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Regular Article |
a Department of Anatomy and Physiology, Kansas State University, Manhattan, Kansas 66506
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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glucocorticoid receptor, male sexual function, steroid hormones, vas deferens
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Vas deferens epithelia express numerous ion transport mechanisms. Cultured porcine vas deferens [5] and rat epididymal [6, 7] epithelial cell monolayers respond to neurotransmitters with HCO3-- and Cl--dependent anion secretion. Alternatively, ex vivo rat epididymis and, to a lesser extent, vas deferens has been reported to secrete acid in the absence of neurotransmitter- or second messenger-mediated stimulation [8, 9]. Systematic studies have not been conducted to determine if chronic steroid hormone treatment modulates ion transport characteristics by direct effects on the epithelial cells in the deferent ducts.
Epithelial cells isolated from the excurrent duct have been cultured both in the presence and in the absence of corticosteroids. Neonatal ovine [10] and fetal human [11, 12] vas deferens epithelial cells have been cultured in the presence of hydrocortisone for ion transport studies. Initially, the authors reported that hydrocortisone along with insulin and cholera toxin were required for maintenance of epithelial cell morphology [11], whereas cellular transformation allowed culture in the absence of these agents [13]. Alternatively, rat epididymal epithelial cells have routinely been cultured in the absence of corticosteroids (but in the presence of 5
-dihydrotestosterone [6]), and we have previously reported that neither corticosteroid nor androgenic steroids are required for the growth of cells isolated from vas deferens of mature pigs [5]. Although steroid hormones may not be required for cell culture, exposure to such hormones may modulate epithelial function.
An aim of the present study was to determine the effects of chronic exposure to steroid hormones on electrophysiologic parameters of epithelial cell monolayers isolated from sexually mature boars. A variety of classes of steroids were employed to test for effects of both naturally occurring (cortisol, cortisone) and synthetic (dexamethasone, prednisolone, prednisone) glucocorticoids and naturally occurring mineralocorticoid (aldosterone), androgens (testosterone, dihydrotestosterone), estrogen (17ß-estradiol), and progesterone. The selection of steroids was developed to allow for testing of distinct structures and to determine if distinct metabolites (e.g., testosterone vs. dihydrotestosterone) were required for effects to be observed. Concomitantly, three different time points were tested to determine if hormone-induced effects on epithelial ion transport were observable at only selected times during monolayer development.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Porcine vas deferens epithelial cells were isolated and cultured as previously described in detail [5]. Briefly, adult reproductive tracts were retrieved from a local slaughterhouse on two occasions. Deferent ducts were isolated from a total of 10 pigs (15 ducts). Those portions of the duct that included convolutions and thus may be identified as transitional epididymis/vas deferens were excluded from the cell isolation protocol. Epithelial cells lining the vas deferens were isolated from the underlying tissues using a collagenase-based dissociation solution and seeded onto tissue culture flasks. Cells were incubated at 37°C at 5% CO2 in a medium containing Dulbecco modified Eagle medium (high glucose) (Invitrogen Corp., Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT) and 1% penicillin/streptomycin. Cells from each isolation grew to confluence within 3–4 days and then were subcultured onto Snapwell permeable supports or to another flask (i.e., passage 1) (Corning, Inc., Corning, NY). Results from the first and second passage were not significantly different from one another; therefore, data from these two passages have been pooled for the present study. Media bathing both the apical and basolateral aspects of the Snapwells and flasks were refreshed on the day after subculturing and ever other day thereafter. Cells growing on permeable supports were exposed to one of 10 steroid treatments in both the apical and basolateral medium beginning 1–3 days after subculture. Treatments included addition of one of the following sex steroids or corticosteroids to the control medium: progesterone, testosterone, dihydrotestosterone, 17ß-estradiol, dexamethasone, cortisol, prednisolone, cortisone, prednisone, or aldosterone.
A single set of experiments was performed to test for the possibility that steroids may be present in FBS in sufficient quantity to affect the outcomes of the experiments. Thus, charcoal-stripped FBS (Hyclone) was employed to prepare the culture medium in the absence and presence of testosterone. Paired monolayers were cultured in medium that contained typical FBS.
