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Regular Article |
and ERß1
a Departments of Animal Science and
b Veterinary Biosciences, University of Illinois at Urbana-Champaign, Urbana, Illinois 61802
ABSTRACT
Earlier studies have shown that the efferent ductules (ED) of the male mouse are a target for estrogen. The loss of estrogen receptor (ER) function through either knockout technology (
ERKO mouse) or chemical interference (pure antagonist, ICI 182 780) results in a failure of a major function of the ED, the reabsorption of testicular fluids. The purpose of this study was to test the hypothesis that estrogen controls fluid (water) reabsorption in the ED by modulating ion transporters important for passive water movement through a leaky epithelium such as the ED. Northern blot analysis was used to detect the mRNA levels for key ion transporters in the following experimental groups: 1) wild-type (WT) control for the 14-day experiment, 2) ER knockout (ERKO) control for the 14-day experiment, 3) WT treated with ICI 182 780 (ICI) for 14 days, 4) ERKO treated with ICI for 14 days, 5) WT control for the 35-day experiment, and 6) WT treated with ICI for 35 days. Estrogen differentially modulated the mRNA levels of key ion transporters. ER mediated carbonic anhydrase II mRNA abundance, and there was a decrease in Na+/H+ exchanger 3 mRNA levels in the ERKO that appeared to be a cellular effect and not a direct estrogen effect. The loss of ER control resulted in an increase in mRNA abundance for the catalytic subunit of Na+-K+ ATPase 1, whereas an increase in the mRNA abundance of the Cl-/HCO3- exchanger and the chloride channel cystic fibrosis transmembrane regulator was significantly ERß mediated. Our results indicate for the first time that estrogen acting directly and indirectly through both ER and ERß probably modulates fluid reabsorption in the adult mouse ED by regulating the expression of ion transporters involved in the movement of Na+ and Cl-.
epididymis, estrogen receptor, male reproductive tract
INTRODUCTION
The efferent ductules (ED) are tubules that form a conduit for the passage of fluids and spermatozoa from the testis to the epididymis. The ED secrete ions and protein, absorb testicular protein, and reabsorb greater than 90% of the testicular fluid, effectively concentrating luminal sperm prior to entry into the epididymis [1]. The major cell types in the ED are nonciliated and to a lesser extent ciliated cells, which chiefly function in absorption/ion secretion and mixing of the luminal contents, respectively.
Morphologic and physiologic analyses of the ED of estrogen receptor (ER) (ER) knockout (ERKO) mice and ICI 182 780 (ICI)-treated wild-type (WT) mice indicate that estrogen has a role in modulating the function of the ED in the mouse [2–5]. The ED of the mouse have a relatively high concentration of ER and a lower concentration of ERß [6]. Although the testis of the ERKO mouse has morphologic abnormalities, such as a very thin seminiferous epithelial layer with grossly dilated seminiferous tubules and greatly enlarged rete testis [2–4], the most notable morphologic aberrations are present in the ED. These include 1) a dilated lumen and a significantly reduced epithelial cell height, 2) nonciliated cells with reduced nuclear and microvilli heights, 3) disrupted homogeneity of microvillus organization, 4) reduction of cilia on ciliated cells, and 5) observable reduction in endocytotic apparatus and lysosomal granules [3, 4]. The male ERKO mouse has disrupted spermatogenesis and is infertile [2]. In contrast, the male ERß knockout mouse is fertile and has a reproductive tract that appears normal [7], whereas the male double ER ( and ß) knockout mouse is infertile, and its reproductive tract has ERKO-like morphology [8]. Collectively, these observations suggest that a functional ER is necessary to maintain fertility and normal morphology of the ED.
We have completed in vitro and in vivo studies investigating the function of estrogen on the ED in ERKO mice and in WT mice treated with ICI, a confirmed ER and ERß antagonist [3, 4]. In the in vitro study, we demonstrated that the ED of the ERKO mouse no longer move fluid out of the lumen but rather allow water to enter, causing the lumen to swell [3]. We also observed that ICI treatment of adult WT mice disrupted fluid reabsorption in the ED [3]. Our more recent in vivo study using ICI-treated WT mice confirmed results obtained in the in vitro study. Most significantly, the in vivo study revealed that the increase in ED luminal volume and the reduction in epithelial cell height observed in the ERKO mice are cellular responses to a functional loss of ER and are not developmental phenomena [4]. Additionally, we determined that the ERKO-like effects on the ED, resulting from a blockade of the ER, were insufficient to directly impair fertility in the mouse [4]. Although estrogen appears to have a principal role in regulating fluid reabsorption in the ED, the failure of this function in the ERKO mouse is likely an indirect contributor to the infertility of this knockout mouse. The accumulation of fluid in the ED of the ERKO mouse, which causes backpressure build up in the testis, probably results in the degeneration of the seminiferous epithelium [3].
