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Male Reproductive Tract |
a Departments of Physiology
b Anatomy, Institute of Biomedicine, University of Turku, FIN-20520 Turku, Finland
c Department of Biology, Åbo Akademi University, BioCity, FIN-20520 Turku, Finland
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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calcium, epididymis, male reproductive tract, signal transduction, sperm maturation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The signals triggered by a number of ligands, including hormones and neurotransmitters, are transferred across the plasma membrane by GPCRs. All GPCRs are structurally similar, although they recognize a specific variety of ligands. GPCRs are integral proteins of the plasma membrane with similar basic structure (i.e., an extracellular N-terminal portion, seven hydrophobic 
-helix segments spanning the membrane, and an intracellular C-terminus [3]). Occupation of the GPCR subtypes by various ligands may lead to one or several of the following intracellular responses: stimulation or inhibition of adenylyl cyclase; activation of phospholipase C (PLC); generation of inositol-1,4,5-trisphosphate (IP3), which releases calcium from intracellular stores; or activation of protein kinase C (PKC), phospholipase A2 (PLA2), or direct G protein activation of inwardly rectifying K+ channels, voltage-dependent N-type channels, or P/Q type calcium channels [3]. Calcium is one of the intracellular messengers that mediates the action of GPCRs. The rise in cytosolic free calcium can activate a variety of cellular responses such as motility, secretion, muscle contraction, gene expression, and cell division [4–6]. Thus, calcium is a second messenger with a wide spectrum of activities.
In the present study, we investigated the expression of purine nucleotide receptors linked to the calcium signaling pathway in mouse epididymis. The importance of purinergic receptors for fertility was indicated by the findings that P2X1 purinergic receptor knockout mice were infertile [7]. However, the infertility was not caused by maturational defects of spermatozoa in the epididymis, but rather by a reduction in the contraction of the vas deferens, thus reducing the amount of ejaculated spermatozoa [7]. It has been previously shown that extracellular purine nucleotides acting on purinergic receptors stimulate Cl- secretion in primary culture of epithelial cells in both rat and mouse epididymis [8, 9]. However, nothing is known about the purinergic receptor subtypes that are responsible for this action. Our goal is to characterize the region and cell specificity of purinergic receptor subtypes in the epididymis, and to study the physiological role of the different receptor subtypes involved in providing a favorable milieu for sperm maturation.
Two families of purine nucleotide receptors have been characterized so far; the P2X receptor family of seven subtypes (P2X1–7) consisting of ligand-gated ion channels, and the P2Y receptor family with nine subtypes (P2Y1–9) consisting of GPCRs, which have been established in vertebrates [10–15]. Extracellular nucleotides can function as paracrine or autocrine mediators after their release into the extracellular fluid during cell lysis or by excocytosis of nucleotide-concentrating granules, or by an efflux through membrane transport proteins [16]. ATP is secreted by skeletal muscle, adrenal chromaffin cells, mast cells, blood cells, fibroblasts, and endothelial cells [17, 18]. The role of ATP as a neurotransmitter or cotransmitter is also well established in the peripheral and central nervous systems [19, 20]. However, the action of ATP released from intracellular sources is limited by several ecto-ATPases, which keep the levels of extracellular ATP very low [21]. In the present study we characterized the ATP-induced Ca2+ signal in the epithelial cells of caput epididymidis using a novel method with vibratome tissue slices, and provided evidence for the expression of multiple purinergic receptor subtypes in various regions of the mouse epididymis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Probenecid (p-[dipropylsulfamoyl]-benzoic acid) and EGTA were purchased from Sigma Chemical Company (St. Louis, MO). Fura-2 acetoxymethyl ester (Fura-2) was purchased from Molecular Probes, Inc. (Eugene, OR).