Experimental Design
Experiments were conducted on the epithelial cell monolayers 5–22 days postseeding. This time segment was divided into an early period (5–8 days postseeding), an intermediate period (11–14 days postseeding), and a late period (18–22 days postseeding) of growth. Monolayers treated with cortisol (500 nM), dexamethasone (10 nM), dihydrotestosterone (5--androstan-17ß-ol-3-one, 100 nM), progesterone (1 µM), and testosterone (100 nM) were tested during all three periods. Monolayers treated with aldosterone (1 nM), 17ß-estradiol (1 nM), cortisone (500 nM), prednisolone (1 µM), and prednisone (1 µM) were tested only in the early and intermediate periods. The treatment concentrations in the cultures are considered to be similar to those seen physiologically or pharmacologically.
Data Acquisition
Data were collected at 39°C in a modified Ussing chamber (Model DCV9; Navicyte, San Diego, CA). Vas deferens epithelial monolayers were bathed in Ringer solution (composition in mM: 120 NaCl, 25 NaHCO3, 3.3 KH2PO4, 0.83 K2HPO4, 1.2 CaCl2, and 1.2 MgCl2) with constant bubbling by a 95% O2 and 5% CO2 gas mixture. Monolayers were clamped to zero transepithelial potential difference (PDte) with a 1- or 5-mV bipolar pulse of 5-sec duration every 100 sec (Model 558C; University of Iowa, Department of Bioengineering, Iowa City, IA). Data were digitally acquired at 1 Hz with a Macintosh computer (Apple Computer, Cupertino, CA) using an MP100A-CE interface and Aqknowledge software (version 3.2.6; BIOPAC Systems, Santa Barbara, CA). Transepithelial electrical resistance (Rte) was calculated using the Ohm law. During each experiment, monolayers were exposed to amiloride (10 µM apical), ATP (10 µM apical), forskolin (2 µM apical and basolateral), bumetanide (20 µM basolateral), and DASU-02 (N-[4-methylphenylsulfonyl]-N'-[4-trifluoromethyl-phenyl]urea, 100 µM, apical and basolateral), and the effects on Isc and Rte were recorded.
Chemical Sources and Stock Solutions
The following chemicals were supplied by Sigma (St. Louis, MO): amiloride, bumetanide, cortisol (hydrocortisone), dexamethasone, prednisolone, cortisone, prednisone, progesterone, 17ß-estradiol, and aldosterone. Forskolin (Coleus forskohlii) was purchased from Calbiochem (La Jolla, CA). Adenosine 5'-triphosphate, disodium salt (ATP), was purchased from Boehringer-Mannheim (Indianapolis, IN). Pharmacia and Upjohn (Kalamazoo, MI) supplied the testosterone (Depo-testosterone). Dihydrotestosterone (5--androstan-17ß-ol-3-one) was purchased from Steraloids, Inc. (Newport, RI). DASU-02 was synthesized de novo in the laboratory.
Stock solutions were prepared as follows: forskolin, 10 mM in ethanol; amiloride, 10 mM in H2O; bumetanide, 20 mM in ethanol; DASU-02, 100 mM in dimethyl sulfoxide (DMSO); and ATP, 10 mM in 10 mM bis-tris propane (pH 6.8–7.0). Forskolin, DASU-02, and bumetanide were stored at -20°C and brought to room temperature immediately before each experiment. Amiloride was stored at 4°C. ATP was dissolved on the day of the experiment.
