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BOR - Papers in Press, published online ahead of print March 5, 2008.
Biol Reprod 2008, 10.1095/biolreprod.107.065508
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BIOLOGY OF REPRODUCTION 78, 1119–1126 (2008)
DOI: 10.1095/biolreprod.107.065508
© 2008 by the Society for the Study of Reproduction, Inc.

Female Reproductive Tract; 

Effect of Basolateral Adenosine Triphosphate on Chloride Secretion by Bovine Oviductal Epithelium1

N. Keating  and L.R. Quinlan  2

Department of Physiology, National University of Ireland Galway, Galway, Ireland

ABSTRACT

The composition of the fluid within the oviduct is largely determined by the secretory and absorptive activities of the oviduct epithelium. The present study explored the effects of basolateral nucleotide stimulation on ion transport in the bovine oviduct using the chamber short-circuit current technique. Basolateral application of ATP induced a rapid transient increase in ion secretion by oviduct epithelial monolayers in a concentration-dependent manner. The ATP-induced short-circuit current (ISC) response was preserved in the presence of amiloride, whereas it was reduced in the absence of extracellular chloride or in the presence of bumetanide. The channels underlying the chloride secretory response were identified as Ca2+-activated Cl– channels and CFTR. The ATP-induced Cl– secretory response was largely preserved in the absence of extracellular Ca2+ but was significantly reduced in the presence of BAPTA-AM (1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid-acetomethoxy ester), thapsigargin, or 2-APB (2-aminoethoxydiphenylborate), demonstrating an important role for intracellular Ca2+ signaling in mediating these effects. A nucleotide potency profile of ATP = UTP (uridine triphosphate) > ADP, sensitivity to suramin, and cross-desensitization by basolateral UTP suggests that ATP exerted its effects on chloride secretion through the purinergic receptor P2Y, G protein-coupled 2, and the presence of the P2RY2 gene was confirmed by RT-PCR. These results provide strong evidence that purinergic signaling constitutes a key mechanism of regulating chloride secretion and thus fluid formation in the bovine oviduct.

ATP, chloride, female reproductive tract, oviduct, purinoceptors, secretory epithelia, signal transducers, signal transduction

INTRODUCTION

The mammalian oviduct forms a dynamic, optimized microenvironment at the site of a wide range of important reproductive events, including sperm and ovum transport, fertilization, the early stages of embryo development, and embryo transport, and it can also act as a sperm reservoir [1]. The fluid formed by the epithelium lining the oviduct tube is essential in creating the appropriate environment for these key events to occur. Fluid formation in the lumen of the tube is fundamental to creating this environment and, furthermore, is critical for the hydration and protection of the mucosal surface. The mechanism and control of oviductal fluid formation are poorly understood in the oviduct and have not been examined in any systematic way or to any great extent. The basic composition of oviductal fluid has been documented, and with respect to ions and by comparison with plasma, it is rich in potassium and bicarbonate [2–4]. In addition, the ionic composition of the oviduct is region specific, with isthmic fluid calcium concentrations greater than those in the ampulla [5].

The formation of oviductal fluid requires transepithelial ion movement. In general, fluid formation as a basic biological principle follows the movement of solutes, particularly ions. In many cases, the flux of chloride ions from the basolateral to apical face of the epithelial layer plays a major role in providing the driving force for fluid formation. Key components in this general process are the basolaterally located Na+K+ ATPase and the Na+K+Cl– cotransporter [6]. Similar components and mechanisms, while suggested by some studies in the oviduct, have not been fully investigated. Nevertheless, numerous studies have reported a chloride flux across the oviduct in a basal to apical direction [7–10]. Downing and coworkers [9] showed that treating cultured human oviduct epithelial cells with a generic chloride channel blocker or using chloride-free conditions reduced the potential difference and short-circuit current (ISC) across a monolayer of oviduct epithelial cells, suggesting that chloride movement was an important component in the generation of transepithelial potential difference and fluid secretion by the human oviduct epithelium.

