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Male Reproductive Tract |
a Department of Anatomy and Physiology, Kansas State University, Manhattan, Kansas 66502-5802
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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cortisol, male reproductive tract, male sexual function, neuroendocrinology, vas deferens
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cultured porcine and ovine vas deferens epithelial cell monolayers respond to both hormones and neurotransmitters with changes in ion transport. Chronic exposure to glucocorticoids, but not mineralocorticoids, has been associated with the expression of amiloride-sensitive ion transport [4, 6]. Vas deferens epithelia receive extensive innervation [8–13]; however, direct effects of neurotransmitters on epithelial electrolyte and fluid transport have not been widely studied. A single report indicates that adenosine, ATP, norepinephrine, vasopressin, and histamine increase anion secretion whereas serotonin and carbamylcholine are without effect [5]. Previous work employing forskolin as a pharmacologic stimulant of adenylyl cyclase indicates that ongoing anion secretion is substantially dependent on the presence of HCO3- in the bathing media, although Cl- is also necessary for a maximal net ion flux [5, 7]. Neurotransmitter receptor subtypes that acutely modulate human vas deferens epithelial function remain to be determined, as do their subcellular locations.
Human vas deferens epithelial cells are known to express selected ion channels that might contribute to ion transport. Human fetal vas deferens epithelial cells have been isolated and grown in culture to evaluate membrane ion permeabilities using whole-cell and isolated membrane patch techniques. Both Cl- [14] and K+ [15] channels are present in the cultured cells, although neither the contributions of these channels to epithelial transport nor control mechanisms has been identified.
Epithelial ion transport generates and maintains an appropriate environment for sperm maturation and storage [3, 16]. Recent reports, however, suggest that acute exposure to neurotransmitters modulates ion transport and thus may contribute to male fertility [5]. Disruption of the cystic fibrosis transmembrane conductance regulator (CFTR), a protein channel that contributes to ion transport, is associated with both obstructive [17–21] and nonobstructive [21–25] male infertility. Thus, it is important to gain a better understanding of the ion transport pathways that are present in human vas deferens and the regulatory pathways that modulate transporter activity.
The focus of the current project was to determine the responsiveness of human vas deferens epithelial cell monolayers to adenosine and related agonists as part of a larger project designed to determine the hormonal or neural mechanisms that modulate ion transport across distal reproductive epithelium. This project required the development of techniques to isolate and culture adult human vas deferens epithelial cells and directly builds on previous success in developing a porcine epithelial cell system that is now being exploited to define subcellular mechanisms of ductile ion transport [5–7]. This report includes compelling evidence that apical adenosine receptors modulate ductile anion secretion in adult human vas deferens.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Small segments (3–4 cm) of abdominal human vas deferens were acquired from a local hospital under procedures approved by both the University and hospital according to Institutional Review Board guidelines. Tissues were collected in sterile saline solution on ice and transported to the laboratory in less than 1 h. Upon arrival, ducts were stripped of connective tissue and flushed with Hanks buffered salt solution (HBSS [composition in mM]: NaCl, 137; KCl, 5.4; KH2PO4, 0.4; Na2HPO4, 0.6; glucose, 5.5) supplemented with 40 µg/ml gentamicin, 100 U/ml penicillin, 100 µg/ml streptomycin, and 4 µg/ml amphotericin B. Ducts were flushed and filled with a collagenase-based dissociation solution (300 U/ml collagenase in HBSS) and incubated at 37°C in HBSS for 30 min. The tissue was blotted dry and massaged to assist epithelial cell disruption. One milliliter of HBSS was then flushed through the lumen to elute the dissociated cells, which were collected in a sterile centrifuge tube. Cells were pelleted at 800 x g for 5 min, the supernatant removed, and the pellet resuspended in 750–1000 µl of prewarmed growth media (Dulbecco modified Eagle medium [DMEM; Gibco, Rockville, MD], 10% heat inactivated fetal bovine serum [FBS; Gibco], 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.2 U/ml insulin). Cells derived from each duct were seeded onto a single well of a 24-well culture plate (Cellstar; Marsh Biomedical Products, Rochester, NY) and incubated at 37°C in a humidified 5% CO2 environment. After 48 h, the initial medium was replaced with fresh medium and changed daily thereafter. Most isolations reached confluency in 3–5 days and were subsequently passed by removing spent media, rinsing the cells in phosphate-buffered saline for cell culture (PBScc [composition in mM]: 140 NaCl, 2 KCl, 1.5 KH2PO4, 15 Na2HPO4) and treated with 0.5 ml dissociation media (0.25% vol/vol trypsin and 2.65 mM EDTA in PBScc) for 30 sec. Dissociation media was removed with the exception of 10–12 µl that remained to prevent desiccation while cells were incubated at 37°C for 5–6 min to achieve detachment. Cells were suspended in 600 µl of media that was divided equally onto two 6.5-mm-diameter permeable supports (Transwell; Corning-Costar, Acton, MA) that had been coated with human placental collagen (Type VI; Sigma, St. Louis, MO) and allowed to incubate for 14–20 days with daily media changes prior to experimental assays.
