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Biology of Reproduction 64, 1653-1659 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Stimulation by N6-Cyclopentyladenosine of A1 Adenosine Receptors, Coupled to G{alpha}i2 Protein Subunit, Has a Capacitative Effect on Human Spermatozoa1

Cinzia Allegruccia, Lavinia Liguoria, and Alba Minelli2,a

a Dipartimento di Scienze Biochimiche e Biotecnologie Molecolari, Sezione Biochimica Cellulare, Università degli Studi di Perugia, 06126 Perugia, Italia

ABSTRACT

The effects of selective A1 receptor agonist on human spermatozoa were examined to verify physiological responses and to investigate the signal transduction pathway. N6-Cyclopentyladenosine on uncapacitated spermatozoa did not induce spontaneous acrosome reaction after 5 h capacitation, whereas the number of capacitated spermatozoa, assessed by lysophosphatidylcholine-induced acrosome reaction with Pisum sativum agglutinin staining, was significantly increased. N6-Cyclopentyladenosine was also added to capacitated human spermatozoa to find out whether the agonist could induce the acrosome reaction. Results, although statistically significant, could not be considered biologically significant. A1-Mediated capacitation was followed by the increase of tyrosine phosphorylation of a protein subset ranging between Mr = 200 000 and 30 000. Stimulation of A1 receptor with the selective agonist elicited an agonist-induced inositol phospholipid hydrolysis leading to a transient rise of inositol triphosphate (IP3). This increase was not induced by A1 receptor antagonist and was blocked by phospholipase C inhibitor. Coimmunoprecipitation experiments showed that the A1 receptor is coupled to G{alpha}i2 subunit suggesting that the activation of phospholipase C is mediated by ß{gamma} subunits. In conclusion, the A1 adenosine receptor in human spermatozoa is coupled to G{alpha}i2, signals via IP3, and affects the capacitative status of ejaculated spermatozoa.

acrosome reaction, gamete biology, male reproductive tract, signal transduction, sperm capacitation

INTRODUCTION

The process of capacitation enables spermatozoa to interact with the zona pellucida and to undergo the acrosome reaction (AR), both crucial steps toward fertilization. Therefore to become fertilization competent, mammalian sperm must spend a period of time in either the female reproductive tract or in suitable in vitro environments.

Capacitation and AR are related to many effectors and signal transduction pathways, but the molecules and the molecular basis of these important events are still only partially known [1–5].

Adenosine is able to elicit biphasic responses in mammalian spermatozoa: stimulatory in uncapacitated cells and inhibitory in capacitated cells [6]. Mammalian spermatozoa have adenosine receptors, either stimulatory (A2) or inhibitory (A1) [7–12].

Specific A2 agonists affected capacitation, enhanced fertilizing ability, and increased the cAMP content of uncapacitated mouse spermatozoa [7]. The same agonist, used with human spermatozoa, confirmed the presence of A2 adenosine receptors responsible for adenosine-mediated enhancement of sperm motility [8], cAMP production, and protein phosphorylation [9].

Recent studies have provided evidence that responses to adenosine in uncapacitated mouse and boar spermatozoa involve stimulatory A2A adenosine receptors, whereas responses in capacitated cells involve inhibitory A1 adenosine receptors (A1AR) [13, 14]. We have solubilized, characterized, and localized the A1AR in mammalian spermatozoa [10–12]. We have also reported [15] that A1ARs are involved in calcium mobilization, which is essential for regulating sperm function [16].

The present study was designed to investigate further A1ARs and their signaling pathways and to assess their role in the mechanisms by which the fertilizing potential of human spermatozoa is enhanced using the highly specific agonist N6-cyclopentyladenosine (CPA) [17].

MATERIALS AND METHODS

Materials

Biotrak [3H]inositol triphosphate (IP3) assay system and protein A Sepharose CL4B were from Amersham Pharmacia Biotech (Rainham, UK). Polyvinylidene fluoride (PVDF) and silver stain plus kit were from Bio-Rad Laboratories (Hercules, CA). Rabbit anti-A1AR antibody PC21 was a gift from Prof. R. Franco (Barcelona, Spain) and elsewhere thoroughly characterized [18]. Rabbit anti-G{alpha}i2 subunit antibody was from Calbiochem Biosciences (La Jolla, CA). All other products were from Sigma Chemical Co. (St. Louis, MO).

Sperm Preparation

Human semen with normal sperm characteristics according to World Health Organization criteria (vol {cong} 2 ml, concentration >=20 x 106 cells/ml; motility >=50%, normal morphology >=15%) was collected by masturbation from healthy donors. After liquefaction at 37°C, motile spermatozoa were selected by centrifugation at 800 x g for 10 min through a two-step Percoll density gradient (90% v/v and 40% v/v). The pellet was washed twice at 800 x g for 10 min and suspended at a concentration of 10 x 106 cell/ml in 95 mM NaCl, 4.8 mM KCl, 1.7 mM CaCl2, 1.2 mM MgSO4, 25 mM NaHCO3, 5.6 mM fructose, 0.25 mM sodium pyruvate, 3.7 ml/L sodium lactate (60% syrup), 20 mM Hepes, 10 U/ml penicillin, 10 mg/ml streptomycin (BWW medium). In capacitation experiments, 0.3% BSA was added to BWW (BWW-BSA) [19].

