Relationship between bicarbonate and cyclic nucleotide in the promoting effects on head-to-head agglutination in boar spermatozoa

Hiroshi Harayama¹, Seishiro Kato²

¹Department of Life Science, Graduate School of Science and Technology, ²Department of Animal Science, Faculty of Agriculture, Kobe University, Kobe 657-8501, Japan

Asian J Androl  2002 Jun; 4:  83-86             

Keywords: cAMP; capacitation; phosphodiesterase; calmodulin

Abstract

Aim: To clarify the relationship between bicarbonate and cAMP in the promoting effects on the sperm agglutination. Methods: Spermatozoa were collected from mature boars, washed and resuspended in a modified Krebs-Ringer HEPES lacking calcium chloride (mKRH). The sperm suspensions were incubated in a water bath (38.5 ) for 60 min and then the percentage of head-to-head agglutinated spermatozoa was determined. Results: Supplementation of the mKRH with sodium bicarbonate (5-10 mM) significantly raised the percentage of head-to-head agglutinated spermatozoa in the samples. The addition of selective inhibitors for calcium/calmodulin-dependent phosphodiesterases (type 1: 8-methoxymethyl-IBMX and vinpocetine, 25-50 mM) or for cAMP-specific phosphodiesterases (type 4: Ro20-1724 and rolipram, 25-50 mM) enhanced the effect of bicarbonate on sperm agglutination as highly as did the addition of non-selective inhibitors for phosphodiesterases (IBMX and papaverine, 25-50 mM). A calmodulin antagonist (W-7, 2 mM), that potentially blocks the stimulator of the calcium/calmodulin-dependent phosphodiesterases, significantly enhanced the effect of bicarbonate on sperm agglutination. Moreover, a phosphodiesterase-resistant cAMP analogue (cBiMPS, 0.1 mM) markedly induced agglutination in more spermatozoa (76%) after the incubation without bicarbonate and phosphodiesterase inhibitors than did a less potent cAMP analogue (dibutyryl cAMP, 1 mM) (21%), while three kinds of cGMP analogues (0.1-1 mM) had no effect on sperm agglutination. In addition, a cAMP antagonist (Rp-cAMPS, 1 mM) significantly reduced the sperm agglutination resulting from the actions of bicarbonate and IBMX. On the other hand, the effect of bicarbonate was abolished by a change of incubation temperature from 38.5 to 25. Conclusion: These findings demonstrate that the bicarbonate-induced agglutination of boar spermatozoa is controlled via the cAMP-mediated, temperature-dependent signaling cascade. This cascade is suppressed by the action of the phosphodiesterase (at least types 1 and 4).

1 Introduction

Boar spermatozoa develop potential fertilizing ability during their transit through the caput and corpus epididymides [1, 2]. However, this ability is temporarily suppressed by the interaction with specific epididymal fluid components while the spermatozoa are stored in the cauda epididymidis, perhaps to prevent premature acrosome reaction before ejaculation [3, 4]. Likewise, boar spermatozoa exhibit similar changes of the potential head-to-head agglutinability during epididymal transit; this agglutinability gradually increases in spermatozoa passing between the caput and corpus epididymides and is suppressed in spermatozoa stored in the cauda epididymidis [5]. This suppression of the sperm potential agglutinability is regulated by the interaction with an epididymal fluid protein known as anti-agglutinin. This agglutination-inhibiting protein is secreted from the epithelia to the luminal fluid in the corpus epididymidis and is accumulated in the cauda epididymidis. Coincidentally, an increasing amount of the anti-agglutinin is bound onto the sperm acrosomes [6-10]. However, neither the fertilizing ability nor the agglutinability seems to be expressed in the spermatozoa stored in the epididymis [5, 6]. After ejaculation, spermatozoa gradually become capable of expressing their fertilizing ability during their stay in the specific environment provided by the female reproductive tract. This event is termed capacitation. Various components on the surface and in the intracellular space of the spermatozoa are altered during capacitation in order to activate molecules required for the expression of the fertilizing ability. The environment within the female reproductive tract can be simulated by incubation in an appropriate medium (a capacitation medium) in vitro [2]. It has also been observed that many spermatozoa become agglutinated at the acrosomes during the incubation [11,12]. Moreover, this agglutination is promoted by capacitation-supporting factors including extracellular calcium [13] and serum albumin [14]. These findings imply the biological significance of head-to-head agglutination in the process of expressing fertilizing ability. On the other hand, the agglutination is likely to cause sedimentation of the spermatozoa. Thus, when spermatozoa become agglutinated in the medium containing oocytes for in vitro fertilization, the sperm concentration is enhanced in the vicinity of the oocytes located at the bottom of the culture dish. However, the agglutinated sperm clusters appear to block access of the remaining free spermatozoa to the oocytes. Moreover, the degree of sperm agglutination is greatly varied among boars [15]. It is possible that the varying degrees of sperm agglutination cause variation in the results of in vitro fertilization in pigs. It is, therefore, significant to clarify the mechanism of head-to-head agglutination in order to improve the in vitro fertilization techniques.