Some experiments were conducted with an alternative growth medium that has previously been employed for fetal human ductile epithelia cells [26]. DMEM was replaced with CMRL 1066 (Gibco) supplemented with 20% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.2 U/ml insulin; and 1 µg/ml cortisol. Primary isolations were either initially seeded using the alternate media or were switched from typical media after 1 day of culture; both methods were considered alternate-media treated. In a single experiment (see Fig. 2), cells were cultured on two Transwell inserts for 14 days in typical media, evaluated in Ussing chambers, and then returned to the incubator in either typical or alternate media for an additional 3 days prior to reevaluation.
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Immunocytochemistry
Immunocytochemical studies were conducted on cells previously evaluated in Ussing chambers to verify transepithelial electrical resistance and responsiveness. Immediately after an Ussing chamber experiment, inserts were removed, fixed in 10% buffered neutral formalin (Fisher, Pittsburgh, PA) for 5–15 min, and stored in PBS for histochemistry (PBSh [in mM]: 5 KH2PO4, 15 K2HPO4, 150 NaCl, pH 7.2–7.4) with 0.02% sodium azide. Standard immunocytochemical techniques were used and have been described previously in detail [5, 7]. A monoclonal antibody to a broad spectrum of human cytokeratins (C-2931, anti-pan cytokeratin, diluted 1:200; Sigma) was employed. A monoclonal antibody (MAB1520, anti-ZO-1, diluted 1:200; Chemicon, Temecula, CA) was used to identify the basis of the observed electrical resistance in confluent monolayers and to further indicate the epithelial nature of the cells in culture. Cells were observed and digital images captured with light microscopy (Leica DM RX microscope; Leica AG, Solms, Germany). Digital images of treated and control monolayers were acquired with identical numerical settings and prepared for publication in parallel using CorelDRAW (version 8.2; Corel Corporation, Ottawa, ON, Canada).
Electrophysiology
Measurements of net ion flux recorded as short circuit current (Isc) and transepithelial electrical resistance (Rte) were conducted using a modified Ussing chamber as described in detail previously [5]. Most experiments were conducted in symmetrical Ringer solution ([composition in mM] 120 NaCl, 25 NaHCO3, 3.3 KH2PO4, 0.83 K2HPO4, 1.2 CaCl2, and 1.2 MgCl2) at 37°C with continuous bubbling of humidified 5% CO2/95% O2. In a single set of experiments, apical Na+ was replaced with K+ to test for the presence of amiloride-sensitive nonselective cation channels.
Confluent monolayers were mounted in modified Ussing flux chambers in symmetrical solutions. Transepithelial electrical potential difference (PDte), an indicator of active ion transport, was directly measured. PDte was then clamped to zero by the imposition of an electrical current (Isc). Isc is a rapid, continual, direct measure of net active ion transport. Standard conventions are used throughout such that elevated Isc is indicative of net anion secretion or cation absorption. Periodically, monolayers were exposed to a bipolar change in clamp potential with the corresponding change in Isc being recorded. These parameters were used to calculate Rte, which provides an indication of the barrier function of the epithelium. Changes in Rte can reflect the modulation of transcellular conductive pathways (i.e., ion channels) or changes in the paracellular pathway.