Cell viability was assessed with propidium iodide by fluorescent microscopy.

Assessment of Capacitation and AR

Sperm were assayed for capacitation by induction of AR with L-{alpha}-lysophosphatidylcholine (LPC) at 100 µg/ml. This concentration of LPC was previously shown to induce the AR in capacitated sperm while having no effects on uncapacitated sperm [20]. To induce the AR with LPC, 10 x 106 sperm were incubated for 5 h in BWW-BSA at 37°C in 5% CO2, 95% humidified air. At the end of the incubation, LPC was added at 100 µg/ml, and the sperm were incubated an additional 15 min. Prior to drying and staining, randomly selected slides containing ~105 cells were examined to verify sperm motility and viability. The percentage of sperm that were acrosome-reacted was determined on air-dried sperm smears with Pisum sativum agglutinin fluorescein isothiocyanate conjugated (PSA-FITC) [21]. At least 200 cells were scored with an Axioplan Zeiss epifluorescence microscope equipped with a 390–525-nm filter (Gottingen, Germany) according to the following patterns: 1) selective staining of the whole acrosome (unreacted cells); 2) no staining or limited to the equatorial segments (reacted cells).

In experiments performed to determine whether specific A1 agonist had a capacitative effect on uncapacitated spermatozoa, the protocol was modified by substituting BSA with CPA. The capacitative status was assayed by induction of AR with LPC, and the capacitative effect of CPA was evaluated by comparing the AR rate promoted by LPC in the control (BWW-BSA) and in the described experimental conditions (BWW-CPA).

In experiments performed to determine whether a specific A1 agonist can act as AR initiator, LPC was substituted by CPA. Sperm were capacitated in BWW-BSA as described. At the end of the incubation, CPA was added and additionally incubated for 15 min. Acrosome-reacted cells were evaluated by PSA-FITC staining. The ability of CPA to induce AR was expressed as the difference between the AR rate promoted by CPA and the spontaneous AR rate observed in the control (BWW-BSA).

The A1 agonist and antagonist were always used in the presence of 0.2 U/ml adenosine deaminase (ADA) because of the role of A1AR as an ADA-binding protein [22].

Inositol Triphosphate Determination

Determination of sperm IP3 concentration was performed with the Biotrak [3H]IP3 assay system (Amersham Pharmacia Biotech, Rainham, U.K.) following the manufacturer's procedure.

Spermatozoa, prepared as described, were suspended in BWW and aliquoted at 3 x 104 cells/vial. Adenosine receptor agonist and antagonist plus ADA at 0.2 U/ml were added and trichloroacetic acid (TCA)-precipitation/diethyl ether extraction initiated at 40 sec. For each sample, IP3 content of the spermatozoa at 0 time was evaluated (control value).

When the effects of other reagents were examined, the products were added at 37°C 15 min before the addition of the A1 agonist.

Immunoprecipitation Procedure

Human sperm (700 x 106 cells), washed in PBS at 800 x g for 10 min at room temperature (RT), was suspended in cold 50 mM Tris-HCl, pH 7.4 containing 0.5% digitonin, 0.5% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), and protease inhibitor cocktail. After sonication (Virsonic 50; Virtis Co., Gardiner, NY) on ice with 15 bursts of 10 sec (10-sec intervals) and extraction for 60 min at 4°C, the supernatant was obtained by ultracentrifugation at 105 000 x g (1 h at 4°C). The preparation was assayed with a Bio-Rad DC protein assay kit (Bio-Rad Laboratories, Richmond, CA) for protein quantitation. Aliquots of the detergent extracts (about 400 µg protein) were incubated with the antibodies (PC21 and anti-G{alpha}i2 at 12 µg/ml) previously coupled to protein A Sepharose CL4B by an overnight incubation at 4°C in constant rotation. Nonspecific immunoprecipitation was assessed by using the same amount of rabbit nonimmune IgG. The mixture was incubated for 3 h at 4°C with constant rotation. Immunoprecipitates were washed twice with 0.5% digitonin, 0.5% CHAPS in Tris-HCl 50 mM, NaCl 140 mM, NaN3 0.025% (TSA buffer), twice with 0.1% digitonin, 0.1% CHAPS in TSA buffer, and once with TSA buffer alone. Pelleted samples were then dissolved in 60 µl 2x SDS-PAGE sample buffer, boiled for 3 min, and centrifuged at 12 000 x g for 2 min. The supernatant was analyzed by SDS-PAGE at 15% [23]. After PAGE, the gels were stained with a silver staining kit (Bio-Rad).

