Biol Reprod
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Luria, A.
Right arrow Articles by Breitbart, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Luria, A.
Right arrow Articles by Breitbart, H.
Agricola
Right arrow Articles by Luria, A.
Right arrow Articles by Breitbart, H.
Biology of Reproduction 66, 429-437 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Extracellular Adenosine Triphosphate Stimulates Acrosomal Exocytosis in Bovine Spermatozoa via P2 Purinoceptor1

Ayala Luriaa, Sara Rubinsteina, Yehudit Laxa, and Haim Breitbart2,a

a Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The presence of ATP in the genital tract fluid of mammals provokes questions regarding its function in the fertilization process. We investigated the effect of extracellular ATP (ATPe) on the activation of bovine spermatozoa. A signal transduction mechanism for ATP involving the receptor-mediated release of second messengers is described. Treatment of spermatozoa with ATP, uridine triphosphate (UTP), or 2-methylthio-ATP resulted in a concentration-dependent increase of acrosomal exocytosis, whereas treatment with either AMP or adenosine induced little exocytosis. This suggested that the receptor involved is of the P2 and not the P1 type. Several lines of evidence also suggest that the ATP purinoceptor is of the P2y and not the P2x type. First, the acrosome reaction was induced by the P2y-agonists ATP, UTP, or 2-methylthio-ATP, but no effects were shown by the P2x-agonists {alpha},ß-methylene-ATP or ß,{gamma}-methylene-ATP. Second, ATP-induced acrosomal exocytosis was inhibited by the P2y antagonists, but not by the P2x antagonists. Third, enhanced Ca2+ uptake into the cells was observed with ATP and 2-methylthio-ATP, but not with ß,{gamma}-methylene-ATP. Additionally, ATP induced elevation of intracellular Ca2+ and cAMP, and the effect on cAMP was predominantly enhanced by including Ca2+ and the Ca2+-ionophore A23187 in the incubation medium. Extracellular ATP also activates protein kinase C{alpha} (PKC{alpha}), and the acrosome reaction, stimulated by ATPe, is inhibited by a PKC-specific inhibitor. In summary, we suggest that ATPe activates the P2 purinoceptor that elevates [Ca2+]i, which leads to PKC{alpha} activation and culminates in acrosomal exocytosis.

acrosome reaction, calcium, gamete biology, signal transduction, sperm


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The sperm acrosome reaction (AR) is an exocytotic event that must occur before the spermatozoon penetrates the zona pellucida [1]. This exocytotic event is mandatory for fertilization, because it enables passage of the sperm through the zona pellucida and its subsequent fusion with the egg oolema. Many artificial stimuli are reported to trigger the AR, either by driving extracellular Ca2+ into the sperm cell (Ca2+ ionophores) or by acting on intracellular second messengers that are involved in the cascade leading to acrosomal exocytosis [2]. Several physiological AR activators usually are present in the female genital tract that act on specific receptors on the sperm surface. In the mouse, it is well established that one of the zona pellucida glycoproteins, ZP3, triggers the AR [3]. In other species, activators such as progesterone [4], atrial natriuretic peptide [5], epidermal growth factor [6], and ATP [7] are also AR inducers. However, the precise physiological role of these inducers remains unknown.

Extracellular ATP (ATPe) has a profound effect on cell responses [8, 9]. Receptors for ATP (P2 purinergic receptors) have been cloned and classified into two main subtypes according to their distinct pharmacological potency and their mode of action in transducing signals. The classification is based on studies showing that ATP can activate either G protein-coupled receptors [10] or ligand-gated ion channels [11, 12]. These two mechanisms are known to be involved in the cascade leading to sperm acrosomal exocytosis [13]. The cascade of ATP-induced exocytosis in other cells involves Ca2+ influx, hydrolysis of inositol phospholipids [14], and triggering of intracellular second messengers leading to exocytosis [15]. Extracellular ATP is also a potent activator of acrosomal exocytosis in human spermatozoa [7].

Foresta et al. [16] suggested that ATP-induced acrosomal exocytosis involves Na+ influx, but not Ca2+ influx [16]. However, the mode of action remains unknown. Adenosine triphosphate is present in the female reproductive tract and increases in concentration at the time of ovulation, although its precise concentration is not known [17]. Thus, it may have a physiological role in fertilization. In this report, we provide evidence that ATP can regulate acrosomal exocytosis via the P2 purinoceptor in a mechanism that involves an increase of intracellular Ca2+ levels and activation of protein kinase (PK) C{alpha}.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Sperm Preparation

Fresh ejaculated bovine sperm were collected in an artificial vagina and diluted (1:1, v:v) in NKM medium (pH 7.4), which consisted of 110 mM NaCl, 5 mM KCl, and 10 mM MOPS (3-[N-morpholino] propane sulfonic acid). The cells were washed three times by centrifugation (780 x g, 10 min), and the final pellet was resuspended in NKM medium to a concentration of 1–3 x 109 cells/ml. Only those samples exhibiting a minimum of 70% motile spermatozoa were used.

Capacitation and AR Evaluation

In vitro capacitation was accomplished according to the method of Parrish et al. [18]. Briefly, washed sperm cells (108 cells/ml) were capacitated for 4 h at 37°C in glucose-free, modified Tyrode medium (m-TALP medium), which consisted of 100 mM NaCl, 3.1 mM KCl, 25 mM NaHCO3, 0.29 mM KH2PO4, 21.6 mM Na-lactate, 1.5 mM MgCl2, 2.0 mM CaCl2, 0.1 mM sodium pyruvate, 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH 7.4), 10 IU/ml of penicillin, 3 mg/ml of BSA (fraction V), and 10 µg/ml of heparin. The AR was evaluated in capacitated sperm (108 cells/ml) that were incubated for an additional 20 min in the presence of various inducers. The inhibitors reactive-blue 2 and pyridoxal-phosphate-6-azophenyl-2'4'-disulphonic acid tetrasodium (PPADS) as well as the ATP analogues used were purchased from RBI (Natic, MA). The PKC inhibitor GF (bisindolylmaleimide I) and the PKA inhibitor RPcAMPS (RP-isomer of cAMPS) were purchased from Calbiochem (San Diego, CA) and Sigma (St. Louis, MO), respectively. The Ca2+-free media contained 0.5 mM EGTA.

