Hum. Reprod. Advance Access published online on September 9, 2008
Human Reproduction, doi:10.1093/humrep/den334
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Enhancement of sperm–zona pellucida (ZP) binding capacity by activation of protein kinase A and C pathways in certain infertile men with defective sperm–ZP binding
1 Department of Obstetrics and Gynaecology, University of Melbourne, Australia 2 Reproductive Services, Royal Women's Hospital, Australia 3 Melbourne IVF, Melbourne, Australia
4 Correspondence address. Tel: +61-3-9344-2042; Fax: +61-3-9347-1761; E-mail: dyl{at}unimelb.edu.au
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Abstract |
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BACKGROUND: Defective sperm–zona pellucida (ZP) binding (DSZPB) is a common cause of failure of fertilization in vitro. This study was to determine if DSZPB is caused by defective pathways upstream of protein kinase A (PKA) and C (PKC), or reduced protein tyrosine phosphorylation (TP).
METHODS: Infertile men with DSZPB and either normal sperm morphology (NSM) 
14% (n = 15) or 
5% (n = 15) were studied. Sperm–ZP binding test was performed by incubation of motile sperm with oocytes for 2 h with or without dibutyryl cyclic AMP (dbcAMP, PKA activator) or phorbol myristate acetate (PMA, PKC activator). TP of capacitated sperm in medium was assessed by immunofluorescence with an anti-phosphotyrosine monoclonal antibody.
RESULTS: For normal sperm with normal sperm–ZP binding, both PMA and dbcAMP significantly enhanced sperm–ZP binding in a dose–response manner. Only dbcAMP, but not PMA, significantly increased TP of capacitated sperm. In DSZPB men with severe teratozoospermia (NSM 5%), neither PMA nor dbcAMP enhanced sperm–ZP binding, despite dbcAMP significantly increasing the TP of capacitated sperm for all samples. In contrast, for DSZPB with NSM 14%, PMA caused significantly increased sperm binding up to normal levels (40 sperm bound/ZP) in five men, and dbcAMP had a similar result in two men. Again TP was significantly enhanced only by dbcAMP, but not by PMA.
CONCLUSIONS: There is defective signalling in pathways upstream of PKC and PKA in some men with DSZPB and normal semen analysis. Stimulation of TP by dbcAMP does not enhance sperm–ZP binding capacity in DSZPB men with low TP, regardless of sperm morphology.
Key words: PKC/PKA/tyrosine phosphorylation/sperm–zona binding/male infertility
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The ability of sperm to bind to the human zona pellucida (ZP) is one of the most important indicators for sperm fertilizing ability (Liu and Baker, 1992
; Franken et al., 1993; Oehninger et al., 1997; Barratt and Publicover, 2001; Liu et al., 2004) and defective sperm–ZP binding (DSZPB) and penetration are the most frequent causes of complete failure of fertilization in conventional in vitro fertilization (IVF, Liu and Baker, 2000). Although most infertile men with DSZPB have abnormal semen analysis results, particularly severe teratozoospermia [strict normal sperm morphology (NSM) 5%], DSZPB is present in 
15% of men with unexplained infertility with normal semen analysis (Liu et al., 2004, 2007). Over 25% of infertile men with severe teratozoospermia have DSZPB (Liu et al., 2007). Obviously with DSZPB, there is a very low or no chance of fertilization in vivo or in conventional IVF. Now patients with this condition can be treated by intracytoplasmic sperm injection (ICSI) as this bypasses the barrier of DSZPB. However, it is important to determine the underlying cause of DSZPB as this would increase understanding of basic sperm pathology, particularly in men with normal semen analysis.
In fertile men each ejaculate contains 200–300 million sperm with on average 50% motility, but we have previously shown that only 14% of these motile sperm are capable of binding to the ZP of human oocytes under in vitro conditions when unlimited ZP receptors are present (Liu et al., 2003). Therefore, the majority of motile sperm in semen do not have the capacity to fertilize, as they are incapable of interaction with the oocyte. That only a very small proportion of motile sperm in the ejaculate are able to bind to the oocyte may be the main reason why conventional semen analysis results provide only a limited prediction of fertility.