Western Blot Procedure

Immunoprecipitates, analyzed at a constant voltage of 150 V for 40 min on 15% polyacrylamide gels, were transferred onto PVDF membranes (Bio-Rad) using a constant voltage of 100 V for 60 min. Nonspecific sites were blocked by incubating the blots overnight at 4°C in 10% (w/v) low-fat dried milk in 50 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20, pH 7.4 (TBST-I). Blots were washed twice with 10 mM Tris-HCl, 500 mM NaCl, 0.5% Tween-20, pH 7.4 (TBST-II), and incubated for 2 h at RT with specific antibodies diluted in TBST-I containing 0.1% NaN3 at a concentration of 10 µg/ml PC21 and 55 µg/ml anti-G{alpha}i2. The PVDF membrane was washed four times with TBST-II and incubated for 60 min at RT with horseradish peroxidase-conjugated goat anti-rabbit IgG diluted1:5000 in TBST-I. The blot was washed twice with TBST-II and twice with TBST-I, then revealed by an ECL-Plus detection system (Amersham Pharmacia Bioteck, Rainham, U.K.). Molecular weight was determined by rainbow-colored protein molecular weight markers. Nonimmune rabbit IgGs at the same concentration were used as negative controls.

Changes in protein tyrosine phosphorylation were analyzed by using an anti-phosphotyrosine antibody. Aliquots of 2 x 106 cells were extracted in 40 µl of 1x SDS-PAGE sample buffer [23] and heated in boiling water for 5 min. Boiled suspensions were loaded in SDS-PAGE gels at 10% acrylamide. After transferring onto PVDF membranes, following the above-described procedure, blots were incubated with mouse monoclonal anti-phosphotyrosine antibody (clone PT-66) diluted 1:2000 and revealed by the ECL-Plus detection system. The specificity of PT-66 was verified by preincubating with 20 mM ortho-phosphotyrosine (30 min at RT) that completely eliminated its reaction on immunoblots (data not shown). After scanning, images were analyzed using an ImageMaster software (Pharmacia Biotech, Uppsala, Sweden) to quantify the changes in intensity of various bands.

Statistical Analysis

Data were expressed as means ± SEM. Statistical significance was determined by using the Student t-test and ANOVA. A P value <0.05 was considered significant.

RESULTS

Effects of A1 Agonist on Capacitation and AR

The effect of A1 selective agonist was tested on uncapacitated human spermatozoa by substituting BSA with 10 nM, 100 nM, and 1 µM CPA (Kd 1 nM) [17] in the capacitative medium (BWW-BSA). Results are shown in Figure 1. Capacitation was measured as the ability of sperm to undergo the AR when LPC, the inducer of AR, is added to the suspension. Pooled sperm samples used in the present study were characterized by 19 ± 4% of spontaneous AR after 5 h incubation in BWW-BSA at 37°C in 5% CO2 and by 56 ± 6% induced AR after 15 min treatment with 100 µg/ml LPC.



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  		FIG. 1. Effect of A1 agonist on uncapacitated human spermatozoa. Pooled human spermatozoa were incubated 5 h at 37°C in 5% CO2 in BWW containing either 0.3% BSA or 10 nM, 100 nM, 1 µM CPA. AR was induced by LPC (100 µg/ml for 15 min at 37°C). Acrosome-reacted cells were determined with PSA-FITC. Results represent the mean ± SEM of n = 5 independent experiments performed in triplicate and 200 sperm counted/sample. Significant differences versus control (BWW/LPC), *P < 0.05. A) Capacitative status of the spermatozoa evaluated with LPC-induced AR. B) Spontaneous AR percentage of the samples after the 5-h incubation under the described experimental conditions

The substitution of BSA with CPA in the capacitative medium affected the capacitative status of human spermatozoa. The percentage of LPC-reacted cells was significantly different from the control (Fig. 1A). However, the incubation of human spermatozoa in the presence of different concentrations of CPA did not affect the rate of spontaneous AR. Results clearly show that for each concentration tested the percentage of cells that underwent the spontaneous AR was not significantly modified (Fig. 1B).

The effects of CPA on capacitated human spermatozoa are shown in Figure 2. CPA, when used at 10 nM and 100 nM, was capable of inducing a moderate AR, whereas, when used at 1 µM, CPA induced an AR rate that did not significantly differ from the control.



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  		FIG. 2. Effect of A1 agonist on capacitated human spermatozoa. Capacitated human spermatozoa (5 h in BWW-BSA at 37°C in 5% CO2) was incubated at 37°C for 15 min with CPA at 10 nM, 100 nM, and 1 µM. The acrosome status was determined by PSA-FITC and results were compared with the spontaneous AR rate of the control. Results represent the mean ± SEM of n = 5 independent experiments performed in triplicate and 200 sperm counted/sample. Significant differences versus spontaneous acrosome-reacted control (BWW-BSA). *P < 0.05

Specific A1 antagonist, N6-cyclopentyl-9-methyladenine (N0840, Ki 10 nM) [24], at 100 nM concentration, did not exert any effect on either uncapacitated or capacitated spermatozoa (data not shown).