At present, the mechanism for the prominent effect of estrogen on fluid reabsorption in the ED has not been identified. Water movement across a leaky epithelium such as the ED is most likely a passive event coupled to active ion transport by the epithelium [9]. As yet, no molecular mechanism has been identified for the control of fluid reabsorption in the ED. Based on our investigations of the role of estrogen in the male reproductive tract, we hypothesized that estrogen regulates gene expression of key molecules involved in ion movement, resulting in the modulation of fluid reabsorption in the ED. To test our hypothesis, we measured differential expression of mRNA for five key epithelial ion transport molecules in six groups of mice.
We found that estrogen affects the differential regulation of mRNA expression for key ion transporter molecules expressed in the mouse ED epithelial cells. These molecules are carbonic anhydrase II (CAII) located in the cytoplasm, Na+-H+ exchanger 3 (NHE3), chloride channel cystic fibrosis transmembrane regulator (CFTR), and the Cl-/HCO3- exchanger downregulated in adenoma (DRA) located on the apical surface, and Na+-K+ ATPase 1 catalytic subunit located in the basolateral surface of the ED cells. Our findings suggest a model in which estrogen, acting directly and indirectly through ER, modulates ion movement across the epithelium and thus controls water reabsorption by the ED. We report here for the first time that estrogen acts through ERß to regulate cellular processes that negatively affect the expression of CFTR and DRA. ERß may be involved in maintaining ion movement homeostasis in the ED.
MATERIALS AND METHODS
Animals and Treatment
Forty-five homozygous ERKO and WT sibling (C57Bl/6) male mice were obtained from a resident breeding colony maintained at the University of Illinois College of Veterinary Medicine. Mice were individually caged under controlled conditions and given food (Teklad mouse chow; Harlan, Madison, WI) and water ad libitum for the entire experimental period. Mice were randomly divided into six experimental groups: 1) WT control for the 14-day experiment (WT-C14, n = 8), 2) ERKO control for the 14-day experiment (ERKO-C14, n = 8), 3) WT treated with ICI for 14 days (WT-ICI14, n = 8), 4) ERKO treated with ICI for 14 days (ERKO-ICI14, n = 8), 5) WT control for the 35-day experiment (WT-C35, n = 5), and 6) WT treated with ICI for 35 days (WT-ICI35, n = 8). Mice of groups 1 and 2, at 60–70 days of age, were injected s.c. with 0.1 ml of castor oil as vehicle (as recommended by Dr. Wakeling, Zeneca Pharmaceuticals, Macclesfield, U.K.). Mice of groups 3 and 4 were injected s.c. with 0.1 ml of ICI as Faslodex long-lasting formulation (corresponding to 5 mg ICI; Zeneca). For groups 1–4, all injections were performed once weekly (as recommended by Dr. Vose, Zeneca) for 14 days. The final age of these mice ranged from 74 to 84 days. Mice of groups 5 and 6, beginning at 30 days of age, were injected s.c. with 0.1 ml of castor oil or ICI, respectively, once weekly for 35 days and killed at 65 days of age.
Tissue Collection and RNA Isolation
At the end of the treatment, mice were killed, reproductive tracts were removed, and the ED were rapidly isolated and washed with ice-cold PBS buffer before being frozen in liquid nitrogen. The ED from within each experimental group were pooled to obtain sufficient amounts of RNA for probe assessment and final Northern blot analysis. Total RNA was prepared by the guanidine isothiocyanate/phenol-chloroform method using a Polytron homogenizer (Fisher Scientific, Pittsburgh, PA). The isolated RNA pellets were dissolved in diethyl pyrocarbonate-treated water and stored at -80°C until used for Northern blot analysis. The purity and yield of the total RNA were determined spectrophotometrically.