A low level of bicarbonate in the epididymal fluid is maintained by the uptake of epithelial anion transporters, including sodium/bicarbonate cotransporters [16, 17] and anion exchangers [18]. Thus, epididymal spermatozoa are likely to be isolated from the actions of a high level of extracellular bicarbonate [19, 20]. At ejaculation, however, spermatozoa are exposed to a large amount of bicarbonate that originates from the accessory glands [20], and consequently sperm movement is initiated [21]. In the female reproductive tract, a relatively higher level of bicarbonate stimulates expression of the fertilizing ability, including capacitation, hyperactivation and acrosome reaction [2]. Moreover, previous reports have revealed that the incubation with bicarbonate induces various alterations in boar spermatozoa, including an increase in the intracellular calcium [22], modification of the lectin-binding affinity [23], raise in the intracellular concentration of cyclic adenosine 3',5'-monophosphate (cAMP) [15], enhancement of the ability to bind to zona pellucida [24] and disorder in the plasma membrane lipid architecture [25, 26]. In our preliminary experiment [13], we observed that replacement of sodium bicarbonate with HEPES in a capacitation medium significantly reduced the occurrence of head-to-head agglutination in boar sper-matozoa. Moreover, the addition of a cell-permeable cAMP analogue (dibutyryl cyclic adenosine 3',5'-monophosphate sodium salt, dbcAMP) attenuates the reduction of sperm agglutination resulting from the lack of calcium in a capacitation medium containing bicarbonate [14]. These suggest that bicarbonate and cAMP might be pivotal factors in regulating the occurrence of sperm head-to-head agglutination. However, there are no available data showing the relationship between bicarbonate and cAMP in the promoting effects on the sperm agglutination. The aim of the present study is to characterize the bicarbonate-mediated signaling cascade leading to the sperm agglutination.

2 Materials and methods

2.1 Collection and washing of spermatozoa

Sperm-rich fractions from ejaculates were collected from 11 Large White boars, 1 Duroc boar and 1 Meishan boar by a manual method. A portion (2 mL) of each sperm-rich fraction was loaded on a discontinuous gradient of 2 mL 90 % and 5 mL 60 % of isotonic Percoll (Amersham Pharmacia Biotech AB, Uppsala, Sweden) that was prepared with a phosphate-buffered saline (PBS: 136.9 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄^.2H₂O and 1.5 mM KH₂PO₄) in a 15-mL plastic centrifuge tube. In each experiment, ten tubes were prepared and centrifuged at 700 g for 10 min. The spermatozoa were recovered and then washed twice in PBS containing 0.1 % polyvinyl alcohol (Molecular Wt. 30,000-70,000, Sigma, St. Louis, MO, USA) by centrifugation at 700 g for 5 min.