Effect of A1 Agonist on Protein Tyrosine Phosphorylation

The appearance of tyrosine phosphorylation of human sperm proteins is correlated with capacitation. Changes in protein tyrosine phosphorylation of uncapacitated, BSA-capacitated, and CPA-capacitated human spermatozoa are reported in Figure 3.



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  		FIG. 3. Tyrosine phosphorylation pattern of human sperm proteins. Human spermatozoa (2 x 106 cells) were capacitated for 5 h at 37°C in 5% CO2 either in BWW-BSA or in BWW-CPA. Cells were suspended in 1x SDS-PAGE sample buffer and samples immunoblotted with monoclonal antiphosphotyrosine antibody PT66. A) Lane 1, uncapacitated spermatozoa; lane 2, BWW-BSA capacitated spermatozoa; lane 3, BWW-CPA capacitated spermatozoa. B) Optical density profiles

In the capacitated samples there was an increase in the tyrosine phosphorylation of a subset of proteins of Mr = 30 000–200 000. The most prominent tyrosine phosphorylated proteins had Mr = 95 000, Mr = 80 000, and Mr = 30 000. A protein of Mr = 62 000 was only present in BSA-capacitated sperm preparations.

Effect of A1 Agonist on IP3 Production

Human spermatozoa responded to the A1 selective agonist CPA with a transient increase of IP3 with a peak rise at 40 sec. The effects of A1AR agonist, antagonist, and phospholipase C (PLC) inhibitor on IP3 production are shown in Table 1.


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  		TABLE 1. Effects of CPA and PLC inhibitor on IP3 production

CPA caused an increase in IP3 approximately 100-fold over the basal. Specific A1 antagonist N0840 did not cause IP3 production, and the response to CPA was blocked by pretreatment with N0840. In the presence of 25 µM 1-[6-[[(17ß)-3-methoxyesha-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5 dione (U73122) [25], a permeable inhibitor of phosphatidyl inositol (PI)-PLC, there was no IP3 accumulation, whereas the use of 2.5 µM U73122 did not affect IP3 accumulation.

The treatment of human spermatozoa with the same concentrations of U73122 for 15 min at 37°C did not affect spermatozoa viability significantly.

Coimmunoprecipitation Experiments

Results of immunoprecipitation studies are shown in Figure 4. Using the PC21 antibody, G{alpha}i2 coprecipitated with A1ARs. Commercial antibody against G{alpha}i2 also coprecipitated a band of Mr = 40 000 corresponding to intact A1AR. Samples in which specific antibodies were substituted with nonimmune rabbit IgG were used as negative controls.



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  		FIG. 4. Immunochemical detection of A1AR and G{alpha}i2 subunit A1AR and G{alpha}i2 were immunoprecipitated from human spermatozoa lysate (700 x 106 cells) using either the polyclonal PC21-antibody and the commercial anti-G{alpha}i2 previously coupled to protein A Sepharose CL4B. Controls were obtained by precipitation with nonimmune rabbit IgG. A) Immunoprecipitation with PC21 and immunoblotting with anti-G{alpha}i2. B) Immunoprecipitation with anti-G{alpha}i2 and immunoblotting with PC21

DISCUSSION

Ejaculated mammalian spermatozoa are not capable of fertilizing the oocyte until they acquire fertilizing potential by undergoing several physiological changes collectively called capacitation [1, 4].

This priming step is a prerequisite that allows the spermatozoa to bind to the zona pellucida and to undergo the exocytotic event occurring before fertilization, i.e., the AR. These events, triggered in vivo by residence for a finite period of time in the female reproductive tract, can be mimicked in vitro by incubating spermatozoa in a number of defined media. Several compounds have been described as modulators of both capacitation and AR [1–5], but, although some progress has been made in understanding modulatory mechanisms, the molecules and the molecular bases of these mandatory events are still little known.

In this study we have shown a capacitative effect of the selective A1AR agonist added to uncapacitated human sperm suspensions. The number of capacitated spermatozoa, assessed by LPC-induced AR, was significantly increased when compared with BSA-capacitated spermatozoa. The capacitative effect showed no secondary inhibition at very high concentration of CPA compared to its Kd value (1 nM). Spontaneous AR was not affected by the selective A1 agonist. Capacitated human spermatozoa responded to the selective A1 agonist, used at nanomolar concentrations, by marginally increasing the rate of the AR, but the result should be taken as of no biological significance. On the other hand, 1 µM CPA reduced the rate of the AR to a value not significantly different from the control. This result agrees with a previous report [13] showing that 1 µM CPA inhibited spontaneous acrosome loss in capacitated mouse sperm suspensions.

It has been reported that adenosine elicits a biphasic response in mouse and boar spermatozoa, first stimulating capacitation and then inhibiting the spontaneous AR [13, 14]. In our case, using agonist concentrations highly selective to A1AR, i.e., in the nanomolar range, we observed only stimulatory effects: CPA was a more efficient capacitative agent than BSA. The effects exerted by A1ARs are most probably common to mammalian species. We therefore suggest that the discrepancies between our data and those in the literature may be attributed to the different methodological and experimental procedures followed to assess capacitation and the AR. However, in the light of our results, and in agreement with Fraser, Funahashi, and colleagues [13, 14], we propose that the stimulation of A1AR is not relevant to the AR. The fertilizing spermatozoa undergo the AR as a strictly controlled response to the oocyte-cumulus complex because spontaneously acrosome-reacted cells are nonfertilizing [26].

