ORIGINAL RESEARCH ARTICLE 3068 JournalJournal ofof Cellular Involvement of a P2X7 Physiology in the Acrosome Reaction Induced by ATP in Rat Spermatozoa

JORGE L. TORRES-FUENTES, MARIANA RIOS, AND RICARDO D. MORENO* Departamento de Fisiologıa, Facultad de Ciencias Biologicas, Pontificia Universidad Catolica de Chile, Santiago, Chile

The acrosome reaction (AR) is the exocytosis of the acrosomal vesicle in response to different physiological and non-physiological stimuli. Particularly in mammals, the AR is needed for sperm to fuse with the oocyte plasma membrane, and it occurs only in capacitated sperm. Previous evidence in the literature indicates that extracellular ATP induces the AR in capacitated human and bovine spermatozoa, but its receptor has not yet been identified. The aim of this work was to define a putative ATP receptor in rat spermatozoa using pharmacological and biochemical approaches. We found that ATP induced the AR only in capacitated rat spermatozoa, which was inhibited in the presence of two general inhibitors of ATP receptors (P2 receptors), , and oxidized ATP (oATP), and one inhibitor of P2X receptor (pyridoxalphosphate-6-azophenyl-20,40-disulfonic acid [PPADS]). In addition, the AR induced by ATP in capacitated rat spermatozoa was inhibited by brilliant blue-G (BB-G) and 17-b-oestradiol, two blockers of P2X7 receptors. Moreover, the ATP analog 20 (30)-O-(4-benzoylbenzoyl) ATP (BzATP) was almost 500 times more potent than ATP to induce the AR, which agrees with the pharmacology of a P2X7 receptor. Here, we show the presence of P2X7 receptor by Western blot and its localization in the tail and acrosome by indirect immunofluorescence. Finally, we quantify the presence of ATP in the rat oviduct during the estrous cycle. We found that the ATP concentration within the lumen of the oviduct is similar to those required to induce acrosome reaction, which agree with its role during in vivo fertilization. Therefore, our results strongly suggest that ATP induces the AR in capacitated rat spermatozoa through a P2X7 receptor, which may be functional during in vivo fertilization. J. Cell. Physiol. 230: 3068–3075, 2015. © 2015 Wiley Periodicals, Inc.

It is known that mammalian ejaculated sperm are not capable of (P2X1, P2X2, P2X3, P2X4, P2X5, and P2X7) or heteromeric fertilizing eggs when they leave the male reproductive tract (P2X2/3 and P2X1/5). They are differentially expressed in (Barros et al., 1996; Yanagimachi, 2011) because they need to different tissues and throughout the entire male reproductive undergo a series of biochemical changes known as tract (Burnstock, 2013). The presence of different P2X “capacitation,” before they bind and fuse with the oocyte receptors can be determined by the use of specific antibodies plasma membrane (Austin, 1951; Chang, 1951). These changes or different agonist or antagonists such as occur during female genital tract transit and can be mimicked in pyridoxalphosphate-6-azophenyl-20,40-disulfonic acid vitro in a chemically defined culture medium or capacitating (PPADS), 20,30-O-(2,4,6-trinitrophenyl)-ATP (oATP), Suramin medium (CM; Visconti et al., 1995a,b; Salicioni et al., 2007). The or Coomassie blue brillant-G (North, 2002). endpoint of capacitation is the acrosome reaction (AR), which Extracellular ATP induces AR at concentrations above is the exocitosis of the acrosomal vesicle localized over the 500 mM in humans (Foresta et al., 1992) and bovine sperm head (Barros et al., 1967; Ramalho-Santos et al., 2002; spermatozoa (Luria et al., 2002). One report shows that Moreno and Alvarado, 2006; Zanetti and Mayorga, 2009). The extracellular ATP activate P2X2 receptors in non-capacitated acrosome is an acid compartment derived from Golgi vesicles mouse spermatozoa causing a large and fast-activating inward and lysosomes containing hydrolytic that supposedly current (Navarro et al., 2011). However, genetic ablation of help the sperm to cross the zona pellucida (Barros et al., 1992; P2X2 receptor does not have any consequence in fertilization, Moreno, 2003; Codelia et al., 2005; Moreno and Alvarado, suggesting the participation of other P2 receptors. In addition, 2006; Yanagimachi, 2011). In addition, the AR changes the ATP can induce intracellular to increase in a post-acrosomal membrane in order to prepare the sperm to concentration-dependent manner in the mouse head fuse with the egg’s plasma membrane (Barros et al., 1992; Inoue et al., 2005; Moreno et al., 2011) During the AR, there is a massive increase in the intracellular free calcium that triggers the membrane fusion machinery between the outer acrosomal membrane and the adjacent Contract grant sponsor: FONDECYT; plasma membrane (Darszon et al., 2005). In addition, the AR Contract grant number: 1110778. dissipates the acid intra-acrosomal pH, and soluble proteins *Correspondence to: Ricardo D. Moreno, Departamento de and active proteases are released to the extracellular milieu Fisiologıa, Facultad de Ciencias Biologicas, Pontificia Universidad (Barros et al., 1992; Codelia et al., 2005; Buffone et al., 2009a,b). Catolica de Chile, Alameda 340, Santiago, Chile. However, recent experiments have cast doubt about the real E-mail: [email protected] physiological role of the zona pellucida as the inducer of the AR Manuscript Received: 27 December 2014 (Hirohashi et al., 2011; Inoue et al., 2011). Manuscript Accepted: 11 May 2015 Purinergic P2X receptors are transmembrane ion channels Accepted manuscript online in Wiley Online Library that open in response to the binding of extracellular ATP and (wileyonlinelibrary.com): 18 May 2015. elicit different physiological responses. Channels form as DOI: 10.1002/jcp.25044 multimers of several subunits, which could be homomeric