Tissue Preparation and Preparation of Vibratome Sections
All mice were handled in accordance with the institutional animal care policies of the University of Turku. Adult male mice (FVB/N strain) were specific pathogen-free, they were fed complete pelleted chow and tap water ad libitum, and were housed in a room with controlled light (12L:12D) and temperature (21 ± 1°C). The mice were killed by CO2 asphyxiation, the epididymides were removed at room temperature and then transferred to a Petri dish containing Dulbecco modified Eagle medium (DMEM)/F12 medium (Gibco, Paisley, U.K.) supplemented with 10% v/v fetal calf serum (Gibco), 100 U/ml penicillin G, 80 U/ml streptomycin sulfate, and 100 nM testosterone (Sigma). Caput epididymides were dissected under a stereomicroscope and separated from the other parts of epididymis, fat was removed, and the tissues were kept at 37°C in an atmosphere of 95% air and 5% CO2 in a fully humidified incubator until sectioning. For sectioning, the tissues were immobilized with 1% low-melting-point agarose in 0.9% NaCl (FMC BioProducts, Rockland, ME) and glued onto the vibratome stage. The stage was placed in a chamber filled with TBM [2-([2-hydroxy-1,1-bis{hydroxymethyl}-ethyl]amino) ethane sulphonic acid (TES)-buffered medium consisting of 137 mM NaCl, 5 mM KCl, 1 mM CaCl2, 10 mM glucose, 1.2 mM MgCl2, 0.44 mM KH2PO4, 4.2 mM NaHCO3, and 20 mM TES] adjusted to pH 7.4 with NaOH at room temperature. Longitudinal sections 150 µm in thickness were cut under a stereomicroscope with a vibratome (752HA; Campden Instruments, Leichestershire, U.K.), carefully removed from the bath with coverslips, and transferred to a Petri dish containing DMEM/F12 medium supplemented with 10% v/v andfetal calf serum, 100 U/ml penicillin G, 80 U/ml streptomycin sulfate, and 100 nM testosterone. Representative sections from different regions of caput epididymides were obtained and kept at 37°C in an atmosphere of 95% air and 5% CO2 in an fully humidified incubator for 16–24 h until calcium measurements were performed. Slices from seven mice were assessed and calcium measurements were performed on 8–12 slices per animal.
Calcium Measurement
The fluorescent calcium indicator Fura-2 was used to monitor changes in intracellular calcium [22]. After the 16- to 24-h preincubation the sections were loaded at 37°C in TBM supplemented with 0.5 mM probenecid and 4 µM Fura-2 AM for 30–60 min. After loading, the sections were washed once with TBM in a Petri dish and transferred to a perfusion chamber. The volume of the perfusion chamber used for calcium measurement was 0.5 ml. In the perfusion chamber the sections were immobilized with a 25-µm-thick membrane filter (Osmonics, Livermore, CA) similar to that recently developed for suspension cells in culture [23]. Thereafter, the cells were perfused for 10 min with TBM at a rate of 2 ml/min before calcium measurements were started. For stimulation, the perfusion was stopped and 100 µl of medium was removed from the chamber with a pipette, and after adding ATP or calcium the medium was readded to the chamber. For stimulations, 100 µM ATP was used because this was found to be the lowest concentration with maximal response in intracellular calcium.
The areas of epithelial cells used to capture fluorescence images were first defined by light microscopy, and from each slice the data were collected from 40 to 50 areas with defined epithelium. Always before starting the experiments a few areas were selected with the imaging program to follow them live. These areas were continuously followed during the stimulations, and when the fluorescence signal after stimulation was sustained, the next substance was added to the chamber or the experiment was stopped. The calcium measurements were performed using a Zeiss Axiovert microscope and Axon 2.2-image workbench. The cells were kept in TBM at 37°C, excited by alternating wavelengths of 340 and 380 nm using a narrow band filter, and fluorescence was measured through a 430 nm dichroic mirror and a 510 nm barrier filter with a charge-coupled device camera. One rationed image was acquired per second. The method we developed is summarized in Figure 1.
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Data Analysis
The image data from the experiments were saved and later analyzed using Calcalc, a recently developed Java program [23] using Metrowerks CodeWarior. This program allows the user to analyze a decided set of cells and to remove unsatisfactory cells from the analysis (poorly loaded cells, cells that had moved during the experiment, or cells with a high basal 340:380 nm ratio). Furthermore, because data were collected on a continual basis (1 image/sec), it allowed us to select the time intervals for data extraction separately for each area analyzed. Briefly, using the Calcalc program, the background fluorescence (the fluorescence value detected in an area without cells) values at 340 and 380 nm at all recorded time points were first subtracted from all the areas used for analysis. Thereafter, the ratios were calculated by dividing the background-subtracted fluorescence values at 340 nm with the background-subtracted fluorescence values at 380 nm for every area analyzed separately. The actual changes in ratios after stimulation were also calculated using the Calcalc program. For all areas analyzed, we first calculated the basal ratio at an interval of 10 sec before agonist addition, and thereafter the maximal ratio values were calculated at an interval of 40 sec after agonist addition separately for each area. The time point for maximal ratio was automatically identified for each area by the program used. The basal and maximal values for each area were then exported to Microsoft Excel and further analyzed. The basal value of each area was then subtracted from its own maximal ratio value. The calcium data presented are the averages of the response of all the areas of epithelial cells examined.