Consistent with data in the literature [4, 27–33], the capacitation induced by selective A1 agonist was correlated with the increase of tyrosine phosphorylation of a protein of Mr = 95 000 that turns out to be the human homologue of the mouse proAKAP82 [28, 32]. In agreement with Brewis et al. [33], in BSA- and CPA-capacitated spermatozoa a capacitation-dependent increase of tyrosine phosphorylation of a protein of Mr = 80 000 was also found. It has been proposed that protein tyrosine phosphorylation and capacitation are regulated through a cAMP/protein kinase A pathway, a signal transduction cross talk probably universal to mammalian spermatozoa. Gs protein subunits are absent in sperm cells [34]. Moreover, sperm adenylyl cyclase, the classical effector coupled to Gs, is not regulated by Gs [35] and is activated by increases of intracellular Ca2+ concentration [36–38].

The capacitative effect of A1 selective agonist cannot be related to receptor-mediated cAMP increase as it is known that A1AR is coupled to Gi/Go and therefore inhibits adenylyl cyclase activity [39]. However, these receptors can stimulate phosphatidyl inositol hydrolysis and release intracellular Ca2+ stores by a pertussistoxin (PTX)-sensitive mechanism, suggesting that they also couple to the Gß{gamma}/PLCß pathway through Gi proteins [40–42]. In addition to directly activating PLC, the A1 receptors are capable of potentiating the PLC response by a universal cross-talk mechanism between adenylyl cyclase inhibitory receptors and a range of Gq-coupled receptors [43]. Whether by direct or indirect stimulation, A1 receptors activate PLC and increase IP3 levels. IP3, in turn, acting on specific IP3 receptors, cooperates in controlling intracellular calcium release, thus eliciting cellular responses. IP3 receptors are present in human spermatozoa [44], and intracellular calcium concentrations are strongly related to the capacitative event [45, 46].

We have previously demonstrated that the G-protein coupled to A1AR in mammalian spermatozoa is PTX sensitive [11]. In addition, we have shown by Ca2+-loading experiments [15] that the treatment of mammalian spermatozoa with selective A1 agonist causes a Ca2+ release similar to that caused by IP3. This finding suggested that A1 receptors in spermatozoa signal via IP3.

In this study we provide evidence for A1-mediated IP3 production through G{alpha}i2 protein. G proteins (G{alpha}i2, G{alpha}i3, G{alpha}q/11) have been identified and localized to discrete regions of human spermatozoa [34]. This subcellular distribution represents an important step to understanding the role of these signaling molecules in the regulation of sperm functions. Furthermore, the identification of receptors and effectors that couple to G proteins will provide additional clues to the role of G protein-coupled pathways in sperm physiology.

G{alpha}i proteins have a variable subcellular location in many cell lines [47, 48]. In human spermatozoa G{alpha}i2 is mainly localized in the acrosomal region [34]. The presence of this subunit together with G protein-coupled receptor kinase GRK4{gamma} [49] suggested their involvement in the signaling pathway to the AR [2]. It has been postulated that the stimulation of G{alpha}i2 causes sperm activation by releasing ß{gamma} subunits that activate PLC [50, 51]. Here we produce evidence for the coupling of A1 receptor with G{alpha}i2 subunit. It is plausible to suggest that ß{gamma} subunits will activate PLC. The ßark1 minigene, a cellular scavenger Gß{gamma} subunit, has been used effectively to identify the involvement of Gß{gamma} subunit in modulating intracellular signal responses [40, 52, 53].

Transfection techniques of spermatozoa are still in a preliminary stage [54] therefore our investigations on IP3 accumulation have been limited to the use of putative PLC inhibitor U73122. The initial aim of this study was to block selectively the Gi-mediated interaction of A1 receptors with PLC. It has been reported [42] that rather than being a selective antagonist of PLC activity, U73122 can produce a number of undesired effects. In pancreatic acinar cells and NG105-15 cells, U73122 produces oscillations in intracellular calcium levels, activates cation channels, activates tyrosine kinases, and inhibits voltage-sensitive Ca2+ channels and Ca2+ entry into cells. Therefore the use of U73122 as a tool in establishing the dependence of stimulated cellular events on PLC activity is greatly limited. After checking the effects of each concentration of aminosteroid on human sperm viability, we observed that high concentrations of U73122 were effective in blocking IP3 accumulation, whereas low concentrations of U73122, reported in the literature [25, 55–57], did not interfere with IP3 production.

In conclusion we have shown that the stimulation of A1AR with selective agonist leads to the capacitation of human spermatozoa without increasing the spontaneous AR rate. Capacitation is related to an increase of tyrosine phosphorylation of two proteins of Mr = 95 000 and 80 000 always found in capacitated spermatozoa.