© 2015 WILEY PERIODICALS, INC. INVOLVEMENT OF A P2X7 RECEPTOR IN THE ACROSOME REACTION 3069 independent of CATSPER channel (Xia and Ren, 2009), The acrosome reaction was evaluated by the Coomassie blue suggesting that other channels could also be involved in the AR. dye technique (Bendahmane et al., 2002; Cisternas and Moreno, Giving the previous antecedents, the major goal of this work 2006). In brief, fixed spermatozoa were washed three times with was to a characterize a putative ATP receptor involved in the 100 mM ammonium acetate by centrifugation at 3,000 rpm, layered AR in rat spermatozoa. into microscope glass slides and dried at 30°C. Slides were washed in methanol, distilled water and methanol for 5 min at room Materials and Methods temperature, submerged in G-250 Animals solution for 2 min, washed with distilled water and mounted with Entellan (Merck). All samples were observed in a phase contrast Adult male Sprague–Dawley rats were acquired from the Animal light microscope with objective 40 (Olympus CX31, Tokyo, Facility of our institution. The rats were housed under a 12L:12D Japan) and at least 200 cells were counted for acrosome negative cycle with water and rat chow ad libitum and they were killed by or positive staining from at least three different rats. high CO2 atmosphere. Investigations were conducted in accordance with the rules laid down by the Consortium for Indirect inmunofluorescence Developing a Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching and by the National Research Rat spermatozoa were extracted in NCM and fixed as indicate Council. All animal protocols were endorsed by the Chilean above. Fixed samples were washed with PBS by centrifugation at National Fund of Science and Technology (FONDECYT). 3,000 rpm for 5 min, layered into microscope glass slides and dried at 30°C. Slides were washed twice with PBS, and cells were Reagents permeabilized with Triton X-100 1%-PBS for 10 min. Non-specific reaction sites were blocked with 3% BSA-PBS for 0 -5 -triphosphoric acid disodium salt (ATP) was 1 h and incubated overnight with a rabbit anti-P2RX7 (dilution purchased from Merck (Darmstadt, Germany), adenosine 1:200) antibody at 4°C in a humidified chamber. Then, samples 1 50-triphosphate, periodate oxidized sodium salt (oATP), and were washed three times in PBS, incubated with Alexa Fluor suramin sodium (Suramin) were purchased from Santa Cruz 488 donkey anti-rabbit antibody for 3 h, washed three times and Biotechnology (Santa Cruz, CA), 20(30)-O-(4-benzoylbenzoyl) incubated with 1 mM PI/0.1%Tween-PBS for 15 min at room adenosine-50-triphosphate triethylammonium salt (BzATP), and temperature in a humidified chamber protected from light. rabbit anti-P2RX7 antibody (ab77413) were obtained from Abcam Finally, samples were washed and mounted in Fluoromount (Cambridge, England); goat anti-rabbit IgG HRP and anti-mouse mounting medium (Sigma) and observed under a Bx51 IgG (H þ L) HRP antibodies were acquired from KPL fluorescence inverted microscopy (Olympus, Center Valley, PA) (Gaithersburg, MD); mouse anti-b-tubulin antibody (32-2600), and photographed with a PM-30 digital camera (Olympus). 1 Alexa Fluor 488 donkey anti-rabbit antibody (A-21206), and Propidium iodide (PI) were acquired from Life technologies Spermatozoa protein extraction and Western blotting (Carlsbad, CA); pyridoxalphosphate-6-azophenyl-20,40-disulfonic acid (PPADS), Coomassie brilliant blue-G (BB-G) (B0770), Sperm protein extraction was performed by homogenizing sperm 17b- (E8875) (17b-E2), BSA, and other reagents were suspensions in a buffer containing 1 M NaCl, 1 mM EDTA, purchased from Sigma (St. Louis, MO). 