Extracellular ATP has been shown to have effects on ion or metabolite transport in essentially every major mammalian organ or tissue thus far studied. Adenosine triphosphate has been demonstrated to regulate transepithelial transport in a variety of cell types, including airway, renal, and bile duct, to name a few (reviewed by Burnstock and Knight [11]). Adenosine triphosphate exerts its biological activity through specific cell surface receptors, the so-called nucleotide P2 receptors, of which there are 15 different subtypes to date. The P2Y family members are G protein-coupled receptors commonly associated with Ca2+ mobilization [11]. The P2X family members function as ligand-gated ion channels [12]. A number of studies have examined the effect of ATP on the oviduct from various species, including rabbit [8], bovine [13], and human [14]. In general, these studies have shown ATP to have the ability to induce transient changes in transepithelial potential difference and elevate intracellular calcium levels. Downing et al. [9] suggested that the observed effects of ATP were, in part, mediated by Cl– ions. Based on comparisons in responses to other nucleotides, the purinergic receptor P2Y, G-protein coupled 2 was suggested as the active receptor most likely to be transducing the observed biological response.

In this study, we examined the effect of extracellular ATP on transepithelial ion movement in cultured polarized monolayers of bovine oviduct epithelial cells (BOECs) in primary culture, and we determined what receptors are involved and the signaling pathways eliciting the cellular response. Our results provide direct evidence that basolateral application of ATP stimulates Cl– secretion by BOECs. Thus, this supports the conclusion that autocrine/paracrine effects of ATP may play a key role in oviduct fluid formation and thus are implicated in optimizing the oviduct luminal environment, facilitating a series of key reproductive events.

MATERIALS AND METHODS

Materials

Adenosine triphosphate, UTP (uridine triphosphate), ADP, BAPTA-AM (1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid-acetomethoxy ester), lanthanum chloride, suramin, thapsigargin, amiloride, bumetanide, NPPB (5-nitro-2-(3-phenylpropylamino)-benzoic acid), niflumic acid (NFA), and glybenclamide were purchased from Sigma. CFTRinh-172, DPC (2,2'-iminodibenzoic acid), and 2-APB (2-aminoethoxydiphenyl borate) were purchased from Calbiochem. Chlorotoxin (CTX) was purchased from Alomone Labs. All general cell culture reagents were purchased from Sigma, with the exceptions of pancreatin, fungizone, and fetal calf serum (FCS), which were purchased from Gibco.

Cell Culture

Bovine oviducts at various stages of the estrous cycle were removed from cattle at a local abattoir within 5 min of slaughter. Epithelial cells were isolated according to the method described by Dickens [14], which itself is a modification of methods devised by Glasser and Mulholland [15] for the isolation of rat uterine epithelium. The oviducts were immediately separated from connective tissue, and fat and major blood vessels and the fimbriae were removed. Oviducts were washed in a Ca2+- and Mg2+-free Hanks balanced salt solution (HBSS). Each oviduct was opened longitudinally to expose the epithelia and cut into 1-cm segments and incubated with 0.5% type I trypsin and 2.7% (w/v) pancreatin for 1 h at 4°C, followed by 1 h at room temperature. The cellular suspension was vortexed for 30 sec followed by centrifugation at 500 x g for 4 min. The resultant pellet was washed three times in Ca2+- and Mg2+-free HBSS. After washing, cells were resuspended in prewarmed, pregassed culture medium.

Culture medium consisted of the nutrient mixture of F12 Ham plus Dulbecco modified Eagle medium in a 1:1 ratio (v/v). The final culture medium also had the following additions: 0.1% BSA, 150 IU/ml penicillin G, 150 µg/ml streptomycin sulphate, 1.25 µg/ml fungizone, 2 mM L-glutamine, and 10% FCS. Isolated cells from multiple oviducts were pooled and plated at a density of approximately 1 x 106 cells/ml. During isolation, ciliated and nonciliated cells are clearly visible. However, over the course of the culture period, the cilia disappear, and the cells are nonciliated epitheliallike cells.

