised against a peptide consisting of amino acids 54-71 in rodent ERbeta. It does not cross-react with ER{alpha} (25, 49, 53). The specificity of these antibodies against the classical nuclear ER{alpha} and ERbeta was validated using ER{alpha}–/–, ERbeta–/–, ER{alpha}/beta–/–, and ER{alpha}/beta+/+ mice (Fig. 1). All extracts from ER{alpha}/beta+/+ and ERbeta–/– mice exhibited a protein triplet of 51-64 kDa using MC-20. 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) or anti-rabbit antibody (111-166-006) were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA) and used for fluorescence immunohistochemistry. Normal rabbit IgG and mouse 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) were used as secondary antibodies in the Western blot analysis. Other reagents were purchased from Sigma or Merck (Darmstadt, Germany) and were of the highest-purity grade available.


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

 

Figure 1
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  		Fig. 1. Characterization of estrogen receptor-{alpha} (ER{alpha})- and ERbeta-specific antibodies; ER{alpha} and ERbeta protein detection in uterine tissues by Western blot (WB) analysis as described in MATERIALS AND METHODS. Proteins were extracted from the uteri of 3-mo-old female wild-type (WT), ER{alpha}/beta, ER{alpha}, and ERbeta knockout (KO) mice (n = 3 in each group). The protein blots were probed with specific antibody against either ER{alpha} (MC-20), ER{alpha} (6F11), or ERbeta (06-629), demonstrating specificity of the antibody for the ER{alpha} and ERbeta isoforms of appropriate molecular weight (MW) in mouse uterus. The molecular weight of the protein is indicated. *Protein bands of a possibly truncated, dysfunctional ER{alpha}. beta-Actin was used as an internal control to show that the ER protein was detected at approximately equal signal intensities in all protein samples.

 
Animals. All experimental procedures and protocols used in the present study were approved by the local ethics committee of Gothenburg University, Gothenburg, Sweden. Intact prepubertal female (21 days old) C57BL/6 mice obtained from Taconic (Copenhagen, Denmark) were used to avoid both the effects of endogenous estrogen and surgical procedures such as ovariectomy to remove the main source of endogenous 17beta-estradiol (E2) production. The generation of ER{alpha}/beta, ER{alpha}, ERbeta, and GHR gene-disrupted or knockout mice is described elsewhere (8, 28, 37, 67, 68). All ER knockout mice used in the study were kept on a C57BL/6J background (64), whereas the GHR-deficient mice were kept on a mixed Sv129Ola-Balb/c background (68). The mice have been characterized previously, confirming the disruption of ER{alpha}/beta, ER{alpha}, ERbeta, and GHR by a variety of molecular and cellular techniques in previous studies (6, 12, 33, 64, 67). All comparisons of each individual knockout strain were done using +/+ littermate controls. All animals were maintained in standard cages under 12-h cycles of light and dark with a 1-h dawn/sunset function at 21 ± 2°C and a relative humidity between 45 and 55% and had ad libitum access to normal rodent chow and water. Animals were allowed to acclimate to the animal facilities for at least 5 days before initiation of treatment and experiments.

Experimental design and tissue preparation in mice. Female mice at 26 days of age with a body weight (BW) ranging from 13 to 15 g were used in this study (experiments 1–4). Selective agonists or antagonists for ER{alpha} were used to elucidate the biological function of ER{alpha} as a valuable alternative and complementary approach to the use of knockout animals (17). Whenever possible, doses and treatment regime were based on published studies or verified when appropriate (14). Experiments 2 and 3 were repeated three times with 5–10 animals per group.

The aim of experiment 1 was to investigate the expression of ER subtypes in female mouse reproductive tissues: ovary, fallopian tube, and uterus from 26-day-old female mice were collected and freed from fat and connective tissue immediately after mice were killed. Tissues were frozen in liquid nitrogen for Western blot analysis.

The aim of experiment 2 was to examine whether exogenous estrogens regulated ER{alpha} isoform expression in mouse fallopian tubes; 26-day-old female mice, in which endogenous concentrations of E2 were expected to be minimal (55, 57), were given a single subcutaneous (sc) injection of 0.5 µg E2 (Sigma)/g BW or a selective ER{alpha} ligand (59), 4,4',4-(4-prophl-[1H])-pyrazole-1,3,5-triyl-trisphenol (PPT), at 100 µg (Tocris Cookson, Bristol, UK)/g BW in 100 µl of sesame oil (vehicle) or vehicle alone for 4 days.

The aim of experiment 3 was to determine the effect of the steroidal ER antagonist ICI 182,780 (Faslodex; fulvestrant) (21) and progesterone (P4) on the regulation of ER{alpha} isoform expression in the fallopian tube; 26-day-old female mice received either E2 (0.5 µg/g BW sc) or PPT (100 µg/g BW sc) for 4 days and, in addition, an intraperitoneal injection of either 8.3 mg ICI 182,780 (Tocris Cookson)/kg BW, 2.5 mg P4/each animal in 100 µl of sesame oil (vehicle), or vehicle alone for 1–2 days.