Data Analysis
Numerical data from Ussing chamber experiments are presented as the arithmetic mean and SEM using culture well as the experimental unit. ANOVA (Statistical Analysis Systems; SAS Institute, Inc., Cary, NC) as well as the Student t-test (Excel; Microsoft Corporation, Redmond, WA) were used to assess the likelihood of differences between control and treatment data. The probability of making a type I error less than 0.05 was considered statistically significant. Statistical analysis was performed to determine whether tissues derived from the two ducts of a boar exhibited less variation than tissues from other animals, and thus should be used as a blocking criteria for statistical analysis. Likewise, analyses were performed to determine if isolation number (1 or 2) or passage number (1 or 2) should be employed as a blocking criterion for the analysis. However, there was no statistical justification for such blocking criteria. Alternatively, Snapwells are packaged by the manufacturer in trays of six, and a six-channel voltage clamp was employed for the experimental paradigm. Experiments were conducted with five treated wells and one untreated control well per plate, with all wells being simultaneously assayed in the Ussing chamber. Thus, after a generalized ANOVA to establish that significant treatment effects were present in the data set, pairwise comparisons using the Student t-test were conducted between responses of the treated monolayers and control monolayers from the same tray.
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RESULTS |
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cm2 (n = 26); this value increased to 3910 ± 330 cm2 (n = 27) by 11–14 days in culture. At both time points, basal Isc was less than 0.6 µA/cm2. Amiloride was used to test for the presence of Na+ transport and was without effect. Exposure to ATP resulted in a rapid and sustained increase in Isc. (ATP was used to test for the presence of purinergic receptor-mediated modulation of Isc. However, subsequent results [data not shown] indicated that the response was mimicked by adenosine, but not by nonhydrolyzable ATP analogues. Thus, it appears that the ATP-stimulated change in Isc resulted from the activity of ecto-ATPases and adenosine receptor stimulation.) Treatment with forskolin, a potent stimulator of adenylyl cyclase, was associated with a transient increase in Isc followed by a prolonged elevated plateau. The sustained current was sensitive to bumetanide, an inhibitor of the Na+-K+-2Cl- cotransporter and by DASU-02, a blocker of the CFTR anion channel. Whereas Rte increased between 5–8 and 11–14 days postseeding, other parameters (i.e., responses to all test compounds) were not significantly different between time points in these conditions (i.e., in the absence of steroid exposure). The responses in control conditions have been discussed in detail elsewhere [5] and are used in the current context for closely paired comparisons.
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Steroids Affect Basal Rte
Chronic exposure to most steroid hormones was associated with a reduced Rte. To assess the Rte, monolayers were mounted in Ussing flux chambers and periodically exposed to a 1- or 5-mV bipolar pulse. The resulting change in Isc was employed to determine Rte using the Ohm law. At the earliest time point tested (5–8 days postseeding), Rte ranged from 57 to 5700 cm2, with a mean value of 2361 ± 129 cm2 (135 observations; Figs. 1 and 2). ANOVA indicated that no treatment resulted in a significant difference from control values (n = 6–16 paired observations per treatment) at the earliest time point. However, significant differences between treatment groups were observed at 11–14 days postseeding (Fig. 2A). Monolayers cultured in the presence of natural and synthetic glucocorticoids (cortisol, cortisone, prednisolone, prednisone, dexamethasone), aldosterone, and 17ß-estradiol exhibited significantly (P < 0.05) lower initial Rte when compared with paired control monolayers. Alternatively, progesterone and the androgens testosterone and dihydrotestosterone exhibited no effect on Rte (P > 0.25). Selected treatments were continued out to 18–22 days postseeding. Control monolayers exhibited an Rte (3770 ± 490 cm2, n = 15) that was slightly lower but not different from the value observed for 11–14 days. Treatment with dihydrotestosterone was also associated with a slight decrease in Rte, whereas treatments with progesterone and testosterone were associated with modest increases in Rte; all three treatments yielded results that were not significantly different from those for control monolayers. Cortisol and dexamethasone treatments continued to be associated with a reduced Rte, although the difference between control and dexamethasone-treated monolayers only approached statistical significance (P = 0.134).
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Taken together, these results suggest that physiological (or pharmacological) concentrations of selected steroid hormones modulate transepithelial resistance of cultured vas deferens epithelium. Perhaps surprisingly, the responses of monolayers treated with androgenic hormones did not differ from those of monolayers exposed to no added hormones. Conversely, treatments with estrogenic steroids and corticosteroids was associated with significant reductions in Rte that became statistically significant during the middle time point tested (with at least 5 days of growth in the presence of steroid hormones).
