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Female Reproductive Tract |
Department of Biology, University of North Carolina at Charlotte, Charlotte, North Carolina 28223
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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estradiol, female reproductive tract, progesterone, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1-protease inhibitor and others (up to a molecular size of 720 kDa) into the uterus [1]. Consequently, this movement would set up a strong osmotic gradient that would then drive the concomitant movement of water into the tissue. Chaves et al. [3] have suggested that uterine edema may be induced by nitric oxide (NO), a potent vasodilator. In these studies, pretreatment with L-nitroarginine methyl ester (L-NAME; a competitive antagonist of NO production) caused a dose-dependent reduction of E2-induced uterine edema, which could be reversed by administration of L-arginine (a NO precursor). This water moves from the vascular supply to the stroma and into the lumen of the uterus [4]. Edema is observed within the stroma, whereas minimal edema is seen in the myometrium. In addition, the stromal edema is continuous and not limited to areas directly around capillaries. Although the effect of E2 on water transport into and within the mammalian uterus is well known, the mechanisms responsible for trafficking this water have not yet been identified. Water movement across cells can occur by diffusion through either the lipid bilayer or protein water channels termed aquaporins (AQPs). These water channels are found in tissues that exhibit rapid and regulated movement of water, such as the kidney and lung [5, 6]. Structurally, the AQPs consist of six transmembrane spanning domains and two hemichannels, each with a highly conserved NPA (asparagine, proline, alanine) motif [7]. These hemichannels are found on loops B and E and form an hourglass-shaped channel through which water may pass [7]. Each AQP monomer appears to constitute a functional water channel, although the proteins are found as homotetramers in the plasma membrane [8].
Previous studies have shown that specific AQP isotypes are expressed in both the male and female reproductive tissues of the rat, mouse, marmoset, and human. For example, AQP-7 and -8 have been shown to be expressed in the rat testis and may have a role in testicular development [9]. In another study, AQP-1 was localized in the apical brush border of the epithelial cells lining the efferent ducts of the epididymis in the male marmoset and rat [10]. In the female reproductive tract, our laboratory has found AQP-7, -8, and -9 to be present in the granulosa cells of the rat ovary [11]. Two separate experiments by Li et al. [12, 13] found AQP-1 mRNA to be present in the human and rat uterus and regulated by E2 in the rat, whereas Gannon et al. [14] conducted protein studies that localized AQP-1 to visceral smooth muscle cells of the fallopian tubes and vagina in the rat. Recently, Richard et al. [15] described the expression profile of AQPs 0–9 in the peri-implantation mouse uterus. This study used in situ hybridization to analyze the expression profile of AQPs on Days 1–8 of pregnancy in the mouse and found expression of AQP-1, -4, and -5 and AQP-5 protein expression in a Day 5 pregnant mouse uterus. They then determined the localization of mRNAs of AQP-1, -4, and -5 in an ovariectomized, hormonally treated mouse uterus by in situ hybridization. However, no studies to date have examined the expression of AQP family members in the ovariectomized mouse uterus at the protein level or changes in the functional activity of these proteins in response to ovarian hormones. Thus, the purpose of this study was to assess the expression profile of the AQP family members in the mouse uterus and to determine if this expression is regulated by E2 or progesterone (P4). Additionally, we will determine if E2 treatment will increase the water permeability of luminal epithelial cells at a time when there is a great reduction in viscosity of the luminal fluid.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Female CD1 mice weighing 20–25 g (Charlas River Co., Raleigh, NC) were ovariectomized and allowed to recover for 1 wk. Short-term effects of ovarian steroid hormones were determined by giving single injections of E2 (100 ng subcutaneously in corn oil; Sigma Chemical Co., St. Louis, MO), P4 (2 mg subcutaneously in corn oil; Sigma), corn oil (0.1 ml subcutaneously; Sigma, vehicle control), or ICI 182,780 (E2 antagonist, 500 ng subcutaneously in corn oil; Tocris Cookson Inc., Ellisville, MO) [16] followed 1 h later by injection with E2 (100 ng). An additional group was injected with P4 (2 mg subcutaneously in corn oil) followed by injections of P4 with E2 (2 and 100 ng, respectively) 24 h later. All uteri were harvested at 2 or 6 h after final injection. Kidney, salivary glands, and peritoneal adipose tissue were harvested from normal CD1 mice as positive controls. All experimental protocols were approved by the Institutional Animal Care and Use Committee at the University of North Carolina at Charlotte and were performed in accordance with the guidelines set forth in the National Institutes of Health Guide for the Care and Use of Laboratory Animals, published by the U.S. Public Health Service.