The aim of experiment 4 was to investigate the effects of E2 on tubal mass, morphology, protein content, IGF-I, IGF-II, and VEGF expression in mouse fallopian tube tissues and fluid; 26-day-old female mice received either E2 (0.5 µg/g BW sc) in 100 µl of sesame oil (vehicle) or an equal volume of sesame oil for 4 days and, additionally, were injected intraperitoneally with 8.3 mg ICI 182,780/kg BW in 100 µl of sesame oil for 2 days. Paraffin tubal sections were stained with Mayer hematoxylin (Histolab Products, Frölunda, Sweden).

The aim of experiment 5 was to determine whether ER{alpha}-mediated regulation of IGF-I and IGF-II production depends on GH signaling in mouse fallopian tube tissue and fluid; 26-day-old female GHR–/– mice and +/+ littermates were injected with either E2 (0.5 µg/g BW sc) in 100 µl of sesame oil (vehicle) or an equal volume of sesame oil for 4 days.

While mouse was under anesthesia, fallopian tube was removed and stripped of fat and connective tissue and weighed. One side of the fallopian tube in each animal was immediately frozen in liquid nitrogen and stored at –70°C for subsequent Western blot analysis. The other side was fixed in 4% formaldehyde neutral buffered solution for 24 h at 4°C and embedded in paraffin for histochemical analysis. The protein from mouse fallopian tubes from 5–10 animals in each group was pooled and processed for Western blot analysis as described below. Protein was harvested from a matched portion of the organ in all experiments. While treatment with oil lacked an estrogenic action on mouse uteri, E2, E2 + ICI 182,780, PPT, PPT + ICI 182,780, and PPT + P4 induced similar uterine ballooning; constant estrogen administration increases in uterine weight, whereas ER antagonist displays an opposite effect. This confirms that ER{alpha} mediates the effect on uterine weight. In addition, the fallopian tube tissues from experimental animals were collected in microcentrifuge tubes (Brand, Wertheim, Germany), and tubal fluid was extracted using a pipette tip (Molecular BioProducts, San Diego, CA). Changes in protein content and growth factor production in the intraluminal tubal fluid were analyzed following in vivo extraction of fluid. Protein concentration of tubal flushing was determined using a bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL).

Protein extraction and Western blot analysis. Whole tissue extracts for protein preparations were essentially carried out as described previously (55, 57); 10 mM iodoacetamide was included in each buffer used for protein preparations to prevent nonspecific disulfide linkage, and 1 µM 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). Western blot analyses were performed using standard procedures to evaluate the abundance and distribution of ER{alpha}, ERbeta, beta-actin, IGF-I, IGF-II, and VEGF (55, 57). For each time point or treatment, equivalent amounts of protein from at least five individual fallopian tube extracts were pooled; 30 µg of protein were directly electrophoresed on either 4–12% Bis-Tris gels or 10–20% Tris-glycine gels (Novex, San Diego, CA). Blots generated with these extracts were probed with primary antibodies. The immunosignal-CDP-Star substrate for alkaline phosphatase system (Tropix, Bedford, MA) 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 enhanced chemiluminescence film (Amersham). Individual bands were quantified directly from membranes by densitometry using Image Gauge software (Fujifilm). Signal intensities of the mouse 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 (55, 57). All steps were carried out at room temperature unless otherwise stated.

Immunohistochemical analyses and microscopy. Two different methods of immunohistochemistry were performed and confirmed in the same mouse fallopian tube tissue. Single-diaminobenzidine (DAB) (55, 57) and dual-fluorescence (56) immunohistochemistries were based on methodology described previously. After deparaffinization and rehydration, antigen retrieval was completed with 10 mM sodium citrate buffer (pH 6.0, 10 min in a 700-W 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 (Japan) under bright-field optics and photomicrographed using Easy Image 1 (Bergström Instrument).

For single- or 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 primary antibody in PBS supplemented with Triton X-100 (PBST) containing 1% BSA-3% fat-free milk overnight at 4°C. After a washing in PBST for 3 x 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, Cy3-conjugated anti-mouse antibody, or Cy3-conjugated anti-rabbit antibody, was employed for different ER subtype and IGF-I and IGF-II 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 (Zeiss, Jena, Germany) equipped with a laser-scanning confocal imaging LSM 510 META system (Carl Zeiss, Jena, Germany) 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 no cross-reactivity between the two staining sequences. Mouse ovarian and uterine tissues served as a positive control and were stained as above for ER subtypes (data not shown). Tissues from a minimum of five animals at each treatment were evaluated to ensure the reproducibility of the results. The immunohistochemical findings illustrated are representative of those observed in random sections from multiple animals. The resulting stain was evaluated blind by two observers.

Serum hormone assay. Trunk blood was collected following cardiac puncture. Serum was collected following centrifugation (14,000 rpm, 4°C, 10 min) and stored at –135°C. Concentrations of estradiol and P4 were measured by radioimmunoassay according to a protocol provided by the manufacturer (PerkinElmer Life and Analytical Sciences, Wallac, Turku, Finland) as described previously (57). The sensitivity of the assay was typically better than 50 pmol/l for estradiol and 0.8 nmol/l for P4. The intra-assay coefficient of variation was 3.8–10% for estradiol and 3.3–7.3% for P4.