10 mg/ml PMSF, 1% Triton X-100, 20 mM Tris–HCl pH 7.4, plus a protease inhibitor cocktail (Sigma) including 2 mM AEBSF fl Rat sperm capacitation [4-(2-Aminoethyl) benzenesulfonyl uoride hydrochloride], 0.3 mM aprotinin, 130 mM bestatin hydrochloride, 14 mME-64, Caudal epididymal sperms were collected from 4-month-old male 1 mM EDTA, 1 mM leupeptin hemisulfatein then centrifuged for Sprague–Dawley rats. Cauda epididymis of each animal was placed 10 min at 10,000g at 4°C. Proteins were resolved on a 7% into 3 ml of non-capacitating medium (NCM), containing 86.8 mM polyacrylamide gel (SDS–PAGE) under reducing and denaturing NaCl, 4.17 mM KCl, 3.5 mM CaCl2 2H2O, 1.22 mM conditions then transferred to nitrocellulose at 400 mA for 2 h. MgSO4 7H2O, 1.22 mM KH2PO4, 50.28 mM HEPES, 5.6 mM The nitrocelullose sheet was blocked with 5% (w/v) non-fat milk, glucose, 521 mM pyruvic acid, 45 mM DL-lactic acid, 0.06 mg/ml 0.1% Tween in PBS, pH 7.4 for 1 h and incubated overnight with penicillin, 0.05 mg/ml streptomicin, and . This medium rabbit anti-P2RX7 antibody (dilution 1:15,000) or mouse was prepared in the absence of bovine serum albumin (BSA) and anti-b-tubulin antibody (0.5 ng/ml) at 4°C. Then, membranes were NaHCO3, and it does not support sperm capacitation (Visconti washed three times and incubated with a goat anti-rabbit IgG HRP et al., 1995a,b; Codelia et al., 2005). Sperms were allowed to freely antibody or a goat anti-mouse IgG HRP antibody in blocking swim into the media for 5 min at 37°C, then the epididymal cauda solution for 2 h at room temperature. Peroxidase activity was tissue was removed and cell concentration was determined using a detected by ECL (Pierce Biotechnology, Rockford, IL). Neubauer chamber. Samples with greater than 80% motility were used for capacitation. Five million sperms were suspended into Oviductal and uterine ATP measurements 200 ml of rat capacitating medium (CM) containing 72 mM NaCl, 4.17 mM KCl, 3.5 mM CaCl2 2H2O, 1.22 mM MgSO4 7H2O, Estrous cycle was determined by examination of vaginal smears 1.22 mM KH2PO4, 23.53 mM HEPES, 25.7 mM NaHCO3, 5.6 mM from 3-month-old female Sprague–Dawley rats. In brief, vaginal glucose, 521 mM pyruvic acid, 45 mM DL-lactic acid, 0.06 mg/ml secretion was collected by vaginal flushing with 100 ml of PBS and penicillin, 0.05 mg/ml streptomicin, 4 mg/ml BSA, and Phenol Red placed on glass slides, permeabilized with absolute for (Kaplan and Kracier, 1978), or NCM (lacking BSA and NaHCO3, 10 min and stained with methylene blue for 3 min at room which was replaced by HEPES) and incubated for upto 5 h at 37°C temperature. Stained cells were mounted with Entellan (Merck) with 5% CO2. and observed under a light microscope (Olympus CX31). The proportion of stained cell types was used for the determination of Acrosome reaction the estrous cycle phases. Then, three rats of each cycle phase were sacrificed by high CO2 atmosphere, and uterus and oviduct were An AR was induced by incubating sperm for 15 min in CM or NCM obtained by abdominal dissection and placed into PBS filled petri with ATP, BzATP, vehicle (Veh) or different P2R pharmacological dishes. For luminal ATP collection, uterus and oviducts were inhibitors. Then, sperm suspensions were collected and fixed with flushed individually with 100 or 600 ml of cooled Hank’s Balanced 4% paraformaldehyde in PBS for 30 min at 4°C. Salts medium (Sigma) and cells were removed by centrifugation at

