Molecular Human Reproduction, Vol. 5, No. 7, 618-626,
July 1999
© 1999 European Society of Human Reproduction and Embryology
Pentoxifylline-stimulated capacitation and acrosome reaction in hamster spermatozoa: involvement of intracellular signalling molecules
1 Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore 560012, and 2 Centre for Cellular and Molecular Biology, Hyderabad, India
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We investigated the role of cAMP/cGMP, protein kinases and intracellular calcium ( [Ca2+]i) in pentoxifylline-stimulated hamster sperm capacitation and the acrosome reaction (AR) in vitro. Treatment with pentoxifylline (0.45 mM) initially increased sperm cAMP values 2.8-fold, compared with untreated controls (396 ± 9.2 versus 141 ± 6.0 fmoles/106 spermatozoa; mean ± SEM, n = 6) after 15 min, although by 3 h, cAMP values were similar (503–531 fmoles/106 spermatozoa). cGMP values (~27 fmoles/106 spermatozoa) were the same in treated and control spermatozoa. Both sperm capacitation and the AR, determined from the absence of an acrosomal cap, were stimulated by pentoxifylline; these were almost completely inhibited by a Cl–/ HCO3– antiporter inhibitor (4,4-diisothiocyanato-stilbene-2,2 disulphonic acid; 1 mM) defined from the degree of sperm motility and by a protein kinase A inhibitor (H89; 10 µM). A protein kinase C inhibitor (staurosporine, 1 nM) did not affect pentoxifylline-stimulated capacitation but inhibited the AR by 50%. A protein tyrosine kinase inhibitor (tyrphostin A-47, 0.1 mM) had no effect on either pentoxifylline-stimulated capacitation or AR. A phospholipase A2 inhibitor (aristolochic acid, 0.4 mM) markedly inhibited the pentoxifylline-stimulated AR but not capacitation. When intracellular sperm calcium [Ca2±]i was measured using fura-2-AM, there was an early rise (271 nM at 0.5 h) in pentoxifylline-treated spermatozoa; this appeared to be due to intracellular mobilization rather than to uptake. In the absence of extracellular Ca2+, sperm motility was maintained in the presence of pentoxifylline, but capacitation did not occur; spermatozoa exhibited a low level of hyperactivated motility and had a poor rate of AR (20.5 ± 2.3%). These results suggest that: (i) the pentoxifylline-stimulated early onset of sperm capacitation may be mediated by an early rise in cAMP and [Ca2±]i and involves protein kinase A activity; and (ii) pentoxifylline-stimulated AR may require phospholipase A2 and protein kinase C activity.
calcium/cAMP/hamster/pentoxifylline action/spermatozoa
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Introduction |
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Acquisition of fertilizing ability by mammalian spermatozoa is dependent on capacitation and the acrosome reaction (AR). These events are mediated by various complex intracellular signal transduction pathways (Breitbart and Spungin, 1997
; de Lamirande et al., 1997; Baldi et al., 1998; Visconti and Kopf, 1998) resulting in changes in sperm motility patterns and acrosomal exocytosis. Certain metabolic stimulants induce sperm capacitation and AR via changes in the values of cAMP and intracellular calcium ([Ca2+]i) and in the activities of protein kinases. Abnormalities in these biochemical events adversely affect sperm function and result in infertility. Stimulators of sperm motility, e.g. pentoxifylline or progesterone, are used in assisted reproductive technology (ART) to enhance the fertilizing potential of inherently poor quality spermatozoa from oligoasthenozoospermic patients (Yovich et al., 1990; Kay et al., 1994; Matson et al., 1995; Tournaye et al., 1995; Baldi et al., 1998). We have shown that 0.45 mM pentoxifylline can stimulate capacitation and AR in hamster spermatozoa (Rupasri et al., 1995; Jayaprakash et al., 1997). Whether this is mediated via changes in the concentrations of cAMP/cGMP, [Ca2+]i and the activity of protein kinases is unknown.