The assay of sperm agglutination was performed as described previously with minor modifications [6]. Briefly, the washed spermatozoa were resuspended in a modified Krebs-Ringer HEPES lacking calcium chloride (mKRH, pH 7.4, Table 1) to give a final sperm concentration of 2.510⁷ cells/mL. They were then incubated in a 38.5 water bath. After the incubation, an aliquot was gently smeared on a slide, dried up, and stained in a phosphate-buffered solution of Giemsa (Merck, Darm-stadt, Germany). More than 300 spermatozoa were counted at random by light microscopy (400 to determine the percentage of head-to-head agglutinated cells.

Table 1. Composition of modified Krebs-Ringer HEPES lacking calcium chloride (mKRH).

Water-soluble reagents, including sodium bicarbonate (Nacalai Tesque, Kyoto, Japan), 3-isobutyl-1-methyl-xanthine (IBMX, Sigma), dbcAMP (Sigma), Rp-adenosine 3',5'-cyclic monophosphorothioate triethylamine salt (Rp-cAMPS, Sigma), dibutyryl cyclic guanosine 3',5'-monophosphate sodium salt (dbcGMP, Sigma), 8-bromogua-nosine 3',5'-cyclic monophosphate sodium salt (8-Br-cGMP, Sigma), 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate sodium salt (8-pCPT-cGMP, Sigma), and N-(6-aminoheyxl)-5-chloro-1-naphthalene-sulfonamide hydrochloride (W-7, Seikagaku Corporation, Tokyo, Japan), were dissolved in the medium and added to the sperm suspensions. Water-insoluble reagents, including papaverine (Sigma), 8-methoxymethyl-IBMX (Sigma), 3a, 16a-eburnamenine-14-carboxylic acid ethyl ester (vinpocetine, Sigma), 4-(3-[cyclopentyloxy]-4-methoxyphenyl)-2-pyrrolidinone (rolipram, Biomol Research Laboratories, Inc., Plymouth Meeting, PA, USA), 4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one (Ro20-1724, Sigma), 1,4-dihydro-5-(2-propoxyphenyl)-7H-1,2,3-triazolo-(4,5-d)pyrimidine-7-one (zaprinast, Sigma), and Sp-5,6-dichloro-1-b-D-ribofuranosyl-benzimidazole-3',5'-monophosphorothioate (cBiMPS, Biomol), were dissolved in dimethyl sulfoxide (DMSO, Nacalai Tesque) and added to the sperm suspensions. In each experiment using water-insoluble reagents, DMSO was added to equalize the final DMSO concentrations (2.5 % for 250 and 500 mM papaverine, or 0.25 % for the other water-insoluble reagents, including 25 and 50 mM papaverine).

2.3 Statistical analysis

Percentages of head-to-head agglutinated spermatozoa were subjected to one-way analysis of variance (ANOVA) or paired t-tests. When F-test results were significant in ANOVA, individual means were further tested by Tukey's multiple range tests [27].

3.1 Induction of sperm agglutination by sodium bicarbonate and non-selective phosphodiesterase (PDE) inhibitor

At first, we examined the effects of supplementation with sodium bicarbonate at different concentrations on agglutination in ejaculated boar spermatozoa incubated in mKRH without PDE inhibitors. The results are presented in Table 2.  Series 1 of the experiments were conducted separately from the following experiments, while series 2 involved the accumulation of the data obtained from the control samples in the following experiments. Thus, some data in series 2 were obtained from spermatozoa that were incubated in mKRH containing DMSO (0.25 or 2.5 %, v/v), as described in Materials and me-thods. In both series of experiments, the percentages of head-to-head agglutinated spermatozoa greatly varied among experiments. However, supplementation with sodium bicarbonate (final concentrations: 5-20 mM) showed a constant tendency to enhance sperm agglutination in all experiments and significantly increased the average percentages of head-to-head agglutinated spermatozoa in a concentration-dependent manner. Next, we examined the effects of supplementation with sodium bicarbonate on agglutination in the spermatozoa incubated in mKRH containing PDE inhibitors. In the presence of non-selective PDE inhibitors, including IBMX and papaverine (25 and 50 mM), the percentages were increased more remarkably by supplementation with sodium bicarbonate (5-10 mM), as compared with the results obtained in the absence of the PDE inhibitors (Figure 1). Moreover, the addition of higher concentrations of IBMX or papaverine (250 or 500 mM) to the sperm samples gave rise to a significant increase in the percentages irrespective of the presence of sodium bicarbonate (Figure 1). These findings can be interpreted as showing that the non-selective PDE inhibitors (25 and 50 mM) support the effect of sodium bicarbonate on the induction of sperm agglutination, and that the supplementation alone with the inhibitors at the higher concentrations (250 or 500 mM) can overcome the reduction in sperm agglutination due to the lack of sodium bicarbonate.