Complementary DNA Probe Preparation
Probes used in this study were prepared by isolating the appropriate restriction fragment from plasmid clones. The probes were a 1.46-kilobase (kb) EcoRI cDNA fragment of rat CAII from pIBI-CAII, a 1.2-kb PstI cDNA fragment of rat NHE3 from pCVMNHE3, a 2.4-kb XbaI cDNA fragment of mouse CFTR from pFLMCFTR, a 2.3-kb XhoI and NotI cDNA fragment of DRA from pClneo-mDRA, and a 332-bp EcoRI and PstI cDNA fragment of rat Na+-K+ ATPase 1 subunit from pRATalpha.
Northern Blot Analysis and Presentation of Data
Northern analysis was performed using 15 µg RNA/lane. RNAs were denatured with formamide, fractionated on a 1.5% agarose gel containing formaldehyde, run at 40 V and 32 mA for 12–15 h, and transferred to a nylon membrane (Duralon-UV membrane, Stratagene, La Jolla, CA) by capillary blotting with 20x standard saline citrate (SSC; 1x SSC = 0.15 M sodium chloride, 15 mM sodium citrate, pH 7.0) for 18 h. Ethidium bromide staining of the gel verified loading in each lane. RNA was fixed to the membranes using an ultraviolet cross-linker (UV Stratalinker 2400; Stratagene). The membranes were prehybridized in QuikHyb solution (Stratagene) for 2 h and hybridized with 32P-labeled cDNA for 3 h. Radiolabeled probes were produced using the Prime-a-gene kit (Promega, Madison, WI). After hybridization, membranes were washed with 2x SSC and 0.1% SDS at room temperature and then 0.1x SSC and 0.1% SDS at 55°C. The blots were exposed to x-ray film at -80°C. Individual Northern blot experiments were repeated multiple times in testing and developing each probe to ensure reproducibility. Final duplicate blots containing all experimental and control samples were prepared for serial hybridization of all probes. The optical densities of the primary autoradiograph signals were measured and quantified using a PDI scanner and RFLPrint software (both from Bio-Rad Laboratories, Hercules, CA). Densitometry values were normalized to 28S rRNA as an internal control. In the figures, bars show mean ± SEM for all hybridizations. The lack of bars indicates insignificant SEMs.
RESULTS
Changes in CAII mRNA Expression
The 1.7-kb transcript of CAII was detected in the ED of all experimental mice (Fig. 1). Treatment of WT mice with ICI for 14 and 35 days produced a similar decrease in the abundance of CAII mRNA as compared with the WT-C14 and WT-C35 mice, respectively. A decrease of 26% and 21% in CAII mRNA was measured for WT-ICI14 (Fig. 1a) and WT-ICI35 (Fig. 1b) mice, respectively. However, in ERKO-C14 and ERKO-ICI14 animals, there was approximately a threefold decrease in CAII mRNA levels as compared with WT-ICI animals (Fig. 1a). A decrease of 67% and 64% in CAII mRNA as compared with WT-C14 mice was observed for ERKO-C14 and ERKO-ICI14 mice, respectively (Fig. 1a).
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Changes in NHE3 and Na+-K+ ATPase 1 mRNA Expression
Although fluid reabsorption in the ED appears to depend mainly on Na+ transport, we looked for estrogen effects on the expression of the key Na+ epithelial ion transporters, NHE3 and Na+-K+ ATPase 1 catalytic subunit. Expression of a 6.6-kb mRNA for the apical membrane-located NHE3 was detected in the ED of all experimental mice (Fig. 2). The pattern of expression was different from that observed for CAII. No reduction in NHE3 mRNA levels was detected for WT-ICI14 and WT-ICI35 mice, respectively (Fig. 2, a and b). In contrast, both ERKO-C14 and ERKO-ICI14 mice showed approximately a 50% reduction in NHE3 mRNA levels as compared with WT-C14 mice (Fig. 2a).
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As with the expression of the other ion transporters, Na+-K+ ATPase 1 subunit was found in the ED of all experimental groups, as indicated by the presence of a 4.5-kb fragment of mRNA (Fig. 3). However, unlike the changes seen in CAII and NHE3 mRNA levels, the mRNA levels for Na+-K+ ATPase 1 increased 44% and 102% in the WT-ICI14 and WT-ICI35 groups, respectively (Fig. 3, a and b). However in both the ERKO-C14 and ERKO-ICI14 groups there was only approximately a 70% increase in the Na+-K+ ATPase 1 subunit mRNA levels (Fig. 3a).