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12,000 rpm at 4°C. ATP quantification was performed using the highly compact nature of the sperm nucleus and the protein Enliten ATP Assay kit (Promega, Madison, WI) following the content of the acrosome, in spermatozoa stained with manufacturer’s instructions. Briefly, 100 ml of sample solution, Coomassie blue, the acrosome is observed as an intense blue blank solution, or standard ATP solution was added to each well of line over the light stained nucleus (Fig. 1A, arrows). In a 96-well microplate, and then 100 ml of reconstituted acrosome reacted spermatozoa, the intense blue line over the luciferin-luciferase reagent solution was added to each well. The sperm head disappears due to the release of acrosomal protein luciferin-luciferase assay was quantified by a luminometer content after the AR, in the absence of any other (Luminoskan Ascent 2.5; Thermo Labsystems, Franklin, MA). morphological alteration (Fig. 1A). Physiological oviductal and uterine ATP concentrations were To investigate whether ATP induces the AR, rat estimated calculating ATP concentration obtained from ATP spermatozoa were incubated for 5 h in capacitating (CM) or standard curve, and uterine and oviductal maximal volume non-capacitating medium (NCM) and then incubated with estimated from serial sections stained of each sample with different concentration of ATP, as described in Materials and hematoxylin-eosin dye by light microscopy (Olympus CX31). Methods. Results revealed that the percentage of acrosome reacted spermatozoa significantly increased at 50 mM or higher Statistical analysis concentrations of ATP when they were incubated in CM (Fig. 1B). Given this result we chose to work with the For mean comparison, we used analysis of variance (ANOVA). When concentration of 500 mM ATP because this concentration has the ANOVA test showed statistical differences, Bonferroni or been found in vivo in several models and has been used in Dunnett’s post hoc test was used to discriminate between groups. previous works related to P2 (North, 2002). Results showed Statistical significance was defined as P < 0.05. Statistical analyses that AR was induced in rat spermatozoa as soon as 2 min after were performed using GraphPad Prism version 5.0 for Windows incubation with 500 mM ATP (Fig. 1C). Interestingly, 500 mM (GraphPad Software, San Diego, CA, www.graphpad.com). ATP did not induce the AR in rat spermatozoa incubated in NCM samples (Fig. 1D). To determine the maximal AR in rats, m Results we used 10 M Ionomycin, which is calcium ionofore that has ATP induces acrosome reaction (AR) in capacitated rat been used previously in spermatozoa from other species. sperm Results showed that the AR induced by Ionomycin in rat spermatozoa incubated in CM was higher than 500 mM ATP The AR consists of the fusion between the plasma membrane (Fig. 1E), suggesting that ATP is not as potent as Ionomycin to and outer acrosome membrane with the subsequent releases induce AR. Sperm viability, as evaluated by motility, was not of the acrosomal content to the extracellular milieu. Given that affected under our experimental conditions with motile sperm