A1ARs are coupled to PTX-sensitive G{alpha}i2 subunits and signal through IP3 production, which follows the activation of PLC.

Several questions of considerable importance are still to be addressed. First, how can a receptor inhibiting adenylyl cyclase activate the cAMP/PKA pathway and how does the stimulation of this pathway lead to cross talk and upregulation of protein tyrosine phosphorylation? Second, is PLC directly activated by ß{gamma} subunits or does a cross-talk mechanism between A1AR and Gq-coupled receptors exist in sperm? The signaling processes underlying capacitation will be of help in elucidating the molecular basis of this extratesticular maturational event.

ACKNOWLEDGMENTS

We thank Dr. M. Kerrigan (Cantab) for valuable linguistic suggestions.

FOOTNOTES

First decision: 7 November 2000.

1 Research funded by Cofin Murst 40%, Italia. Back

2 Correspondence: Alba Minelli, Dipt. Scienze Biochimiche Biotecnologie Molecolari, Sezione Biochimica Cellulare, Via del Giochetto, 06126 Perugia, Italia. FAX: 39 075 585 7442; albami{at}tin.it Back

Accepted: January 16, 2001.

Received: October 12, 2000.

REFERENCES

  1. de Lamirande E, Leclerc P, Gagnon C. Capacitation as a regulatory event that primes spermatozoa for the acrosome reaction and fertilization. Mol Hum Reprod 1997; 3:175–194[Abstract/Free Full Text]
  2. Breitbart H, Spungin B. The biochemistry of the acrosome reaction. Mol Hum Reprod 1997; 3:195–202[Abstract/Free Full Text]
  3. Tulsiani DR, Abou-Haila A, Loeser CR, Pereira BM. The biological and functional significance of the sperm acrosome and acrosomal enzymes in mammalian fertilization. Exp Cell Res 1998; 240:151–164[CrossRef][Medline]
  4. Visconti PE, Kopf GS. Regulation of protein phosphorylation during sperm capacitation. Biol Reprod 1998; 59:1–6[Free Full Text]
  5. Wassarman PM. Fertilization in animals. Dev Genet 1999; 25:83–86[CrossRef][Medline]
  6. Green CM, Cockle SM, Watson PF, Fraser LR. A possible mechanism of action for fertilization promoting peptide, a TRH-related tripeptide that promotes capacitation and fertilizing ability in mammalian spermatozoa. Mol Reprod Dev 1996; 45:244–252[CrossRef][Medline]
  7. 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]
  8. Shen MR, Linden J, Chen SS, Wu SN. Identification of adenosine receptors in human spermatozoa. Clin Exp Pharmacol Physiol 1993; 20:527–534[Medline]
  9. Fenichel P, Ghabir A, Emiliozzi C, Donzeau M, Menezo Y. Stimulation of human sperm during capacitation in vitro by an adenosine agonist with specificity for A2 receptors. Biol Reprod 1996; 54:1405–1411[Abstract]
  10. Minelli A, Miscetti P, Allegrucci C, Mezzasoma I. Evidence of A1 adenosine receptor on epididymal bovine spermatozoa. Arch Biochem Biophys 1995; 322:272–276[CrossRef][Medline]
  11. Minelli A, Allegrucci C, Mezzasoma I. 3H-R-N6-phenylisopropyladenosine agonist binding to the solubilized A1 adenosine receptor from bovine epididymal spermatozoa. Arch Biochem Biophys 1997; 337:54–61[CrossRef][Medline]
  12. Minelli A, Allegrucci C, Mezzasoma I, Ronquist G, Lluis C, Franco R. CD26 and adenosine deaminase interaction: its role in the fusion between horse membrane vesicles and spermatozoa. Biol Reprod 1999; 61:802–808[Abstract/Free Full Text]
  13. Fraser LR, Adeoya-Osiguwa S. Modulation of adenylyl cyclase by FPP and adenosine involves stimulatory and inhibitory adenosine receptors and G proteins. Mol Reprod Dev 1999; 53:459–471[CrossRef][Medline]
  14. Funahashi H, Fujiwara T, Nagai T. Modulation of the function of boar spermatozoa via adenosine and fertilization promoting peptide receptors reduce the incidence of polyspermic penetration into porcine oocytes. Biol Reprod 2000; 63:1157–1163[Abstract/Free Full Text]
  15. Minelli A, Allegrucci C, Piomboni P, Manucci R, Lluis C, Franco R. Immunolocalization of A1 adenosine receptors in mammalian spermatozoa. J Histochem Cytochem 2000; 48:1163–1171[Abstract/Free Full Text]
  16. 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]
  17. Bruns RF, Lu GH, Pugsley TA. Characterization of the A2 adenosine receptor labeled by 3H-NECA in rat striatal membranes. Mol Pharmacol 1986; 29:331–346[Abstract]