Human ejaculated sperm, like many other mammalian sperm, require capacitation before they are capable of interaction with oocytes during the process of fertilization, either in vivo or in vitro (Yanagimachi, 1994). Although the mechanisms of sperm capacitation are currently not fully understood, it involves a complex series of events including increases in membrane fluidity, cholesterol efflux, calcium influx, bicarbonate uptake, protein tyrosine phosphorylation (TP) and hyperactivated motility (Leclerc et al., 1996; Galantino-Homer et al., 1997; Visconti and Kopf, 1998; Nassar et al., 1999; Urner and Sakkas, 2003, Asquith et al., 2004). The TP is mainly regulated by protein kinase A (PKA) and incubation of sperm with dibutyryl cyclic adenosine monophosphate (dbcAMP) in vitro significantly promotes TP of capacitated sperm (Visconti and Kopf, 1998). Recent studies found that pp60sc-src (SRC) is present in both the tail and the head of acrosome-intact sperm and it plays an important role as a key intermediate kinase in promoting TP events associated with human sperm capacitation (Lawson et al., 2008; Mitchell et al., 2008). Inhibition of SRC kinase can suppress TP but not hyperactivation of capacitated sperm (Mitchell et al., 2008).
TP of human sperm can be detected in the principle piece of the tail using immunofluorescence (IF) with an anti-tyrosine monoclonal antibody (Sakkas et al., 2003; Buffone et al., 2005). There is an association between reduced TP of capacitated sperm and the poor motility in asthenozoospermic men (Buffone et al., 2005). TP of capacitated human sperm is also significantly related to sperm–ZP binding and fertilizing capacity in vitro (Sakkas et al., 2003). Similarly in the mouse, all the sperm bound to the ZP were found to have TP, despite only a small proportion of sperm in the medium having TP (Asquith et al., 2004). Our recent study, involving a large number of infertile men, found that TP of capacitated sperm is highly correlated with sperm–ZP binding capacity but not with the ZP-induced AR (Liu et al., 2006). These results suggest that TP of capacitated sperm detected by IF in vitro may be a useful indicator for sperm–ZP binding capacity. However, it is still unknown if enhancement of TP of capacitated sperm can improve sperm–ZP binding capacity in infertile men with DSZPB associated with a low level of TP.
In the literature, the ZP receptors for binding sperm have been well characterized. For example, the mouse ZP (mZP) contains three major glycoproteins (mZP1, mZP2 and mZP3, Bleil and Wassarman, 1980), and the human ZP (hZP) is composed of four major glycoproteins (hZP1, hZP2, hZP3 and hZP4, Lefievre et al., 2004). The ZP3 of both mouse and human oocytes is believed to be a primary ZP receptor for binding capacitated acrosome-intact sperm (Bleil and Wassarman, 1980; Lefievre et al., 2004). In contrast, the exact nature of human sperm receptors for the ZP has not been established. Although a number of candidate sperm proteins have been found to be able to interact with either solubilized or intact ZP, it is not clear whether or not they are the primary receptors for sperm binding to the ZP (Bleil and Wassarman, 1990; Wassarman, 1999; Lasserre et al., 2003; van Gestel et al., 2007). It is likely that sperm–oocyte binding involves multiple proteins and other factors including PKA and PKC signal transduction pathways (Thaler and Cardullo, 1996).
Activation of PKC pathways by the PKC activator phorbol 12-myristate 13-acetate (PMA) can significantly enhance ZP-induced AR in mouse sperm (Lee et al., 1987). Tollner et al. (1995) found that both PKA and PKC activators, dbcAMP and PMA, can enhance sperm–ZP binding and the ZP-induced AR in macaque sperm in vitro. In human sperm, PMA induces acrosomal ruffling and also enhances the ZP-induced AR of ZP-bound sperm (Liu and Baker, 1997; Liu et al., 2002a,b). As DSZPB is a common sperm defect causing male infertility, we have investigated whether defects in PKC or PKA pathways or reduced TP of capacitated sperm are common causes of DSZPB in infertile men either with, or without, severe sperm morphological defects.