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Changes of DRA and CFTR mRNA Expression
To study estrogen effects on the Cl- ion transporters, mRNA expression of CFTR and DRA was examined. A 3.4-kb DRA mRNA was detected in the ED of all experimental groups (Fig. 4). A slight increase of 10% was seen in DRA mRNA levels for mice treated with ICI for just 14 days. However in contrast to CAII and NHE3, DRA mRNA levels after 14 days of ICI treatment showed a large increase in of 200% in WT-ICI35 mice (Fig. 4b). DRA mRNA levels only increased by 63% for ERKO-C14 mice (Fig. 4a) but showed a 156% increase in ERKO-ICI14 mice (Fig. 4a), a finding similar to that observed for WT-ICI35 mice (Fig. 4b).
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Expression of the 7.6-kb CFTR mRNA was detected in the ED (Fig. 5). In a manner similar to DRA, CFTR mRNA level changes were in contrast to those seen for CAII and NHE3. A small increase of 18% in CFTR mRNA was seen in WT-ICI14 mice (Fig. 5a), but a large increase of 145% was seen in WT-ICI35 mice (Fig. 5b). In the ERKO mice, there was a moderate increase of 61% in CFTR mRNA in ERKO-C14 mice and a 117% increase in ERKO-ICI14 mice (Fig. 5a), which was also similar to the results for the WT-ICI35 group (Fig. 5b).
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DISCUSSION
In the current study, we sought to determine whether the disruption of fluid reabsorption in the ED of mice lacking ER function could be a result of changes in gene expression for key ion transporter molecules. Using vehicle-treated ERKO and WT mice as controls for comparison to ERKO and WT mice treated with the pure estrogen antagonist ICI, we examined the affect of a lack of ER ( and ß) function on the mRNA levels for five key molecules suspected to be involved in ion movement in leaky epithelial cell tissues, such as proximal tubules of the kidney and the ED.
In designing these experiments, we used 14 days of ICI treatment as a relatively short time period of ER inhibition for comparison to the ERKO mouse, which lacks ER from conception. With this short treatment period, we expected that inhibition of ER would produce early changes in mRNA levels for genes likely to be directly under estrogen control or significantly affected by estrogen-regulated cellular processes. However, the detection of changes in mRNA levels using adult ERKO mice versus adult WT mice cannot simply be interpreted as showing ER regulation. Because the ERKO lacks ER from conception, changes seen in mRNA levels could result from a combination of processes. Observed changes could be due in part to direct ER regulation, to the degeneration of normal cellular physiology as a result of a long-term lack of ER, or to developmental abnormalities. Experimental results based solely on comparisons between WT and ERKO mice should not be construed as evidence of ER-mediated regulation because any differences could be the result of general changes due to an aberrant physiologic state as seen in the ED epithelium in the ERKO mouse [3, 4]. The use of a pure ER antagonist such as ICI for a defined period of time, in conjunction with the ERKO, allowed us to separate estrogen-mediated changes from changes likely due to an altered physiologic state. To determine whether the observed results after 14 days of ICI treatment were maximal, we also looked at a group of animals treated with vehicle or ICI for 35 days. These mice began treatment at 30 days of age and were treated until 65 days of age, making them similar in adult age to the first group of adult mice, which were treated for 14 days with ICI. This present study shows that mRNA expression of ion transporters in the ED of adult mice are differentially regulated by estrogen in the following manner: 1) estrogen upregulates expression of CAII mRNA through ER, 2) NHE3 mRNA expression is not directly regulated by estrogen through ER, 3) regulation of DRA mRNA expression is strongly downmodulated by the influence of estrogen acting through ERß, 4) estrogen likely also downregulates CFTR mRNA expression through the function of ERß, and 5) mRNA expression of Na+-K+ ATPase 1 subunit seems to be downmodulated by estrogen through ER.