The values of cyclic nucleotides, protein kinase activities, Ca2+ and phosphoinositide metabolites are the most important of the cell signalling events which control capacitation and AR of mammalian spermatozoa (Brucker and Lipford, 1995; Breitbart and Spungin, 1997; de Lamirande et al., 1997; Baldi et al., 1998; Bonaccorsi et al., 1998; Visconti and Kopf, 1998). Elevation of sperm cAMP content by membrane permeant cAMP analogues or phosphodiesterase inhibitors increases the hyperactivated motility of mammalian spermatozoa (Stein and Fraser, 1984; de Lamirande et al., 1997; Calogero et al., 1998). Moreover, capacitation of hamster spermatozoa is associated with a progressive increase in cAMP concentrations that precede the onset of hyperactivated motility (White and Aitken, 1989). A rise in cAMP values during capacitation is a consequence of an increase in bicarbonate-stimulated adenylate cyclase activity (Stein and Fraser, 1984). The role of cGMP in sperm function is unclear. Dibutyryl cGMP treatment accelerates capacitation of mouse and AR of guinea pig spermatozoa. However, the concentrations of sperm cGMP remain unchanged during capacitation (Cohen-Dayag and Eisenbach, 1994).
The role of protein kinases during capacitation and AR in mammalian spermatozoa is well documented (Garbers et al., 1973; Doherty et al., 1995; Visconti et al., 1995; Bonaccorsi et al., 1998). Recently, we demonstrated that several protein kinases, e.g. cAMP-dependent and -independent protein kinases, casein kinase II and protein kinase C (PKC) are associated with the maturation-related differential phosphorylation of sperm proteins (Uma Devi et al., 1996a,b). Inhibition of cAMP-dependent protein phosphorylation by protein kinase A (PKA) inhibitors blocks sperm capacitation in mice (Visconti et al., 1995) and humans (Leclerc et al., 1996). Activators of PKC such as phorbol myristoyl acetate potentiate human sperm capacitation, whereas PKC inhibitors such as staurosporine (STP) cause its inhibition (Furuya et al., 1993; Bonaccorsi et al., 1998).
Ca2+ has been shown to play a pivotal role in sperm capacitation and AR (Bielfeld et al., 1994; Brucker and Lipford, 1995; Meizel et al., 1997; Bonaccorsi et al., 1998) and [Ca2+]i increases during hamster sperm capacitation and AR (Suarez and Dai, 1995). This leads to phospholipase C (PLC) activation which converts phosphatidyl inositol bisphosphate to diacylglycerol and inositol trisphosphate (O'Toole et al., 1996). The latter is involved in sperm capacitation and AR (Breitbart and Spungin, 1997). Diacylglycerol activates phospholipase A2 (PLA2) and PKC, which are involved in AR (Bonaccorsi et al., 1998) and are able to activate plasma membrane Ca2+ channels (Spungin and Breitbart, 1996).
Pentoxifylline is a methylxanthine derivative which inhibits phosphodiesterase, thereby elevating the concentrations of intracellular cAMP (Calogero et al., 1998) and/or cGMP. Alterations in cyclic nucleotide values regulate the activities of certain protein kinases involved in sperm function. Methylxanthines have also been implicated in [Ca2+]i mobilization in frog muscle (Rall, 1982). Hence, pentoxifylline may act via mobilization of intracellular sperm Ca2+, and the activation of Ca2+-mediated signalling pathways. The present study was designed to investigate the molecular mechanism of pentoxifylline-stimulated capacitation and AR in hamster spermatozoa and the role of key cell signalling molecules, i.e. cyclic nucleotides, protein kinases and [Ca2+]i.
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Animals and reagents
Sexually mature golden hamsters (Mesocricetus auratus) were maintained on a lighting schedule of 14 h light:10 h dark (lights on at 06:00) at a temperature of 24–26°C. Experiments were conducted in accordance with the Guiding Principles for the Care and Use of Research Animals promulgated by INSA (New Delhi, India).
Reagents were obtained as follows: cAMP enzyme immunoassay kit from Amersham International (Buckinghamshire, UK), PKA inhibitor H89 from LC laboratories (Woburn, USA) and 45Ca2+ from Bhaba Atomic Research Center (Mumbai, India). All other reagents were from either Sigma Chemical Company (St Louis, MO, USA) or HiMedia Lab Pvt Ltd (Mumbai, India).
