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BOR - Papers in Press, published online ahead of print October 15, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.020487
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BIOLOGY OF REPRODUCTION 70, 518–522 (2004)
DOI: 10.1095/biolreprod.103.020487
© 2004 by the Society for the Study of Reproduction, Inc.

Gamete Biology

Role of Hydrogen Peroxide in Sperm Capacitation and Acrosome Reaction1

J. Rivlin, J. Mendel, S. Rubinstein, N. Etkovitz, and H. Breitbart2

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The generation of reactive oxygen species (ROS) has been implicated in the regulation of sperm capacitation and acrosome reaction; however, the mechanisms underlying this regulation remain unclear. To examine the cellular processes involved, we studied the effect of different concentrations of hydrogen peroxide (H2O2) on protein tyrosine phosphorylation under various conditions. Treatment of spermatozoa with H2O2 in medium without heparin caused a time- and dose-dependent increase in protein tyrosine phosphorylation of at least six proteins in which maximal effect was seen after 2 h of incubation with 50 µM H2O2. At much higher concentrations of H2O2 (0.5 mM), there is significant reduction in the phosphorylation level, and no protein tyrosine phosphorylation is observed at 5 mM H2O2 after 4 h of incubation. Exogenous NADPH enhanced protein tyrosine phosphorylation similarly to H2O2. These two agents, but not heparin, induced Ca2+-dependent tyrosine phosphorylation of an 80-kDa protein. Treatment with H2O2 (50 µM) caused approximately a twofold increase in cAMP, which is comparable to the effect of bicarbonate, a known activator of soluble adenylyl cyclase in sperm. This report suggests that relatively low concentrations of H2O2 are beneficial for sperm capacitation, but that too high a concentration inhibits this process. We also conclude that H2O2 activates adenylyl cyclase to produce cAMP, leading to protein kinase A-dependent protein tyrosine phosphorylation.

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


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mammalian sperm undergo the acrosome reaction before fertilization in order to penetrate the oocyte zona pellucida (ZP). This process will occur following binding to the ZP only if the spermatozoa have previously undergone a poorly-defined maturation process known as capacitation [1]. The binding of the capacitated spermatozoa to the oocyte ZP activates a low-voltage Ca2+ channel [2] and a store-operated Ca2+ channel, which causes a sustained elevation of intracellular Ca2+, leading to the acrosome reaction [3]. The priming of spermatozoa to such calcium signals during capacitation involves many changes, including cholesterol efflux from the plasma membrane [4, 5] and increases in intracellular free Ca2+ [6–8], cAMP [9–11] pH [12], protein tyrosine phosphorylation [13, 14], and actin polymerization [15]. It has been suggested that reactive oxygen species (ROS) such as hydrogen peroxide and superoxide anion are involved in the regulation of human sperm capacitation and protein tyrosine phosphorylation [16–18]. We show that hydrogen peroxide is also involved in actin polymerization in bovine sperm [15], and in elevation of intracellular Ca2+ levels and fertilizing potential of mouse sperm [19]. Other lines of evidence have implicated cAMP and protein kinase A (PKA) as regulators of protein tyrosine phosphorylation [13, 14, 20].

However, the relation between the production of ROS, the elevation of cAMP and the protein tyrosine phosphorylation is not clear. Some authors suggest that ROS generation must lie upstream from cAMP in the reaction cascade [11, 17, 21], whereas others believe that ROS is located downstream from cAMP in the reaction sequence [22]. In the present study, the temporal sequence of signaling events was investigated. It is known that the soluble adenylyl cyclase (AC) present in sperm cells is activated by HCO3- [23, 24]. We suggest that bovine sperm AC can be activated by 50 µM hydrogen peroxide in the absence of added HCO3-. The process of cAMP/PKA-dependent protein tyrosine phosphorylation can be induced either by HCO3- or by H2O2. Thus, HCO3- and H2O2 can substitute for each other or work together under physiological conditions in which the concentration of one of them is reduced in the female reproductive tract.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials

BSA (Fraction V), FITC-Phalloidin, monoclonal antiphosphotyrosine (clone PT-66), and all chemicals were purchased from Sigma (St. Louis, MO). Horseradish peroxidase (HRP)-linked goat anti-mouse IgG was from Bio-Rad Laboratories (Hercules, CA).