Steroids Affect Basal Isc
Chronic exposure to corticosteroids elevated basal Isc (Fig. 1, H, I, and K, and Fig. 2B). At the earliest time point tested, no significant differences in basal Isc were observed between treatment groups. However, by Days 11–14, prednisolone treatment was associated with significantly greater (P < 0.01) baseline Isc. Both dexamethasone and cortisol treatment resulted in elevated Isc, although these differences were not statistically significant (P = 0.22 and 0.11, respectively). The effects of both dexamethasone and cortisol were significant at the last time point tested. Thus, chronic exposure to both natural and synthetic glucocorticoids was associated with elevated basal Isc, whereas exposure to aldosterone and sex steroids was without effect.
Corticosteroid-Induced Isc Is Amiloride Sensitive
Glucocorticoid-induced changes in basal Isc were sensitive to amiloride. Figure 2C provides a virtual mirror image of Figure 2B, showing that all treatments (i.e., cortisol, dexamethasone, and prednisolone) that resulted in significantly elevated Isc were also sensitive to amiloride. Even at the earliest time point tested (5–8 days postseeding), inhibitory effects of amiloride were readily observed in monolayers that had been exposed to these glucocorticoids. These results strongly support the conclusion that glucocorticoids induce the expression or increase the activity of the amiloride-sensitive epithelial sodium channel (ENaC) in vas deferens epithelial cells. Amiloride was effective only from the apical aspect of the monolayer, indicating that vas deferens epithelial cells target the ion channel to the apical membrane.
Steroid-Induced Reduction in Rte Is Partially Explained by Increased Transcellular Conductance
The steroid-induced reduction in Rte could reflect an increase in paracellular permeability, transcellular permeability, or a combination of the two. After observing that glucocorticoids both reduced Rte and induced the expression of an amiloride-sensitive conductance, we speculated that transcellular permeability might account for the reduced Rte. Therefore, comparisons were made for effects of steroids on Rte in the presence of amiloride. The results presented in Figure 2D (with amiloride present) differ from those presented in Figure 2A (recorded in the absence of amiloride) at both the intermediate and late time points. Importantly, the Rte of monolayers cultured in the presence of dexamethasone, cortisol, and prednisolone was significantly increased by amiloride treatment so that differences from control monolayers were reduced. However, even in the presence of amiloride, many of the steroid treatments (dexamethasone, cortisol, prednisolone, aldosterone, and 17ß-estradiol) were associated with significantly reduced Rte when compared with control monolayers at the intermediate time point. At the latter time point, no significant differences in Rte were observed in the presence of amiloride (effect of cortisol approached statistical significance, P < 0.07). Thus, at least two effects of steroid treatment were observed. First, treatment with aldosterone, 17ß-estradiol, cortisone, and prednisone reduced Rte without affecting amiloride-sensitive current; second, treatment with dexamethasone, cortisol, and prednisolone reduced Rte, at least in part, by increasing the apical expression of an amiloride-sensitive conductance in vas deferens epithelia.
Anion Secretion Is Unaffected by Chronic Steroid Exposure
As shown in Figure 1, vas deferens epithelial monolayers cultured in the presence of each of the steroids responded to ATP and forskolin with prototypical increases in Isc and reductions in Rte. Subsequently, both bumetanide and DASU-02 reversed these stimulatory effects. The average effects of each acute treatment on monolayers exposed to each of the culture treatments are presented in Table 1. Our previous work has shown that the ATP- and forskolin-induced increases in Isc can be attributed to both Cl-- and HCO3--dependent anion transport [5]. Statistical analysis provided no indication that any of the steroid treatments significantly modified the stimulatory or inhibitory effects of any of these four compounds. Thus, the significant effects of steroids on Rte and of glucocorticoids on amiloride-sensitive ion transport occur via mechanisms that do not affect second messenger-stimulated anion transport.