At the end of the incubation period, cells were pelleted by centrifugation (12 930 x g for 5 min), and occurrence of the AR was determined by measuring the activity of acrosin released into the supernatant fluid as previously described in ram and bovine spermatozoa [19–21]. Briefly, the supernatant fluid was adjusted to pH 3.0 with 3 M HCl, and the acrosin activity was measured by esterolytic assay using benzoylarginine ethyl ester as substrate. The increase was recorded in A259 with time. The molar absorption coefficient was taken as 1150. We then subtracted the spontaneous acrosin release from each value obtained. To be sure that acrosin activity itself is not affected by the various agonists or antagonists, their effect on acrosin activity was tested directly, and no such effect was evident in the described experiments. Occurrence of the AR was determined using two methods: first, assay of the release of acrosin from acrosome-reacted cells [20]; and second, staining of cells by Pisum sativum agglutinin (PSA), whereby intact cells are stained and acrosome-reacted cells are not [22].

Staining with PSA was performed using a modification of the method described by Mendoza et al. [22]. Samples of cells treated to induce the AR were smeared on microscope slides. After air-drying, sperm smears were dipped in absolute methanol for 30 sec and allowed to dry rapidly. Methanol-fixed smears were incubated with blocking solution (PBS containing 1% [w/v] BSA) for 10 min, then with biotin-conjugated PSA (50 µg/ml) in PBS containing 1% BSA for 30 min, and finally with peroxidase-conjugated extravidin (1:400) for 10 min. All incubations were performed in a humid chamber. The slides were washed between incubations by dipping in PBS for 5 min. The substrate (aminoethyl carbazole from Histostain-SP kit; Zymed Laboratories, South San Francisco, CA) was then added for 10 min; this was followed by washing with distilled water. Hematoxylin was usually used for counterstaining (3 min). The slides were mounted with GVA mounting medium (Zymed) and examined under a bright-field microscope. Cells with red staining over the acrosomal cap were considered to be acrosome intact, and cells with equatorial red staining or no staining at all were considered to be acrosome reacted. Two hundred cells were counted per slide. Occurrence of the AR was also confirmed by observing thin sections of spermatozoa in the transmission electron microscope [20].

Determination of Intracellular Calcium

The spatial distribution of intracellular calcium was determined using a fluorescence-microscopy imaging system as recently described [21, 23]. Capacitated sperm cells were incubated on 25-mm glass coverslips (covered with poly-L-lysine) with Fluo-3 AM (2 µM) and pluronic acid (1.5 µM) for 2 h in m-TALP at 37°C in 5% CO2. After incubation, each dish was rinsed twice with m-TALP, and the coverslip was placed in the microscope holder. Cells were covered with 1 ml of m-TALP. Intracellular calcium measurements were performed using a fluorescence-microscopic system consisting of an inverted epifluorescence microscope (Axiovert 135M; Zeiss, Oberkochen, Germany), an intensified charge-coupled device C2400 camera (Hamamatsu, Hamamatsu City, Japan), and frame-grabbing software (Galai, Migdal-Haemek, Israel). A 75-W xenon lamp served as the source for excitation. A 510 long-pass emission filter was used to select fluorescence emission. The first pictures were taken to estimate basal levels of fluorescence. Afterward, ATP was added, and the fluorescence was measured every 30 sec for 10 min. Analysis of intracellular calcium levels was performed employing Scan Array 2 software (Galai).

Calcium Uptake Measurements

Uptake of 45Ca2+ by the cells was determined using the filtration technique. Capacitated sperm cells (108 cells/ml) were incubated in the capacitation-medium m-TALP containing 2 mM CaCl2 and 5 µCi 45CaCl2. At appropriate time intervals, 0.1-ml samples were removed and immediately vacuum-filtered on glass fiber/C filters. The cells trapped on the filter were washed three times with an ice-cold solution composed of 150 mM NaCl, 10 mM Tris (pH 7.4), and 2 mM EGTA. The dry filters were counted for ß-radioactivity.

cAMP Measurement

Capacitated cells (108 cells/ml) were incubated for the last 10 min of capacitation with 0.1 mM 3-isobutyl-L-methylxanthine (IBMX), and the inducers were added according to the assay. The reaction was stopped by boiling the samples for 10 min. Then, the samples were sonicated (three times for 10 sec at power setting 4 with a Vibra cell; Sonics & Materials, Inc., Danbury, CT) and centrifuged (10 000 x g, 10 min, 4°C). The supernatant fluids were collected and frozen at -70°C. Cellular cAMP was determined by radioimmunoassay using acetylated cAMP [24]. Rabbit anti-ScAMP (generous gift from Dr. D.C. Klein, NIH/NICHD, Bethesda, MD), iodinated cAMP, standards, and samples were mixed with 50 mM sodium acetate buffer (pH 6.0) and incubated for 18–24 h at 4°C. Free iodine and unbound, iodinated cAMP were precipitated by adding dextran-coated charcoal. Tubes were centrifuged (2000 x g, 20 min, 4°C), and radioactivity was counted in the supernatant fluid.

Subcellular Fractionation

Sperm cells were fractionated into separate cytosol, membranes, and particulate fraction proteins. The cells (1.5 x 109) were resuspended in 20 mM Tris-HCl (pH 7.5), 0.25 M sucrose, 2 mM EGTA, 2 mM EDTA, 1 mM benzamidine, 1 mM Na3VO4, 10% glycerol, 25 µg/ml of leupeptin, 4 µg/ml of aprotinin, and 1 mM PMSF (homogenization buffer) and then sonicated (three 10-sec pulses; power setting 4) with a Vibra cell. The homogenate was centrifuged for 10 min at 10 000 x g to pellet the particulate fraction containing the head and tail fragments. The resulting supernatant was centrifuged at 100 000 x g (60 min, 4°C) to recover the cytosolic fraction (supernatant) and the membrane fraction (pellet). The cytosolic fraction was concentrated to at least one-tenth the original volume using a microconcentrator 30 (Amicon, Lexington, MA). The membrane fraction was resuspended in the homogenization buffer supplemented with 0.6% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate [25]. The particulate fraction was solubilized with SDS-lysis buffer. The protein concentrations of the cytosolic and membrane fractions were determined according to the method of Bradford [26] using Bio-Rad (Richmond, CA) reagents. For immunoblot analysis, the cell lysates were boiled for 5 min in SDS-PAGE sample buffer [27] and separated on a 7.5% SDS-polyacrylamide gel.