In general a complete understanding of the cellular mechanisms involved in fluid movement is lacking, but information is especially scarce for mouse ED, an often overlooked tissue. However, significant work has been done in the rat that suggests that, as in the kidney, Na+ movement is of primary importance for passive fluid movement. An earlier comprehensive review by Clulow et al. [10] on fluid and electrolyte flow using micropuncture/perfusion techniques on the rat ED suggests that reabsorption in the ED is mainly mediated through Na+-H+ exchangers. Whereas 95% of luminal fluids are reabsorbed by the ED, 70% appears to be sodium dependent, as determined by luminal amiloride treatments [11, 12]. In recent studies by Leung et al. [13], polymerase chain reaction, Western blots, and activity measurements were used to confirm the presence of Na+-H+ exchanger isoforms (NHE1, NHE2, and NHE3) in cultured rat ED epithelial cells. These authors found that NHE3 was apically located, NHE2 was in the cytoplasm, and based on earlier work, NHE1 was likely basolaterally located [14]. They suggested that Na+ and fluid reabsorption at the apical surface, which accounts for 24% of all NHE activity in their cell culture system, is due to NHE3. Morphologic observation of NHE3 knockout (NHE3KO) mice shows a greater swelling of the ED lumen than in the ERKO mouse [15]. The apical import of Na+ coupled with basolateral Na+-K+ ATPase activity moves Na+ across the cell. Basolaterally located NHE1 works to maintain "housekeeping" functions, which include regulating cellular volume and possibly transepithelial electrolyte transport [13]. Located in the cytoplasm, NHE2 had no apparent role, but it may be involved in controlling endosomal intracellular pH [13]. Although greater than 95% of fluid and electrolytes are reabsorbed by the ED, the secretion of bicarbonate and chloride anions into the lumen (HCO3- and Cl-) has been documented in the ED [16, 17]. Leung et al. [13] suggested that in concert, basolaterally located Na+-K+ ATPase, NHE1, Na+/K+/2Cl- cotransporter, and a basolateral K+ channel support the accumulation of Cl- and HCO3- within the cell. Release to the lumen occurs through apical channels, opened in response to a luminal stimulus [13]. As yet, it is unclear what role anion secretion plays in the ED. Our current findings support the hypothesis that NHE3, CAII, and Na+-K+ ATPase are expressed in the mouse ED. We also detected for the first time the presence of anion transporters DRA and CFTR in the mouse ED. Our data further suggest that estrogen, acting through both ER and ERß, modulates ion movement homeostasis.
Based on our Northern analysis data and results of recent studies [13], we suggest a partial model for ion movement across the major absorptive cell (nonciliated) of the ED (Fig. 6). This model illustrates the potential functional changes that occur at the cellular level of the ED epithelium with nonfunctional ER that result in the observed morphology of a swollen lumen and compressed epithelial cells [4].
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Functional Role of ER in Na+ and Fluid Movement Across the ED Epithelium
When the function of ER is disrupted in the mouse ED, we suggest that the following occurs (Fig. 6b). The function of apical NHE3 is reduced because of a decrease in NHE3 function rather than NHE3 level (Fig. 6b, step 2), as indicated by the fact that ICI treatments do not produce a significant change in NHE3 mRNA levels after 14 or 35 days of treatment. In a recent study, 35 days of ICI treatment of WT mice was sufficient to cause a greater swelling of the ED lumen than that seen in the ERKO mice [4]. Although analysis of NHE3 mRNA levels in the ERKO ED (treated or not treated with ICI) shows a significant decrease, we attribute this to indirect cellular effects due to the long-term loss of ER function and the altered physiologic condition of the ERKO epithelium. The function of NHE3 could be reduced because of a combination of factors, including diminished availability of intracellular H+ (changes in intracellular pH) or modifications of the NHE3 protein due to direct or indirect estrogen affects on cellular regulatory mechanisms such as protein kinase A (PKA), cAMP, and protein kinase C (PKC). Other recent studies have indicated that the activity level of NHE3 is quickly and strongly regulated by its degree of phosphorylation and by that of its associated proteins [18–20]. The results of the current study show that CAII mRNA levels are quickly but moderately affected by blocking ER for 14 days. The levels observed at 14 days probably represent maximal repression, because no further change was seen with 35 days of treatment. Intracellular H+ would decrease if the change seen in CAII mRNA expression (Fig. 6b, step 1) were significant enough by itself to affect translational levels of CAII. The greater decreases in CAII mRNA levels in the ERKO mice (treated or not treated with ICI) probably also reflect a general decline in the normal physiologic condition of the ED epithelium. If the decline seen is insufficient to cause a significant reduction of CAII enzyme activity, then there may be other intracellular H+ transporters/producers that are dependent on estrogen for expression. Significant increases or decreases in their function could reduce H+ availability for NHE3 Na+ exchange. One such transporter could be NHE1, a basolaterally located Na+-H+ exchanger (Fig. 6b, shaded oval) reported to be in rat ED [13], or possibly a basolaterally and/or apically located H+ ATPase pump. Future experiments will address this issue of potential loss of NHE3 function through depletion of available H+ or through cellular modifications (phosphorylation) of NHE3 function.