Immunocytochemistry

Isolated bovine oviduct epithelial cells were cultured for 5–7 days in glass-bottomed six-well plates to achieve confluency. Cells were washed three times in rinse buffer (20 mM Tris HCl, 150 mM NaCl, 0.05% Tween-20), fixed with 4% paraformaldehyde (Sigma) for 20 min, and then permeabilized with 0.1% Triton X-100/PBS for 20 min at room temperature. Cells were then washed three times in rinse buffer, followed by a blocking solution (4% goat serum; Sigma) for 15 min and stained with monoclonal anti-pan cytokeratin (Sigma) for 1 h. Cells were washed three times with rinse buffer and incubated with a TRITC-labeled goat anti-mouse immunoglobulin M secondary for 60 min. Cells were then washed, nuclei counterstained with 4',6'-diamidino-2-phenylindole (DAPI; Sigma), washed three times in rinse buffer, and visualised on a Zeiss Axiovert 200 inverted microscope.

Electrophysiological Studies

Cells were plated at a density of 1 x 106 cells/ml on Snapwell inserts (Corning Costar) and achieved confluence after approximately 4 days. A volume of 250 µl of the cell suspension was added on top of the inserts, and 4 ml of culture medium added underneath. The cells were incubated in a humidified incubator at 37°C and gassed with 5% CO2 in air. The medium above and below the filters was replaced every 48 h. The development of an appreciable transepithelial resistance across these filters was used an indicator of the formation of a polarized monolayer. Monolayers were only considered for use in electrophysiological studies if the resistance across the monolayer was greater than 1 K{Omega}/cm2. Filters were placed in modified Ussing chambers (World Precision Instruments Inc., Sarasota, FL), with both surfaces of the cells bathed with normal Krebs Ringer bicarbonate solution and gassed with 95% O2/5% CO2 at 37.5°C. The normal Krebs Ringer bicarbonate solution contained 118 mM NaCl, 25 mM NaHCO3, 4.74 mM KCl, 1.19 mM MgSO4, 1.17 mM KH2PO4, 1.17 mM CaCl2, and 1 mM glucose, gassed with 95% O2/5% CO2. In chloride-free medium, NaCl, KCl, and CaCl2 were replaced with sodium gluconate, potassium gluconate, and calcium gluconate, respectively.

The spontaneous transmembrane potential was measured using a voltage clamp amplifier (DVC 1000; World Precision Instruments) and clamped to 0 mV by the application of an ISC. The output from the amplifier was digitized using a MacLab (AD Instruments) and analyzed using Chart software (AD Instruments). Cells were allowed to equilibrate for 20 min to achieve a stable ISC before beginning an experiment. Adenosine triphosphate, UTPm, and ADP were prepared in normal Krebs Ringers and added to the experimental chamber in 100-µl aliquots. All other agents/antagonists were prepared in dimethyl sulfoxide (maximum final volume of 0.1%). Where ion channel inhibitors were used, the cells were incubated in the presence of the inhibitor for 10 min prior to ATP addition. In this case, all results were expressed as a percentage of the control ATP response.

RNA Isolation

Briefly, oviduct epithelial cells (up to 5 x 106) cultured on Snapwell inserts were collected by scraping the cells from the growing surface using a Pasteur pipette. The cellular suspension was then centrifuged, and RNA isolation was achieved using a Nucleospin RNA II kit from Macherey-Nagel per manufacturer's instructions. The absorbance ratio at 260/280 nm of the resultant purified RNA was measured to determine quality and the RNA concentration from its absorption at 260 nm.

Reverse Transcriptase-Polymerase Chain Reaction

Reverse transcriptase-PCR analysis was performed using an enhanced avian HS RT-PCR kit from Sigma (catalogue no. HSRT-ZO) using a set of specific primers designed to amplify regions highly conserved in bovine purinoceptors by RT-PCR. The amplification of the housekeeping gene 18S ribosomal RNA was used as an internal control. Primer sequences and locations from published cDNA sequences in GenBank are shown in Table 1. The one-step RT-PCR reaction was carried out per the manufacturer's instruction, with no modifications. The conditions for PCR were as follows: 45°C for 45 min; 94°C for 2 min, followed by 35 cycles (94°C for 15 sec, 55°C for 30 sec, and 68°C for 1 min), and finally 68°C for 5 min. As negative controls, reactions were carried out in the absence of reverse transcriptase or primer, and experiments were repeated at least three times. The PCR products were analyzed by agarose (1.5%) gel electrophoresis and stained with ethidium bromide. Bands were visualized using an Alpha DigiDoc system. Bands of interest were excised and sequenced commercially by MWG biotech AG Germany.