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Chemicals and culture medium
Human tubal fluid (HTF, Irvine Scientific, Irvine, CA, USA) supplemented with 2% human serum albumin (Irvine Scientific) was used as culture medium for all the experiments. Anti-phosphotyrosine monoclonal antibody (PY20, mouse IgG) labelled with fluorescein isothiocyanate (FITC) was obtained from ICN Pharmaceuticals Inc. (Costa Mesa, CA, USA). Dilbutyryl cyclic adenosine monophosphate (dbcAMP), phorbol 12-myristate 13-acetate (PMA), 4
-phorbol 12,13-didecanoate (4 PDD), dimethyl sulphoxide (DMSO) and Pisum sativum agglutinin conjugated with FITC (PSA-FITC) were purchased from Sigma Chemical Company (St Louis, MO, USA). A stock solution of 200 mM dbcAMP was made using HTF medium, and aliquots were stored at –20°C. Stock solutions of 5 mM PMA or 4 PDD were made using DMSO, and aliquots were stored at –70°C.
Human oocytes
Oocytes used for the ZP binding test were obtained from the clinical IVF programme. They showed no evidence of pronuclei or cleavage at 48–60 h after insemination or were immature (with either germinal vesicle or metaphase I). All the oocytes were obtained on Day 3 after collection and the oocytes were pooled from several patients and used for the test on the same day or kept in a 5% CO2 incubator and used within the next 3 days. Degenerate, activated or morphologically abnormal oocytes, as well as oocytes with >10 sperm penetrating the ZP, were not used for the test. Prior to the sperm–ZP binding test, any remaining cumulus and corona cells or sperm bound to the ZP (from the IVF insemination) were removed by repeated aspiration of the oocyte using a fine glass pipette with an inner diameter 120 µm (Liu et al., 2004).
Sperm samples and preparation
Semen samples were obtained by masturbation after 3–5 days abstinence from 30 infertile male partners with diagnosis of DSZPB. The DSZPB was defined as average <20 sperm bound/ZP after incubation of 2 x 106/ml motile sperm with a group of four oocytes for 2 h [under this experimental condition, fertile men will have consistently >100 sperm bound/ZP, and <40 sperm bound/ZP has been used as the threshold for diagnosis of low ZP binding: DSZPB (Liu et al., 2004; 2007)]. All men with DSZPB had been confirmed by repeated testing of sperm from a different ejaculate. Routine semen analysis results showed that all 30 men had sperm counts >20 x 106/ml and progressive motility >30%, 15 had strict NSM 14% and 15 had severe teratozoospermia (NSM 5%). In our clinic, men diagnosed with DSZPB were recommended for treatment by ICSI regardless of sperm morphology results because of the high risk of low or zero fertilization rates with conventional IVF. However, one couple with unexplained infertility and normal semen analysis had one cycle of conventional IVF with 1 of 16 oocytes fertilized before the man was diagnosed with DSZPB. Seventeen of 30 couples (nine with normal semen and eight with severe teratozoospermia) underwent ICSI treatment with an average of 51% (range 38–75%) of oocytes normally fertilized and developed to usable embryos (transferred and cryopreserved). Clinical characteristics of these couples were: mean age: males: 37 (range 27–54) years, females: 34 (range 24–44) years; duration of infertility: 2.7 (range 1–10) years. Infertility was primary in 23 and secondary in seven men (three with normal semen and four with severe teratozoospermia). Two females had endometriosis and one had tubal disease. Fifteen men had unexplained infertility (normal semen) and 15 men had male factor (severe teratozoospermia) without other features.