Statistical analysis. Data are presented as means ± SE of the number of independent experiments indicated. Multiple comparisons among data were performed using one-way ANOVA with correction of P values using the Bonferroni multiple range test in the Analyse-It program (Analyse-It software). A value of P < 0.05 was set as the limit of statistical significance.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Different expression levels of ER{alpha} isoforms in the reproductive tissues of prepubertal mice. The pattern of ER{alpha} isoform protein levels was determined in prepubertal mouse (26 days old) ovary, fallopian tube, and uterus (Fig. 2). Western blot analysis showed that three immunoreactive bands of the correct molecular mass of ER{alpha} isoforms were differently expressed in several tissues (43). The immunoreactivity of these bands was absent in uterine tissues of ER{alpha}/beta–/– and ER{alpha}–/– mice, confirming the specificity of the reaction (Fig. 1). ER{alpha}1 was the most abundant isoform expressed in all tissues examined, in agreement with the predicted molecular mass of the full-length ER{alpha} (~66 kDa) (43). Densitometric analysis indicated that levels of ER{alpha}1 expression in the fallopian tube ≥ uterus >>> ovary. Furthermore, two short forms of ER{alpha}, ER{alpha}2 and ER{alpha}3, were distinctly expressed in the fallopian tube and uterus. In contrast, both ER{alpha}2 and ER{alpha}3 expression remained extremely low in the ovary (Fig. 2).


Figure 2
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  		Fig. 2. Expression of ER{alpha} isoforms in mouse reproductive tissues. Western blot analysis of ovary, fallopian tube, and uterus from 26-day-old (26d old) mice (n = 3). Protein extracts from total lysates (30 µg/lane) were analyzed using antibodies against ER{alpha} (MC-20) and beta-actin as described in MATERIALS AND METHODS. The correct loading was evaluated by both expressing beta-actin and staining the gels with Coomassie blue. Relative levels of ER{alpha} protein isoforms were expressed as a ratio of ER densitometric value to whole proteins in Coomassie blue staining. ADU, arbitrary densitometric units.

 
ER{alpha} protein was localized in the nuclei of fallopian tube principal cells. A previous study from our laboratory demonstrated distinct cellular localization of ER{alpha} and ERbeta in rat fallopian tubes (56). To clearly visualize the differential expression of these two ER subtypes in different cell types in mouse fallopian tube, we performed dual immunofluorescence on paraffin sections of prepubertal mouse fallopian tube. No difference of ER{alpha} localization was detected in any particular tubal region (the infundibulum, the ampulla, and the isthmus). Specific immunostaining of ER{alpha} was seen in the principal cells of the fallopian tube, including epithelial cells, as well as smooth muscle cells. Moreover, the immunostaining of ER{alpha} was found in both secretory and ciliated epithelial cells, and no difference in the expression intensity was noted between those two cell types. ER{alpha} immunofluorescence was particularly intense in cell nuclei (Fig. 3, left). In contrast, intense ERbeta immunoreactivity was mainly detected in the ciliated epithelial cells (Fig. 3, left and right), in keeping with findings from studies performed in rats (56). These histological demonstrations together with our previous study (56) suggest that the two ER subtypes are differentially expressed depending on the cell type in rodent fallopian tube.


Figure 3
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  		Fig. 3. Localization of endogenous ER proteins in mouse fallopian tubes. Tubal sections were immunolabeled for ER{alpha} (6F11) 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. Confocal microscopy revealed that the patterns of ER{alpha} and ERbeta expression were not overlapping. Whereas ER{alpha} was ubiquitously expressed in the nuclei of all cell types, ERbeta was mainly detected in the surface of tubal epithelium. Sections were subsequently counterstained with DAPI to visualize cell nuclei (right). Using tubal tissues from different mice (n = 5), the same results were obtained for replicate experiments. Scale bar is shown; epi and m indicate epithelial cells and smooth muscle cells, respectively.

 
Downregulation of ER{alpha} isoforms by exogenous estrogens in mouse fallopian tube. Next, we determined whether the expression levels of ER{alpha} isoforms in mouse fallopian tube would be specifically regulated by long-term estrogen stimulation. Following daily treatment of prepubertal mice with E2 or PPT, a synthetic ER{alpha} agonist (for longer periods of time; up to 4 days), the relative abundance of the ER{alpha} protein levels with all isoforms was gradually decreased (Fig. 4A). Three tubal ER{alpha} isoforms displayed a similar regulation pattern after different treatment. Additionally, fallopian tubes from mice treated with oil (Fig. 4B) or diarylpropionitrile (a synthetic ERbeta agonist) (41) (data not shown) for the same time periods did not change the levels of ER{alpha} isoforms or ERbeta expression. No appreciable changes in serum estradiol and P4 levels were observed between control mice (oil treatment) and E2/PPT-treated mice (Fig. 4C).