Incubation of spermatozoa with pentoxyfilline, inhibitors and progesterone
Cauda epididymal spermatozoa were used throughout the study. Briefly, 2 µl of neat cauda epididymal content were incubated in 2 ml of pre-equilibrated TALP–PVA (Tyrode's solution supplemented with 3 mg/ml bovine serum albumin), 10 mM sodium lactate, 0.25 mM sodium pyruvate and 1 mg/ml polyvinyl alcohol) and incubated in a humidified atmosphere of 5% CO2 in air at 37°C. The following reagents were included in the culture medium as indicated in the results: pentoxifylline [9-(5-oxohexyl)-3,7-dimethylxanthine], 0.45 mM (Rupasri et al., 1995; Jayaprakash et al., 1997); 4,4-diisothiocyanatostilbene-2,2 disulphonic acid (DIDS), 1 mM (Visconti et al., 1990); N-[2-(p-bromocinnamylamino)ethyl]5-isoquinolinesulphonamide (H89);10 µM (Leclerc et al., 1996); STP 1 nM (Lax et al., 1994); tyrphostin A-47, 0.1 mM (Leyton et al., 1992); aristolochic acid, 0.4 mM (Garde and Roldan, 1996); and progesterone, 0.1 mM (Kay et al., 1994). All test compounds were added from the start of the culture period.
Sperm motility
The percentage of motile sperm and the quality of motility (score: 0–5) were assessed hourly for 8 h using Olympus stereo zoom microscope. The sperm motility index (SMI) was defined as: percentage motilityx[quality score, 0–5]2. An SMI value 
1200 indicated that the spermatozoa were capacitated. Sperm motility was determined using a standard procedure (Bavister and Andrews, 1988; Rupasri et al., 1995; Jayaprakash et al., 1997).
CASA analysis
Sperm kinematic analysis was carried out using computer-aided sperm analysis (CASA) (HTM-S Motility Analyzer, version 7.2; Hamilton Thorn Research, Danvers, MA, USA), as detailed elsewhere (Jayaprakash et al., 1997). Briefly, a 20 µl aliquot of each sperm incubation was transferred to a slide chamber (80 µm depth) for analysis. Using the `play back' and `zoom function', the following kinematic parameters were determined for 100–150 individual spermatozoa from each treatment: straight line velocity (VSL), average path velocity (VAP), beat cross frequency (BCF), curvilinear velocity (VCL), amplitude of lateral head displacement (ALH), straightness (STR) and linearity (LIN). As detailed elsewhere (Shivaji et al., 1995; Jayapraksh et al., 1997), the sort function of the analyser was used to determine the populations of spermatozoa exhibiting planar (VSL >80 µm/s, STR >80%, LIN >50%, ALH <20 µm), or helical or circular (VSL >30 µm/s, STR <80%, LIN <50%, ALH >15 µm) patterns of motility, by visualizing individual sperm tracks. Hyperactivated spermatozoa, defined as those with helical or circular motility patterns, were expressed as a percentage of the total sperm population.
Acrosome reaction
The proportion of acrosome-reacted spermatozoa was assessed after 4 h of culture, from the presence or absence of acrosomal cap under light microscope at x400 magnification (Jayaprakash et al., 1997). The proportion of viable spermatozoa was determined using eosin Y staining (World Health Organization, 1992).
Assay of sperm intracellular cyclic nucleotides
cAMP
This was determined using an enzyme immunoassay kit, in accordance with the manufacturer's specifications. Briefly, sperm cyclic nucleotides were extracted with 1 N HCl, lyophilized and stored at –70°C until assay. The lyophilized samples were reconstituted in assay buffer. Samples or standards and rabbit anti-cAMP antisera were added to microtitre wells precoated with secondary antibody (donkey anti-rabbit) and incubated at 4°C for 2 h. Cyclic AMP-peroxidase conjugate was then added, plates were incubated for an additional 1 h at 4°C. The amount of peroxidase labelled-cAMP bound to the antibody was then determined by adding a tetramethylbenzidine/hydrogen peroxide substrate. The reaction was stopped by adding 1 N H2SO4 and the opical density was determined at 450 nm. The sensitivity of the assay was 12.5 fmoles/well.