Table 2. Effects of supplementation with sodium bicarbonate on head-to-head agglutination in ejaculated boar spermatozoa incubated in mKRH.

Ejaculated boar spermatozoa were washed and incubated in mKRH supplemented with sodium bicarbonate (0-20 mM) at 38.5 for 60 min. Series 1 of the experiments were conducted independently of the following experiments. Series 2 of the experiments were conducted as the control experiments of the following experiments.  ^a-cValues within the same line with different superscripts are different significantly, P<0.05 (Tukey's multiple range tests).

The supplementation with sodium bicarbonate was also accompanied by slight changes in the pH and osmolarity of the media. In order to confirm whether these changes have some influence on sperm agglutination, ejaculated boar spermatozoa were washed and then incubated in mKRH containing 25 mM IBMX that was alkalized within the pH ranges between 7.4 and 8.0 (Figure 2A) or that was further supplemented with 2.5-10 mM sodium chloride (Figure 2B). Neither modifications of the medium resulted in a significant rise in the percentage of head-to-head agglutinated spermatozoa.

Figure 2. Effect of pH and sodium chloride concentration of the media on head-to-head agglutination in ejaculated boar spermatozoa.  The basic medium was mKRH (pH 7.4, 94.6 mM sodium chloride, 0mM sodium bicarbonate) supplemented with 25 mM IBMX. ^aIn the panel A, pH of the medium was adjusted to 7.4 -8.0 with 1 NNaOH. ^bIn the panel B, the medium was supplemented with sodium chloride (2.5-10 mM). In the columns with asterisks (positive controls) of both panels, the basic media were supplemented with 5 mM sodium bicarbonate. The supplementation slightly raised the pH of media. Sperm samples were incubated at 38.5 for 60 min.  Values are meansSEM.  ^c,dValues within the same panels with different letters are significantly different, P<0.05 (Tukey's multiple range tests).

In order to disclose the incubation conditions under which bicarbonate will work, we examined the effects of incubation time and temperature on sperm agglutination induced by sodium bicarbonate in the presence of IBMX (25 mM). Figure 3A shows incubation time-related changes in the percentages of head-to-head agglutinated spermatozoa incubated with or without sodium bicarbonate (5 mM). In the control samples without sodium bicarbonate, the percentages gradually rose to 31% throughout the incubation period of 180 min. Supplementation with sodium bicarbonate (5 mM) accelerated and enhanced the rise during the incubation period between 5 and 30 min, after which only minor changes occurred. However, sodium bicarbonate (5 mM) was observed to have almost no enhancing effect in the sperm samples incubated at 25 (Figure 3B).

Figure 3. Effect of incubation time and temperature on head-to-head agglutination induced by sodium bicarbonate in ejaculated boar spermatozoa.  The medium was mKRH supplemented with IBMX (25 mM) and 5 mM sodium bicarbonate (panel A: filled circles, panel B: filled columns). In the control experiments, the addition of sodium bicarbonate was omitted (panel A: open circles, panel B: open columns). Ejaculated boar spermatozoa were washed and incubated in the media at 38.5 for 5-180 min (panel A) or at 38.5 or 25 for 60 min (panel B).  Values are meansSEM.  In the panel A, values within the same lines with asterisks are significantly different from values obtained in the samples before incubation (time zero), P<0.05 (Tukey's multiple range tests).  ^a,bIn the panel B, values with different letters are significantly different, P<0.05 (Tukey's multiple range tests).