Figure 4
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  		Fig. 4. Time-course effect of exogenous ER{alpha} agonists on ER{alpha} isoform expression as well as serum estradiol and progesterone (P4) concentrations in mouse fallopian tubes; a representative Western blot of tubal proteins for ER{alpha} isoforms of 3 experiments carried out with different tubal preparations. Extracts were prepared following 17beta-estradiol (E2) or PPT (A) or oil (B) treatment at the time intervals indicated. Blot was probed with specific antibody against ER{alpha} (MC-20) as described in MATERIALS AND METHODS. The proper loading was evaluated by both expressing beta-actin and staining the gels with Coomassie blue. All 3 tubal ER{alpha} isoforms display a similar regulation pattern following E2 or PPT treatments. Relative levels of ER{alpha}1 and total ER{alpha} (ER{alpha}1 + ER{alpha}2 + ER{alpha}3) proteins were expressed as a ratio of ER{alpha} densitometric value to whole proteins in Coomassie blue staining. Graphed results are means ± SE of 3 independent determinations, each performed in 5 mice. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. each control animal (day 0), respectively. Statistical analysis was performed as described in MATERIALS AND METHODS. Trunk blood was obtained from heart puncture. Sera were collected after clotting and centrifugation. Serum estradiol and P4 concentrations (C) were measured in duplicate aliquots of each sample. The no. of mice per group is indicated.

 
ICI 182,780 and P4 reversed the effect of exogenous estrogens in mouse fallopian tube. To verify whether regulation of the ER{alpha} isoform expression was estrogen dependent in estrogen-primed mouse fallopian tubes, we investigated the effect of ICI 182,780, a synthetic ER antagonist, which blocks the estrogen-induced transcriptional activity of the receptor (21). Western blot analysis showed that subsequent treatment with ICI 182,780 resulted in upregulation of ER{alpha} isoform expression in E2- and PPT-primed mice (Fig. 5A, left and right), suggesting the importance of autologous regulation of ER{alpha} protein in mouse fallopian tube. However, the blocking capability of ICI 182,780 (at comparable levels at 1 day) in PPT-primed mice was considerably less than that observed in E2-primed mice. This difference may be explained by the possibility that ICI 182,780 has different anti-estrogen activity regarding E2 and PPT or that the effect of ICI 182,780 in diminishing estrogen activity is time dependent. While treatment of E2- and PPT-primed mice with ICI 182,780 resulted in significantly higher serum E2 levels, it was noted that serum levels of P4 were increased only in E2-primed mice treated with ICI 182,780 (Fig. 5B). To evaluate the specificity of estrogen action in mouse fallopian tube, we demonstrated the effect of P4 on ER{alpha} isoform expression. Treatment with P4 also completely abolished the estrogenic effect on ER{alpha} isoform expression (Fig. 5A, right), an effect similar to that observed in mice treated with ICI 182,780. Furthermore, fallopian tubes from mice treated with the same dose of P4 alone for the longer period of time had upregulated levels of ER{alpha} isoform expression (data not shown). These results suggest that the effect of P4 is antagonistic to that of estrogen in the regulation of ER{alpha} isoform expression in mouse fallopian tube.


Figure 5
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  		Fig. 5. Effect of ICI 182, 780 (ICI), P4, or oil on ER{alpha} isoform expression as well as serum estradiol and P4 concentrations in E2- or PPT-primed mouse fallopian tubes; a representative Western blot of tubal proteins for ER{alpha} isoforms of 3 experiments carried out with different tubal preparations. Extracts were prepared following combined treatment with ICI 182, 780, oil, or P4 and E2 (A, left)- or PPT-primed (A, right) mice. Blot was probed with specific antibody against ER{alpha} (MC-20) as described in MATERIALS AND METHODS. The proper loading was evaluated by both expressing beta-actin and staining the gels with Coomassie blue. All 3 tubal ER{alpha} isoforms display a similar regulation pattern following different treatments. Relative levels of ER{alpha}1 and total ER{alpha} (ER{alpha}1 + ER{alpha}2 + ER{alpha}3) proteins were expressed as a ratio of ER{alpha} densitometric value to whole proteins in Coomassie blue staining. Trunk blood was obtained from heart puncture. Sera were collected after clotting and centrifugation. Serum estradiol and P4 concentrations (B) were measured in duplicate aliquots of each sample. The no. of mice per group is indicated. Graphed results are means ± SE of 3 independent determinations, each performed in 5 mice. **P < 0.01 and ***P < 0.001 vs. animals treated with oil (4 days), respectively. Statistical analysis was performed as described under MATERIALS AND METHODS.