Expression of Na+-K+ ATPase in male rat reproductive tract, including the ED, has been well documented by Byers and Graham [21]. In conjunction with apical transporters of Na+, basolaterally located Na+-K+ ATPase exchanges three intracellular Na+ for two extracellular K+ at the expense of metabolic energy. The net movement of Na+ from the lumen to the intracellular space creates an electrochemical gradient between the inside and outside of the cell, resulting in a passive movement of water secondary to the active transport of Na+ [22]. In contrast to our findings for CAII and NHE3, mRNA levels for the catalytic subunit of Na+-K+ ATPase was much greater in all ER-repressed tissues. Increases were seen in the 14-day ICI treatment group, and large increases were found in the 35-day ICI treatment group, indicating a strong dependence on estrogen for suppression of expression. Animals treated with ICI for 35 days showed a much greater swelling than did ERKO mice of the same age. An increase in Na+-K+ ATPase levels in the absence of apical Na+ import could likely lead to a hypo-osmotic environment within the cell. This condition could force water to move out of the cell, resulting in a reduction in cellular volume and epithelial cell height. Additionally, Na+-K+ ATPase transcript levels were similar between the ERKO groups, suggesting that ERß has little or no effect on the expression of this gene. Again, the smaller increases in mRNA, seen in the ERKO mice as compared with the 35-day ICI mice, suggest that the ERKO epithelium has an altered physiologic condition due to the long-term loss of ER function.
Functional Role of ERß in Cl- and Fluid Movement Across the ED Epithelium
In looking at fluid movement in colon epithelium in NHE3KO mice, Melvin et al. [23] reported the increased expression of the apically located Na+-independent DRA, postulating that its upregulation compensated for the loss of fluid due to a lack of NHE3. Using a probe for DRA, we detected the transcript in the mouse ED. To our knowledge, this is the first molecular evidence showing the mRNA expression of this Cl-/HCO3- exchanger in the mouse ED. Newcombe et al. [17] showed that the ED of the rat does secrete HCO3- into the lumen. Our results place at least one of the molecules likely responsible for luminal HCO3- secretion in the mouse ED. DRA expression showed a small increase in the ED after 14 days of ICI treatment but showed a significant increase after 35 days of ICI treatment. As reported for the NHE3KO mouse colon epithelium, an increase in DRA expression could be due in part to a cellular compensatory mechanism responding to a reduction of Na+ movement [23]. We noted a small increase in DRA expression in the ERKO mouse ED that might reflect this compensatory effect. However, even more intriguing is the observation in the ED from ERKO ICI-treated mice,where there was a large increase in DRA mRNA expression more similar to that in the WT-ICI35 group. Assuming that the ERKO ED shows changes due to cellular effects rather than direct ER effects, it is surprising that DRA increased in the ERKO-ICI14 group. Because ICI is both a pure ER and pure ERß antagonist, this result suggests that ERß is involved in the modulation of DRA expression in the ED. In our model, this modulation would likely lead to an increase in DRA function, predicting an increase in HCO3- secretion and Cl- import (Fig. 6b, step 4).