In addition, light microscopic examination of the sperm preparations for the agglutination assay confirmed that most spermatozoa still possessed acrosomes after the incubation (data not shown), indicating that this sperm agglutination is not a result of the acrosome reaction.

Previous reports [28-31] have revealed that PDEs 1 and 4 are apparently present in mammalian testicular germ cells and spermatozoa. PDE1 is characterized by calcium/calmodulin-dependent activity and dual substrate specificity for cAMP and cyclic guanosine 3',5'-monophosphate (cGMP), while PDE4 specifically catalyzes the hydrolysis of cAMP to 5'-AMP. In the present study, in order to reveal whether these types of PDEs have functions of modulators of sperm agglutination, we examined the effects of selective inhibitors for PDEs 1 and 4 on sperm agglutination induced by sodium bicarbonate. As shown in Figure 4, supplementation with the inhibitors for PDE1 (8-methoxymethyl-IBMX and vinpocetine: 25 and 50 mM) markedly enhanced the percentages of head-to-head agglutinated spermatozoa in the samples incubated with sodium bicarbonate (1-10 mM). Moreover, similar results were obtained by supplementation with the inhibitors for PDE4 (Ro20-1724 and rolipram: 25 and 50 mM). In the additional experiments performed to confirm the specific effects of selective inhibitors on sperm agglutination, however, an inhibitor for PDE5 (zaprinast) did not enhance the percentages of head-to-head agglutinated spermatozoa as the inhibitors for the PDEs 1 and 4 did at the same concentrations (25 and 50 mM, data not shown).

As shown in Figure 4, the selective inhibitors for calcium/calmodulin-dependent PDE (PDE1) stimulated the sperm agglutination that was induced by the actions of sodium bicarbonate. In order to get further data supporting involvement of this enzyme, we examined the effects of a calmodulin antagonist (W-7, Figure 5) on head-to-head agglutination in spermatozoa incubated with 0-10 mM sodium bicarbonate and without PDE inhibitors. At 2 mM, W-7 significantly enhanced the percentage of head-to-head agglutinated spermatozoa in the samples incubated with sodium bicarbonate (1-10 mM), though no significant effect of the antagonist was observed in the samples incubated without sodium bicarbonate. Moreover, at 10 mM, W-7 highly induced sperm agglutination irrespective of the presence of sodium bicarbonate.

As mentioned above, the selective inhibitors for cAMP-specific PDE4 were more effective than that for cGMP-specific PDE5 on the enhancement of sperm agglutination. Moreover, in our previous report [14], dbcAMP was shown to enhance bicarbonate-induced sperm agglutination, while dbcGMP had no effect. Thus, in order to confirm the specificity of effects of cAMP analogues, we examined the effects of several kinds of more potent cyclic nucleotide analogues on head-to-head agglutination in spermatozoa incubated with 0-10 mM sodium bicarbonate and without PDE inhibitors (Table 3). In series 1 of the experiments, two kinds of cAMP analogues (dbcAMP and cBiMPS) were examined; the latter is much more potent than the former in the term of resistance to the hydrolysis by the PDEs [32,33]. Both of these cAMP analogues significantly enhanced the percentages of head-to-head agglutinated spermatozoa in the samples incubated with sodium bicarbonate (5-10 mM). In the samples incubated without sodium bicarbonate, however, only more potent analogue (cBiMPS) produced a significant effect on the enhancement of sperm agglutination. In series 2 and 3 of the experiments, three kinds of cGMP analogues (dbcGMP, 8-Br-cGMP and 8-pCPT-cGMP; 8-Br-cGMP and 8-pCPT-cGMP are more potent than dbcGMP, as described in catalogue from Sigma-RBI [34]) were examined. None of these cGMP analogues were significantly effective in enhancing the percentages of head-to-head agglutinated spermatozoa irrespective of the presence of sodium bicarbonate.