 
ER{alpha} protein location was oppositely regulated by E2 and ICI 182,780 in tubal epithelial cells. Although no significant differences were observed in the expression level of ER{alpha} in all fallopian tube regions in immature mice, it was important to analyze possible differences in the localization patterns of ER{alpha} in different tubal regions under estrogen and/or ER antagonist treatment(s) because of the specialized functions of distinct tubal regions (24) and the substantial effects of estrogen on tissue histology. Cellular localization of ER{alpha} was performed by immunohistochemistry on fallopian tube sections from mice treated with oil, E2, or E2 + ICI 182,780. The predominant nuclear localization pattern of ER{alpha} seen in mice treated with oil for 4 days (Fig. 6, A–C) closely resembled those seen in prepubertal mice (Fig. 3), although two different antibodies were used in these respective studies. Treatment with E2 led to a marked decrease of ER{alpha} immunoreactivity in the nuclei of epithelial cells, and there were no regional differences observed (Fig. 6, D–F). Similar localization changes were also found in mice treated with PPT for 4 days (data not shown). However, compared with E2 treatment, immunoreactivity of ER{alpha} was only increased in the epithelial cell nuclei of the ampullary region after additional ICI 182,780 treatment (Fig. 6H), whereas no changes were observed in the infundibulum (Fig. 6G) and isthmus (Fig. 6I) regions. Moreover, it is surprising to note that both estrogens and ICI 182,780 had no effect on ER{alpha} expression in the smooth muscle cells (Fig. 6, E, F, H, and I). The presence of neutralizing ER{alpha} antibody was also examined in an adjacent tubal section (Fig. 6J).


Figure 6
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  		Fig. 6. Effect of E2 and ICI 182,780 on ER{alpha} localization in mouse fallopian tubes. Tubal sections were immunolabeled for ER{alpha} (MC-20) as described in MATERIALS AND METHODS. Bright-field microscopy revealed that lower intensity of ER{alpha} staining in the epithelial cells was seen after 4 days of E2 treatment without any regional differences, whereas ICI 182, 780 only blocked the E2-induced decreases in ER{alpha} immunoreactivity in the epithelial cells within the ampullary region. There were no obvious changes in ER{alpha} immunoreactivity observed in smooth muscle cells during E2 and ICI 182, 780 treatment. With the use of tubal tissues from different mice (n = 5), the same results were obtained for replicate experiments. Scale bar is shown. BP, a specific blocking peptide.

 
ER{alpha} is functionally connected to tubal growth and regulation of protein content and growth factor expression. In general, the distribution of a protein in a certain tissue/cell is closely associated with its function in vivo. Since the importance of estrogen-mediated effects on fallopian tube function has been acknowledged (24), we focused our attention on long-term E2-induced regulation of ER{alpha} and its implications in the biological changes in mouse fallopian tube. We observed a gain of tubal weight following daily treatment with E2 for 4 days in mice compared with treatment with/without oil, whereas subsequent treatment with ICI 182,780 resulted in a loss of tubal weight (Fig. 7A). Histological examination of sections from the E2-treated fallopian tubes showed an increase in tubal diameter, luminal space, and epithelial cell height compared with fallopian tubes treated with oil (Fig. 7B). It is likely that the long-term estrogenic effect on regulation of ER{alpha} was accompanied by important modifications in tubal epithelium composition, morphology, and formation of the intraluminal tubal fluid. Immunoreactivity for IGF-I and IGF-II was predominantly localized in the epithelial cell cytoplasm and was uniformly present in both secretory and ciliated cells (Fig. 7D). Additionally, smooth muscle cells were negative for both IGF-I and IGF-II (Fig. 7D). Since ER{alpha} was the most prominently expressed ER subtype in multiple cell types (Figs. 3 and 5, A–C) within the fallopian tube, including epithelial cells, which are the main cell type to product and secrete proteins into tubal lumen, we next considered the possibility that regulation of ER{alpha} may be responsible for the changes in tubal protein production and secretion. Analysis of protein content in the intraluminal tubal fluid could provide an indirect measure of protein secretion from epithelial cells in the fallopian tube. Treatment with E2 resulted in a significant increase in protein content in the tubal fluid (Fig. 7C). Importantly, estrogen treatment was able to increase IGF-I and IGF-II expression in tubal epithelial cells (Fig. 7D) and also induce IGF secretion into the tubal fluid (Fig. 7E). These estrogenic effects were abolished by subsequent treatment with ICI 182,780 (Fig. 7, C and E), suggesting that estrogen acting via its receptor controls IGF regulation.


Figure 7
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  		Fig. 7. Relative effects of E2 and ICI 182, 780 on tubal weight (A); cross-sectional histology (B); protein content (C); and IGF-I, IGF-II, and VEGF expression (D and E) in mouse fallopian tube tissues and fluid. Histological appearance of representative fallopian tubes treated with oil and E2 (B), demonstrating that increases in lumen spaces in the ampullary region were observed in mice treated with E2. Samples from mouse fallopian tube fluid were subjected to SDS-PAGE, and the gel was stained with Coomassie blue. Gel lanes were loaded with either equivalent volumes (C, left) or equivalent concentration (C, right) of pooled tubal fluid from untreated mice (n = 20) or mice treated with oil (n = 20), E2 (n = 15), oil + ICI 182, 780 (n = 15), or E2 + ICI 182, 780 (n = 15). Tubal fluid with equivalent concentration was also used for Western blot analysis as described in MATERIALS AND METHODS to determine IGF-I, IGF-II, and VEGF protein expression (E). Experiments were performed independently twice, and similar results were observed. D: effect of E2 on IGF-I and IGF-II localization in mouse fallopian tubes. Confocal microscopy revealed that both IGF-I and IGF-II were specifically localized in the epithelial cell cytoplasm, and higher intensity of IGF-I and IGF-II staining in the epithelial cells was seen after 4 days of E2 treatment, evident throughout mouse fallopian tubes. With the use of tubal tissues from different mice (n = 5), the same results were obtained for replicate experiments. Scale bar is shown in B and D; lu, epi, and m indicate lumen, epithelial cells, and smooth muscle cells, respectively.