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Charcoal-Stripped Serum Does Not Affect Experimental Outcomes
To test for the possibility that serum in the culture medium may include sufficient quantities of steroids to mask the effects of any exogenously added androgens, charcoal-stripped serum was used. Epithelial monolayers were cultured in three conditions for 14 days: typical medium containing 10% FBS, typical medium containing 10% charcoal-stripped FBS, and typical medium containing 10% charcoal-stripped FBS with testosterone (8 ng/ml). The results showed that there were no statistically significant differences in Rte, basal Isc, or the responses to ATP or forskolin. These results suggest that serum in the medium does not significantly contribute to the overall steroid exposure of the epithelial cells.
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DISCUSSION |
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The vas deferens is a steroid-responsive organ. Initially, its development is triggered by testosterone [14, 15]. Vas deferens epithelial cells have been reported to express both androgen [16, 17] and estrogen [18] receptors, although any direct physiological effects on epithelial activity have not been clearly defined. In mice, androgens stimulate the secretion of an aldo-keto reductase, mouse vas deferens protein, from cultured epithelia (although the physiological function of this protein remains poorly defined), and this protein has not been described in nonrodents [19]. We observed no effects of androgens on epithelial resistance or basal or stimulated ion transport in the current studies. However, the possibility remained that serum in the culture medium may have included sufficient quantities of steroids to mask the effects of any exogenously added androgens. This possibility was ruled out by a single set of experiments in which media containing charcoal-stripped serum was used. Our results suggest that fully differentiated epithelial cells recovered from adult porcine vas deferens do not require androgens to adhere to and proliferate in tissue culture flasks and that androgens are not required to modulate epithelial formation on permeable supports. Furthermore, androgens are not required to observe neurotransmitter-stimulated ion transport that would acutely alter the volume or composition of luminal fluids to which sperm are exposed.
Porcine vas deferens epithelial monolayers exhibit an extremely high Rte when grown in the absence of exogenous steroid hormones. We previously reported that the resistance increases over the first 2 wk postseeding and then remains stable. Monolayers exposed to most of the tested steroids did not exhibit the increased Rte observed in control monolayers by the intermediate time point (11–14 days postseeding). These results suggest either that glucocorticoids, aldosterone, and 17ß-estradiol slow the rate at which monolayers develop resistance, or that they simply cause transepithelial resistance to fall. Unfortunately, not all treatments were carried through the third week of culture. Of the five treatments that were carried through the third week, only dexamethasone and cortisol treatments were associated with reduced resistance at the intermediate time point. At the latter time point, only cortisol treatment was associated with an Rte that was significantly lower than that of controls, although the difference between control and dexamethasone-treated monolayers approached statistical significance. When the effect of the glucocorticoids on transcellular conductance was blocked by amiloride, neither treatment resulted in an Rte that was statistically different from that of the control. Thus, it appears that the selected steroids reduce the rate at which maximal Rte is achieved in vas deferens epithelium. Additional experiments are required to definitively rule out the possibility that steroid hormone treatments reduce Rte after a maximum resistance has been reached.
The possibility that the concentration of steroid (regardless of structure) was related to changes in Rte was also considered, since concentrations varying from 1 nM to 1 µM were used. This, however, seems not to be the case since both aldosterone and 17ß-estradiol were associated with significant effects on Rte and were used at only 1 nM, whereas progesterone was without effect and was employed at 1000 times that concentration. Taken together, these results suggest that glucocorticoids, aldosterone, and 17ß-estradiol reduce the rate at which cultured vas deferens epithelial cells establish a highly resistant monolayer. The mechanism by which this occurs remains to be determined.
Steroid modulation of Rte is not without precedence. It has been known for some time that dexamethasone increases Rte of cultured mouse mammary epithelial monolayers [20, 21], and it is now commonly included in cell culture media to induce or maintain mammary epithelial differentiation. The human endocervical cell Rte is unaffected by testosterone or cortisol treatment, but is reduced by 17ß-estradiol or diethylstilbestrol treatment [22]. Alternatively, neither estrogen nor progesterone treatment affect Rte of porcine uterine epithelial monolayers [23]. As in these systems, Rte of porcine vas deferens epithelial monolayers is modulated by steroid hormones. However, modulation of vas deferens Rte appears to differ from that any of the previously described systems.