cGMP
This was measured by radioimmunoassay (Brooker et al., 1979). Briefly, samples or standards were incubated with cGMP antiserum and [125I]-TME-ScGMP (tyrosine methyl ester of succinyl cGMP; 12 000 cpm per tube) in 50 mM acetate buffer (pH 4.5) at 37°C for 4 h. A 2 mg/ml charcoal suspension in phosphate buffer (pH 6.3) containing 2.5 mg/ml bovine serum albumin was then added, centrifuged and the pellet radioactivity was determined in a 
-counter. The sensitivity of the assay was 30 fmoles/tube.
Measurement of intracellular calcium
The concentration of intracellular free, extra-mitochondrial Ca2+ was assessed using a fluorescent Ca2+ indicator, fura-2-AM, as previously described (White and Aitken, 1989). Briefly, spermatozoa (4x106/ml) were incubated with 5 µM fura-2-AM at 25°C for 30 min. After washing, Fura-2-AM-loaded spermatozoa were incubated in TALP medium at 37°C in the presence or absence of pentoxifylline. Aliquots (2 ml) were taken at 0.5, 1, 2, 4 and 6 h for [Ca2+]i measurements using a Hitachi fluorimeter (model no. 605–105; excitation: 340 nm and emission: 480 nm). [Ca2+]i was calculated using:
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To measure the uptake of Ca2+, spermatozoa (10x106/ml) were cultured in the presence of 5 µCi/ml 45Ca2+ (~2 mM Ca2+), at 37°C in 5% CO2 in air in the presence or absence of pentoxifylline. At 0.25, 1, 2, 4 and 6 h, 100 µl aliquots were transferred to 1 ml of 10 mM EGTA in 0.9% saline to chelate the extracellular Ca2+. After passage through GF-C filters and rapid washing with 5x1 ml of ice-cold 10 mM EGTA solution in 0.9% saline, the radioactivity of the filters was determined. Measurements were performed in duplicates and results were expressed as cpm/1x106 cells.
Statistical analysis
The data were analysed using Student's t-test or by one-way analysis of variance, followed by Newman–Keuls multiple comparison test, as appropriate. Each experiment was repeated six times with sperm samples from six different animals.
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Pentoxifylline-stimulated capacitation and modulation of cyclic nucleotide values
Pentoxifylline generally reduced the time taken to achieve sperm capacitation (SMI
1200) by ~1 h. Consistently, higher SMI values were observed at all time points in pentoxifylline-treated versus untreated samples (Figure 1A). Pentoxifylline-treated spermatozoa also exhibited an earlier and increased accumulation of cAMP compared with untreated controls (Figure 1B). The highest concentration of cAMP was achieved at 3 h in both control (503 ± 10.1) and pentoxifylline-treated spermatozoa (531 ± 23.5). Furthermore, the cAMP concentrations in the pentoxifylline-treated group remained elevated and declined more slowly than in the controls. In contrast to cAMP, the concentration of cGMP was not significantly altered by the presence of pentoxifylline (Figure 1C).
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) or presence (
) of pentoxifylline. Values with identical superscripts are significantly different: aP <0.001 in (A); a,b.c,eP <0.005, dP <0.01 in (B). Results are expressed as mean ± SEM (n = 6).Influence of inhibitors on pentoxifylline-stimulated capacitation
To determine the role of bicarbonate (HCO3–)-stimulatable adenylate cyclase, spermatozoa were cultured in the absence or presence of DIDS, a specific Cl–/HCO3– antiporter inhibitor. The presence of DIDS caused inhibition of capacitation (Figure 2A
), which was not reversed in the presence of pentoxifylline. Moreover, in the absence of extracellular HCO3–, pentoxifylline failed to induce capacitation (data not shown).