Immunohistochemistry
Immunohistochemistry for P2X purinergic receptors was performed using polyclonal antibodies (Sigma-Aldrich, St. Louis, MO) against rat P2X1, P2X2, P2X4, and P2X7 receptor subtypes. The antibody binding was visualized using the avidin-biotin technique (ABC Kit, Vector Laboratories, Burlingame, CA) and Liquid DAB-Plus Substrate Kit (Zymed Laboratories, San Francisco, CA). For this, FVB/N strain adult male mice were killed with CO2 asphyxiation and the epididymides were removed and fixed in 4% paraformaldehyde in PBS at room temperature for 12–16 h. Two- to three-micrometer-thick paraffin sections were used for immunohistochemistry. Nonspecific protein binding sites were blocked by a 1-h incubation in 10% goat serum in PBS. The sections were then incubated at 4°C overnight in a humidified chamber using the primary antibodies at a dilution of 1–3 µg/ml in PBS and 1% goat serum. The slides were then washed three times for 5 min with PBS and incubated with biotinylated anti-rabbit secondary antibody at room temperature for 30 min. After washing three times for 5 min in PBS, the specimens were incubated for 30 min at room temperature with the ABC solution (prepared as described by the manufacturer). After this, the 3,3'-biaminobenzidine (DAB) reaction was performed for 1 min. Control experiments with no primary antibody were performed routinely and showed no staining.
Reverse Transcriptase-Polymerase Chain Reaction
Total RNA was isolated from caput, corpus, and cauda epididymides of FVB/N strain adult mice by using a single-step method [24], and expression of various P2X and P2Y receptor mRNAs was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). Mouse-specific primers were generated for the subtypes present in GenBank that were known to be expressed in epithelial cells. The primers used are listed in Table 1. One microgram of DNAse I-treated (Gibco, Paisley, U.K.) total RNA was reverse-transcribed with avian myeloblastosis virus RT (Promega, Madison, WI) and amplified using Dynazyme II-polymerase (Finnzymes, Espoo, Finland) in the same reaction tube. Experiments were also performed in the absence of RT to control for possible DNA contamination in the reaction. The PCR conditions for the P2X receptors consisted of 35 cycles of denaturation at 96°C for 1 min, annealing at 58°C for 1 min, and extension at 72°C for 1.5 min. The PCR conditions for the P2Y receptors consisted of 35 cycles of denaturation at 96°C for 1 min, annealing at 60°C for 1 min, and extension at 72°C for 1.5 min. All PCR reactions were terminated with a long extension at 72°C for 10 min and finally transferred at 4°C.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study we investigated ATP-induced Ca2+ signaling using a novel method (Fig. 1). To assure that the measured changes in intracellular calcium concentrations upon stimulation were not due to a contribution of other than epithelial cells, the sections were first inspected with light microscopy. This analysis confirmed the existence of intact epithelial cells of the ducts on the sections used (Fig. 2A). Thereafter, the changes in fluorescence intensity upon ATP stimulation was analyzed from 40–50 defined areas in the epididymal ductules present in the slice (Fig. 2B and C). The Fura-2 AM-loaded vibratome sections of the caput were stimulated with 100 µM ATP, which induced a rapid transient Ca2+ signal that was sustained after 60 sec (Fig. 2D). We further noticed that the Ca2+ signal was equally expressed in the epithelial cells of the different subregions of mouse caput epididymidis (Fig. 3A and B). Preincubating the vibratome sections in Ca2+-free media in the presence of 0.5 mM EGTA for 2 min did not affect the amplitude of ATP-induced Ca2+ signal, but the sustained phase/plateau was completely abolished (Fig. 4A). This indicates the presence of P2Y type purinergic receptors and PLC activity. However, extracellular Ca2+ was also found to be responsible for the ATP-induced Ca2+ signal. This was demonstrated by the results showing that when 1.5 mM CaCl2 was added after ATP stimulation in Ca2+-free media in the presence of 0.5 mM EGTA, there was an influx of calcium into the cells (Fig. 4A). This influx is likely to consist mainly of store-evoked calcium entry, which opens calcium channels in the plasma membrane as the calcium stores are depleted with ATP in a calcium-free buffer, but it could also be partially dependent on P2X channels. In control experiments, 1.5 mM CaCl2 was added as described above before ATP stimulation, and no influx of calcium into the cells was noticed (Fig. 4B). This indicates that the influx of calcium into the cells after calcium addition (Fig. 4A) is not caused by prior depletion of extracellular calcium. The fluorescence imaging studies could not distinguish between calcium entry through P2X channels and store-operated calcium entry, but in later experiments we could confirm the expression of various purinergic receptors in epithelial cells of the caput.