 
Estrogen is a major GH-independent regulator of IGF synthesis in mouse fallopian tube tissues and fluid. Previous studies have demonstrated that GH is the main regulator of IGF expression and secretion in most tissues including the female reproductive organs (22, 23). We wanted to determine whether estrogen-induced IGF production and secretion in mouse fallopian tube tissues and fluid (see Fig. 7, D and E) require GH acting via GHR signaling. Histological examination of tubal sections from wild-type and GHR knockout mice did not reveal obvious changes in tubal morphology or ER{alpha} distribution and localization (data not shown). Furthermore, there were no differences in ER{alpha} isoform expression between wild-type and GHR knockout mice (Fig. 8A). Western blot analysis showed that treatment with E2 resulted in downregulation of all ER{alpha} isoforms in both wild-type and GHR knockout mice (Fig. 8A). Additionally, increases in protein content and IGF production in the intraluminal tubal fluid after exposure to long-term estrogen were observed in both wild-type and GHR knockout mice (Fig. 8, B and C), establishing that GH signaling is not essential for estrogen-induced regulation of IGF-I and IGF-II production in the fallopian tube tissues and fluid.


Figure 8
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  		Fig. 8. Relative effects of E2 on tubal ER{alpha} isoform expression (A); protein content (B); and IGF-I, IGF-II, and VEGF expression (C) in WT and growth hormone receptor (GHR) KO mice. Representative Western blots of tubal proteins for ER{alpha} isoforms (A). Nos. 1 and 2 represent sample preparation using different mice. All extracts were prepared during E2 treatment at the time intervals indicated in WT and GHR KO mice. Blot was probed with specific antibody against ER{alpha} (MC-20) as described in MATERIALS AND METHODS. The proper loading was evaluated by both expressing beta-actin and staining the gels with Coomassie blue. Samples from fallopian tube fluid were subjected to SDS-PAGE, and the gel was stained with Coomassie blue. Gel lanes were loaded with either equivalent volumes (B, left) or equivalent concentration (B, right) of pooled tubal fluid from E2-primed WT mice (n = 10) and GHR KO mice (n = 10). Tubal fluid with equivalent concentration was also used for Western blot analysis as described in MATERIALS AND METHODS to determine IGF-I, IGF-II, and VEGF protein expression (C).

 

   DISCUSSION
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Although evidence from both clinical studies and animal experiments indicates that estrogen controls several fundamental aspects of fallopian tube function, including regulation of tubal protein synthesis and secretion (1, 4, 29, 30, 34, 63), the molecular mechanisms behind these estrogen-dependent effects are not well defined. This is partly due to the presence of different ER subtypes in the fallopian tube. Moreover, multiple isoforms for both ER subtypes have been identified in different tissues and cell types (2, 3, 32, 42). In the present study, we demonstrate for the first time that three ER{alpha} isoforms are expressed in mouse fallopian tube, and the estrogen-dependent regulation of these isoforms is closely associated with changes in tubal morphology and protein content in the intraluminal tubal fluid. In particular, a clear upregulation of IGF-I and IGF-II expression is observed in both tubal tissues and fluid after E2 stimulation. Furthermore, we provide direct evidence that regulation of tubal tissue and fluid IGFs is estrogen dependent through activation of ER{alpha} and does not depend on GH/GHR signaling.

We first investigated the cell type-specific expression of ER{alpha} and ERbeta subtypes in mouse fallopian tube. Using two different ER{alpha} antibodies (MC-20 and 6F11), a strong immunopositive signal for ER{alpha} was detected in the nuclei of epithelial and smooth muscle cells, consistent with previous studies in rat fallopian tube (39, 50, 54, 56). Moreover, ER{alpha} protein was localized in both ciliated and secretory epithelial cells, whereas distribution of ERbeta was restricted mainly to the ciliated epithelial cells. Dual-fluorescence immunohistochemistry revealed that the co-localization of ER{alpha} and ERbeta expression in mouse fallopian tube is similar to that in rat fallopian tube (56), and that the distinct expression patterns of ER{alpha} and ERbeta may reflect the diversity of their biological functions in rodent fallopian tube. Mammalian fallopian tube epithelium, which consists of two cell types, ciliated and secretory cells, performs critical functions in reproductive events leading up to the establishment and maintenance of pregnancy (24). While ciliated epithelial cells support gamete transport, the functional properties of secretory cells are especially suited to regulate protein synthesis and contribute to the formation of the intraluminal tubal fluid (1, 30). In fact, the effect of short-term treatment with E2 (within 24 h) on ciliated ERbeta expression strongly suggests that ERbeta might be crucial for the regulation of calcium-dependent ciliary beating (56). Since ERbeta was not found in the secretory epithelial cells, and the level of ERbeta expression was not regulated by long-term estrogen stimulation (4 days, data not shown), one possibility would be that ER{alpha} is the primary receptor subtype involved in E2-induced tubal secretory processes.