Sperm samples from 14 healthy men with normal semen analysis and normal sperm–ZP binding were used to study the dose–response effect of dbcAMP and PMA on sperm-ZP binding and TP. Semen analysis was performed after liquefaction within 1 h of collection according to World Health Organization (1999). Per cent NSM was assessed according to strict criteria on Shorr stained smears under oil immersion with magnification x1000 and bright field illumination (Kruger et al., 1988; World Health Organization, 1999).
Motile sperm were selected by colloidal silica gradient centrifugation (PureSperm, Nidacon International AB, Molndal, Sweden) using two layers of 1 ml of 40% and 1 ml of 80% PureSperm. The pellet of motile sperm obtained from PureSperm was washed once with 1 ml of HTF supplemented with 2% human serum albumin (HSA). The washed sperm pellet was resuspended with HTF medium to a sperm concentration of 2 x 106/ml for the ZP binding test.
All patients signed consent forms permitting use of their gametes (unfertilized oocytes and sperm samples) for research. The Royal Women Hospital Research and Ethics Committees approved the project.
Effect of dbcAMP and PMA on sperm–ZP binding and TP of sperm from men with normal semen analysis and normal sperm–ZP binding
To determine the effect of dbcAMP and PMA on normal sperm–ZP binding capacity and TP, sperm samples from men with normal semen analysis and normal sperm–ZP binding were used. Motile sperm (2 x 105 in 0.5 ml) were incubated with a group of four oocytes for 2 h with a range of concentrations of dbcAMP (0–2 mM) or PMA (0–15 µM). This sperm concentration (2 x 105) was used to avoid saturated binding of sperm to the ZP which might occur with sperm from men with normal semen analysis and normal sperm–ZP binding. After incubation, the oocytes were flushed several times to dislodge loosely adherent sperm using a pipette approximately twice the diameter of the oocyte (250 µm) in three separate wells containing 0.5 ml phosphate buffered saline (PBS) with 0.2% bovine serum albumin (BSA). Since many oocytes had more than 100 sperm bound under these experimental conditions, it was not possible to measure sperm binding accurately by counting sperm bound to the ZP directly under an inverted phase-contrast microscope. Therefore, to allow accurate counting of the number of sperm bound to the ZP, the sperm tightly bound to the four oocytes were then removed by repeated vigorous aspiration using a narrow gauge micropipette with an inner diameter that was slightly smaller than that of the oocyte (120 µm, Liu et al., 2004). This was performed on a glass slide with 2–3 µl PBS containing 0.2% BSA and the detached sperm were smeared in a limited area (4 x 4 mm). The smear was then air-dried and marked with a glass pen on the back of the slide to help find the sperm under the microscope. Before counting, the sperm heads were stained red with 2 µl of eosin Y (0.5% in PBS) added to the smear and covered with a small coverslip (6 x 6 mm). The total number of sperm in the smear was counted and the number of sperm bound/ZP was calculated from the group of four oocytes (Liu et al., 2003).
Sperm in the medium were washed with 10 ml normal saline and centrifuged. The sperm pellet was resuspended in 20 µl saline and then 3 µl of this was smeared on a glass slide for assessment of TP by IF as described below.
Effect of dbcAMP and PMA on sperm–ZP binding in infertile men with defective sperm–ZP binding
As the above dose–response experiments showed that 2 mM dbcAMP and 15 µM PMA had large enhancement effects on sperm–ZP binding, these concentrations were used for testing all samples from infertile men with defective sperm ZP–binding. Motile sperm (2 x 106 in 1 ml medium) selected by PureSperm were incubated with a group of four oocytes with or without (control) dbcAMP or PMA in 4-well culture plates (Nunc, Rosilde, Denmark) for 2 h at 37°C in 5% CO2 in air. After 2 h incubation, each group of four oocytes was transferred to PBS containing 2 mg/ml BSA. The oocytes were then flushed several times in three separate wells containing 0.5 ml PBS with 0.2% BSA to dislodge loosely adherent sperm. The number of sperm bound to each of the four oocytes was counted using an inverted contrast microscope and the average number of sperm bound per ZP was used as end-point. Under these experimental conditions, an average number of 40 sperm bound/ZP is used as the threshold for normal sperm–ZP binding since sperm from fertile men would have >100 sperm bound per ZP (Liu et al., 2004, 2007).