Immunoblot Analysis

For immunoblotting, proteins derived from equivalent cell numbers were separated on 7.5% SDS-polyacrylamide gels and then electrophoretically transferred to nitrocellulose membranes (200 mA, 1 h) using a buffer composed of 25 mM Tris (pH 8.2), 192 mM glycine, and 20% methanol. For Western blot analysis, nitrocellulose membranes were blocked with 5% BSA in Tris-buffered saline (pH 7.6) containing 0.1% Tween 20 (TBST) for 30 min at room temperature. The PKC{alpha} isoform was immunodetected using rabbit polyclonal anti-PKC{alpha} (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:1000 and incubated overnight at 4°C. Then, the membranes were washed three times with TBST and incubated for 1 h at room temperature with specific horseradish peroxidase-linked secondary antibody (Jackson Laboratories, West Grove, PA) diluted 1:10 000 in TBST. The membranes were washed three times with TBST and visualized by enhanced chemiluminescence (Amersham, Little Chalfont, UK). Specificity of the antibodies was determined by preabsorbing the antibodies with 10 µg of their peptide antigens for 1 h before incubating the antibodies with the membrane.

Data Analysis

Results are shown as the mean ± SEM. Statistical analyses were performed using the ANOVA test and t-test with multiple comparison. Statistical significance was as indicated in each figure legend.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Induction of AR by ATPe and Various Nucleotides

To study the effect of ATP on the AR, capacitated bovine sperm were triggered by different concentrations of ATP, and the AR was determined by two separate methods: first, by measuring the release of the trypsin-like acrosin from the cells (Fig. 1A); and second, by staining the cells with PSA (Fig. 1B). The effect of ATPe on the AR could be observed only in capacitated cells at ATPe concentrations as low as 0.1 mM, whereas a maximal response was obtained at concentrations between 1 and 2.5 mM (Fig. 1A). A very similar effect of ATPe on AR was obtained when Ca2+ was omitted from the incubation medium (data not shown). At 1 mM ATPe, approximately 30% of the sperm underwent the AR, compared to the 15% AR that was observed in the absence of nucleotides (Fig. 1B). No detectable change in sperm motility (65–75% motile cells) could be observed during the various treatments. The kinetics for ATPe-induced AR revealed maximal effect after 20 min of incubation (data not shown).



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 1. Effect of ATP on acrosome reaction in capacitated bovine spermatozoa. Bovine sperm (108 cells/ml) were capacitated for 4 h in m-TALP medium containing 20 µg/ml of heparin and 2 mM CaCl2, followed by 20 min of incubation in the presence of increased concentrations of ATP. A) Activity of released acrosin from cells as a measure for acrosomal exocytosis. B) Percentage of acrosomal-reacted cells as determined by staining with PSA. Values are mean ± SEM of duplicate determinations from at least three independent experiments. Significant difference from control: #, P < 0.0001; *, P < 0.005

Several receptors for extracellular nucleotides are known, some of which bind ATP/ADP and are classified as P2 purinoceptors and some of which recognize adenosine and are defined as P1 purinoceptors [28, 29]. To find which of these receptors is involved in sperm activity, we examined the effect of various nucleotides, analogues, and inhibitors on bovine sperm AR. Our results demonstrate that the AR could be partially induced by ADP and AMP, whereas two other nucleotides, such as adenosine and 5'-N-ethylcarboxamidoadenosine (NECA), were slightly effective or completely ineffective, respectively (Fig. 2). These results may suggest that the acrosomal exocytosis is mediated by a P2 purinoceptor that responds primarily to ATP and ADP rather than by a P1 receptor that responds to adenosine.



View larger version (24K):
[in this window]
[in a new window]
  		FIG. 2. Acrosomal exocytosis induction by various nucleotides. Capacitated bovine sperm were incubated for 20 min in the presence of 1.0 mM of the various nucleotides (see Fig. 1). Acrosomal exocytosis was determined by following the activity of acrosin released from the cells. Measurements of acrosin activity in the absence of each nucleotide were subtracted from each point. Values are mean ± SEM of duplicate determinations from three independent experiments

A large number of P2-receptor subtypes have been described and divided into two families, P2x and P2y, according to the relative potency of various nucleotide agonists and their mechanisms of action in signal transduction. To evaluate which family participates in bovine sperm cells, we used various ATP analogues known to interact with P2 receptors. Our data shown in Figure 2 reveal that the AR is highly stimulated by the agonist 2-methylthio-ATP, which is considered to be a very efficient agonist for activating the subtype P2y purinoceptor and very weak for the P2x type [30–32]. In contrast, the ATP-analogues {alpha},ß-methylene-ATP and ß,{gamma}-methylene-ATP, which are more potent agonists for the P2x purinoceptor [33, 34], did not induce the AR (Fig. 2). The P2y family is composed of seven subfamilies, including receptor types P2y1–7 [35]. The P2y2 type, for example, is more specific for pyrimidine nucleotides, in which the order for activation is: uridine triphosphate (UTP) = ATP >> 2-methylthio-ATP [35]. Because purinergic receptors of the type P2u, which are specific for UTP, have been identified in rat sperm [36], we determined the effect of UTP on bovine sperm AR. As shown in Figure 2, UTP (1.0 mM) stimulated the AR most prominently. Because ATP does not affect P2u receptors, and both ATP and UTP similarly affect the P2y2 type of receptors, it is suggested that ATP might affect the AR by activating P2y receptors.

To further evaluate which of the purinoceptors are involved in AR, we used the antagonists reactive-blue 2, which in some cells showed higher specificity for inhibiting the P2y purinoceptor [11], and PPADS, which is considered to be more specific for the P2x purinoceptor [37]. As shown in Figure 3, an AR induced by ATP is 90% inhibited by reactive-blue 2, and no significant inhibition was seen with PPADS. Reactive-blue 2 did not affect AR induced by 12-O-tetradecanoyl phorbol-13-acetate (PMA) or by lysophosphatidic acid (data not shown). These results further indicate that ATP-induced ARs are mediated by the subtype P2y purinoceptor.



View larger version (24K):
[in this window]
[in a new window]
  		FIG. 3. Effect of purinoceptor antagonists on ATP-induced acrosomal exocytosis. Capacitated bovine sperm were incubated for 10 min in the presence of the P2y-antagonist reactive-blue 2 (RB; 100 µM) or the P2x-antagonist PPADS (100 µM). Then, ATP (1.0 mM) was added for an additional 20 min of incubation. Acrosomal exocytosis was determined by following the activity of acrosin released from the cells. Values are mean ± SEM of duplicate determinations from three independent experiments. Significant difference from control: #, P < 0.0001

ATPe Affects Intracellular Calcium

The preceding data show that ATP (1.0 mM) added to capacitated sperm stimulates the AR, probably by activating P2y purinergic receptors. It is well accepted that AR depends on elevation of [Ca2+]i [38], and that P2 receptors are active in regulating this activity [11, 12]. The effect of ATPe on the [Ca2+]i was evaluated in Fluo-3 AM-loaded capacitated sperm using a fluorescence-microscopy imaging system (Fig. 4). On addition of ATP (1.0 mM) to the cell suspension, 30% of the sperm cells showed a rapid, significant increase in [Ca2+]i, mainly in the heads. In 50% of the responding cells, an elevation of [Ca2+]i appeared within 30 sec of ATP application, peaked within 1 min, and then decreased. In the other responding cells, the transient response was slower, and the time from initiation to peak was 60–90 sec.