As previously shown for ER{alpha} isoforms in rat tissues in vivo (2, 3, 52), Western blot analysis from the present study revealed that three ER{alpha} protein isoforms were present in mouse fallopian tube. While sequence analyses of ER{alpha} isoforms reveal that they differ in the pattern of single or multiple exon deletion(s) and NH2/COOH-terminal truncations (43), the question as to the whether individual ER{alpha} isoforms in the estrogen target tissues/cells remains open. To determine the functional importance of these ER{alpha} isoforms in mouse fallopian tube, we demonstrated time-dependent regulation of ER{alpha} isoform expression and localization. Long-term treatment (4 days) with either E2 or PPT, an ER{alpha} agonist, resulted in a parallel downregulation of all three ER{alpha} isoforms in mouse fallopian tube, demonstrating that regulation/activation of estrogen-induced ER{alpha} is not isoform specific in mouse fallopian tube. The time-dependent downregulation of ER{alpha} isoforms was also evidenced in the fallopian tube from immature mice treated with diethylstilbestrol (a nonsteroid synthetic estrogen) or equine chorionic gonadotropin, which induced endogenous E2 levels (55, 57) (data not shown), indicating a specific estrogenic regulation of ER{alpha} isoform expression. Moreover, immunohistochemical analysis demonstrated that the number of ER{alpha}-immunoreactive nuclei in the epithelial cells within all tubal regions was decreased after estrogen treatment. However, subsequent treatment with ICI 182,780 reversed this estrogenic effect on ER{alpha} isoform expression, with the number of labeled epithelial cell nuclei being increased in the ampullary region compared with the infundibulum and isthmus regions. In addition, in the same tubal tissues, smooth muscle cell ER{alpha} immunoreactivity was unchanged, regardless of estrogen and/or ICI 182,780 treatment. These observations suggest cell type- and region-specific estrogenic regulation of ER{alpha} expression in mouse fallopian tube. In our studies, we note that the expression pattern of ER{alpha} isoforms in mouse fallopian tube is similar to that in rat fallopian tube (58), but it differs from other tissues previously studied. For example, in rat skeletal muscle and adipose tissue, ER{alpha} proteins with molecular masses of 80, 66, and 46 kDa (but not 55 kDa) were detected (3). However, the anti-ER{alpha} antibody used in the present study revealed a double band at 67 and 54 kDa in rat placenta (2) and four molecular masses (67, 61.8, 53, and 45 kDa) in rat pituitary (52). Since the rat ER{alpha} and mouse ER{alpha} share a high degree of sequence homology, it is plausible that the antibody used would also recognize all corresponding mouse ER{alpha} isoforms. Thus it is unlikely that there are differences in the expression of ER{alpha} isoforms between rat and mouse. With these findings in mind, it is very likely that the expression pattern of ER{alpha} isoforms is specific to each tissue. This possibility merits investigation in the future. Traditionally, estrogen acts in tissue/cells through the genomic (i.e., nuclear initiated) activities of ER{alpha} and/or ERbeta (20, 48). Recent evidence has emerged indicating the nongenomic (i.e., membrane initiated) activation of ER by estrogen in a variety of cell types (48). Although it is still controversial as to whether one or both of the nuclear ERs are similar to the putative membrane ER, a rapid nongenomic response to E2 was characterized in osteoblasts and endothelial cells and was shown to occur via one ER{alpha} isoform (46 kDa) (43). Such a mechanism for E2-mediated cell signaling has not been established in the fallopian tube. Further study is needed to dissect the specific ER{alpha} isoform-mediated signaling pathway from ER-signaling pathways that regulate fallopian tube function.

Regarding physiological function, several studies have shown that the morphology and the functional integrity of the fallopian tube is estrogen dependent (24), but the mechanism by which simultaneous specific inputs from estrogen lead to ER subtype-dependent biological outputs remains unresolved. We therefore were interested in determining the physiological relevance of the tight regulation of ER{alpha} expression after long-term estrogen stimulation. Successive treatment with E2 in mouse fallopian tube induced increases in tubal wet weight with considerable cellular hypertrophy and protein synthesis in the tubal fluid, consistent with the fact that, under estrogen regulation, the tubal epithelial cells produce proteins, components of intraluminal tubal fluid (1, 4, 29, 30, 34). These results suggest that estrogens and ER can exert important actions on metabolism. It has previously been reported that more tubal fluid is produced in the ampullary region than in other tubal regions; the ampullary region is associated with estrogen levels in several animal species in vivo (30). In this study, increased ER{alpha} expression following ICI 182,780 treatment was only observed in the epithelial cells of the ampullary region. Thus we hypothesized that the decrease in fluid volume may result from blocking of long-term estrogenic actions on ER{alpha} in the ampullary region. Furthermore, the inhibition of an estrogenic effect on protein content in the intraluminal tubal fluid supports our theory that estrogen-dependent regulation of cell type- and region-specific ER{alpha} expression is relevant for the biological processes of tubal protein synthesis and secretion.