Immunofluorescence
Uteri were excised and fixed in 4% paraformaldehyde for 24 h at 4°C. Fixed uterine pieces were dehydrated in ascending grades of ethanol, washed in Hemo-De (Fisher Scientific, Pittsburgh, PA), and embedded in paraffin. Paraffin sections (4 µm) were adhered to positively charged glass slides (SuperFrost Plus; Fisher Scientific), deparaffinized in Hemo-De, and rehydrated in descending grades of ethanol followed by a final incubation in PBS. Sections were incubated in blocking solution (10% normal goat serum) for 20 min before incubation with primary antibodies.
Anti-peptide AQP-1, -2, -4, -5, -6, -8, and -9 antibodies (Alpha Diagnostic, San Antonio, TX) and anti-peptide AQP-3 and -7 antibodies (Chemicon, Temecula, CA) were diluted 1:100 with PBS, applied to sections, and allowed to incubate for 12 h at room temperature in a humidified chamber. The slides were then washed in PBS (3x for 5 min each), and an R-Phycoerythrin conjugated goat anti-rabbit IgG secondary antibody (1:100 dilution; Sigma Aldrich, St. Louis, MO) was added to sections and allowed to incubate at room temperature for 1 h. The slides were again washed in PBS (3x for 5 min each), coverslips mounted, and the slides stored at 4°C. Slides were examined using confocal microscopy. Negative control sections were incubated in an identical manner, substituting PBS for primary antibody. Appropriate positive control tissues were harvested from oil-treated CD1 mice and incubated in an identical manner as the treatment groups for each specific AQP family member. Positive controls included kidney for AQP-1, -2, -3, -4, and -6, adipose tissue for AQP-7 and -9, and lung for AQP-8.
Immunoprecipitation and Western Blotting
Left uterine horns of mice ovariectomized and injected with steroid hormones (E2, P4, or corn oil, see above) were homogenized in 1x RIPA buffer and centrifuged at 10 000 x g for 10 min and insoluble fraction was discarded. The protein concentrations of the supernatants were determined using a Bradford Assay (Biorad Laboratories, Hercules, CA) with BSA as a standard. Samples with 200 µg of protein each were incubated with 5 µl of primary antibody (see above) overnight at 4°C on a shaker. The samples were then incubated with protein A/G agarose beads for 3 h at 4°C with shaking and then centrifuged at 10 000 x g to collect beads. Supernatants were removed, and 20 µl of SDS sample buffer and 1 µl of ß-mercaptoethanol (Sigma) were added to the beads and boiled for 10 min. Equal amounts of protein samples were separated on a 15% SDS-PAGE gel and then transferred to a nitrocellulose membrane. After incubating for 1 h with blocking buffer (5% milk and 0.1% Tween 20 in PBS), blots were incubated with anti-AQP antibodies diluted 1:500 in blocking buffer for 3 h at room temperature. The membranes were then washed and incubated with anti-rabbit horseradish-peroxidase-conjugated IgG for 30 min at room temperature. The bound antibody was detected using an ECL chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ) . Kidney homogenates harvested from oil-treated CD1 mice were used as a positive control.