View larger version (91K):
[in this window]
[in a new window]
  		FIG. 4. Effect of ATP on [Ca2+]i. Spermatozoa were capacitated for 2 h. Then, the Ca2+-indicator Fluo-3 AM was added, and the cells were incubated for an additional 2 h to complete capacitation. The cells were photographed, and after 30 sec, ATP (1.0 mM) was added (arrow) and pictures taken again every 30 sec for 150 sec. The data show a representative experiment that was repeated four times. The colors represent the following: green, low [Ca2+]i; yellow, higher [Ca2+]i; and red, highest [Ca2+]i

Moreover, ATPe significantly stimulated 45Ca2+ uptake by the sperm in a dose-dependent manner, with a range between 0.05 and 1.0 mM (Fig. 5). This ATP-dose response was similar to the ATP effect observed for the AR (Fig. 1). To further characterize the purinoceptor involved in this Ca2+ uptake, the response to the ATP-analogues 2-methylthio-ATP and ß,{gamma}-methylene-ATP were also measured (Fig. 6). As shown, the time curve for 2-methylthio-ATP stimulated Ca2+ uptake is similar to ATP, whereas ß,{gamma}-methylene-ATP had no agonistic effect. The maximal enhancement of Ca2+ uptake by ATP precedes the occurrence of AR. These results further support our notion regarding involvement of the P2y purinoceptor in sperm-Ca2+ transport that leads to acrosomal exocytosis.



View larger version (10K):
[in this window]
[in a new window]
  		FIG. 5. Effect of ATP concentrations on calcium uptake by bovine spermatozoa. Capacitated bovine sperm were incubated for 10 min in the presence of 45Ca2+ and increased concentrations of ATP. Uptake of 45Ca2+ into the cells was determined as described in Materials and Methods. Values are the mean ± SEM of duplicate determinations from three independent experiments



View larger version (21K):
[in this window]
[in a new window]
  		FIG. 6. Time curve of calcium uptake induced by ATP and ATP analogues. Capacitated bovine sperm were incubated with 45Ca2+ and 1.0 mM of ATP (circles) or the ATP-analogues 2-methylthio-ATP (2CH3SATP; balanced) or ß,{gamma}-methylene-ATP (ß,{gamma} CH2ATP; triangles). The squares represent the control without nucleotides. Uptake of 45Ca2+ by the cells was determined as described in Materials and Methods. Values are the mean ± SEM of duplicate determinations from three independent experiments. Significant difference from control: A vs. B, P < 0.001

Effect of ATPe on Intracellular cAMP Levels

The P2y purinoceptor is characterized as a G protein-coupled receptor that is involved in the activation of phospholipase C (PLC) and the mobilization of intracellular Ca2+ [39]. It is well known that G proteins are involved in the regulation of adenylyl cyclase activity and, therefore, influence the intracellular levels of cAMP. Current evidence suggests that cAMP is an important factor in sperm AR [40, 41], but the mechanism regulating sperm adenylyl cyclase is not yet understood. The possible involvement of the P2y purinoceptor in ATP-induced sperm AR and in [Ca2+]i elevation encouraged us to examine the effect of ATP on sperm cAMP levels. We demonstrated that, in the absence of extracellular Ca2+, ATP (1.0 mM) enhances the level of intracellular cAMP by 2.6-fold (Fig. 7). This effect of ATP was further enhanced (up to 4.5-fold) by supplementing Ca2+ into the incubation medium (Fig. 7), and prominent levels of cAMP (an 11-fold increase) were found when the Ca2+-ionophore A23187 was added to the cells (Fig. 8A). These data clearly demonstrate that ATPe can stimulate cAMP production, and this effect is amplified by elevating the [Ca2+]i. Moreover, because the Ca2+-ionophore A23187 is a common pharmacological inducer of the AR, we examined its effect on the ATP-induced AR. Indeed, addition of the ionophore additively enhanced the ATP-induced exocytosis (Fig. 8B).



View larger version (26K):
[in this window]
[in a new window]
  		FIG. 7. Effect of ATP on intracellular sperm cAMP. Capacitated bovine sperm were incubated for the last 10 min of the capacitation with 0.1 mM IBMX. Then, they were further incubated for 5 min in the presence (+) or absence (-) of either ATP (1 mM) or CaCl2 (2 mM), and the levels of cellular cAMP were determined as is described in Materials and Methods. Values are the mean ± SEM of duplicate determinations repeated three times. Significant difference from control: *, P < 0.005; #, P < 0.0001



View larger version (17K):
[in this window]
[in a new window]
  		FIG. 8. Effect of ATP and Ca2+ ionophore on cAMP and acrosomal exocytosis. Capacitated bovine sperm were incubated for the last 10 min of the capacitation with 0.1 mM IBMX. Then, 1.0 mM ATP and 10 µM Ca2+-ionophore A23187 (Iono) were added as indicated. A) Levels of cAMP were determined after 5 min of incubation. B) Acrosomal exocytosis was determined after 20 min of incubation by following the activity of released acrosin. Values are the mean ± SEM of duplicate determinations repeated three times. Significant difference from control: *, P < 0.05; #, P < 0.001

Involvement of PKC{alpha} in ATP-Induced AR

In our recent publications, we showed that the Ca2+-dependent PKC{alpha} isoform is involved in the mechanism of bovine sperm AR [42, 43]. The involvement of the P2y receptor suggests that the activation of PLC results in the hydrolysis of phosphatidyl inositol to produce diacylglycerol (DAG) and inositol 1,4,5-tris-phosphate (IP3). Production of DAG, a known activator of PKC, and of IP3, which activates intracellular Ca2+ channels to mobilize Ca2+, would cause the activation of Ca2+-dependent PKC (cPKC) isoforms to initiate the AR. It was previously demonstrated that PKC, on activation, translocates from the cytosol to the cell membrane; thus, translocation may serve as an indicator of PKC activation [44]. Here, we followed the translocation of PKC{alpha} from the cytoplasm to the cell plasma membrane in ATP-treated bovine sperm. The sperm cells were fractionated into three fractions: cytosol, plasma membrane, and particulate fractions (see Materials and Methods). As shown in Figure 9, PKC{alpha} is localized in the cytosolic fraction only in unstimulated cells, and a fraction of the PKC{alpha} was translocated to the membrane fraction in response to ATP (1.0 mM, 10 min). The phorbol ester PMA, a known activator of PKC, also activated the translocation of PKC{alpha} to the membrane fraction. Furthermore, we demonstrated approximately 70% inhibition of ATP-stimulated AR by the specific PKC-inhibitor bisindolylmaleimide (GF), but no inhibition was seen with the PKA-inhibitor RPcAMPS (Fig. 10). These data suggest that PKC, but probably not PKA, is involved in ATP-induced sperm AR.