Fig. 1. ATP induces AR in capacitated rat sperm. A: Coomasie blue stained rat spermatozoa before (left) and after (right) acrosome reaction induced by 500 mM ATP. The intact acrosomal vesicle (arrows) is visualized as a dark blue line, which is lost after the AR. B: Capacitated rat sperms were incubated in presence of increasing concentrations of ATP as described in “Methods.” C: Epididymal rat spermatozoa were incubated for 5 h in capacitating medium. The acrosome reaction was assessed at different times after addition of 500 mM ATP. D: Epididymal rat spermatozoa were incubated for 5 h in capacitating (CM, black bar) or non-capacitating medium (NCM, white bar) and then the AR was induced with 500 mM of ATP. Gray bar indicate AR induced with vehicle. E: Epididymal rat spermatozoa were incubated for 5 h in capacitating medium and then the AR was assessed 10 min after addition of 500 mM ATP or 10 mM Ionomycin, as a positive control of AR inducer. Data are expressed as average and SD. *P < 0.05; **P < 0.01; ***P < 0.001.

JOURNAL OF CELLULAR PHYSIOLOGY INVOLVEMENT OF A P2X7 RECEPTOR IN THE ACROSOME REACTION 3071 remained above 80%. Thus, these results indicate that ATP and P2X2 receptors (Murgia et al., 1993; North, 2002). On the induces AR in capacitated but not in non-capacitated rat other hand, BB-G, which blocks rat P2X7 at nanomolar spermatozoa. concentrations (typically 10 nM), also blocks rat P2X2 at the micromolar range (about 10 mM; Jiang et al., 2000; North, 2002). P2X receptors participates in ATP-induced AR To investigate the contribution of P2X receptors in ATP-induced AR, we incubated capacitated spermatozoa with It is well established that there are two families of receptors different concentrations of oATP or BB-G, prior to adding activated by extracellular ATP, namely P2X receptors (P2X) 500 mM ATP into culture medium, and then we evaluated AR. and P2Y receptors (P2Y; Coddou et al., 2011) P2X are a family Results showed that 100 mM oATP exhibited an effective of ligand-gated receptor channels, while P2Y are coupled to G reduction of ATP-induced AR, which was even more proteins and activate intracellular second messenger signaling pronounced at 1 mM concentration (Fig. 2C). By the same pathways (Baroja-Mazo et al., 2013) To know the P2 token, 10 mM BB-G prevented the ATP-induced AR in participation in ATP-induced AR, capacitated samples were capacitated rat spermatozoa to levels similar of spontaneous incubated with different concentrations of Suramin, a blocker reaction (Fig. 2D). oATP or BB-G did not exert any effect on of P2X and P2Y (Abbracchio and Ceruti, 2006) prior to the spontaneous AR of capacitated rat spermatozoa inducing AR with 500 mM ATP. Results showed that the AR was (Fig. 2C and D, respectively). prevented with concentrations between 250 mM and 1 mM There are several features that allow distinguishing P2X7 Suramin in capacitated rat spermatozoa (Fig. 2A). On the other activation from other P2X receptors. One of them is the hand, 1 mM suramin alone, without ATP, had no effect in requirement of ATP at concentrations greater than 100 mM spontaneous AR as compared with vehicle (spermatozoa and the other is that BzATP, and ATP analog, is about 10–30 incubated without ATP and containing only the vehicle used to times more potent than ATP to induce P2X7 currents (North, dissolve Suramin; Fig. 2A). In the same way, ATP-induced AR 2002). Our results showed that BzATP induced the acrosome was blocked with PPADS, an inhibitor of P2X (North, 2002), at in capacitated rat spermatozoa between 50 nM and 100 mM concentrations as low as 1 mM, without affecting spontaneous (Fig. 3A). Next, we wanted to know whether ATP induced the AR (Fig. 2B). AR in capacitated rat spermatozoa to the same extent as its There is no report so far suggesting the presence of a P2Y or analog BzATP. Results showed that capacitated rat P2X receptor in rat sperm. However, while studying the spermatozoa showed the same percentage of AR with 500 mM presence and localization of P2Xs during rat spermatogenesis, ATP or 1 mM BzATP, suggesting that the latter is about 500 Glass and colleagues observed that elongated spermatids have times more potent than ATP to elicit the signaling machinery in immunoreactivity for three P2Xs (P2X2, P2X3, and P2X7), rat sperm to induce AR (Fig. 3B). In addition, the AR induced by suggesting that mature spermatozoa could also harbor these 1 mM BzATP or 500 mM ATP was prevented by 10 nM BB-G same types of receptors (Glass et al., 2001). Regarding the (Fig. 3C and D). pharmacology of these receptors, it is known that 100 mM It has also been shown that 3 mM17b-oestradiol inhibits oxidized ATP (oATP) block currents induced by P2X7, P2X1, the P2X7 receptor cation current by a non-genomic pathway