Voltage-Sensitive Vibrating Probe
The vibrating probe technique was identical to that described previously [27]. Briefly, Isc,probe was monitored by vibrating (200–400 Hz) a Pt-Ir wire microelectrode with Pt-black tip. The probe was positioned 20–30 µm from the apical surface of the epithelium with computer-controlled, stepper-motor manipulators (Applicable Electronics, Forest Dale, MA) and probe software (ASET, version 1.05; Science Wares, East Falmouth, MA). The bath references were 26-gauge Pt-black electrodes. Signals from the phase-sensitive detectors were digitized (0.5 Hz, 16 bit) and the output expressed as current density at the electrode. The perfusion solution had a composition of (in mM) 140 NaCl, 3.3 KH2PO4, 0.83 K2HPO4, 1.2 CaCl2, 1.2 MgCl2, and 10 HEPES, pH 7.4. The solution in the chamber was exchanged 1.1 times per minute and maintained at 37°C.
Cyclic AMP Assay
A commercially available enzyme immunoassay kit (Biotrak; Amersham Pharmacia Biotech, Inc., Piscataway, NJ) was used to detect ligand-induced differences in cellular cAMP. Epithelial monolayers were grown on Transwell permeable culture supports as described above. Assays were conducted with supports suspended in a 24-well culture plate as packaged by the manufacturer. Culture media was replaced with PBScc and the cells returned to the incubator for 30 min to allow cells to acclimate to the fluid environment. Ro 20-1724 (100 µM), a phosphodiesterase inhibitor that has previously been shown not to interfere with the assay [28], was added to both the apical and basolateral compartments. Four monolayers derived from a single vas deferens were then exposed to one of four treatment conditions: vehicle control, apical adenosine (1 µM), apical and basolateral 8-phenyltheophylline (10 µM), or 8-phenyltheophylline and adenosine. Five minutes after the addition of the treatments, cells were rapidly lysed by the addition of dodesyltrimethylammonium bromide (0.25% final concentration) as per manufacturer's instructions. The remainder of the assay was conducted as per manufacturer's instructions, including the addition of 1.0 M sulphuric acid, with absorbance being assessed at 450 nm. Results are expressed as fmol cAMP per Transwell as determined from the standard curve.
Chemical Sources
Forskolin (Coleus forskohlii) was purchased from Calbiochem (La Jolla, CA). Adenosine-5'-triphosphate (ATP) was purchased from Boehringer-Mannheim (Indianapolis, IN). Collagenase, gentamicin, penicillin-streptomycin solution, and trypsin-EDTA (0.5% trypsin, 5.3 mM EDTA, 10x liquid) were purchased from Invitrogen-Life Technologies (Carlsbad, CA). 8-Phenyltheophylline (8-PT), adenosine, amiloride, amphotericin B, ß,
-methyleneadenosine-5'-triphosphate (AMP-PCP), bumetanide, cortisol, insulin, 5'-(N-ethylcarboxamido)adenosine (NECA), and sodium azide were purchased from Sigma (St. Louis, MO). All other chemicals were of reagent grade or better and were purchased from reputable sources.