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  		TABLE 1. Primer sequences for RT-PCR.

Experimental Design

Results are expressed as means ± SEM. Each cell isolation is an experimental block, and each well/monolayer a replicate within the block. Thus, each block contained cells pooled from two or more animals. For all treatments, n indicates the number of wells/monolayers examined, and in all cases replicates were examined over two or more experimental blocks. In all cases, each treatment was paired with a vehicle control. The vehicle control was found to have no effect on basal or ATP-stimulated ISC responses. All results were examined statistically by analysis of variance and, where appropriate, the Bonnferroni/Dunn posthoc test was used. P values < 0.05 were considered to be statistically significant.

RESULTS

Immunocytochemistry—Characterization of the Cell Population

Freshly isolated cells contain a mixture of ciliated and nonciliated cell types. Cell cultures reached confluency after approximately 5–6 days. At the end of the culture period, all cells stained intensely for cytokeratins, confirming the culture to be dominated by epithelial cells (Fig. 1).


Figure 01
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  		FIG. 1. Immunocytochemical characterization of BOECs after 6 days in culture. Representative image of BOECs stained for cytokeratins; the nuclei are counterstained with DAPI as described in Materials and Methods. Bar = 25 µM.

The Effect of ATP on Bovine Oviduct ISC

Application of ATP to the basolateral surface of the oviduct cell monolayer induced a rapid transient increase in ISC with no detectable latency, reaching a peak after approximately 3.5 sec (Fig. 2A). The increase in ISC was dose dependent over the range 1 µM to 2 mM ATP (Fig. 2B). Adenosine triphosphate at a dose of 100 µM induced a substantial ISC response that was below the maximum measured and was chosen as the concentration of ATP for all further studies.


Figure 02
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  		FIG. 2. Effect of ATP on ISC across a polarized BOEC monolayer. A) Representative trace of 100-µM ATP challenge to basolateral membrane of the monolayer. B) Dose-response curve to ATP challenge to basolateral membrane. Bars represent the peak change in current postchallenge. All levels are significantly different from control (P < 0.001), and bars with common symbols (*) are not significantly different from each other. All values represent at least seven replicates.

To investigate the possibility of ATP desensitization, the basolateral membrane was challenged with a repeat equimolar dose of ATP 5 min after the initial challenge. The second challenge was 90% less effective at altering the ISC (Fig. 3A). Washout of the bathing solution between doses (30-min duration) improved the efficacy of the second challenge, but only up to a maximum of 50% (Fig. 3B).


Figure 03
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  		FIG. 3. Desensitization of ATP response. A) Representative trace of 100 µM ATP rechallenge to basolateral membrane of a polarized BOEC monolayer. B) Plot of peak current in response to ATP, rechallange, and rechallange after washout. Both washout (*) and rechallenge in the absence of washout (**) were significantly different from ATP alone and each other (P < 0.001). All values represent at least seven replicates.

Involvement of Purinoceptors in the ATP Response

Pretreatment of the cells with the general P2 receptor blocker suramin inhibited the ATP response by more than 50% (Fig. 4A). To determine the P2 subtype activated, a panel of nucleotides was examined as to their ability to activate an ISC. The rank order of potency was determined as ATP = UTP > ADP (Fig. 4B). This profile is most consistent with the P2RY2 subclass of purinoceptors. Cross-desensitization experiments were also carried out to explore interaction between ATP and UTP. Prestimulation with ATP almost completely inhibited the response to UTP (Fig. 5A). The same was true in the reverse: prestimulation with UTP almost completely inhibited the response to ATP (Fig. 5B). Prestimulation with ATP completely blocked the ADP response (Fig. 5C). The same was partially true in the reverse: prestimulation with ADP attenuated the ATP response but did not fully block it (Fig. 5D).


Figure 04
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  		FIG. 4. Effect of P2 blockers and nucleotides on the ATP response. A) Effect of suramin (500 µM) on the ATP response. *P < 0.001 compared with control. B) Plot of peak current in response to equimolar challenges with ATP, UTP, and ADP. *P < 0.001 compared with ATP. Values with common symbols (**) are not significantly different from each other. All values represent at least seven replicates.