Fig. 2. P2X receptors participates in ATP-induced acrosome reaction. Epididymal rat spermatozoa were incubated for 5 h in a capacitating medium and then they were incubated for 15 min with 500 mM in order to induce the AR. When increasing concentrations of: (A) Suramin, (B) PPADS or (C) oATP were added 15 min previous the treatment with 500 mM ATP, it was observed a gradual decrease in the AR. The incubation with 1 mM Suramin, PPADS or oATP (white bars) did not influence the spontaneous acrosome reaction (Gray bars). D: The graph shows that the AR induce by ATP (black bar) is prevented in the presence of 10 mM BB-G. The spontaneous AR (without ATP, gray bar) was not affected in the presence of 10 mM BBG. Data are expressed as average and SD. *P < 0.05; **P < 0.01; ***P < 0.001.

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Fig. 3. Pharmacological evidence that a P2X7 receptor participates in the acrosome reaction induced by ATP. A: The percentage of acrosome reaction in capacitated rat sperm incubated for 15 min in presence of increasing concentrations of BzATP. B: The acrosome reaction (AR) of capacitated rat spermatozoa incubated with 500 mM ATP (black bar) is similar to that induced with 1 mM BzATP (white bar). Epididymal rat spermatozoa were incubated for 5 h in a capacitating medium and then they were incubated for 15 min with 500 mM BzATP (C) or 1 mM ATP (D) in order to induce the AR. When capaciated rat spermatozoa were incubated with 3 mM 17-b-oestradiol or 10 mM BB-G 15 min prior the treatment with 1 mM BzATP (C) or 500 mM ATP (D) a significant reduction of AR was observed, which was similar to the basal level (vehicle, gray bar). The treatment with 3 mM 17-b-oestradiol or 10 mM BB-G alone did not influence the AR. Data are expressed as average and SD. *P < 0.05, **P < 0.01, ***P < 0.001.

and that it prevents the AR induced by ATP in human Presence and localization of P2X7 in rat spermatozoa spermatozoa (Cario-Toumaniantz et al., 1998; Rossato et al., 2005) We observed that in rat spermatozoa, 3 mM To demonstrate the presence of P2X7 in rat spermatozoa, we b fi performed immunoblot analysis. We detected the presence of 17 -oestradiol signi cantly prevented the AR in capacitated rat spermatozoa incubated with 1 mM BzATP or 500 mMATP a 70 kDa in epididymal spermatozoa from three different (Fig. 3C and D). BB-G or 17b-oestradiol did not exert any animals (Fig. 4A). Pre-incubation of the antibody with the blocking peptide abolish the 70 kDa band. Next, we performed effect in the spontaneous AR (Fig. 3C and D). Thus, our fl pharmacological approach suggests the participation of a immuno uorescence in order to investigate the P2X7 receptor P2X7 receptor in the AR induced by ATP in capacitated rat subcellular localization, in epididymal spermatozoa. Positive spermatozoa. staining for P2X7 was observed mostly localized in the principal

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Fig. 4. P2X7 receptor is present in rat spermatozoa. A: Western blot showing the presence of P2X7 receptors, each lane represent indivual samples of caudal epididymal spermatozoa from different mice. Pre-incubation with the antibody blocking peptide (P2X7þ peptide) completely removed the label observed at 70 kDa. Anti-tubulin and red ponceu (lower blots) were used as loading controls. B: Inmunofluorescence images of P2XR7 receptor, a strong stain is observed in the principal piece (arrowhead) and acrosomal region (white dot) of rat spermatozoa. C: Bright field of the spermatozoon showed in B). The nucleus in red was stained with propidium iodide (IP). D: Bright field of a rat spermatozoon incubated without the primary antibody and stained with IP. Rat spermatozoon stained with the antibody against P2X7 receptor preincubated wth the blocking peptide.

piece and acrosomal region of head (Fig. 4B, arrowhead and white dot, respectively). No immunoreaction was detected in the negative controls without secondary antibody (Fig. 4D) or when incubated with the primary antibody pre-adsorbed with the peptide antigen (Fig. 4E).