An increase in the expression of an amiloride-sensitive current accounts for a portion of the glucocorticoid-induced reduction in Rte. The effects of corticosteroids (including aldosterone) on Na+ transport across colon and kidney epithelia have been documented for many years [24, 25]. Advances in molecular biology have demonstrated that this amiloride-sensitive absorptive cation current can be attributed to a steroid-induced increase in expression of ENaC [26]. Alternatively, airway epithelium has been shown to express ENaC, but this expression is not modulated by corticosteroid exposure [27]. Thus, it became important to determine which corticosteroids affected amiloride-sensitive Na+ transport across vas deferens epithelia. The results show that both naturally occurring and routinely prescribed synthetic glucocorticoids, but not aldosterone, induce the expression of amiloride-sensitive current in vas deferens epithelial cells that we interpret to be ENaC. The present results suggest that stimulation by glucocorticoids was not likely due to crossover effects on mineralocorticoid receptors since aldosterone was without effect. These results are particularly important because they suggest that states with high circulating levels of cortisol or that treatment with dexamethasone, prednisolone, or related glucocorticoids could dramatically affect the luminal environment of the deferent duct. Increased Na+ absorption could reduce fluid volume, leaving a more viscous environment in the lumen, or could reduce the concentration of Na+ available for other ion transporters. Both the Na+-H+ exchanger (NHE) and the Na+-HCO3- cotransporter (NBC) are reportedly present in epithelia of the male reproductive tract and are thought to regulate luminal pH [5, 28–30]. Thus, the luminal pH could also be substantially changed by increased expression of ENaC. Either of these effects, increased viscosity of luminal contents or altered pH, might be expected to reduce fertility.
Both estrogens and androgens are known to have acute effects on ion transport. We previously showed inhibitory effects of estrogens on forskolin-stimulated anion secretion by T84 colonic epithelial cells [31]. Similarly, estrogens were shown to reduce secretion by female, but not male, rat colon [32]. Testosterone has been shown to stimulate fluid and anion secretion by a canine kidney cell line, MDCK cells [33]. Most importantly, it has been shown that testosterone and dihydrotestosterone, but not estrogen, dexamethasone, or aldosterone, reduce Cl--dependent, forskolin-stimulated ion transport across cultured rat efferent ductile epithelium [34]. Although these effects of steroid hormones on ion transport are intriguing, it must be noted that their rapid time course, the requirement for high concentrations, and a pharmacological profile that is inconsistent with known estrogen or androgen receptors argue strongly that these effects are unrelated to the receptor-mediated events for which we were testing in the present experiments.
Chronic effects of sex steroids on ion transport in vas deferens have not been studied previously. 17beta-Estradiol was reported to up-regulate the expression of CFTR in rat uterine epithelial cells [35] and thus might have been expected to modulate DASU-02-sensitive anion secretion across vas deferens epithelial monolayers. Given that the male, but not the female, reproductive duct is adversely affected by the loss of CFTR, one might have expected an effect of either testosterone or 17ß-estradiol on vas deferens epithelia. The fact that we observed no effects of these steroids on anion secretion does not rule out the possibility that this tissue can be affected by these hormones, but indicates that anion secretion by fully differentiated epithelial cells is unaffected; there may be profound effects of these hormones at other stages of development. CBAVD is closely, but not universally, associated with abnormalities in CFTR [36]. Differences in development thus reflect a variety of influences that may include the actions of cortical hormones that disturb the development of tight junctions within the epithelium or may modulate cation transport, leading to changes in volume, composition, or pH of the vas deferens lumen. Any such changes in the luminal environment would be expected to affect fertility, regardless of the developmental stage at which they occur.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the Cystic Fibrosis Foundation (SCHULT99P0) and the Kansas State University College of Veterinary Medicine Dean's Research Fund. Contribution 02-39-J from the Kansas Agricultural Experiment Station. 
2 Correspondence: Bruce D. Schultz, Department of Anatomy and Physiology, Kansas State University, 1600 Denison Ave., Coles Hall 228, Manhattan, KS 66506. FAX: 785 532 4557; bschultz{at}vet.ksu.edu
Accepted: November 5, 2001.
Received: July 30, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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