Cell Swelling Assay
Two cell swelling assays were used to investigate water permeability of luminal epithelial cells in response to E2 and P4. For the flow cytometry-based cell swelling assay, a population of cells enriched for luminal epithelial cells were harvested [17] and resuspended in PBS. The cell size distribution was then analyzed by flow cytometry. Water was then added to adjust the osmolarity to 210 mOsM. Following a 30-sec incubation, the cell size distribution was again analyzed by flow cytometry, and histograms of initial and final cell volumes were overlaid for comparison.
Water permeability was also determined using a Z2 Coulter Counter (Beckman Coulter, Miami, FL), which provides the specific mean cell volume of a population of cells. Luminal epithelial cells were harvested from the right horns of ovariectomized/steroid hormone-treated mice and resuspended at a concentration of 2 x 104 - 5 x 104 cells/ml in 15 ml of Isotone buffer (Beckman Coulter) and the initial mean cell volume measured. The Isotone buffer was then diluted to 210 mM by the addition of water and the resulting mean cell volume measured after 30 sec. The relative water permeability of the epithelial cells was then ascertained by calculation of the water permeability coefficient (Pf) [18]:
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Osm is the osmotic gradient of the buffer to the inside of the cell. Statistics
Statistical analysis was performed using a one-way ANOVA for repeated measures and the Tukey post hoc test. Statistical significance was set at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To identify AQP family members, ovariectomized mice were treated with corn oil or E2 and uteri were harvested at 6 h. AQP-1, -3, and -8 were expressed in both the vehicle- and E2-treated mouse uterus, although they differed in their cell-specific localizations, whereas AQP-2 was present only after E2 exposure (Fig. 1). AQP-1 was localized in both longitudinal and circular smooth muscle cells of the myometrium (Fig. 1). AQP-2, following E2 treatment, was found in the luminal and glandular epithelial cells and myometrium of the uterus. Expression of AQP-3 was reduced from other family members and was primarily localized in the luminal epithelial cells of the uterus with scattered immunolocalization in the glandular epithelial cells and the myometrium, similar to the expression pattern of AQP-2 (Fig. 1). Expression of AQP-8 was observed in the stromal cells of the endometrium and the myometrium (Fig. 1). Expression of these AQP family members was observed primarily on the plasma membrane. AQP-4, -5, -6, -7, and -9 were not present in the vehicle- or E2-treated mouse uterus (Fig. 1). Appropriate positive and negative control slides were used for all immunohistochemical experiments used in this study. Representative micrographs of both the positive and negative controls are shown in Figure 1.
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To further demonstrate the constitutive expression of AQP-1, we performed immunohistochemical analysis on uteri harvested from animals treated with E2 (harvested after 2 h), E2 antagonist, P4 alone, or P4 in combination with E2 (Fig. 2a). We found that in all treatment groups, AQP-1 protein was present and localized to the myometrium of the uterus. We performed immunoprecipitations with AQP-1 in homogenates from mice injected with oil, E2, or P4 to confirm our immunofluorescent results (Fig. 2b). AQP-1 protein was present in the oil-treated controls and slightly increased in homogenates from E2-treated mice. Furthermore, in response to P4, this expression was slightly decreased compared with oil-treated controls. These slight changes may have been undetectable in the immunohistochemical experiments shown in Figures 1 and 2a. AQP-1 protein is known to be glycosylated in many cell types, and the top band at 38–40 kDa, which did not change with steroid treatment, may represent the glycosylated form of this family member.
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Of all the AQP family members tested, only AQP-2 was found to be strongly regulated by E2 (Figs. 1 and 3). Minimal expression of AQP-2 was observed in uteri isolated 2 h after E2 injection (Fig. 3a). However, at 6 h, when maximum water imbibition occurs, AQP-2 expression was pronounced and localized as seen in Figure 1. Because P4 is known to antagonize E2-induced responses, ovariectomized mice were treated with P4 alone and expectedly showed no positive staining for AQP-2, whereas mice injected with both P4 and E2 also exhibited no AQP-2 expression (Fig. 3a). To determine the dependence of this estrogenic effect on the E2 receptor, ovariectomized mice were injected with the potent E2 antagonist, ICI 182,780, 1 h before E2 treatment. Uteri isolated from these animals 6 h after the E2 injection demonstrated no expression of AQP-2 (Fig. 3a).