Extracellular ATP is present in rat estrus oviduct fluid One of the questions raised by our results is, what is the source of extracellular ATP during in vivo fertilization? AR normally occurs in the female reproductive tract, but the precise time at which AR occurs is still controversial. To know if the concentration of extracellular ATP is in the range to elicit AR in rat sperm, we estimated extracellular ATP concentration in oviduct and uterine fluids at different stages of the estrous cycle. Data showed that ATP concentration did not change in the uterus during the estral cycle (Fig. 5A). On the other hand, in ovidutal fluid, ATP was raised during the estrus phase at a concentration near 50 mM (Fig. 5B), which is sufficient to elicit AR in rat sperm samples (see Fig. 1).

Discussion The AR is required for mammalian spermatozoa in order to fuse with the oocyte plasma membrane under in vitro and in vivo conditions. Despite that in vitro the AR can be induced by different stimuli, the physiological inducer is still under debate. One of the proposed natural (physiological) inducers of AR is ATP; however, its receptor in the mammalian spermatozoa has not yet been determined. Here, we have used biochemical and Fig. 5. Presence of ATP in the female rat reproductive tract. pharmacological approaches in order to determine that ATP A: ATP concentration in the uterus remains unchanged during the induces the AR only in capacitated rat spermatozoa, and its estrous cycle. B: ATP concentration in the oviduct significantly cognate receptor is P2X7. increases during the the oestrous phase of the estrous cycle. In this work, we proposed the participation of P2X7 *P < 0.05, n ¼ 3. receptors in the AR induced by ATP of rat spermatozoa based