Table 3. Effects of cyclic nucleotide analogues on head-to-head agglutination induced by sodium bicarbonate in ejaculated boar spermatozoa.

Values are meansSEM. Ejaculated boar spermatozoa were washed and incubated at 38.5 for 60 min in mKRH supplemented with sodium bicarbonate (0-10 mM) plus with cyclic nucleotide analogues (0-1 mM).
^aDibutyryl cyclic adenosine 3',5'-monophosphate
^bSp-5,6-Dichloro-1-b-D-ribofuranosyl-benzimidazole-3',5'-monophosphorothioate
^cDibutyryl cyclic guanosine 3',5'-monophosphate
^d8-Bromo cyclic guanosine 3',5'-monophosphate
^e8-(4-Chlorophenylthio) cyclic guanosine 3',5'-monophosphate
^A,BValues within the same column in each experimental series with different superscripts are different significantly, P<0.05 (Tukey's multiple range tests).

Rp-cAMPS is a cAMP antagonist that interferes with the cAMP-mediated signaling cascade [34]. In the present study, the addition of this antagonist (1 mM) significantly reduced the percentages of head-to-head agglutinated spermatozoa after the incubation with 5 mM sodium bicarbonate and 25 mM IBMX (control vs. 1 mM Rp-cAMPS, meanSEM: 47 %6 % vs. 27 %5 %, P< 0.05, paired t-tests, n=3). This strongly indicates that the promoting effects of bicarbonate are mediated by the cAMP-mediated signaling cascade.

Bicarbonate is brought into the intracellular space of spermatozoa by anion transporters in the plasma membrane [35], including anion exchangers [36, 37] and sodium/bicarbonate cotransporters [16]. Bicarbonate then regulates intracellular pH. This anion also activates the sperm adenylyl cyclases, resulting in increased generation of the signaling second messenger [21]. The intracellular concentration of the second messenger (cAMP) is regulated by the balance between the activity of two kinds of enzymes: adenylyl cyclase and PDE [38]. The sperm adenylyl cyclase is characterized by a soluble form with a unique molecular structure, and is apparently distinct from the classical transmembrane form usually found in somatic cells. Regulation of the enzyme activity is also unique in the spermatozoa; the sperm adenylyl cyclase does not require G-protein for its activation, but is activated by direct interaction with bicarbonate [39, 40]. The generated cAMP is subsequently hydrolyzed to adenosine 5'-monophosphate by the catalysis of PDEs. In mammalian somatic cells, at least 11 families of PDEs of Class I have been already identified; e.g., some PDEs (PDE4, PDE7 and PDE8) are highly specific for cAMP, some (PDE5, PDE6 and PDE9) are highly specific for cGMP, and some (PDE1, PDE2, PDE3, PDE10 and PDE11) have dual specificity [41-43]. To our knowledge, two families of PDEs (PDE1 and PDE4) have been identified in testicular germ cells of rats [28, 31, 44] and mice [29, 30, 45]. These types of PDEs are also contained in mature spermatozoa of rats [31, 46] and bulls [47]. Interestingly, a calcium/calmodulin-dependent PDE (PDE1) of the testes and spermatozoa seems to have a higher affinity for cAMP than for cGMP [47, 48]. For other types of PDEs, Richter et al. [49] have detected mRNA transcripts of several PDE subtypes (PDE1A/B/C, PDE2, PDE3A/B, PDE4A/B/C, PDE5 and PDE8) in ejaculated human spermatozoa. Moreover, Soderling et al. [42] have reported a high expression of PDE10 in mouse testes. However, no data are available on boar spermatozoa. In boar spermatozoa, it is apparent that adenylyl cyclase is stimulated by the actions of bicarbonate [21]. Specifically, the generation of cAMP increases in boar spermatozoa incubated at 38 for 30 min in the medium containing bicarbonate, but this increase is nearly abolished by either a change in the incubation temperature to 25 or the omission of bicarbonate [15]. In our present study, a stimulator of adenylyl cyclase (bicarbonate) promoted the head-to-head agglutination of boar spermatozoa in a concentration-dependent manner (Table 2). This event was apparently enhanced by the addition of cAMP-specific PDE (PDE4) inhibitors (Figure 4) as well as of non-selective PDE inhibitors (Figure 1), but was only slightly affected by the coexisting effects of bicarbonate, including changes in the pH and osmolarity of the medium (Figure 2). Moreover, the effect of bicarbonate on sperm agglutination appeared within 30 min incubation period was dependent on the incubation temperature (Figure 3). These findings suggest that the bicarbonate-induced head-to-head agglutination is regulated via the activity of the adenylyl cyclase and PDE in boar spermatozoa. This suggestion is further supported by the results indicated in Table 3 and the text (Results): 1) only cAMP analogues promoted the sperm agglutination, 2) a PDE-resistant analogue (cBiMPS) was more effective for the promotion of sperm agglutination than was a PDE less resistant analogue (dbcAMP), and 3) cAMP antagonist (Rp-cAMPS) significantly reduced the sperm agglutination resulting from the actions of bicarbonate and IBMX.