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An immunoprecipitation was performed on homogenates from oil-, P4-, or E2-treated uteri, which confirmed the E2 up-regulation of AQP-2 (Fig. 3b). Basal expression of AQP-2, which was undetectable through immunohistochemical analysis, is seen in uterine homogenates from oil-treated mice, and this expression is up-regulated in response to E2. In homogenates from P4-treated animals, this hormone abolished both the E2 up-regulated and basal levels of AQP-2.
Estrogen Increases Plasma Membrane Permeability of Luminal Epithelial Cells
Cell swelling assays were performed on luminal epithelial cells to determine their plasma membrane permeability in response to E2 and P4. The cell volumes were analyzed by flow cytometry before and after they were introduced to a hypotonic insult. Figure 4 shows representative histograms of luminal epithelial cells harvested from oil- and E2-treated uteri, where the shaded peak is indicative of the initial volume and the solid line is the cell volume 30 sec after exposure to hypotonic media. These results show that oil-treated epithelial cells display similar initial and final volumes, whereas epithelial cells from E2-treated uteri swell substantially after 30 sec. To define the plasma membrane permeability of these cells in terms of a permeability coefficient, we also performed cell swelling assays on the Z2 Coulter Counter. As seen in Figure 5, E2 increased the Pf value from 5.43 ± 1.5 µm/sec seen with oil to 124.58 ± 25.3 µm/sec. Treatment with P4 caused a slight but not significant increase in water permeability over oil-treated controls.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Of all the AQP family members tested, only AQP-2 is up-regulated by E2, and expression of this family member increased with time after E2 exposure. Minimal expression of AQP-2 was observed at 2 h, whereas by 6 h, when maximal imbibition is observed, AQP-2 is highly expressed throughout the luminal and glandular epithelium and in the myometrium of the uterus.
Water movement in the uterus is not restricted to imbibition. The imbibed water, in response to E2, accumulates in the lumen of the uterus, leading to a watery phenotype of uterine luminal fluid. The presence of tight junctions between the epithelial cells leads us to anticipate that water will cross this layer through transcellular pathways rather than through pericellular routes. Both AQP-2 and -3 are found in epithelial cells in both the apical and basolateral portions of the membrane. Uterine levels of AQP-2 protein substantially increased following treatment with E2 compared with oil- and P4-treated controls. Furthermore, in luminal epithelial cells, E2 increased the Pf value to 124.6 µm/sec compared with 5.4 µm/sec with oil, suggesting the increase in AQP-2 levels contributes to this increase in permeability. Given the constitutive expression of AQP-3 and the extremely low Pf of the controls, it is possible that AQP-3 is not active in oil- or P4-treated epithelial cells but becomes functionally available in response to E2. Activation of AQP-3 may then cooperate with the up-regulated AQP-2 to move water across the cell membrane and into the uterine lumen.
Classically, P4 is known to antagonize estrogenic effects in the uterus, including partial blockage of E2-induced edema [19]. We have shown that E2-induced AQP-2 expression was completely blocked by pretreatment with P4, suggesting the edema that remains following P4 exposure is mediated by water movement through the constitutively expressed AQP family members. One potential mechanism for the antagonistic effects of P4 is the reduction of estrogen receptor (ER) mRNA and protein levels in the uterus within 24 h after P4 exposure [20]. To further substantiate that the E2-mediated increase in AQP-2 occurred in response to ER activation, uteri were collected after exposure to ICI 182,780 followed by E2. AQP-2 expression was not observed in these mice.