View larger version (49K):
[in this window]
[in a new window]
  		FIG. 9. Subcellular redistribution of PKC{alpha} in response to ATP. Sperm cells were incubated either alone (C) or with either 100 ng/ml of PMA (P) or 1.0 mM ATP (A) for 10 min. Cells were fractionated into cytosol, plasma membrane, and particulate fractions as described in Materials and Methods. Proteins were extracted from 1.5 x 107 cells, and cell fractions equal to protein aliquots of 15 µg of total protein per lane were separated by SDS-electrophoresis, transferred onto nitrocellulose, immunoblotted with anti-PKC{alpha}-specific polyclonal antibody, and visualized. The blot shown is representative of four separate experiments. The molecular weight of PKC{alpha} is 80 KDa



View larger version (25K):
[in this window]
[in a new window]
  		FIG. 10. Effect of PKA and PKC inhibitors on ATP-induced acrosomal exocytosis. Capacitated bovine sperm were incubated for 20 min with 1.0 mM ATP in the presence or absence of either 0.1 mM of the RPcAMPS (PKA inhibitor) or 0.1 mM GF (PKC inhibitor). Acrosomal exocytosis was determined by following the activity of released acrosin from the cells. Values are the mean ± SEM of duplicate determination from three independent experiments. Significant difference from control: #, P < 0.001


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Molecular cloning and characterization of ATP receptors has provided strong proof for some pharmacological and physiological data suggesting that ATP, and possibly other nucleotides, play important roles as extracellular messengers. In the present report, we describe the role of ATPe in triggering bovine sperm acrosomal exocytosis in capacitated cells, which is an essential step in the fertilization process. Within this framework, three major findings are reported. First, bovine sperm cells express the P2y purinergic receptor. Second, extracellular nucleotides elevate [Ca2+]i and cAMP levels. Third, the receptor is coupled to intracellular signaling pathways leading to AR.

It is well established that the two types of purinergic receptors, P1 and P2, are responsive to adenosine and ATP, respectively [35]. The presence of stimulatory A2 adenosine receptors on mouse spermatozoa was shown during the initial stages of capacitation [45]. Furthermore, the addition of exogenous adenosine analogues, including NECA, accelerated mouse sperm capacitation with an even greater sensitivity than that to adenosine, which is consistent with the action at A2 receptors during capacitation [46]. Adenosine receptors present in bovine and human sperm are also involved in capacitation and not in AR [47, 48].

In the present study, we tested the effect of ATP and adenosine on bovine sperm AR. As an activator, ATP (1 mM) was sixfold more potent than adenosine. Thus, the data suggest that P2 purinoceptors, rather than P1 receptors, are involved in the bovine sperm AR. Similar conclusions were demonstrated in a previous study, in which the occurrence of human sperm AR was fully stimulated in the presence of the nonhydrolyzable ATP-analogue AMP-PNP, suggesting that ATP hydrolysis is not necessary and that P1 receptors are not involved during the AR [7].

To characterize the receptor in the present study, various P2-receptor agonists were determined based on their ability to induce acrosomal exocytosis and to increase Ca2+ uptake. Acrosomal exocytosis is a Ca2+-dependent process that occurs following the induced elevation of [Ca2+]i after the sperm binds to the zona pellucida of the egg [49].

The similarities between nucleotide-induced AR and induced Ca2+ uptake by the cells strongly suggest that the purinoceptors described are involved in the two processes. The correlation between the two is based on three observations. First, the ATP-dose response for inducing AR is very similar to that of Ca2+ uptake. Second, the ATP-analogue 2-methylthio-ATP stimulates AR and Ca2+ uptake, whereas ß,{gamma}-methylene-ATP seems to have no effect on either process. Third, the maximal enhanced effect of ATP or 2-methylthio-ATP on Ca2+ uptake is seen after 15 min of incubation, indicating that this phenomenon precedes the AR, which shows maximal effect after 20 min of incubation. The agonist 2-methylthio-ATP, a potent activator of the P2y receptor subtype, showed a high activation of bovine sperm AR as well as Ca2+ uptake by the cells (Figs. 2 and 6). Moreover, the agonist ß,{gamma}-methylene-ATP, which is a potent activator of the P2x receptor, was not active in either the acrosomal exocytosis or the Ca2+ uptake (Figs. 2 and 6). Furthermore, we showed that UTP and ADP, both of which are activators of P2y receptors, cause high stimulation of the AR. These findings suggest that the purinoceptor involved in sperm AR is of the P2y subtype.

The involvement of P2y receptors was further clarified using specific inhibitors. The ATP-induced acrosomal exocytosis was inhibited by reactive-blue 2, an inhibitor for P2y receptors. Moreover, no inhibition was achieved by PPADS, which is considered to be more specific for P2x receptors (Fig. 3). Conversely, Foresta et al. [7] have suggested that the purinergic receptor in human sperm is of the P2x and not P2y type, because P2y is usually linked to intracellular Ca2+ release, which they could not detect. Nevertheless, we have found that ATP can evoke the AR in the absence of added extracellular Ca2+, similar to results accepted by Foresta et al. Because the AR depends on elevation of the [Ca2+]i [49], we assume that, in the absence of extracellular Ca2+, intracellular Ca2+ mobilization occurs, which is needed for acrosomal exocytosis [50]. We show here that ATPe induces elevation of [Ca2+]i, but only when Ca2+ is present in the incubation medium (Fig. 4). It is possible that ATPe induces intracellular Ca2+ mobilization, resulting in the operation of a plasma membrane-capacitative, Ca2+-entry mechanism, also known as a store-operated Ca2+ channel (SOC) [51]. This Ca2+-entry mechanism, which is involved in the cascade, leads to AR [21, 52].