Calmodulin has been known as a ubiquitous regulatory protein that mediates calcium signals in a variety of cell functions. For example, this calcium-binding protein modulates the activity of important enzymes including adenylyl cyclase, PDE, protein phosphatase 2B (calcineurin) and calcium-dependent ATPase (calcium pump) [50]. Mammalian spermatozoa possess a large amount of this protein in the head and flagellum. Calmodulin plays important roles in the calcium-dependent control of sperm capacitation and hyperactivated motility during the process of the expression of fertilizing ability [51, 52]. Since calmodulin antagonists accelerate the process of capacitation in spermatozoa [52-55], calmodulin seems to participate in the down-regulation of the expression of fertilizing ability. Indeed, it has been observed that the concentration and binding affinity of this protein decrease in bull spermatozoa during the incubation in capacitation media containing calcium [56, 57]. In the present study, the effect of bicarbonate on sperm agglutination was apparently enhanced by the addition of calcium/calmodulin-dependent PDE (PDE1) inhibitors (Figure 4). Moreover, a calmodulin antagonist (W-7), which could potentially block the regulator of PDE1, stimulated the sperm agglutination (Figure 5). These results suggest that a PDE1-mediated signaling cascade is present in boar spermatozoa and is involved in the suppression of head-to-head agglutination.

In summary, our data demonstrate that the bicarbonate-induced agglutination in boar spermatozoa is controlled via a cAMP-mediated and temperature-dependent signaling cascade. This cascade is suppressed by the action of PDE (at least types 1 and 4). Our previous studies [13, 14] have shown that extracellular calcium and serum albumin also promote head-to-head agglutination in boar spermatozoa. Several lines of evidence have demonstrated that sperm capacitation is modulated by these regulatory factors, including bicarbonate, calcium, serum albumin and cAMP [58-60]. Moreover, the occurrence of head-to-head agglutination is positively correlated with spermatozoa classified as the B pattern by the chlortetracycline assay [14]. These findings are consistent with our suggestion that head-to-head agglutination is associated with capacitation.

We thank Hyogo Prefectural Agricultural Institute for cooperation in sample collection.

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Correspondence to: Dr. Hiroshi Harayama, Department of Life Science, Graduate School of Science and Technology, Kobe University, 1 Rokko-dai, Nada, Kobe 657-8501, JAPAN
Tel: +81-78-803 5805, Fax: +81-78-803 5807
E-mail: harayama@ans.kobe-u.ac.jp
Received 2002-02-08      Accepted 2002-04-18