Sperm Preparation

Ejaculated bull spermatozoa were obtained using an artificial vagina. The semen was washed three times by centrifugation (780 x g, 10 min at 25°C) in NKM buffer containing 110 mM NaCl, 5 mM KCl and 10 mM N-morpholinopropanesulfonic acid (pH 7.4). The washed cells were suspended in NKM buffer to a concentration of 109 cells/ml and were maintained at room temperature until use. Investigations were conducted in accordance with the Guide for the Care and Use of Agricultural Animals.

Capacitation and Acrosome Reaction

In vitro capacitation of bull sperm was induced by the method of Parrish et al. [25]. Briefly, sperm pellets were resuspended to a final concentration of 108 cells/ml in glucose-free Tyrode medium (TALP) containing 100 mM NaCl, 3.1 mM KCl, 1.5 mM MgCl2, 25 mM NaHCO3, 0.29 mM KH2PO4, 21.6 mM sodium lactate, 0.1 mM sodium pyruvate, 2 mM CaCl2, 20 mM Hepes (pH 7.4), 30 µg/ml BSA, 10 U/ml penicillin and 20 µg/ml heparin. The cells were incubated in this capacitation medium for 4 h at 39°C with 5% CO2.

Whole Cell Lysates

Washed sperm cells (109 cells) were solubilized in SDS-lysis buffer consisting of 125 mM Tris (pH 7.5), 4% SDS, 1 mM sodium orthovanadate, 1 mM benzamidine, and 1 mM PMSF added just before use. Cells were lysed for 10 min at room temperature and centrifuged at 12 930 x g for 5 min at 4°C. The supernatant was supplemented with 0.05% bromophenol blue, 5% glycerol, and 2% ß-mercaptoethanol and boiled for 5 min.

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 mAmp; 1 h), using a buffer composed of 25 mM Tris (pH 8.2), 192 mM glycine, and 20% methanol. For Western blotting, 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 membranes were incubated overnight at 4°C with the antibody diluted 1:10 000. Next, the membranes were washed three times with TBST and incubated for 1 h at room temperature with specific HRP-linked secondary antibody diluted 1:10 000 in TBST. The membranes were washed three times with TBST and visualized by enhanced chemiluminescence (Amersham, Little Chalfont, UK).

Measurement of Intracellular cAMP

Spermatozoa (108 cells/ml) were incubated for 1.5 h in capacitation medium without NaHCO3, CaCl2, and heparin. The cAMP-dependent phosphodiesterase (PDE) inhibitor 3-iso-butyl-methylxantine (IBMX) (100 µM) was added during the last 15 min of the incubation period. Then the inducers were added according to the assay for 15 min. The cells were diluted in NKM buffer and centrifuged at 500 x g for 10 min. The cell's pellets were resuspended and the amount of cAMP produced in the cells was determined after lysis using a nonradioactive enzyme immunoassay kit (RPN 255; Amersham) according to the manufacturer's instructions.

Statistical Analysis

Statistical analyses were performed using the ANOVA test and t-test with multiple comparisons. Statistical significance is indicated in the figure legends.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of Exogenous H2O2 on Protein Tyrosine Phosphorylation

It was previously shown that bovine sperm capacitation is correlated with the tyrosine phosphorylation of various proteins [14]. A direct association between ROS generation and tyrosine phosphorylation has been demonstrated in human spermatozoa [20, 26]. Here, the effect of increased concentrations of H2O2 on protein tyrosine phosphorylation was determined in bovine sperm by immunoblotting (Fig. 1). In these experiments, heparin, which is usually present in bovine sperm capacitation medium, was replaced by increased concentrations of H2O2, and cells were incubated for the regular capacitation time (4 h). Without adding H2O2, two proteins of about 120 and 145 kDa are phosphorylated on tyrosine residue, and their phosphorylation level is enhanced by increasing H2O2 concentration (Fig. 1). At 50, 100 and 200 µM H2O2, proteins of different molecular weights are highly phosphorylated, whereas at 500 µM there is a decrease in protein phosphorylation rate, and dephosphorylation occurs at 5 mM H2O2. According to this phosphorylation pattern, 50 µM H2O2 was used further in our experiments.



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  		FIG. 1. Effect of increased concentrations of H2O2 on protein tyrosine phosphorylation. Bovine spermatozoa were incubated for 4 h in heparin-free TALP medium with increased concentrations of H2O2. Proteins were extracted from the cells and protein tyrosine phosphorylation was determined by Western blot analysis. The blot shown is representative of two experiments. Numbers to the left of blot indicate size in kDa

Time-Dependent Increase in Protein Tyrosine Phosphorylation by H2O2

In human spermatozoa, the production of ROS starts at the very beginning of the capacitation process and reaches its maximum after 15–25 min [27]. In bovine sperm, capacitation takes 4 h [28], and during this time maximal tyrosine phosphorylation was reached (Fig. 2). When heparin was replaced by 50 µM H2O2, the phosphorylation rate increased more rapidly, reaching maximum after 2 h of incubation. It was interesting to find that a protein of about 80 kDa is tyrosine phosphorylated after 4 h in H2O2 treated cells, but not under regular capacitation conditions (with heparin). Protein phosphorylation was completely blocked by incubating the cells with 10 µM herbimycin, a known tyrosine kinase-specific inhibitor (data not shown).