Regarding Ca2+ mobilization, to the best of our knowledge, no published information shows this phenomenon in spermatozoa. However, several publications have shown that the acrosomal membrane contains IP3 receptor, suggesting that Ca2+ can be mobilized from the acrosome to the cytoplasm [21, 53]. We recently showed the activation of capacitative Ca2+-entry activity in bovine sperm by inhibiting intracellular Ca2+-ATPase using thapsigargin [21]. Entry of Ca2+ via SOC in mouse sperm is initiated by the ZP3 glycoprotein in the egg [52]. This suggests that Ca2+ mobilization does occur in the sperm cell, but for unknown reasons, neither we nor others have found evidence of Ca2+ mobilization, meaning an increase in [Ca2+]i in a Ca2+-deficient medium.

Electrophysiological studies have shown that P2x receptors mediate responses via the opening of intrinsic cation-selective channels [54], and that P2y receptors are involved in the activation of PLC and in the mobilization of intracellular calcium [11]. It is known that PLC is involved in acrosomal exocytosis [55]. The possible activation of PLC by a ATP-P2y receptor interaction will result in an increase in [Ca2+]i and DAG, two factors necessary for the activation of cPKC. Our data, which show an increase in [Ca2+]i and activation of PKC{alpha} by ATPe, probably via the activation of PLC, further support our notion regarding the involvement of P2y receptors in ATPe-induced AR. Activation of PKC by ATPe was also found in CHO cells [56]. In recent papers, we showed that one of the cPKC isoforms, PKC{alpha}, is involved in the bovine sperm AR [42, 43]. Here, we provide further support for this notion by showing that ATPe activates PKC{alpha}, and that the AR induced by ATPe is inhibited by a PKC-specific inhibitor.

It is possible that the increase in Ca2+ uptake induced by ATP (Figs. 7 and 8) may be due to the opening of a plasma membrane ligand-gated channel of the P2x subtype. Nevertheless, that the Ca2+ uptake is enhanced by 2-methylthio-ATP and not by ß,{gamma}-methylene-ATP and the transient change in [Ca2+]i is induced by ATP (Fig. 6) strongly argue against P2x being the ATP receptor involved in Ca2+ influx.

In our previous study, we showed that cAMP can activate Ca2+ channels in outer acrosomal membranes [57] and the present data demonstrate the stimulation of cAMP production by ATPe (Fig. 9). Thus, ATPe activates the cAMP-dependent Ca2+ channel in the outer acrosomal membrane to mobilize Ca2+ from the acrosome to the cytoplasm. If this is the case, then the purinoceptor involved in the AR of bovine sperm has more of a resemblance to the P2y subtype. We showed here that ATPe stimulates cAMP production by the cells, with an additional major enhancement seen when Ca2+ and the Ca2+ ionophore are also present in the incubation medium (Fig. 10). Although an increase in the intracellular Ca2+ by the Ca2+ ionophore in the absence of ATPe caused a significant increase in AR, no increase in cAMP was seen under these conditions, indicating the importance of ATPe in this process. Thus, when [Ca2+]i is increased artificially by the Ca2+ ionophore, cellular cAMP need not be increased to induce the AR. These data indicate that cAMP may be involved in elevating the level of [Ca2+]i needed for AR, as suggested by us previously [57].

The additive effect of ATP and the Ca2+ ionophore on AR and cAMP levels (Fig. 10) suggest that cAMP itself might be important in other steps leading to AR in addition to its effect of increasing [Ca2+]i. It was suggested that cAMP is involved in acrosomal exocytosis [58], but how its biosynthesis by adenylyl cyclase is regulated remains unclear. Solubilized ZP from mouse eggs caused an elevation in sperm cAMP that is dependent on the presence of extracellular Ca2+ [41]. This ZP-induced cAMP increase appears to be mediated by the activation of sperm membrane-bound adenylyl cyclase [59]. Thus, it is possible that, under physiological conditions in the female reproductive tract, ATPe might ensure occurrence of the AR after the sperm-ZP interaction. Because cAMP-dependent PKA is an important regulator of sperm capacitation [60] and does not involve ATPe-induced AR (Fig. 10), it is possible that ATP in the female reproductive tract physiologically induces cAMP elevation during capacitation. This point should be clarified in the future.

Taken together, the data presented here suggest the following sequence of events in the mechanism of ATPe-induced acrosomal exocytosis: ATP activates the P2y receptor, which leads to an elevation in [Ca2+]i and to activation of PKC{alpha}, which phosphorylates proteins participating in the cascade leading to the AR.


   FOOTNOTES
 
First decision: 2 August 2001.

1 Supported by the Israel Science Foundation, funded by the Academy of Sciences and Humanities, and by Ihel Foundation to H.B. Back

2 Correspondence. FAX: 972 3 534 4766; breith{at}mail.biu.ac.il Back

Accepted: September 19, 2001.