Figure 05
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  		FIG. 5. Cross-desensitization of ATP response with UTP and ADP. Representative trace of (A) ATP (100 µM) followed by UTP (100 µM), (B) UTP (100 µM) followed by ATP (100 µM), (C) ATP (100 µM) followed by ADP (100 µM), and (D) ADP (100 µM) followed by ATP (100 µM) (n ≥ 4). The x-axes show time measured in seconds.

Investigating the Ionic Basis of the ATP-Induced ISC

The polarity and direction of the measured current suggested that the current was either due to cation absorption or anion secretion. Amiloride, the epithelial sodium channel (ENaC) blocker, had no effect on ATP-stimulated ISC (Fig. 6A), but inhibited baseline current by approximately 50% (Fig. 6B). To examine the potential role of chloride in the ATP-induced ISC, chloride ions were replaced by gluconate in the solution bathing either one or both faces of the epithelium. Removal of chloride from the basolateral surface resulted in the complete inhibition of the ATP response. When chloride was removed from both surfaces of the monolayer, the ATP response was inhibited by approximately 90% (Fig. 6A). Furthermore, addition of bumetanide to the basolateral bathing solution reduced the ATP response in excess of 80% (Fig. 6A).


Figure 06
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  		FIG. 6. Effect of ENaC inhibition, chloride removal, and Na+/K+/Cl– transporter inhibition on the ATP response. A) Effect of apical amiloride, chloride removal bilaterally, chloride removal basolaterally only, and addition of bumetanide on the ATP response. Bars with symbols (*, **) indicate significance, P < 0.001 compared with control. Values with common symbols are not significantly different from each other. B) Representative trace of effect of amiloride on baseline current and on the ATP-stimulated ISC. All values represent at least seven replicates.

Role of Apical Chloride Channels in the ATP Response

To investigate the type of chloride channel involved in the generation of the ATP-stimulated response, a panel of chloride channel inhibitors was used. The chloride channel blockers are generally placed into one of two categories: cystic fibrosis transmembrane regulator (CFTR) blockers (GLYB, DPC, Cl-172, and NPPB) or calcium-activated chloride channel (CLCA) blockers (NFA and CTX). All CFTR-specific blockers employed inhibited the ATP response, but only up to a maximum of 25% (Fig. 7). The nonspecific chloride channel blocker NPPB was more effective at inhibiting the ATP response (approximately 50% inhibition). The CLCA blockers were far more effective in blocking the ATP response. Both NFA and CTX had similar effects and blocked the ATP response by approximately 65% (Fig. 7).


Figure 07
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  		FIG. 7. Effect of chloride channel inhibitors on the ATP response. The CFTR and nonspecific chloride channel blockers GLYB (100 µM), DPC (100 µM), CI-172 (100 µM), and NPPB (500 µM), and the calcium-activated chloride channel blockers NFA (100 µM) and CTX (1 µM) were added apically 10 min prior to ATP challenge. Bars with symbols indicate significance, P < 0.001 compared with control. Values with common symbols are not significantly different from each other. All values represent at least seven replicates (P < 0.001).

Role of Calcium in the ATP Response

Based on the chloride channel inhibitor experiments, it is clear that CLCAs play a major role in the ATP response. Therefore, we examined the contribution of intracellular and extracellular calcium in this phenomenon. Preincubation with thapsigargin or BAPTA-AM essentially abolished the ATP response (Fig. 8). In addition, 2-APB, a blocker of calcium release from IP3-sensitive stores, inhibited the ATP response by approximately 60% (Fig. 8). We also examined the contribution of external calcium using the trivalent metal ion lanthanum (La3+), which inhibits the entry of external calcium into the cell. La3+ alone blocked the ATP response by approximately 20%; however, the combination of La3+ and 2-APB blocked the ATP response by more than 90% (Fig. 8).


Figure 08
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  		FIG. 8. Effect of thapsigargin, BAPTA, 2-APB, and La3+ on the ATP response. Thapsigargin (THAPS, 1 µM added to the basolateral solution), BAPTA-AM (BAPTA, 25 µM added to both apical and basolateral solutions), 2-APB (APB, 150 µM), and La3+ (La3+, 250 µM) were added 20 min prior to ATP challenge. All treatments were significantly different from control (P < 0.001); bars with common symbols are not significantly different from each other. All values represent at least four replicates.