We found a similar response for the mRNA expression of CFTR. CFTR is an apical channel responsible for secreting Cl- in epithelial cells [24]. A deficiency of CFTR results in disrupted NaCl transport and thus water movement across the epithelium [24, 25]. Cystic fibrotic males are infertile and frequently have dilated ED and sometimes lack a vas deferens and epididymis [26]. The data presented here are the first molecular evidence of CFTR mRNA expression in the mouse ED. Regulation of CFTR mRNA expression by estrogen has been studied in the female reproductive tract [27, 28] and other tissues [29]. CFTR is thought to functionally interact with other ion transporter molecules, such as apical DRA [30]. The coupling of CFTR with Cl-/HCO3- transporters such as DRA would allow the regulation of luminal Cl-, HCO3- content, NaCl, and fluid absorption in the duodenum [30]. This regulation may be happening in the ED as well, and along with a basolateral Na+/K+/2Cl- cotransporter (Fig. 6b, shaded oval) [31], Cl- and HCO3- content could be regulated. As seen for DRA, the increased expression of CFTR in the ERKO-ICI14 group indicates that ERß likely modulates CFTR mRNA expression in the ED. The result may maintain cellular Cl- levels by removing increased Cl- uptake due to an increase of DRA expression. In addition, if the increase in CFTR expression results in greater levels of Cl- secretion (Fig. 6b, step 5), this effect could explain the greater luminal dilation in WT-ICI35 mice compared with ERKO mice reported previously [3, 4]. We speculate that the reduction of epithelial cell heights by up to 76% in the ED of ERKO and WT-ICI35 mice [4] is due to the lack of apical Na+ transport and an increase in basolateral Na+ export by increased Na+-K+ ATPase levels. Under these conditions, the cell could be depleted of Na+ and higher concentrations could develop on both the luminal and intercellular sides of the epithelium, drawing water out of the cell and reducing cell volume, which results in the observed reduction in cell height. This reduction in cell height is not seen in the NHE3KO mouse even though the lumen is greatly swollen [15], suggesting that a block in apical Na+ transport alone will not alter the ability of the cell to manage internal electrolyte levels. However, when estrogen effects are disrupted, the results are many fold, some direct and some indirect, the result of which is likely an inability of the epithelium to regulate normal ion movement and ion homeostasis.
Estrogen, working through ER, directly modulates cellular processes important for supporting the reabsorption of Na+ from the lumen by NHE3 but does not directly affect NHE3 expression. The net result is likely to be a lack of luminal Na+ reabsorption and an increase in cellular loss of Na+. Estrogen also acts through ERß to modulate other ion transporters involved in anion secretion into the lumen. Thus, epithelial ion homeostasis is controlled by the differential action of estrogen through ER and ERß.
ACKNOWLEDGMENTS
We are grateful to Drs. A.E. Wakeling and B.M. Vose (Zeneca Pharmaceuticals) for their support of this work through the generous gift of Faslodex (ICI 182 780). We are also grateful to Dr. C.A. Stolle for providing rat CAII cDNA, Dr. G.E. Shull for providing rat NHE3 cDNA, Dr. D. Lunn for providing mouse CFTR cDNA, Dr. J. Melvin for providing mouse DRA cDNA, and Dr. R.W. Mercer for providing rat Na+-K+ ATPase 1 cDNA. We also thank Ki-Jun Lee for help with tissue collecting.
FOOTNOTES
1 This work was supported in part by USDA grant AG9735203-4615 and NIH grant HD-35126. 
2 Correspondence: David Bunick, Department of Veterinary Bioscience, University of Illinois, 2001 South Lincoln Ave., Urbana, IL 61802. FAX: 217 244 1652; d-bunick{at}uiuc.edu
Accepted: June 25, 2001.
Received: April 10, 2001.
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 Q. Zhou, R. Nie, G. S. Prins, P. T. K. Saunders, B. S. Katzenellenbogen, and R. A. Hess Localization of Androgen and Estrogen Receptors in Adult Male Mouse Reproductive Tract J Androl, November 1, 2002; 23(6): 870 - 881. [Abstract] [Full Text] [PDF]
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 M. A. Yorek, L. J. Coppey, J. S. Gellett, E. P. Davidson, X. Bing, D. D. Lund, and J. S. Dillon Effect of treatment of diabetic rats with dehydroepiandrosterone on vascular and neural function Am J Physiol Endocrinol Metab, November 1, 2002; 283(5): E1067 - E1075. [Abstract] [Full Text] [PDF]
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 C. A. Oliveira, Q. Zhou, K. Carnes, R. Nie, D. E. Kuehl, G. L. Jackson, L. R. Franca, M. Nakai, and R. A. Hess ER Function in the Adult Male Rat: Short- and Long-Term Effects of the Antiestrogen ICI 182,780 on the Testis and Efferent Ductules, without Changes in Testosterone Endocrinology, June 1, 2002; 143(6): 2399 - 2409. [Abstract] [Full Text] [PDF]
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N. Pastor-Soler, C. Isnard-Bagnis, C. Herak-Kramberger, I. Sabolic, A. Van Hoek, D. Brown, and S. Breton Expression of Aquaporin 9 in the Adult Rat Epididymal Epithelium Is Modulated by Androgens Biol Reprod, June 1, 2002; 66(6): 1716 - 1722. [Abstract] [Full Text] [PDF]
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