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Complete inhibition of capacitation was observed in the presence of PKA inhibitor H89, regardless of the presence or absence of pentoxifylline (Figure 2B
). In contrast, the PKC inhibitor, STP, did not affect SMI either in the control or in the pentoxifylline-treated spermatozoa (Figure 2C). Similarly, the tyrosine kinase inhibitor, tyrphostin A-47, had no effect on SMI irrespective of the absence or presence of pentoxifylline (Figure 2D). The SMI profile of progesterone-treated spermatozoa was similar to that of control spermatozoa and tyrphostin A-47 had no effect on sperm SMI, either in the presence or absence of progesterone (Figure 2D).
Effect of inhibitors on pentoxifylline-stimulated AR
Pentoxifylline addition leads to stimulation of the AR, which was almost completely abolished (82%) in the presence of the Cl–/ HCO3– antiporter inhibitor, DIDS (Figure 3A). Similarly, in the absence of HCO3–, spermatozoa did not undergo AR despite the presence of pentoxifylline (data not shown). Inhibition of AR was also observed when spermatozoa were cultured in the presence of the PKA inhibitor H89, both in the presence or absence of pentoxifylline (Figure 3B). Although the PKC inhibitor, STP, decreased pentoxifylline-stimulated AR by ~50%, it did not affect 8-bromo-cAMP-mediated AR (Figure 3C). H89 inhibited 8-bromo-cAMP-induced capacitation and AR (data not shown). The protein tyrosine kinase inhibitor, tyrphostin A-47, did not inhibit AR in control spermatozoa (10%), but had no effect on pentoxifylline-treated spermatozoa. Under similar conditions, tyrphostin A-47 markedly inhibited AR in progesterone-treated spermatozoa (Figure 3D).
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Inhibition of PLA2 by aristolochic acid had no effect on pentoxifylline-stimulated capacitation (data not shown) but markedly inhibited pentoxifylline-stimulated AR (Figure 4
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Changes in pentoxifylline-stimulated sperm motility in the presence and absence of calcium
Spermatozoa were cultured in a Ca2+-free medium, either in the presence or absence of pentoxifylline (Figure 4
). In the absence of both Ca2+ and pentoxifylline, spermatozoa soon demonstrated either loss of motility or poor quality motility (Figure 5A and B). In Ca2+-free, pentoxifylline-supplemented medium, the quality and percentage of motility were sustained for up to 4 h and then decreased gradually. Addition of Ca2+ either at 1 or 3 h to spermatozoa cultured in Ca2+-free medium, containing pentoxifylline caused a great increase in SMI (Figure 5C).
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), open triangle (
) correspond to Ca2+-free, pentoxifylline-supplemented culture media. Calcium was absent either throughout the culture period (O) or for the first 1 (CASA analysis showed that there was no appreciable change in VSL, VAP and BCF of spermatozoa cultured in Ca2+-containing medium either in the absence or presence of pentoxifylline (data not shown). However, profiles of VCL, ALH, STR and LIN changed significantly when spermatozoa were cultured in the presence of pentoxifylline when Ca2+ was removed from the medium (Figure 6
), and the increase in VCL and ALH and decrease in STR and LIN (which are characteristic of hyperactivated spermatozoa), were not observed. After 2 h of culture in pentoxifylline-containing, Ca2+-free medium, only ~9 and 12% of spermatozoa exhibited helical and circular motility patterns, respectively (Figure 7). In medium containing both pentoxifylline and Ca2+ there was a significant increase in the percentage of spermatozoa exhibiting circular and helical motility. In the presence of pentoxifylline, only a small population of spermatozoa underwent hyperactivation in Ca2+-free medium, compared with Ca2+-containing medium (Figure 7). In Ca2+-free medium, pentoxifylline caused only a low level of AR (20.5 ± 2.3%).