Although the protein composition of fallopian tube fluid has been characterized in different species (4, 60), the regulation of the production and secretion of individual components remains largely unknown. In the present study, we focused on long-term estrogen-induced regulation of IGF-I and IGF-II in mouse fallopian tube tissues and fluid. Immunochemical analysis demonstrated localization of IGF-I and IGF-II in the cytoplasm of all epithelial cells. In combination with the results from immunostaining of ER subtypes, it is likely that IGF-I/II is co-localized with ER{alpha} in the tubal epithelial cells. Furthermore, treatment with E2 increased immunoreactivity for both IGF-I and IGF-II, partly confirming previous findings in both mouse and rat fallopian tubes (5, 10). In addition to upregulation of IGF-I and IGF-II in tubal tissues, we also found a significant increase in IGF-I protein level in the intraluminal tubal fluid in response to E2 treatment. Since there were no changes in expression of VEGF in the intraluminal tubal fluid after E2 and/or ICI 182,780 treatment, these data indicate the specificity of the IGF response to estrogenic stimulation. Furthermore, the release of IGF-I into luminal fluid was abolished by ICI 182,780 treatment, demonstrating an ER-mediated regulation of IGF-I production and secretion. Although ICI 182,780 usually results in an attenuated cellular response of E2 via antagonism at both ER subtypes (21), extensive work has been performed suggesting that estrogen-mediated regulation of IGF-I is mainly through ER{alpha} and not through ERbeta in culture cell lines in vitro (13, 62) and in rat uterus in vivo (27). Thus our findings indicate that the functional significance of ER{alpha} is closely related to IGF-I regulation in mouse fallopian tube tissues and fluid in vivo. Unfortunately, our data do not allow us to determine whether one specific ER{alpha} isoform acts as a major mediator regulating IGF-I and IGF-II expression or whether all ER{alpha} isoforms are required for this regulation. On the other hand, IGF-I is able to regulate uterine epithelial cell function via ER{alpha} in the absence of E2 (27). This complex interaction and additional effect of IGF-I on the regulation of ER{alpha} expression in the fallopian tube warrant further study.

It is well know that GH, an important regulator for endocrine and local IGF-I stimulation, conveys its action by binding to and activating the GHR (22, 23). Deletion of GHR in mice decreases circulating and ovarian IGF-I expression associated with lower levels of endogenous E2 compared with wild-type animals (67, 68). One important finding that came out of this study was that no obvious differences in ER{alpha} isoform expression and morphological changes (data not shown) in the fallopian tube were found between wild-type and GHR gene-disrupted mice (26 days old). Furthermore, we observed that immature GHR gene-disrupted mice responded to E2 stimulation in a manner similar to that of wild-type mice, including stimulation of tubal growth and increases in protein content and IGF production in the intraluminal tubal fluid. The present data provide valuable direct evidence that estrogen rather than GH is a major determinant of GH-independent IGF synthesis in mouse fallopian tube.

The present study has provided evidence of the existence and functionality of ER{alpha} isoforms in mouse fallopian tube. From a physiological perspective, we have identified a molecular mechanism for ER{alpha}-mediated tubal protein synthesis and secretion that involves the maturation of gametes and initial embryonic development. Furthermore, the full recovery of IGF-I and IGF-II production in long-term E2 treatment of GHR gene-disrupted mice supports the concept that ER{alpha} regulates IGF synthesis and secretion in mouse fallopian tube. Our findings provide a mechanistic link among estrogens, ER{alpha} signaling, and formation of tubal fluid. ERs are important therapeutic targets, and while estrogenic agents are widely used for emergency contraception (18) and menopausal hormone replacement therapy (36), anti-estrogens are in clinical trials for the first-line treatment of infertility (31), although they increase the rate of tubal ectopic pregnancies (26, 40). Given the differences in the regulation of ER subtypes in the fallopian tube, it will be critical to distinguish which ER subtype is obligatory for fallopian tube physiology.


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
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This work was supported by grant no. 10380 from the Swedish Medical Research Council (to H. Billig) and by the Svenska Sällskapet för Medicinsk Forskning and Göteborgs Läkaresällskap; the Scientific Foundation of Fred G. and Emma E. Kanolds, Eva and Oscar Ahrens, Iwan and Eleonore Ljunggrens, Wilhelm and Martina Lundgrens, Hjalmar Svenssons, and Emil and Maria Palms; and the Adlerbertska Research Foundation (to R. Shao). J. J. Kopchick is supported by the State of Ohio's Eminent Scholars Program, which includes a gift by Milton and Lawrence Goll.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. Shao, Dept. of Physiology/Endocrinology, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at Gothenburg Univ., 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.


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