  18. Ciruela F, Casado V, Mallol J, Canela EI, Lluis C, Franco R. Immunological identification of A1 adenosine receptors in brain cortex. J Neurosci Res 1995; 42:818–828[CrossRef][Medline]
  19. Naz RK. Involvement of protein serine and threonine phosphorylation in human sperm capacitation. Biol Reprod 1999; 60:1402–1409[Abstract/Free Full Text]
  20. Parrish JJ, Susko-Parrish J, Winer MA, First NL. Capacitation of bovine sperm by heparin. Biol Reprod 1988; 38:1171–1180[Abstract]
  21. 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]
  22. Ciruela F, Saura C, Canela EI, Mallol J, Lluis C, Franco R. Adenosine deaminase affects ligand-induced signalling by interacting with cell surface adenosine receptors. FEBS Lett 1996; 380:219–223[CrossRef][Medline]
  23. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227:680–685[CrossRef][Medline]
  24. Barrett RJ, May JM, Martin PL, Miller JR. In vitro and in vivo pharmacological chracterization of N6-cyclopentyl-9-methyladenine(N-0840): a selective, orally active A1 adenosine receptor antagonist. Pharmacol Exp Ther 1993; 265:227–236[Abstract/Free Full Text]
  25. Yule DI, Williams JA. U73122 inhibits Ca2+ oscillation in response to cholecystokinin and carbachol but not to JMV-180 in rat pancreatic acinar cells. J Biol Chem 1992; 267:13830–13835[Abstract/Free Full Text]
  26. Yanagimachi R. Mammalian fertilization. In: Knobil E, Neill J (eds.), Physiology of Reproduction. New York: Raven Press; 1994: 189–317
  27. Leclerc P, de Lamirande E, Gagnon C. Cyclic adenosine 3',5' monophosphate-dependent regulation of protein tyrosine phosphorylation in relation to human sperm capacitation and motility. Biol Reprod 1996; 55:684–692[Abstract]
  28. Carrera A, Moos J, Ning XP, Gerton GL, Tesarik J, Kopf GS, Moss SB. Regulation of protein tyrosine phosphorylation in human sperm by a calcium/calmodulin-dependent mechanism: identification of A kinase anchor protein as major substrates for tyrosine phosphorylation. Dev Biol 1996; 180:284–296[CrossRef][Medline]
  29. Galantino-Homer H, Visconti PE, Kopf GS. Regulation of protein tyrosine phosphorylation during bovine sperm capacitation by a cyclic adenosine 3',5'-monophosphate dependent pathway. Biol Reprod 1997; 56:707–719[Abstract]
  30. Emiliozzi C, Fenichel P. Protein tyrosine phosphorylation is associated with capacitation of human sperm in vitro but is not sufficient for its completion. Biol Reprod 1997; 56:674–679[Abstract]
  31. Kalab P, Peknicova J, Geussova G, Moos J. Regulation of protein tyrosine phosphorylation in boar sperm through a cAMP-dependent pathway. Mol Reprod Dev 1998; 51:304–314[CrossRef][Medline]
  32. Osheroff JE, Visconti PE, Valenzuela JP, Travis AJ, Alvarez J, Kopf GS. Regulation of human sperm capacitation by a cholesterol efflux-stimulated signal transduction pathway leading to protein kinaseA-mediated up-regulation of protein tyrosine phosphorylation. Mol Hum Reprod 1999; 5:1017–1026[Abstract/Free Full Text]
  33. Brewis IA, Clayton R, Browes CE, Martin M, Barrat CL, Homby DP, Moore HD. Tyrosine phosphorylation of a 95 kDa protein and induction of the acrosome reaction in human spermatozoa by recombinant human zona pellucida glycoprotein 3. Mol Hum Reprod 1998; 4:1136–1144[Abstract/Free Full Text]
  34. Merlet F, Weinstein LS, Goldsmith PK, Rarick T, Hall JL, Bisson JP, de Mazancourt P. Identification and localization of G protein subunits in human spermatozoa. Mol Hum Reprod 1999; 5:38–45[Abstract/Free Full Text]
  35. Hildebrandt JD, Codina J, Tash JS, Kirchick HJ, Lipschultz L, Sekura RD, Birnbaumer L. The membrane-bound spermatozoal adenylyl cyclase system does not share coupling characteristics with somatic cell adenylyl cyclases. Endocrinology 1985; 116:1357–1366[Abstract]
  36. Ward CR, Kopf GS. Molecular events mediating sperm activation. Dev Biol 1993; 158:9–34[CrossRef][Medline]
  37. Leclerc P, Kopf GS. Mouse sperm adenylyl cyclase: general properties and regulation by the zona pellucida. Biol Reprod 1995; 52:1227–1233[Abstract]
  38. Parrish JJ, Susko-Parrish J, Uguz C, First NL. Differences in the role of cyclic adenosine 3',5'-monophosphate during capacitation of bovine sperm by heparin or oviduct fluid. Biol Reprod 1994; 51:1099–1108[Abstract]
  39. Fredholm BB, Abbracchio MP, Burnstock G, Daly JW, Harden TK, Jacobson KA, Leff P, Williams M. Nomenclature and classification of purinoceptors. Pharmacol Rev 1994; 46:143–156[Medline]