Received: June 29, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Yanagimachi R. Mammalian fertilization. In: Knobil E, Neil JD (eds.), The Physiology of Reproduction, vol. 1. New York: Raven Press; 1994: 189–317
  2. Zaneveld LJD, De Jonge CJ. Mammalian sperm acrosomal enzymes and the acrosome reaction. In: Dunbar BS, O'Ran MG (eds.), Mammalian Fertilization. New York: Plenum Press; 1991: 63–79
  3. Wassarman PM. Early events in mammalian fertilization. Annu Rev Cell Biol 1987; 3:109-142[CrossRef]
  4. Barnard EA, Burnstock G, Webb TE. G protein-coupled receptors for ATP and other nucleotides: a new receptor family. Trends Pharmacol Sci 1994; 15:67-70[CrossRef][Medline]
  5. Zamir N, Barkan D, Keynan N, Naor Z, Breitbart H. Atrial natriuretic peptide induces acrosomal exocytosis in bovine spermatozoa. Am J Physiol 1995; 269:E216-E221[Abstract/Free Full Text]
  6. Lax Y, Rubinstein S, Breitbart H. Epidermal growth factor induces acrosomal exocytosis in bovine sperm. FEBS Lett 1994; 339:234-238[CrossRef][Medline]
  7. Foresta C, Rossato M, Di Virgilio F. Extracellular ATP is a trigger for the acrosome reaction in human spermatozoa. J Biol Chem 1992; 267::19443-19447[Abstract/Free Full Text]
  8. Neary JT, Van Reemen C, Laskey R, Blicharska J, Norenberg LOB, Norenberg MD. ATP evoked calcium flux, protein phosphorylation and stellation in astrocytes. Ann N Y Acad Sci 1989; 560:473-475
  9. Soltoff SP, McMillian MK, Lechleiter JD, Cantley LC, Talamo BR. Elevation of [Ca2+]i and fluxes by extracellular ATP and phospholipase C-linked agonists in rat parotid acinar cells. Ann N Y Acad Sci 1989; 560:76-90
  10. O'Conner JL, Wade MF, Zhou Y. Control of buffer pH during agarose gel electrophoresis of glyoxylated RNA. Biotechniques 1991; 10:300-302[Medline]
  11. Bean BP. Pharmacology and electrophysiology of ATP-activated ion channels. Trends Pharmacol Sci 1992; 13:87-90[CrossRef][Medline]
  12. Bean BP. Whole-cell recording of calcium channel currents. Methods Enzymol 1992; 207:181-193[Medline]
  13. Florman HM, Arnoult C, Kazam IG, Li C, O'Toole CM. A perspective on the control of mammalian fertilization by egg-activated ion channels in sperm: a tale of two channels. Biol Reprod 1998; 59:12-16[Free Full Text]
  14. Kozawa O, Miwa M, Tokuda H. Cross-talk between Ca2+ influx and phosphoinositide hydrolysis by extracellular adenosine triphosphate in rat PC12 pheochromocytoma cells. Endocrinology 1993; 133:166-170[Abstract]
  15. Schofl C, Rossig L, von zur Muhlen A, Beil W, Jahne J, Manns MP, Wagner S. Extracellular nucleotides increase cytosolic free calcium by activating P2u-receptors in single human gastric mucous cells. Biochem Biophys Res Commun 1995; 216:636-641[CrossRef]
  16. Foresta C, Rossato M, Chiozzi P, Di Virgilio F. Mechanism of human sperm activation by extracellular ATP. Am J Physiol 1996; 270::C1709-C1714[Abstract/Free Full Text]
  17. Karuhn RF. Method of predetermining time of ovulation in women and in animals to control conception. U.S. Patent 4,036,212; 1977
  18. Parrish JJ, Susko-Parrish J, Winer MA, First NL. Capacitation of bovine sperm by heparin. Biol Reprod 1988; 38:1171-1180[Abstract]
  19. Breitbart H, Lax Y, Rotem R, Naor Z. Role of protein kinase C in the acrosome reaction of mammalian spermatozoa. Biochem J 1992; 281::473-476
  20. Ben-Av P, Rubinstein S, Breitbart H. Induction of acrosomal reaction and calcium uptake in ram spermatozoa by ionophores. Biochim Biophys Acta 1988; 939:214-222[Medline]
  21. Dragileva E, Rubinstein S, Breitbart H. Intracellular Ca2+-Mg2+-ATPase regulates calcium influx and acrosomal exocytosis in bull and ram spermatozoa. Biol Reprod 1999; 61:1226-1234[Abstract/Free Full Text]
  22. Mendoza C, Carreras A, Moos J, Tesarik J. Distinction between true acrosome reaction and degenerative acrosome loss by a one-step staining method using Pisum sativum agglutinin. J Reprod Fertil 1992; 95::755-763[Abstract]
  23. Cohen N, Lubart R, Rubinstein S, Breitbart H. Light irradiation of mouse spermatozoa: stimulation of in vitro fertilization and calcium signals. Photochem Photobiol 1998; 68:407-413[CrossRef][Medline]
  24. Harper JF, Brooker G. Femtomole sensitive radioimmunoassay for cyclic AMP and cyclic GMP after 2 acetylation by acetic anhydride in aqueous solution. J Cyclic Nucleotide Res 1975; 1:207-218[Medline]
  25. Rush JS, Klein J, Fanti P, Bhat NR, Waechter CJ. Direct assay of membrane-associated protein kinase C activity in B lymphocytes in the presence of brij 58. Anal Biochem 1992; 207:304-310[CrossRef][Medline]
  26. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 1976; 72:248-254[CrossRef][Medline]
  27. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227:680-685[CrossRef][Medline]
  28. Boarder MR, Weisman GA, Turner JT, Wilkinson GF. G protein-coupled P2 purinoceptors: from molecular biology to functional responses. TiPS 1995; 16:133-139
  29. Boarder MR, Turner JT, Erb L, Weisman GA. Classification of P2 purinoceptors. Trends Pharmacol Sci 1994; 15:280-281[Medline]
  30. Janssens R, Communi D, Pirotton S, Samson M, Parmentier M, Boeynaems JM. Cloning and tissue distribution of the human P2Y1 receptor. Biochem Biophys Res Commun 1996; 221:588-593[CrossRef][Medline]
  31. Leon C, Hechler B, Vial C, Leray C, Cazenave JP, Gachet C. The P2Y1 receptor is an ADP receptor antagonized by ATP and expressed in platelets and megakaryoblastic cells. FEBS Lett 1997; 403:26-30[CrossRef][Medline]
  32. Leon C, Vial C, Cazenave JP, Gachet C. Cloning and sequencing of a human cDNA encoding endothelial P2Y1 purinoceptor. Gene 1996;; 171:295-297[CrossRef][Medline]
  33. Garcia-Guzman M, Stuhmer W, Soto F. Molecular characterization and pharmacological properties of the human P2X3 purinoceptor. Brain Res Mol Brain Res 1997; 47:59-66[Medline]
  34. Chen CC, Akopian AN, Sivilotti L, Colquhoun D, Burnstock G, Wood JN. A P2X purinoceptor expressed by a subset of sensory neurons. Nature 1995; 377:428-431[CrossRef][Medline]
  35. Abbracchio MP, Burnstock G. Purinoceptors: are there families of P2x and P2y purinoceptors. Pharmacol Ther 1994; 64:445-475[CrossRef][Medline]