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on: (1) the AR induced by ATP is prevented by general P2 Navarro et al., 2011), whereas in rats it is dependent on P2X7 receptor inhibitors such as Suramin and oATP; (2) BzATP is 500 (this work), and in mice, it has not been yet determined. In times more potent than ATP to induce AR; (3) the AR is addition, the AR in bovine and human spermatozoa is prevented by 17b-oestradiol and BB-G, two blockers of P2X7 stimulated with higher concentrations of ATP than in rats, ion currents in other cell types; (4) it is present in the mature suggesting that the pharmacology of purinergic receptors is spermatozoa and localized in the tail and in the acrosomal different between these species (Foresta et al., 1992; Luria domain of the head. Our results agree with previous reports in et al., 2002). On the other hand, the presence of a human spermatozoa that 17-b-oestradiol prevents the AR 50-nucleotidase has been identified in human and mouse induced by ATP (Foresta et al., 1992; Rossato et al., 2005). On spermatozoa (Takayama et al., 2000; Martin-Satue et al., 2010), the other hand, a previous study showed a bivalent ion current which could degrade extracellular ATP, and in this way, lower evoked by ATP in epidydimal non-capacitated mouse the availability of this ligand. The presence and high activity of spermatozoa (Navarro et al., 2011). By using pharmacological this could explain that long exposure of ATP to and genetic approaches, they reached the conclusion that a non-capacitated mammalian spermatozoa does not induce AR P2X2 receptor was responsible for that current. However, (Rodriguez-Miranda et al., 2008). Thus, it remains to be shown genetic ablation of P2X2 receptors does not prevent AR or whether ATP is a universal AR inducer or depends on the fertilization but prevents the bivalent ion current evoked by ATP species-specific reproductive biology of each species. in non-capacitated mouse sperm. Even more, this receptor is The final question is, where does ATP come from in the localized in the tail and not in the head, reinforcing the idea that it female reproductive tract? One of the possibilities is that cells is not involved in the acrosome reaction. from the uterus, oviduct or cumulus oophorus secrete ATP to We think that since those experiments were never the extracellular milieu. In this work, we have measured the ATP performed in capacitated sperm samples, this could explain why content of the oviduct and uterus during the four stages of the they were not able to detect the P2X7 currents in mouse sperm, estrous cycle. Our results showed that ATP content in the which may explain the fertility of mice lacking P2X2 receptors uterus is low and does not change over the estrous cycle. On the (Navarro et al., 2011). In fact, one of the interesting results in the contrary, in the oviduct, ATP significantly increases during the present work is that ATP induced the AR only in capacitated oestrous stage, at concentrations enough to induce AR in rat spermatozoa, and likewise and solubilized zona sperm. This result suggests that by the time of fertilization, pellucida. P2X7 receptors are trimeric proteins where each spermatozoa could find an environment with the amount of ATP subunit has a C- and N-terminal in the cytoplasm (North, 2002). high enough to stimulate the AR. Alternatively, it is possible that Interestingly, ATP induces the AR in a fraction of the total sperm mammalian spermatozoa could secrete ATP in an autocrine way population, which strengthens the idea that only capacitated once they become capacitated. In fact, a recent report suggests spermatozoa respond to these stimuli. In the same way, in the that pannexin are present in the male tract and germ cells presence of Ionomycin, potent as a non-specific AR inducer, (Turmel et al., 2011). These are channel forming proteins, that around 60% of the spermatozoa undergo this exocytic process. allow the efflux of ATP and in same cell type they are coupled to These results suggest that not all the cells are prone to undergo P2X7 receptors (Pelegrin and Surprenant, 2006). In this way, it is the acrosome reaction, and that ATP was almost as potent as possible that upon capacitation, autocrine stimulation of ATP Ionomycin. Interestingly, the P2X7 C-termini contains receptors could induce AR in spermatozoa in areas nearby conserved phosphorylation sites for cAMP-dependent protein and/or within the cumulus oophorus, which could potentiate kinase A (PKA) and protein kinase C (PKC) and other important and/or facilitate the effect of progesterone. regions that are responsible for the regulation of P2XRs; and Previous works have shown that ATP is important to inhibitors of PKA prevent P2X activation in dorsal root ganglia increase the motility in cultured human and mouse neuron (Wang et al., 2012; Stojilkovic et al., 2013). spermatozoa, without affecting the capacitation and/or the AR It is well known that PKA is essential during sperm (Edwards et al., 2007; Rodriguez-Miranda et al., 2008). capacitation from different species, and that this process However, other studies have shown that extracellular ATP required the presence of bicarbonate in order to activate the induces the AR in bovine and human sperm (Foresta et al., soluble adenyl cyclase and produce cyclic AMP (Visconti et al., 1992; Luria et al., 2002; Rossato et al., 2005). Thus, the role of 1995a,b; Salicioni et al., 2007). Thus, it is possible that P2X7 ATP regarding its role as AR inducer still remains controversial. receptor activation relies somehow upon PKA activity, which Here, we show that ATP induces the AR through a putative could explain the effect of ATP upon AR only in capacitated rat P2X7 receptor that is activated only in capacitated rat spermatozoa. Interestingly, P2X7 receptor activation requires spermatozoa, suggesting that in vivo or in vitro ATP could the presence of bicarbonate, reinforcing the idea of that this participate during the fertilization process. However, our data receptor becomes prone to activation only in capacitated does not preclude the participation of other P2 receptors that spermatozoa, and it also explains the lack of an ATP effect in rat may not have been affected by the pharmacological approaches spermatozoa cultured for 5 h in non-capacitating medium used in this study. (Wang et al., 2002). Thus, we think that P2X7 are present in rat spermatozoa, and they participate in the AR induced by ATP. In Acknowledgments this way, it has been shown that P2X7 receptors participate in the exocytosis of other secretory vesicles in neurons and Our special thanks to the Manuel Villalon laboratory for the gift salivary gland, suggesting that similar intracellular pathways of P2XR7 antibody. We also thank Gisela Eller and Veronica could be elicited in spermatozoa (Leon et al., 2008; Qu and Aguirre for support of immunoblot techniques and the Dubyak, 2009; Adler et al., 2013). manuscript, respectively. This work was funded by a grant from It has been reported that in vitro ATP induces the acrosome FONDECYT 1110778 to RDM. reaction in bovines (Luria et al., 2002), humans (Foresta et al., 1992), rats (the present work) but not in mice (Rodriguez-Miranda et al., 2008). 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