Statistics
Numerical values are reported as the mean ± SEM using each Transwell insert as an experimental unit. Unless otherwise indicated, experiments were conducted on a paired basis using two culture inserts derived from a single epithelial isolation for comparison. The Student t-test for paired or unpaired assays was used to determine statistical significance. Because of the precious nature of the primary human cell cultures, monolayers were frequently recovered following Ussing chamber experiments, returned to the incubator for 3 or more days (with daily media changes), and used for an additional assay. Statistical analysis indicates that previous acute exposure to forskolin or neurotransmitters does not alter the outcome of subsequent experiments.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Initial tests were conducted to verify that isolation and culture protocols yield human vas deferens cells that exhibit cytochemical and functional epithelial markers. Indirect immunofluorescence was employed to test for the presence and distribution of ZO-1 and cytokeratin immunoreactivity, both of which would be expected in epithelial cells. When probed with a commercially available ZO-1-specific antibody, a reticular pattern is observed (Fig. 1A). This pattern is consistent with the expected localization of ZO-1 at cell margins, where it contributes to the tight junctions. All cells are observed to be fully circumscribed by ZO-1 immunoreactivity, indicating that all cells are epithelial in nature and that a confluent monolayer capable of performing a barrier function develops in culture. Figure 1B shows intense cytokeratin immunoreactivity in the cytoplasm of all cells in culture. No immunoreactivity is observed when either primary antibody is omitted from the assay protocol (Fig. 1, C and D). These immunocytochemical results indicate that cells isolated from adult human vas deferens exhibit prototypical epithelial morphology when cultured on permeable supports. Functional tests confirmed that an epithelial barrier is formed and that cell monolayers exhibit polarity.
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The epithelial nature of the cultured cells was further evaluated with electrophysiological techniques. Monolayers maintain PDte of 8.2 ± 1.0 mV, lumen negative, and display a basal Isc of 9.2 ± 0.7 µA/cm2 (n = 76 Transwell inserts derived from 33 ducts from 21 donors). The observed PDte and Isc are consistent with ongoing active anion secretion and/or cation absorption. Rte was determined using Ohms Law by observing changes in Isc while exposing the epithelial monolayer to a 1-mV bipolar pulse. Initial Rte was 660 ± 51 
cm2. Monolayers with Rte ranging from 146 to 1956 cm2 were employed for these studies. Taken together, the observed morphological and electrophysiological parameters provide compelling evidence that cells isolated from adult human vas deferens grow in culture to form an epithelium that functions both as a barrier to ion and particle movement and as an active transporter of ions to generate a transmural electrical gradient.
Media Comparison
We sought to determine how media composition might affect epithelial function. A relatively simple medium has been employed previously to culture cells derived from adult porcine vas deferens [5–7], whereas it was reported that hormone supplements are required to maintain typical morphology of human fetal ductile epithelial cells [26]. The latter medium has also been employed for the culture of freshly isolated perinatal ovine ductile epithelial cells [4]. One essential difference between the two media is the presence of cortisol, an agent shown to dramatically affect basal ion transport across cultured porcine vas deferens epithelial cell monolayers [6]. To test for media effects on the human epithelial cell system, paired monolayers were grown in typical medium and assessed in the Ussing chamber with particular attention to the magnitude of amiloride-sensitive ion transport, which is interpreted to be epithelial Na+ channel (ENaC)-mediated absorption (Fig. 2, A and C). After an initial assessment to determine that paired monolayers exhibited similar responses, monolayers were recovered, placed in either typical or alternate media, and returned to the incubator for 3 days prior to a second evaluation. As predicted, exposure to the alternate medium that includes cortisol markedly increases the amiloride-sensitive Isc (compare Fig. 2B to Fig. 2A). Conversely, when recovered and grown in typical medium, basal, amiloride-sensitive Isc was slightly increased when compared with the earlier evaluation of the same monolayer (compare Fig. 2D to Fig. 2C) but was substantially less than that observed in the paired monolayer that was exposed to the alternate medium (compare Fig. 2D to Fig. 2B). Results from eight monolayer pairs exposed to typical or alternate media are summarized in Figure 2E. The magnitude of amiloride-sensitive current is more than 2.5-fold greater in monolayers exposed to cortisol-containing medium than in monolayers exposed to the typical medium (P < 0.05). Additional evidence that amiloride is affecting a conductive pathway such as ENaC is presented in Figure 2F. The addition of amiloride causes a significantly greater increase in Rte across cortisol-treated cell monolayers (655 ± 251 cm2) versus nontreated monolayers (71 ± 44 cm2; P < 0.05). The concomitant reduction in Isc and increase in Rte indicates that the block of a conductive pathway, likely ENaC, mediates the amiloride-sensitive effects. The possibility remained, however, that amiloride-sensitive current might result from the activity of a nonselective cation channel [29]. To test for this possibility, amiloride-sensitive Isc was determined in paired monolayers (n = 3) that were evaluated in the presence of an apical bath that contained either Na+ or K+. Amiloride-sensitive current was observed only in Na+-containing media (
Isc = -11.64 ± 1.52 µA cm-2 versus -0.84 ± 0.56 µA cm-2 in K+), although forskolin-stimulated Isc was indistinguishable in the two conditions. Thus, amiloride-sensitive Isc is Na+ selective. As shown in Figure 2, B and D, growth media has no apparent effect on responsiveness to forskolin and other agonists (e.g., NECA) that acutely modulate anion secretion. Such acute modulation is the focus of ongoing studies. All subsequent experiments in this report employ cells cultured in typical media and are conducted in the presence of amiloride (10 µM) to minimize the contribution of ENaC to the overall ion transport activities of the human vas deferens epithelial cell monolayers.