  36. Wu WL, So SC, Sun YP, Chung YW, Grima J, Wong PY, Yan YC, Chan HC. Functional expression of P2U receptors in rat spermatogenic cells: dual modulation of a Ca(2+)-activated K+ channel. Biochem Biophys Res Commun 1998; 248:728-732[CrossRef][Medline]
  37. Ziganshin AU, Hoyle CH, Bo X, Lambrecht G, Mutschler E, Baumert HG, Burnstock G. PPADS selectively antagonizes P2X-purinoceptor-mediated responses in the rabbit urinary bladder. Br J Pharmacol 1993; 110:1491-1495[Medline]
  38. Florman HM. Sequential focal and global elevations of sperm intracellular Ca2+ are initiated by the zona pellucida during acrosomal exocytosis. Dev Biol 1994; 165:152-164[CrossRef][Medline]
  39. Motte S, Communi D, Pirotton S, Boeynaems JM. Involvement of multiple receptors in the actions of extracellular ATP: the example of vascular endothelial cells. Int J Biochem Cell Biol 1995; 27:1-7[CrossRef][Medline]
  40. Kopf GS, Woolkalis MJ, Gerton GL. Evidence for a guanine nucleotide-binding regulatory protein in invertebrate and mammalian sperm. Identification by islet-activating protein-catalyzed ADP-ribosylation and immunochemical methods. J Biol Chem 1986; 261:7327-7331[Abstract/Free Full Text]
  41. Noland TD, Garbers DL, Kopf G-S. An elevation in cyclic AMP concentration precedes the zona pellucid-induced acrosome reaction of mouse spermatozoa. Biol Reprod 1988; 38: (suppl): 94
  42. Lax Y, Rubinstein S, Breitbart H. Subcellular distribution of protein kinase C{alpha} and ß, in bovine spermatozoa, and their regulation by calcium and phorbol esters. Biol Reprod 1997; 56:454-459[Abstract]
  43. Garbi M, Rubinstein S, Lax Y, Breitbart H. Activation of protein kinase Ca in the lysophosphatidic acid-induced bovine sperm acrosome reaction and phospholipase D1 regulation. Biol Reprod 2000; 63::1271-1277[Abstract/Free Full Text]
  44. Kraft AS, Anderson WB, Cooper HL, Sando JJ. Decrease in cytosolic calcium/phospholipid-dependent protein kinase activity following phorbol ester treatment of EL4 thymoma cells. J Biol Chem 1982; 257:13193-13196[Free Full Text]
  45. Monks NJ, Fraser LR. Inhibition of adenosine-metabolizing enzymes modulates mouse sperm fertilizing ability: a changing role for endogenously generated adenosine during capacitation. Gamete Res 1988; 21:267-276[CrossRef][Medline]
  46. Fraser LR, Duncan AE. Adenosine analogues with specificity for A2 receptors bind to mouse spermatozoa and stimulate adenylate cyclase activity in uncapacitated suspensions. J Reprod Fertil 1993; 98:187-194[Abstract]
  47. Minelli A, Miscetti P, Allegrucci C, Mezzasoma I. Evidence of A1 adenosine receptor on epidydimal bovine spermatozoa. Arch Biochem Biophys 1995; 322:272-276[CrossRef][Medline]
  48. Allegrucci C, Liguori L, Minelli A. Stimulation by N6-cyclopentyladenosine of A1 adenosine receptors, coupled to G{alpha}i2 protein subunit, has a capacitative effect on human spermatozoa. Biol Reprod 2001; 64:1653-1659[Abstract/Free Full Text]
  49. Florman HM, Carron ME, Kim TDH, Babcock DF. Activation of voltage dependent calcium channels of mammalian sperm is required for zona pellucida induced acrosomal exocytosis. Dev Biol 1992; 152::304-314[CrossRef][Medline]
  50. Meizel S, Turner KO. Initiation of the human sperm acrosome reaction by thapsigargin. J Exp Zool 1993; 267:350-355[CrossRef][Medline]
  51. Putney JWJ. Capacitative calcium entry revisited. Cell Calcium 1990;; 11:611-624[CrossRef][Medline]
  52. O'Toole CM, Arnoult C, Darszon A, Steinhardt RA, Florman HM. Ca2+ entry through store-operated channels in mouse sperm is initiated by egg ZP3 and drives the acrosome reaction. Mol Biol Cell 2000; 11:1571-1584[Abstract/Free Full Text]
  53. Walensky LD, Snyder SH. Inositol 1,4,5-trisphosphate receptors selectively localized to the acrosomes of mammalian sperm. J Cell Biol 1995; 130:857-869[Abstract/Free Full Text]
  54. Bean BP, Friel DD. ATP-activated channels on excitable cells. In: Narahashi T (ed.), Ion Channels. New York: Plenum Press; 1990: 169–203
  55. Roldan ERS, Harrison RAP. Polyphosphoinositide breakdown and subsequent exocytosis in the Ca2+/ionophore-induced acrosome reaction in mammalian spermatozoa. Biochem J 1989; 259:397-406[Medline]
  56. Ohmori S, Shirai Y, Sakai N, Fujii M, Konishi H, Kikkawa U, Saito N. Three distinct mechanisms for translocation and activation of the delta subspecies of protein kinase C. Mol Cell Biol 1998; 18:5263-5271[Abstract/Free Full Text]
  57. Spungin B, Breitbart H. Calcium mobilization and influx during sperm exocytosis. J Cell Sci 1996; 109:1947-1955[Abstract]
  58. Garbers DL, Kopf GS. The regulation of spermatozoa by calcium and cyclic nucleotides. Adv Cyclic Nucleotide Res 1980; 13:251-305[Medline]
  59. Leclerc P, Kopf GS. Mouse sperm adenylyl cyclase: general properties and regulation by the zona pellucida. Biol Reprod 1995; 52:1227-1233[Abstract]
  60. Visconti PE, Moore GD, Bailey JL, Laclerc P, Connors SA, Pan D, Olds-Clarke P, Kopf G-S. Capacitation in mouse spermatozoa. II. Protein tyrosine phosphorylation and capacitation are regulated by a cAMP-dependent pathway. Development 1995; 121:1139-1150[Abstract]



This article has been cited by other articles:


Home page
 Biol. Reprod.Home page
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]

Home page
 Reproductive SciencesHome page
S. E. Edwards, M. G. Buffone, G. R. Knee, M. Rossato, G. Bonanni, S. Masiero, S. Ferasin, G. L. Gerton, S. B. Moss, and C. J. Williams
Effects of Extracellular Adenosine 5'-Triphosphate on Human Sperm Motility
Reproductive Sciences, October 1, 2007; 14(7): 655 - 666.
[Abstract] [PDF]

Home page
 Mol Hum ReprodHome page
L. Liguori, E. de Lamirande, A. Minelli, and C. Gagnon
Various protein kinases regulate human sperm acrosome reaction and the associated phosphorylation of Tyr residues and of the Thr-Glu-Tyr motif
Mol. Hum. Reprod., March 1, 2005; 11(3): 211 - 221.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Luria, A.
Right arrow Articles by Breitbart, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Luria, A.
Right arrow Articles by Breitbart, H.