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Expression of mRNAs for P2X and P2Y Purinergic Receptor Subtypes in Mouse Epididymis
Identification of P2X and P2Y receptor subtypes expressed in mouse epididymis was carried out by RT-PCR using primers designed against the mouse P2X and P2Y receptor sequences available in GenBank (P2X4, P2X6, P2Y1, and P2Y2; Table 1), and by using antibodies available for certain subtypes (P2X1, P2X2, P2X4, and P2X7) of rat purinergic receptors. The analyses revealed that transcripts for P2X4 and P2Y1 were detected in all regions of epididymides, including caput, corpus, and cauda. In contrast, the P2Y2 transcript was not detected in cauda epididymides, but it was present in corpus and caput regions (Fig. 5). No transcript could be detected for P2X6 receptor.
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Immunohistochemical analyzes revealed that all subtypes (P2X1, P2X2, P2X4, and P2X7) were expressed in the clear cells of corpus and proximal cauda epididymides, but not in distal cauda. In the caput region, however, there were differences between the expression of the subtypes: P2X1 and P2X7 were not at all expressed in the caput region, and P2X4 was expressed only in the principal cells of the proximal caput, and not in the initial segment. It was interesting that in contrast to the other subtypes, P2X2 receptor was expressed in narrow cells of the intermediate zone of caput epididymidis, and it was the only subtype analyzed that was also expressed in the initial segment (Figs. 6 and 7, Table 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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With all the information available today, it is evident that the epididymis provides the local environment needed for spermatozoa to mature. However, little is known about the proteins involved, and currently, there are only a few gene modified mouse models with a defect in posttesticular sperm maturation and fertility [28, 29]. These in vivo models provide new tools for studying the role of the epididymis in sperm maturation and for testing strategies of nonhormonal male contraceptives. One approach for screening novel targets for generating nonhormonal contraceptives could be the characterization of the maturational events of spermatozoa in epididymis that are dependent on GPCRs. Therefore, in the present study we developed a novel method to investigate the presence of functional GPCRs in the ductus epididymis by analyzing intracellular calcium on living tissue slices.
Functional purinergic receptors on epididymal cells have earlier been reported from studies performed on primary monolayer cultures of rat epididymal cells [9]. In vivo and in vitro studies have also shown that an ATP-induced rise in intracellular calcium concentration in epididymal cells activates Cl- channels on apical membrane, leading to an increase in transepithelial Cl- secretion [9, 30]. In line with these earlier findings, our data with Ca2+ imaging revealed the presence of functional P2Y purinergic receptors in epithelial cells of the mouse caput epididymis, and the signal was equally expressed in epithelial cells of the different epididymal subregions. However, when the subtypes of P2X and P2Y receptors were analyzed with RT-PCR and immunohistochemistry, differences in their expression in different epididymal regions were detected. It was interesting to find differences in their expression in the caput epididymidis and initial segment, which are known to be the most important epididymal regions in terms of sperm maturation [29]. In corpus and proximal cauda regions, all P2X receptors analyzed (P2X1, P2X2, P2X4, and P2X7) were expressed in the clear cells, whereas in the distal caput, only P2X2 and P2X4 were expressed in a cell-specific manner. The P2X4 receptors were expressed in the principal cells, whereas P2X2 receptors were also expressed in narrow cells in the initial segment and intermediate zone. Narrow cells have been identified in the epididymis of many species, including humans [31, 32]. In the mouse epididymis, narrow cells are present in the initial segment and intermediate zone corresponding to segment 1 [33]. They are characterized by an apically located nucleus, which is usually elongated, and an apical cytoplasm, which often bulges into the lumen [31]. In the mouse caput epididymidis, narrow cells also express carbonic anhydrase II and vacuolar proton adenosine triphosphatase (H+V-ATPase) pump [32]. These cells produce and deliver protons to the epididymal lumen for acidification, an event that is essential for spermatozoa as they traverse and are stored in the epididymis. It has been proposed that low pH, in conjunction with specific proteins, weak acids, and other ions maintain spermatozoa in an immobile state during their transit through the epididymis and vas deferens [34], and the role of the P2X2 receptors expressed in narrow cells needs further investigation.