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  		FIG. 2. Time-dependent protein tyrosine phosphorylation in TALP medium containing heparin or H2O2. Bovine sperm were incubated for 4 h in TALP medium containing 20 µg/ml heparin or 50 µM H2O2. Proteins were extracted from the cells and tyrosine phosphorylation was determined by Western blot analysis. The blot shown is representative of three experiments. Numbers to the left of blot indicate size in kDa. Arrow indicates 80 kDa protein. C, control (zero time)

Involvement of Endogenous H2O2 in Protein Tyrosine Phosphorylation

The stimulation of protein tyrosine phosphorylation during human sperm capacitation depends on redox-regulated events enhanced by the cellular generation of ROS [16, 17, 26]. The addition of catalase, which decomposes H2O2, to human sperm revealed a marked decline in the level of phosphotyrosine expression induced under capacitation conditions or by exogenous NADPH [21]. This suggests that H2O2 is generated during capacitation of human spermatozoa.

In bovine sperm we found that exogenous NADPH enhances protein tyrosine phosphorylation similarly to H2O2 (Fig. 3). It is known that NADPH activates NADPH oxidase to generate superoxide anion, which later dismutates to H2O2 by superoxide dismutase [21]. Thus, stimulation of tyrosine phosphorylation by NADPH indicates that endogenous H2O2 might be produced under these conditions. Further, NADPH, like H2O2, but not regular capacitation conditions induced specifically the tyrosine phosphorylation of an 80-kDa protein (Fig. 3), which also supports endogenous H2O2 production.



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  		FIG. 3. Effect of NADPH on protein tyrosine phosphorylation. Bovine sperm were incubated for 4 h in TALP (Hep.), in heparin-free TALP (Con.), or in heparin-free TALP containing 50 µM H2O2 (H2O2) or 10 mM NADPH (NADPH). Proteins were extracted and tyrosine phosphorylation was determined by Western blot analysis. The blot shown is representative of three experiments. Numbers to the left of blot indicate size in kDa

Mechanism of Action of H2O2

We mentioned above that H2O2 and NADPH induced tyrosine phosphorylation of an 80-kDa protein (Fig. 3). When intracellular Ca2+ was chelated by treating the cells with 1,2-bis-(0-aminophenoxy)-ethane-N,N,N1,N1-tetraacetic acid tetra-acetoxymethyl)-ester (BAPTA-AM), the phosphorylation of this protein but not of others was prevented (Fig. 4, lanes 3 and 5). This indicates that H2O2 also specifically induced protein phosphorylation in a Ca2+ dependent manner. In addition, H2O2-induced tyrosine phosphorylation is completely inhibited by the PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]5-isoquinolinesulfonamide-dihydrochloride (H89) (compare lanes 4 and 5 to lanes 8 and 9 in Fig. 4). These results suggest that H2O2 might directly activate sperm AC or inhibit tyrosine phosphatase [29]. Activation of AC was determined by following intracellular production of cAMP. It was shown that exogenous H2O2, NADPH, or HCO3- enhanced intracellular cAMP production. The values for cAMP (pmol/108 cells) are as follows: control, 2.5 ± 0.6; 25 mM NaHCO3, 6.2 ± 1.0; 50 µM H2O2, 5.3 ± 0.8; and 10 mM NADPH, 11.25 ± 1.2. This stimulation occurred in the presence of cAMP-dependent PDE inhibitor IBMX, indicating that H2O2 activated AC rather than inhibiting PDE activities. To determine the possible involvement of H2O2 in tyrosine phosphatase inhibition, we compared its effect on tyrosine phosphorylation to the effect of sodium vanadate, a known tyrosine phosphatase inhibitor. It was shown that tyrosine phosphorylation induced by sodium vanadate is not affected by H89 (Fig. 5, lanes 4 and 6) conditions in which H2O2-dependent phosphorylation is completely blocked (Fig. 5, lanes 3 and 5). This indicates that H2O2 and vanadate enhance tyrosine phosphorylation in different ways.