Expression of Purinoceptors

Using RT-PCR, mRNA expression for each of the P2Y purinoceptors was investigated. Reverse transcriptase-PCR was performed on total RNA isolated from bovine oviduct epithelial cells, which had been grown to confluence on Snapwell inserts under the same conditions used for standard electrophysiology experiments. Polymerase chain reaction products were derived using specific primers corresponding to the published sequences for P2RY1, P2RY2, P2RY4, P2RY6, P2RY11, P2RY12, P2RY13, and P2RY14 purinoceptors. Specific primers for 18S rRNA were used as houskeeping controls. Specific bands of predicted size were identified for P2RY2, P2RY4, and P2RY12 (Fig. 9). These bands were excised and sequenced commercially by MWG Biotech, which confirmed their identity matching GenBank accession numbers XM_612432.2, AY540307.1, and AJ623293.1, respectively.


Figure 09
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  		FIG. 9. Reverse transcriptase-PCR analysis of total RNA (in bp) isolated from BOECs using gene-specific primers for P2RY1, P2RY2, P2RY4, P2RY6, P2RY11, P2RY12, P2RY13, and P2RY14 purinoceptor subtypes. Bands of predicted size are marked with an asterisk (*). M, Molecular weight markers.

DISCUSSION

The present study investigated the mechanisms underlying basolateral ATP-activated chloride secretion across the bovine oviduct epithelium. The results have clearly demonstrated that the basolateral membranes of BOECs express functional purinoceptors which, when activated, cause rapid transient increases in chloride secretion, primarily through activation of apically located Ca2+-activated Cl– channels. Furthermore, we propose that ATP exerts its effects by binding to the purinergic receptor P2RY2 on the basolateral cell membrane. Several lines of evidence support this: first, we have shown a dose-response relationship between ATP stimulation and changes in ISC. Second, the effect of ATP shows clear desensitization and is reduced by suramin, a P2 receptor antagonist. Nucleotide potency profile reveals that UTP and ATP are equipotent in stimulating an increase in ISC, with ADP much less potent. This is consistent with the presence of the P2RY2 subtype [16]. Third, cross-desensitization experiments revealed that ATP and UTP cross-desensitized each other, suggesting these agonists act at the same receptor subtype. These results are in broad agreement with findings observed in other polarized epithelial monolayer preparations from a number of other tissues, including airways [17], kidney [18], pancreatic duct [19], and gallbladder epithelium gland [20].

Although the potency profile from our work strongly suggests the involvement of the purinergic receptor P2Y, G-protein coupled, 2 subtype, it is important to note that in the rat, purinergic receptor P2RY4 shows equal sensitivity for ATP and UTP (like the classical P2RY2). Since the nucleotide selectivity of bovine P2RY4 is unknown, the possibility that it has some involvement in the response to ATP in cattle cannot be ruled out.

The lack of effect of amiloride on the ATP-induced increase in ISC rules out a possible stimulation of sodium absorption via ENaC by ATP. The transient ATP-induced increase in ISC was markedly reduced by the removal of Cl– from the bathing media, providing the first clear evidence that the source of the ISC increase is chloride secretion. The inhibitory effect of basolateral bumetanide on the ATP response further confirmed the stimulation of Cl– secretion by ATP and also illustrated the importance of the Na+/K+/2Cl– cotransporter in this process. Similar observations of the effects of ATP on chloride secretion in the human [9] and bovine [13] oviducts have been made previously; however, these studies did not investigate the type of chloride channels responsible for this response.

It has been shown in airway epithelial cells that extracellular ATP or UTP could stimulate CFTR-dependent anion secretion through a calcium-independent pathway subsequent to the activation of P2RY2 [21]. Expression of CFTR in the oviduct has been observed in the mouse [22] and also in humans [23]. In this study, the inhibitory effects of CFTR inhibitors on the basal ISC (data not shown) suggest the presence of open CFTR channels in the apical membrane of BOECs. However, these channels appear to play only a minor role in the ATP response.