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There was an accumulation of [Ca2+]i in pentoxifylline-treated spermatozoa (Figure 8
), which was significantly (P < 0.05) higher than in the respective controls. However, after 4 h, the concentrations of [Ca2+]i did not significantly differ (P > 0.1) between control and pentoxifylline-treated spermatozoa. At early time points (<0.5 h), the [Ca2+]i was very low and there was no significant difference between the control and pentoxifylline-treated samples. In Ca2+-free, pentoxifylline-supplemented medium, [Ca2+]i remained at ~350–400 nM for up to 6 h. Addition of pentoxifylline to non-capacitated spermatozoa lead to an increase in [Ca2+]i which was significantly (P < 0.05) higher than that obtained following the addition of progesterone (2.7 ± 0.2 versus 1.5 ± 0.1) at the same time. However, there was no difference in the level of increase of [Ca2+]i when pentoxifylline or progesterone were added to capacitated spermatozoa (~1.5, P > 0.1). There was no significant difference in the uptake of 45Ca2+ in the presence or absence of pentoxifylline up to 2 h. However, at 4 h, the influx of 45Ca2+ was significantly higher (P < 0.01) in the presence of pentoxifylline (Figure 8). There was an apparent reversal in the massive influx of Ca2+ in the control versus pentoxifylline-treated sample.
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Discussion |
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The present study was initiated to investigate the mechanism of action of pentoxifylline during capacitation and AR of hamster spermatozoa. The early onset of pentoxifylline-stimulated sperm capacitation and AR (Jayaprakash et al., 1997
; present study) was associated with an early rise in cAMP concentrations (Figure 1) and intracellular mobilization of Ca2+ but not with a rise in cGMP concentrations or with uptake of Ca2+. The results suggest that PKA activity but not PKC activity is necessary for pentoxifylline-stimulated capacitation, while PKC and PLA2 activities are necessary for pentoxifylline-stimulated AR.
The early, increased accumulation of cAMP observed in pentoxifylline-treated spermatozoa may be due to inhibition of phosphodiesterase (Calogero et al., 1998) and/or to activation of Ca2+-activatable adenylate cyclase. Since pentoxifylline completely failed to reverse the inhibition of sperm capacitation and AR induced by the presence of the Cl–/HCO3– antiporter inhibitor (DIDS) or the absence of extracellular HCO3–, it is possible that cAMP synthesis may be mediated by HCO3-stimulatable adenylate cyclase (Visconti et al., 1995), rather than by the calcium-stimulatable adenylate cyclase. Hence, the accumulation of cAMP in the presence of pentoxifylline may be due to both inhibition of phosphodiesterase and activation of adenylate cyclase. A pentoxifylline-induced increase in intra-sperm cAMP, associated with a change in motility parameters, has been observed in both the hamster (Jayaprakash et al., 1997; present data), and the human (Calogero et al., 1998). The guanylate cyclase system has also been implicated in sea urchin spermatozoa (Bently, 1991), but does not appear to play a role in hamster spermatozoa.
The action of cAMP is mediated via PKA, an important modulator of sperm capacitation (Garbers et al., 1973). The inhibition of capacitation by H89 (a PKA inhibitor) despite the presence of pentoxifylline suggests that protein phosphorylation by PKA may be essential for hamster sperm capacitation, as has been observed in mice (Visconti et al., 1995) and that pentoxifylline acts upstream of PKA, perhaps by modulating the concentrations of second messengers. Our data suggest that the activities of PKC and PTK may not be required for in-vitro sperm capacitation. The results suggest that the protein phosphorylation normally required to increase membrane fluidity during capacitation may be highly specific and predominantly dependent on PKA activity.
Sperm AR appears to be inhibited by DIDS and H89, even in the presence of pentoxifylline. This may be a consequence of the lack of capacitation. The inhibition of AR (but not capacitation) by STP (a PKC inhibitor), both in the absence and presence of pentoxifylline, suggests that PKC action is required for pentoxifylline-stimulated AR but not for capacitation. Activation of PKC may occur via the Ca2+-dependent activation of PLC
. The failure of PKC inhibitors to completely abolish AR suggests that there are parallel pathways controlling AR. A previous report has suggested that PKC induction of AR via a Ca2+-independent pathway may occur (Bonaccorsi et al., 1998).