Adenosine Modulation of Ion Transport
Cultured human vas deferens epithelial cell monolayers respond to adenosine agonists and other stimulants (e.g., forskolin) with prototypical changes in Isc that are characterized by a rapid (<2 min) increase in Isc that reaches a peak value and declines to a sustained plateau over a 5–10-min period (e.g., see Fig. 2, D and B). A sustained plateau has been observed in the ongoing presence of stimulant for periods in excess of 60 min (not shown). Agonist-stimulated Isc is inhibited by pharmacologic modulators (bumetanide, DASU-02, and DNDS) that closely parallel previous reports of anion secretion across porcine vas deferens epithelium [5–7]. These results are consistent with the presence of NKCC, CFTR, and NBC1, respectively, and suggest that human vas deferens secrete both Cl- and HCO3- when stimulated by these ligands [7].
Experiments were conducted to functionally determine the receptor subtype(s) that accounts for stimulation by adenosine analogs. Possibilities included both P1 (i.e., adenosine receptors; AR) and P2 purinergic receptors (P2R). To test for contributions from these receptor families, monolayers were exposed to selective agonists and antagonists. As shown in Figure 3A, the nonhydrolyzable analog of ATP that acts as an agonist of some P2R subtypes, AMP-PCP, is without effect, although both adenosine and NECA, AR agonists, caused prototypical responses characterized by transient and sustained components (Fig. 3, A and B). Results summarizing 4–11 observations for each agonist are presented in Figure 3C. Adenosine and NECA are associated with significantly greater changes in Isc than AMP-PCP (P < 0.02–0.05), although responses were not different from one another (P > 0.2). These results provide substantial functional evidence that ion transport across human vas deferens epithelia is modulated by ARs.
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Adenosine Receptors Reside in the Apical Membrane of Vas Deferens Epithelia
Experiments were conducted both to functionally identify the subcellular localization of the adenosine-mediated effects and to further verify that ARs modulate vas deferens ion transport. Apical (Fig. 4A) but not basolateral (Fig. 4B) exposure to adenosine stimulates anion secretion, clearly demonstrating that ARs are restricted to the apical membrane of this polarized epithelium. 8-Phenyltheophylline, an AR antagonist, fully blocks the effect of adenosine, although forskolin, a direct adenylyl cyclase activator, stimulates anion secretion (Fig. 4C). Results from these and 4–8 additional observations for each condition are summarized in Figure 4D. Significant effects of adenosine are observed only when exposed to the apical aspect of the epithelium and then only in the absence of the AR antagonist.