The P2Y receptors were identified throughout the epididymis with RT-PCR and Ca2+ imaging. Recently, it has been shown that the purine nucleotide-activated P2Y receptors can cooperate with 2-adrenoceptors to mobilize intracellular free calcium [18]. Furthermore, ATP has been shown to regulate anion transport in rat Sertoli cells [35]. Because extracellular ATP has multiple effects on cells by activating several receptor signaling pathways, the action of ATP released from cells is strictly regulated by several ecto-ATPases, which reduce the levels of extracellular ATP [21]. Studies on ecto-ATPase activity in the epididymis have not yet been performed, but for example, in the vas deferens of guinea pig, ecto-ATPase activity is high [36].
There are three putative resources for ATP in the epididymis: the epithelial cells themselves (paracrine/autocrine stimulation), spermatozoa, and the neurons innervating the epididymis. Mouse spermatozoa contain a large number of mitochondria that produce high amounts of ATP. Hence, the local concentration of ATP released from spermatozoa is likely to be very high. As the spermatozoa move forward in the ductuli epididymides, ductal epithelial cells being in close contact to the spermatozoa could be locally stimulated by the high intraluminal ATP provided by the spermatozoa. It has been previously proposed that ATP released from degenerating spermatozoa could stimulate fluid secretion locally [9]. An increase in fluidity in the vicinity of the sperm would help in the reabsorption of sperm fragments by the epididymal cells. This mechanism may be involved in the disposition of unejaculated aged sperm by the cauda epididymis. Nevertheless, we should not exclude the possibility that the extracellular purine nucleotides in the epididymis would cooperate with other ligands as well, similar to that shown in other model systems [18, 20, 37, 38]. Whether ATP released from spermatozoa in connection with such a ligand is capable of stimulating the epididymal epithelial cells remains to be analyzed; however, this would be a feasible model for cell-to-cell communication between sperms and the epididymal epithelium. Recently, muscarinic acetylcholine receptors have been identified in rat caput and cauda epididymides, and morphologic data were provided for the presence of a multisynaptic circuit of neurons innervating the epididymis [39, 40]. These neurons could function as a third putative source of extracellular ATP, and in this model, ATP is co-released with a neurotransmitter [20, 37, 38].
The wide expression of the different types of functional purinergic receptors and the evidently cell-specific expression of the subtypes in the caput and initial segment of epididymides is interesting. The physiological role of purinergic receptors in the regulation of luminal ion concentration in the epididymis is evident [8]. However, the physiological role of different subtypes involved in producing the proper environment in the epididymal luminal fluid essential for posttesticular sperm maturation needs to be elucidated. Furthermore, the method developed provides a novel tool for observing dynamic changes in epididymal cells in vitro, and in other reproductive tissues as well.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Matti Poutanen, Department of Physiology, Institute of Biomedicine, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland. FAX: 358 2 250 2610; matti.poutanen{at}utu.fi
Received: 5 June 2002.
First decision: 25 June 2002.
Accepted: 15 October 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2-adrenoceptor signalling. FEBS Lett 1998 430:209-212[CrossRef][Medline],ß-methylene-ATP: further evidence for ATP and noradrenaline as cotransmitters. Eur J Pharmacol 1984 100:85-90[CrossRef][Medline]This article has been cited by other articles:
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  E. Rodriguez-Miranda, M. G Buffone, S. E Edwards, T. S Ord, K. Lin, M. D Sammel, G. L Gerton, S. B Moss, and C. J Williams Extracellular Adenosine 5'-Triphosphate Alters Motility and Improves the Fertilizing Capability of Mouse Sperm Biol Reprod, July 1, 2008; 79(1): 164 - 171. [Abstract] [Full Text] [PDF]
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 R. Hayato, Y. Ohtubo, and K. Yoshii Functional expression of ionotropic purinergic receptors on mouse taste bud cells J. Physiol., October 15, 2007; 584(2): 473 - 488. [Abstract] [Full Text] [PDF]
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F. C.L. Banks, G. E. Knight, R. C. Calvert, M. Turmaine, C. S. Thompson, D. P. Mikhailidis, R. J. Morgan, and G. Burnstock Smooth Muscle and Purinergic Contraction of the Human, Rabbit, Rat, and Mouse Testicular Capsule Biol Reprod, March 1, 2006; 74(3): 473 - 480. [Abstract] [Full Text] [PDF]
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