  40. Dickenson JM, Hill SJ. Involvement of G-protein ß{gamma} subunits in coupling the adenosine A1 receptor to phospholipase C in transfected CHO cells. Eur J Pharmacol 1998; 355:85–93[CrossRef][Medline]
  41. Peakman MC, Hill SJ. Adenosine A1 receptor-mediated changes in basal and histamine-stimulated levels of intracellular calcium in primary rat astrocytes. Br J Pharmacol 1995; 115:801–810[Medline]
  42. Walker EM, Bispham JR, Hill SJ. Nonselective effects of the putative phospholipase C inhibitor, U73122, on adenosine A1 receptor-mediated signal transduction events in Chinese hamster ovary cells. Biochem Pharmacol 1998; 56:1455–1462[CrossRef][Medline]
  43. Tomura H, Itoh H, Sho K, Sato K, Nagao M, Ui M, Kondo Y, Okajima F. ß{gamma} subunits of pertussis toxins—sensitive G protein mediate A1 adenosine receptor agonist-induced activation of phospholipase C in collaboration with thyrotropin. A novel stimulatory mechanism through the cross-talk of two types of receptors. J Biol Chem 1997; 272:23130–23137[Abstract/Free Full Text]
  44. Kuroda Y, Kaneko S, Yoshimura Y, Nozawa S, Mikoshiba K. Are there inositol 1,4,5 trisphosphate (IP3) receptors in human sperm? Life Sci 1999; 65:135–143[CrossRef][Medline]
  45. Baldi E, Casano R, Falsetti C, Krausz C, Maggi M, Forti G. Intracellular calcium accumulation and responsiveness to progesterone in capacitating human spermatozoa. J Androl 1991; 12:323–330[Abstract/Free Full Text]
  46. DasGupta S, Mills CL, Fraser LR. Ca2+-related changes in the capacitation state of human spermatozoa assessed by a chlortetracycline fluorescence assay. J Reprod Fertil 1993; 99:135–143[Abstract]
  47. Lewis JM, Woolkkalis MJ, Gerton GL, Smith RM, Jarrett L, Manning DR. Subcellular distribution of the {alpha} subunit(s) of Gi: visualization by immunofluorescent and immunogold labeling. Cell Regul 1991; 2:1097–1113[Medline]
  48. Ward CR, Storey BT, Kopf GS. Selective activation of Gi1 and Gi2 in mouse sperm by the zona pellucida, the egg's extracellular matrix. J Biol Chem 1994; 269:13254–13258[Abstract/Free Full Text]
  49. Sallese M, Mariggio S, Collodel G, Moretti E, Piomboni P, Baccetti B, De Blasi A. G-protein-coupled receptor kinase GRK4. Molecular analysis of the four isoforms and ultrastructural localization in spermatozoa and germinal cells. J Biol Chem 1997; 272:10188–10195[Abstract/Free Full Text]
  50. Camps M, Carozzi A, Schnabel P, Scheer A, Parker PJ, Gierschik P. Isozyme-selective stimulation of phospholipase C ß2 by G protein ß{gamma} subunits. Nature 1992; 360:684–686[CrossRef][Medline]
  51. Katz A, Wu D, Simon MI. Subunits ß{gamma} of heterotrimeric G protein activate ß2 isoform of phospholipase C. Nature 1992; 360:686–689[CrossRef][Medline]
  52. Dorn GW, Oswald KJ, McCluskey TS, Kuhel DG, Liggett SB. {alpha}2A-Adrenergic receptor stimulated calcium release is transduced by Gi-associated Gß{gamma}-mediated activation of phospholipase C. Biochemistry 1997; 36:6415–6423[CrossRef][Medline]
  53. Herrlich A, Kuhn B, Grosser R, Schmid A, Schultz G, Gudermann T. Involvement of Gs and Gi proteins in dual coupling of the luteinizing hormone receptor to adenylyl cyclase and phospholipase C. J Biol Chem 1996; 271:16764–16772[Abstract/Free Full Text]
  54. Huguet E, Esponda P. Generation of genetically modified mice by spermatozoa transfection in vivo: preliminary results. Mol Reprod Dev 2000; 56:243–247
  55. Hildebrandt JP, Plant TD, Meves H. The effects of bradykinin on K+ currents in NG108-15 cells treated with U73122, a phopholipase C inhibitor, or neomycin. Br J Pharmacol 1997; 120:841–850[CrossRef][Medline]
  56. Mogami H, Mills CL, Gallacher DV. Phospholipase C inhibitor, U73122, relases intracellular Ca2+, potentiates Ins(1,4,5)P3 mediated Ca2+ release and directly activates ion channels in mouse pancreatic acinar cells. Biochem J 1997; 324:645–651
  57. Macrez-Lepretre N, Morell JL, Mionneau J. Effects of phopholipase C inhibitors on Ca2+ release from intracellular stores evoked by {alpha}1A- and {alpha}2A-adrenoceptors in rat portal vein myocytes. Biochem Biophys Res Commun 1996; 218:30–34[CrossRef][Medline]



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