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Adenosine Stimulates Cellular cAMP Generation
Adenosine stimulation of ion transport precludes subsequent stimulation by forskolin, an activator of adenylyl cyclase, suggesting that adenosine might affect the cAMP second messenger pathway. To test for this possibility, cellular generation of cAMP was assessed following exposure of epithelial monolayers to adenosine in the absence or presence of the adenosine receptor antagonist, 8-phenyltheophylline. As shown in Figure 5, basal cAMP generation is low and unchanged by 8-phenyltheophylline but clearly measurable by the assay. Apical adenosine caused a 5-fold increase in cellular cAMP, an effect that fully blocked 8-phenyltheophylline. Results presented in Figure 5 are representative of those from two donor tissues. These results indicate that apical adenosine receptors are positively linked to the cAMP second messenger cascade, a linkage that can account for the adenosine-dependent changes in the magnitude of forskolin-stimulated anion secretion. Taken together, results presented in Figures 2–5 provide compelling evidence that epithelial cells isolated from adult human vas deferens express adenosine receptors on their apical membrane and that stimulation of these receptors leads to anion secretion that, if it occurred in vivo, would be expected to acutely alter the environment to which sperm are exposed.
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Freshly Excised Human Vas Deferens Recapitulates Results Observed with Cultured Epithelial Cells
The voltage-sensitive vibrating probe was employed to test for ion transport across freshly excised vas deferens epithelium. Typical results presented in Figure 6 demonstrate that freshly excised vas deferens exhibits ongoing ion transport that is consistent with anion secretion and/or cation absorption. In this system, tissue orientation is such that there is free access of the perfusion solution to the apical aspect of the epithelium whereas the basolateral aspect retains 
0.2 mm of submucosal tissues that reduce diffusional access to the basolateral membrane. Exposure to amiloride is associated with an immediate and rapid reduction in net ion transport that suggests that native epithelium absorbs Na+ on an ongoing basis. Subsequent exposure to adenosine resulted in an increase in current that is consistent with anion secretion. Adenosine-stimulated ion transport is rapidly and completely blocked by exposure to 8-phenyltheophylline. The effects of all compounds, 8-phenyltheophylline, adenosine, and amiloride fully reverse with washout, further supporting the conclusion that the effects result from interaction of ligands with apical channels or receptors. In other experiments, forskolin-stimulated anion secretion was inhibited by DASU-02, an inhibitor of apical CFTR anion channels (not shown). Results presented in Figure 6 are representative of seven recordings from tissues of three donors. Like adenosine, NECA also reversibly stimulated anion secretion (not shown). These results demonstrate that human vas deferens epithelium actively participates in generating and modulating the luminal environment by chronically and acutely adjusting ion transport activity in vivo.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Approximately 1 in 6 couples experiences primary or secondary infertility [30–32]. Male factors contribute to 50% of these cases, with a surprisingly high proportion of male-factor infertility diagnosed as idiopathic [30–33]. Present results clearly demonstrate that ion transport across vas deferens epithelium is under both acute and chronic control and the clinical pathology associated with cystic fibrosis (CF) indicates that ductal ion transport is imperative for normal reproductive function. Causal links between ion transport disorders and infertility are not fully appreciated because neither the transport pathways nor the regulatory pathways have been defined in human vas deferens. The present results demonstrate that multiple ion transport mechanisms are present and that these transport processes are tightly regulated by extracellular messengers. The relationship of ductal ion transport to infertility remains to be determined.
Amiloride-sensitive ion transport has not been previously described in distal human vas deferens, although it has been reported in cultured porcine [6] and ovine vas deferens [4]. In each of these reports as well as the present study, a relatively low concentration of amiloride (10 µM) was employed, allowing for the conclusion that ENaC is present and active in the defined conditions. Cation substitution studies were employed in this study to further rule out the possibility that amiloride affects a nonselective cation channel in this epithelium. Other reports of amiloride-inhibitable transport in the male reproductive tract employed higher amiloride concentrations, with corresponding conclusions that Na+/H+ exchangers (NHEs) are present and contribute to net ion flux [34]. The present results allow for the possibility that NHEs are present in the human vas deferens because the electrophysiological systems employed would not necessarily detect the activity of an electroneutral exchanger. A contrast to previous studies is that amiloride-sensitive current is always observed in the human epithelia whereas porcine epithelia exhibit this current only following glucocorticoid exposure [6]. Thus, human vas deferens cells either intrinsically express some given amount of ENaC or the cells have some ‘memory’ of expression modulators that were present in vivo. Cultured human and porcine epithelial cells are similar in that cortisol exposure increases the magnitude of amiloride-sensitive current in both, suggesting similar regulatory mechanisms for this component of the overall ion transport pathway. It is particularly important to note that amiloride-sensitive current is present in freshly excised human tissues, ruling out the possibility that electrogenic Na+ absorption is an artifact of cell culture and indicating that this pathway is present, active, and regulated in vivo.