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  		FIG. 4. Role of intracellular Ca2+ and PKA in protein tyrosine phosphorylation. Bovine sperm were incubated for 4 h in TALP medium containing the specified compounds in the presence or absence of 50 µM H89. Chelation of intracellular Ca2+ was performed by incubating the cells with 10 µM BAPTA-AM for 1 h and washing before the incubation in Ca2+-free TALP medium. Proteins were extracted and tyrosine phosphorylation was determined. The blot shown is representative of three experiments. The concentrations used are: 20 µg/ml heparin, 25 mM NaHCO3 2 mM CaCl2, 50 µM H89. Numbers to the left of blot indicate size in kDa



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  		FIG. 5. Effect of vanadate on protein tyrosine phosphorylation. Incubation conditions as in Figure 4. The concentration of sodium vanadate is 0.1 mM. Numbers to the left of blot indicate size in kDa


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
At present, little is known regarding the signal transduction involved in sperm capacitation [30]. Several lines of evidence suggest that ROS-like superoxide anion and hydrogen peroxide can induce human sperm capacitation [17, 18, 21, 26, 28]. A key element of this process is the spontaneous increase in tyrosine phosphorylation of several proteins as well as actin polymerization observed during capacitation. These findings, which have been shown in several species, including mouse [13, 15], human [15, 20], boar [31], and bovine [14, 15] spermatozoa, present unusual signal transduction events involving the stimulation of tyrosine phosphorylation and actin polymerization via a cAMP/PKA mediated pathway. In a recent study, we showed that factors known to stimulate PKA-dependent protein tyrosine phosphorylation in sperm were able to enhance actin polymerization, whereas inhibition of tyrosine kinase prevented F-actin formation [15]. Thus, we suggest that PKA-dependent protein tyrosine phosphorylation occurred before polymerization in the cascade leading to sperm capacitation. The role of intracellular Ca2+ in these processes is not yet clear. In the present study, we suggest that there is significant cross-talk between this unique signal transduction cascade and ROS in bovine spermatozoa. The pattern of protein tyrosine phosphorylation observed in bovine sperm under capacitation conditions could be mimicked by replacing heparin or HCO3- in the incubation medium with hydrogen peroxide (Figs. 1 and 4). The optimal H2O2 concentration for inducing protein phosphorylation is 50–200 µM;, whereas at higher concentrations (0.5 and 5 mM) inhibition was observed. In a recent paper, we showed that H2O2 can substitute for heparin in inducing F-actin formation during bovine sperm capacitation [15].

These observations may suggest that H2O2 is generated by spermatozoa during capacitation. However, all our efforts to measure H2O2 production during bovine sperm capacitation failed. We did find H2O2 production (not shown) as well as stimulation of protein tyrosine phosphorylation (Fig. 3) by treating the cells with exogenous NADPH; these effects, as well as the effect of exogenous H2O2, were completely inhibited by adding catalase to the cell suspension (not shown). These data suggest that bovine sperm are potentially able to generate H2O2 by activating NADPH oxidase. Although H2O2 could substitute for heparin in stimulating protein tyrosine phosphorylation and actin polymerization, it cannot cause sperm capacitation, as revealed by its inability to induce the acrosome reaction [15]. Thus, H2O2 could induce protein tyrosine phosphorylation as well as actin polymerization, two processes which are essential but not sufficient for capacitation. In human spermatozoa, capacitation, sperm-oocyte fusion [26, 28], and protein tyrosine phosphorylation [21] are inhibited by the addition of catalase and stimulated by the addition of H2O2. Because there is no direct evidence yet for a genuine production of H2O2 by capacitating human spermatozoa, it was hypothesized that H2O2 may originate from the dismutation of superoxide anion generated by sperm [27]. In mouse sperm, we showed that light induced H2O2 production, leading to an enhanced rate of in vitro fertilization, and that these effects are completely blocked by catalase treatment [19]. It was also shown that protein tyrosine phosphorylation in mouse sperm is inhibited by addition of catalase to the cells [32]. We also found that light-induced H2O2 production in sperm is completely blocked by the electron transport inhibitor antimycin A (not shown), indicating the mitochondria as a source of this H2O2 generation. These data further support our notion regarding the endogenous generation of H2O2 in spermatozoa.