Interestingly, uterine water imbibition stimulated by E2 can be inhibited by puromycin and actinomycin D, which block translation and transcription, respectively [2], demonstrating a need for new protein synthesis for the process of water movement to occur. One such de novo protein may be AQP-2. Future studies will focus on the mechanism by which E2 regulates the increase in AQP-2 protein production in epithelial cells of the uterus.
Our data potentially conflict with previous studies concerning AQP-1. Li et al. [12] have shown that AQP-1 mRNA is up-regulated in response to E2 in the rat uterus. Gannon et al. [14] found AQP-1 protein localized to the visceral smooth muscle cells of the myosalpinx and vaginal smooth muscle but surprisingly not in the myometrium of the rat uterus. However, in the mouse, we found AQP-1 to be constitutively expressed exclusively in the myometrium and slightly regulated by E2 through both immunohistochemical and immunoprecipitation techniques (Fig. 1). Although the reasons underlying these variations in results are unknown, they may be attributed to species differences or changes in experimental procedures. For example, Gannon et al. [14] used mature, cycling rats to determine AQP-1 expression, whereas our study used ovariectomized and hormonally stimulated mice.
Interestingly, our data concerning AQP family members in the uterus correlate with a recently published article by Richard et al. [15]. In this article, the authors explored AQP expression in the peri-implantation mouse uterus. AQP-1 mRNA levels are constitutively expressed in the myometrium of uteri treated with oil or P4. Slight regulation of this mRNA expression was seen with E2 at different time points, as well as P4 and E2 together, but the authors did not evaluate expression of AQP-1 at the protein level in either the ovariectomized/steroid-treated or peri-implantation models. These results may correlate with the small amount of steroid regulation we observed in the immunoprecipitations of steroid-treated mouse uteri probed for AQP-1 (Fig. 1). The authors did not find AQP-2, -3, or -8 mRNA in their pregnancy model, nor did they evaluate the mRNA levels of these family members in ovariectomized/steroid hormone-injected mice. We have found AQP-2 protein levels to be down-regulated by P4, which would explain why expression of this family member was not seen in the authors' pregnancy model. Although they found AQP-4 mRNA on Days 4 and 5 after pregnancy, they failed to see any expression in ovariectomized mice, which correlates directly with our data. Significant AQP-5 expression was seen on Days 4, 4.5, and 5 of pregnancy and in the uterus of ovariectomized mice following extended periods of P4 exposure compared with those used in our study. Interestingly, our data combined with the data of Richard et al. [15] seem to set up an intricate model of regulation of these AQP family members both before and after pregnancy at times when water movement into and through the uterus seems to be critical.
In summary, we have demonstrated that AQP-1, -3, and -8 are constitutively expressed in the ovariectomized mouse uterus. AQP-1 is localized to the myometrium of the uterus and may be slightly E2 regulated, whereas AQP-3 is expressed most highly in the luminal epithelial cells with limited expression in the myometrium and glandular epithelium. AQP-8 is primarily located in the stromal cells and the myometrium. AQP-2 expression is induced by E2 in the luminal and glandular epithelium and the myometrium, potentially leading to E2-induced stromal edema. Furthermore, we also showed that plasma membrane permeability of luminal epithelial cells increases with exposure to E2 when compared with oil- and P4-treated epithelial cells, which may be mediated through the up-regulation of AQP-2 and the functional availability of AQP-3 found in this cell type. In conclusion, AQPs are expressed and regulated in the uterus, where they appear to mediate water imbibition and movement of water into the luminal cavity of the uterus.
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FOOTNOTES |
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2 Correspondence: Yvette M. Huet-Hudson, Department of Biology, University of North Carolina at Charlotte, Charlotte, NC 28223. FAX: 704 687 3457; ymhuet{at}email.uncc.edu
Received: 3 June 2003.
First decision: 18 June 2003.
Accepted: 26 June 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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