Corticosteroid-modulated Na+ transport has significant ramifications as one considers fertility. Increases in Na+ absorption would change the composition and/or the volume of the luminal contents, which could, in turn, affect sperm transit efficiency. Additionally, vas deferens epithelia express both NHE and Na+/HCO3- cotransporters (NBCs) [7, 35–37]. Thus, any change in luminal or cytosolic Na+ activity would be expected to change the luminal pH, a factor known to modulate sperm activity [38]. The results suggest that administration of immunosuppressive glucocorticoids would be expected to increase ductile Na+ absorption, whereas treatment with glucocorticoid antagonists (e.g., mifepristone) or amiloride would decrease ductile Na+ absorption and secondarily affect luminal pH. Furthermore, the results provide a possible mechanism that might explain the association between cortisol excess and male factor infertility [39].
Errant anion secretion is clearly associated with male infertility. CF, a genetic disease characterized by reduced HCO3- and Cl- secretion and elevated Na+ absorption, is associated with reduced sperm quality in some cases [21–25] while being associated with gross changes in ductal anatomy in others [17–21]. CF is the most common lethal recessive genetic disease of Caucasians with approximately 1 in 25 North Americans being carriers of an affected allele. Epithelial tissues throughout the body are compromised to varying degrees, with >97% of confirmed CF cases in men including reproductive dysfunction [40]. Thus, the male reproductive tract and especially the vas deferens appear to be more sensitive to the loss of a single ion channel, CFTR, than any other epithelium throughout the body. Numerous studies have shown that a disproportionately high percentage of men seeking assistance at reproductive health centers have at least one affected CFTR allele [22, 23, 25]. It is interesting to note that males diagnosed with idiopathic infertility also had a significantly elevated proportion of CFTR mutations [24].
The present results support a model of vas deferens function that deviates from the currently accepted model by including neurotransmitter- and hormone-induced changes in the luminal environment. Recent publications reiterate observations from an early report that rat vas deferens has an acid luminal pH [41] and go on to show that mechanisms are present in the rat epididymis to maintain an acidic environment that is thought to be required for sperm maturation and storage [16, 35–37, 42–45]. Vas deferens epithelia receive extensive innervation [8–13], and we have shown that porcine epithelium is responsive to a variety of neurotransmitters [5] and steroid hormones [6]. Neurotransmitter-induced stimulation of HCO3- secretion would be expected to activate sperm during the period of sexual arousal and thus to deliver fully activated sperm at ejaculation [38]. Adenosine may be released by cells lining the vas deferens, by cells in a more proximal portion of the duct, or perhaps by sperm. As stated above, modulation of Na+ transport via glucocorticoids would be expected to affect luminal fluid volume and pH. Most important, the present results show that vas deferens epithelium is a dynamic component of the male reproductive system and that epithelial activity can be modulated to therapeutically affect fertility.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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; e-mail: bschultz{at}vet.ksu.edu
Received: 17 July 2002.
First decision: 23 August 2002.
Accepted: 2 October 2002.
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REFERENCES |
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T. M Hagedorn, R. W Carlin, and B. D Schultz Oxytocin and Vasopressin Stimulate Anion Secretion by Human and Porcine Vas Deferens Epithelia Biol Reprod, September 1, 2007; 77(3): 416 - 424. [Abstract] [Full Text] [PDF]
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