The CLCA1 is the primary candidate to mediate the Cl– secretory response induced by ATP. Indeed, the stimulation of CLCA1 through the activation of P2RY2 receptors has been reported in a number of epithelial cell types [24]. The substantial inhibitory effect of CLCA1 inhibitors NFA and CTX on the ATP response demonstrates the importance of these channels in mediating the chloride secretory effect. Our data demonstrate that the change in ISC in response to ATP in BOECs is largely independent of extracellular calcium but is consistent with calcium being released from intracellular stores. Pretreatment with thapsigargin or BAPTA or 2-APB showed the response to be dependent on internal calcium mobilization via IP3 receptor-mediated stores. This is consistent with the known effects of G protein-coupled P2RY2 receptor activation in stimulating IP3 formation [16]. Furthermore, the expression of the P2RY2 was confirmed by RT-PCR. The expression of the P2Y receptor subtypes P2RY4 and P2RY12 was also demonstrated. The functional role of P2RY2 and P2RY4 in regulating epithelial ion transport is well documented; for example, activation of potassium secretion [25, 26], chloride secretion [27, 28], and inhibition of sodium absorption [18, 28] are well-established effects of P2RY2 and P2RY4 activation, which correlate well with our findings in the oviduct.

An obvious question arises as to the physiological role of ATP based on the transient nature of the response. The transient nature of calcium-dependent chloride secretory responses in epithelia has been suggested to subserve an important physiological role by limiting secretion only to when it is acutely needed [29]. The purinergic regulation of chloride secretion within the oviduct may facilitate local changes in luminal fluidity in response to signals provided by the physicochemical status of the luminal contents (i.e., presence of sperm, oocyte, or embryo) as well as neurohormonal stimuli. Thus, by controlling chloride secretion, ATP plays a key role in the production of oviductal fluid, thereby controlling the luminal microenvironment.

In this scenario, however, the source of ATP acting upon the oviduct is unknown, and it is possible that sperm and/or embryos may release ATP, providing a means by which these gametes may communicate with the maternal tract. A number of studies have shown that oviduct ciliated cells increase beat frequency when spermatozoa [30] are added and that embryos are transported down the tube more quickly than unfertilized eggs [31]. Considering ATP has been shown to increase ciliary beat frequency in humans via a calcium-dependent mechanism, it is clear that ATP may be an important signaling molecule acting on the luminal epithelium.

Alternative sources may account for basolateral acting ATP; these include ATP released from the epithelial cells themselves, an event that is well documented in other epithelia. A variety of stimuli are capable of initiating ATP release, including inflammatory signals [32], steroid hormones [33], and mechanical perturbations [34]. Thus, in the context of the oviduct it is possible that mechanical stimuli, such as the shear presence of an embryo or sperm within the oviduct or the resultant increased ciliary activity of the oviduct cells, may trigger release of ATP both apically and basolaterally. Furthermore, ATP is a well-known cotransmitter at nerve endings. The role of the nervous stimulation in oviductal function is not well studied; however, it is known that the oviduct is well supported by both the circulatory and nervous systems, either of which could act as a rich source of ATP.

Given that the fluid within the oviduct provides the essential microenvironment for a number of important reproductive processes, it is very surprising that so little is known about the specific ion transport mechanisms involved that determine the formation of this fluid within the oviduct. Our current study has demonstrated the clear role for purinergic regulation of ion transport and subsequent fluid formation in the oviduct, providing valuable new information that may help in the treatment of the diseases associated with oviduct malfunction. Also, gaining a better understanding of oviductal fluid formation may provide a potential therapeutic focus, which could benefit current and future in vitro fertilization treatment regimes by augmenting any positive effect of the oviduct on fertilization and embryo development.

ACKNOWLEDGMENTS

The authors wish to thank Professors M.T. Kane NUI Galway Ireland and H.J. Leese University of York UK for their comments in preparing the manuscript.

FOOTNOTES

1Supported in part by the Millennium Research Fund, National University of Ireland Galway. Back

Correspondence: 2FAX: 353 91 750544; e-mail: leo.quinlan{at}nuigalway.ie

Received: 10 September 2007.

First decision: 4 October 2007.

Accepted: 26 February 2008.

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