The mechanism by which H2O2 stimulates protein tyrosine phosphorylation could involve the inhibition of tyrosine phosphatase activity [29], the activation of tyrosine kinase, or both. The similar patterns of protein phosphorylation obtained by H2O2 and the known tyrosine phosphatase inhibitor vanadate (Fig. 5) suggest that H2O2 may act as a tyrosine phosphatase inhibitor. When both H2O2 and vanadate are present in the incubation medium, there is a dramatic enhancement in tyrosine phosphorylation [15] due to the generation of pervanadate, which is a very potent inhibitor of tyrosine phosphatase [33]. However, we found that protein tyrosine phosphorylation stimulated by vanadate is not inhibited by the PKA inhibitor H-89 conditions in which H2O2- or HCO3-- dependent phosphorylation are completely blocked (Figs. 4 and 5). These data suggest that HCO3- and H2O2 stimulate tyrosine phosphorylation by activating PKA-dependent tyrosine kinase rather than by inhibiting tyrosine phosphatase. This notion is supported further by showing that H2O2 and NADPH stimulate cAMP production in cells incubated in HCO3--deficient medium, indicating activation of AC. These data are supported by other data that show enhanced effects of exogenous NADPH on ROS and cAMP production in human and rat spermatozoa [21, 34]. It was also shown in other cell types that H2O2 enhances AC activity [35, 36], indicating that cAMP production occurs under oxidant stress, an observation which supports our data. The fact is that exogenous NADPH-induced cAMP production, and tyrosine phosphorylation, which is inhibited by catalase, represent the sperm's ability to produce H2O2 via activation of NADPH oxidase. The similarity between H2O2 and NADPH activities was also demonstrated by showing that each of them induced tyrosine phosphorylation of an 80-kDa protein that is not phosphorylated during regular capacitation (Fig. 3). Because the phosphorylation of the 80-kDa protein depends on Ca2+ and PKA we suggest that H2O2 activates PKA and Ca2+ dependent tyrosine kinase in addition to its direct activation of sperm AC.

It is not clear whether the phosphorylation of the 80-kDa protein is important for sperm capacitation, because this protein is not phosphorylated under regular capacitation conditions. We found that this phosphoprotein disappeared after inducing the acrosome reaction by Ca2+ ionophore (not shown), indicating its possible role in sperm capacitation and/or acrosome reaction. Because tyrosine phosphorylation of the 80-kDa protein is Ca2+-dependent (Fig. 4) and is seen at relatively high concentrations of H2O2 (Fig. 1) and after a relatively long time of incubation (Fig. 2) in comparison to other cases of H2O2-dependent protein phosphorylation, it is possible that this phosphorylation occurs under stress conditions only.

The fact that H2O2 can substitute for HCO3- in stimulating tyrosine phosphorylation (Fig. 4) suggests that HCO3- and H2O2 are both important for achieving higher protein tyrosine phosphorylation leading to sperm capacitation. These two components activate AC to trigger cAMP/PKA activity leading to protein tyrosine phosphorylation. They can work in concert or partially substitute for each other under conditions in which one of them is present in a limited amount in the female reproductive tract.

In conclusion, it appears that ROS has both beneficial and detrimental effects on spermatozoa, and that the accurate balance of the amount of ROS produced and scavenged at any moment will determine whether a given sperm function will be promoted or jeopardized. We suggest that relatively low concentrations of H2O2 in the µM range are beneficial for sperm capacitation, as revealed by its stimulating effect on protein tyrosine phosphorylation; however, this effect is prevented by high H2O2 concentrations in the mM range.


   FOOTNOTES
 
1 This research was supported by the Ihel Research Fund. Back

2 Correspondence: Haim Breitbart, Faculty of Life Sciences, Bar-Ilan University, Ramat Gan 52900, Israel. Fax: 972-3-5344766; breith{at}mail.biu.ac.il Back

Received: 22 June 2003.

First decision: 18 July 2003.

Accepted: 10 October 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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H.J. Chi, J.H. Kim, C.S. Ryu, J.Y. Lee, J.S. Park, D.Y. Chung, S.Y. Choi, M.H. Kim, E.K. Chun, and S.I. Roh
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Characterization of NADPH oxidase 5 in equine testis and spermatozoa
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A. M. Benoit, H. A. LaVoie, G. L. McCoy, and C. A. Blake
Expression of Sperm Protein 22 (SP22) in the Rat Ovary During Different Reproductive States
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Redox Regulation of Sperm Surface Thiols Modulates Adhesion to the Fallopian Tube Epithelium
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M. A. Baker, L. Hetherington, and R. J. Aitken
Identification of SRC as a key PKA-stimulated tyrosine kinase involved in the capacitation-associated hyperactivation of murine spermatozoa
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Regulation of sperm function by reactive oxygen species
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