In several species, cAMP-modulated protein tyrosine phosphorylation is a key event in sperm capacitation and AR (Duncan and Fraser, 1993; Leclerc et al., 1996; Tesarik et al., 1996). The failure of the inhibition of tyrosine phosphorylation (using tyrphostin A-47) in hamster spermatozoa to abolish capacitation suggests that, unlike AR, tyrosine phosphorylation may not be essential for capacitation. An alternate mechanism may involve PKA-dependent indirect tyrosine phosphorylation (Visconti et al., 1995; Visconti and Kopf, 1998). The lack of inhibition of AR by tyrphostin A-47 in the presence of pentoxifylline was in contrast to the inhibition of progesterone-stimulated AR by PTK inhibitor. This implies that PF and progesterone may invoke different requirements for tyrosine phosphorylation. This is consistent with a previous report on the involvement of tyrosine phosphorylation in progesterone-stimulated calcium influx and AR in human spermatozoa (Meizel and Turner, 1996; Tesarik et al., 1996). Furthermore, pentoxifylline-induced [Ca2+]i influx may be differentially controlled by phosphorylation-mediated activation of GABA or glycine receptor/Cl– channels, as shown with human spermatozoa (Meizel, 1997; Meizel et al., 1997).
Unlike other methylxanthines, e.g. isobutyl methylxanthine (White and Aitken, 1989), pentoxifylline is able to maintain sperm motility, induce AR and also permit gamete fusion in Ca2+-free medium. This suggests that pentoxifylline may mobilize Ca2+ from the sperm intracellular pool. This is confirmed by the finding that pentoxifylline causes an early and increased accumulation of [Ca2+]i but does not influence the uptake of Ca2+. Sperm motility was also maintained in Ca2+-free, pentoxifylline-supplemented medium, suggesting that pentoxifylline mobilizes sufficient concentrations of free Ca2+ from intra-sperm stores to maintain motility. Furthermore, Ca2+ may contribute to, but may not be requisite for, AR induction. This is consistent with a similar observation made with human spermatozoa (Bielfeld et al., 1994). Although the mechanism of Ca2+ action during capacitation remains unclear (de Lamirande et al., 1997; Visconti and Kopf, 1998), the process of AR is critically dependent on Ca2+-mediated intracellular events (Breitbart and Spungin, 1997), e.g. activation of PKC (Breitbart et al., 1992) and PLA2 (Roldan et al., 1992). Our results suggest that PLA2 is an indispensable component of pentoxifylline-stimulated AR, whereas PKC plays a role in pentoxifylline-stimulated AR.
Figure 9 suggests possible biochemical mechanism of action of pentoxifylline during sperm capacitation and AR. Pentoxifylline may elevate the concentrations of the second messengers, cAMP and [Ca2+]i. In turn, accumulation of cAMP may lead to PKA activation, bringing about the membrane protein phosphorylation associated with capacitation. Moreover, increased [Ca2+]i may activate PLA2 resulting in the synthesis of fusogenic compounds such as arachidonic acid and lysophospholipids, required for AR-associated membrane fusion. Increased [Ca2+]i may also lead to PKC activation which may induce AR independently, via membrane protein phosphorylation.
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In the present study, we investigated pentoxifylline action during the capacitation and AR of spermatozoa. We did not attempt to determine direct biochemical changes including phosphorylation of specific sperm proteins or the measurement of kinase or phosphodiesterase activities. The mechanism of pentoxifylline-stimulated sperm capacitation and AR was investigated using specific metabolic inhibitors routinely employed elsewhere (Leyton et al., 1992
; Lax et al., 1994; Garde and Roldan, 1996; Leclerc et al., 1996). The results provide evidence for the possible action mechanism of pentoxifylline, and the involvement of key cell-signalling molecules, during capacitation and AR of hamster spermatozoa (Figure 9).
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Acknowledgments |
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This work was supported by grants from the Department of Atomic Energy (Mumbai). PBS would like to thank the Department of Science and Technology (New Delhi) for providing initial funds to establish the basic infrastructure. SS would like to thank the Central Zoo Authority of India, Government of India for the financial assistance provided for the purchase of HTMS-IVOS. K.Uma Devi is a recipient of a research associateship from CSIR, Government of India. Authors thank Dr Sandhya S.Visweswariah for providing reagents for cGMP assay and Manjiri Bakre and Archana Mishra for help. Our thanks are also due to M.S.Padmavathi for her help during the preparation of the manuscript.
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3 To whom correspondence should be addressed
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References |
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Submitted on September 2, 1998; accepted on February 17, 1999.
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