Sperm in the medium were washed with 10 ml normal saline and centrifuged. The sperm pellet was resuspended in 20 µl saline and then 3 µl of this was smeared on a glass slide for assessment of TP by IF as described below.
Assessment of sperm tyrosine phosphorylation
Sperm smears were prepared as described above, dried in air and fixed in 90% ethanol for 30 min to permeabilize the plasma membrane. TP of sperm was assessed by direct IF using the monoclonal antibody PY20 labelled with FITC (Liu et al., 2006). The antibody was diluted 1:300 in PBS containing 10 mg/ml BSA. Each smear (10 x 10 mm) was covered with 50 µl of diluted PY20 and incubated for 4 h in a humidified box at 37°C. After incubation, the slide was washed and mounted with distilled water and covered with a coverslip (22 x 22 mm). Sperm reacting with PY20 antibody had bright fluorescence on the principle piece of the tail, and no fluorescent staining on the sperm head was observed (Liu et al., 2006). The percentage of sperm stained (equivalent to tyrosine phosphorylated sperm) was determined by scoring 200 sperm for each sample using a fluorescence microscope (Liu et al., 2006).
Statistical analysis
Differences in semen analysis results between the two groups of men with NSM 14% and 5% were analysed by the Wilcoxon rank-sum test. The significance of differences in numbers of sperm bound and percentage of sperm with TP at various concentrations of dbcAMP and PMA were examined by non-parametric two-way ANOVA (Friedman) test. Paired t-tests were used for comparison of several variables between test (PKA and PKC activators treatments) and control samples. Correlations between enhanced sperm–ZP binding and enhanced TP of capacitated sperm by dbcAMP were examined by the Spearman test.
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Semen analysis results
Sperm test results for 30 infertile men with DSZPB are summarized in Table I. According to the design of the study, all men had sperm concentrations
20 x 106/ml with reasonable motility. The men were subjectively divided into two groups based on results of NSM in semen. Fifteen men had normal semen analysis (NSM 14%) and a further 15 men had severe teratozoospermia (NSM 5%). There was a significantly higher sperm concentration and motility in men with normal semen analysis than in those with teratozoospermia.
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Enhancement of sperm–ZP binding by dbcAMP and PMA for normal sperm samples with normal sperm–ZP binding
In normal sperm samples with normal ZP binding capacity, incubation of sperm with either dbcAMP or PMA significantly increased the number of sperm bound per ZP in a dose-responsive manner (Figs 1A and 2A). The effect was dose-dependent up to the highest concentrations used: 2 mM dbcAMP and 15 µM PMA, and a maximal effect was not obtained. A similar effect by dbcAMP was observed on TP of sperm in the medium with highest results obtained from 2 mM dbcAMP (Fig. 1B). In contrast, PMA had no effect on TP (Fig. 2B). The concentrations of 2 mM dbcAMP and 15 µM PMA had previously been shown to have no adverse effects on sperm motility (Liu and Baker, 1997
). The biologically inactive phobol ester (4PDD) had no adverse or positive effect on either sperm–ZP binding or ZP-induced AR in our pre-trial experiments, which is similar to previous reports (Tollner et al., 1995; Liu et al., 2002a,b).
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Enhancement of sperm–ZP binding by dbcAMP and PMA in infertile men with defective sperm–ZP binding
Of the 15 DSZPB patients with normal semen analysis (NSM
14%), incubation with PMA increased sperm–ZP binding to normal levels (>40 sperm bound per ZP) in five men (Table II, Fig. 3). In two of these five men, sperm–ZP binding was also increased to normal levels by dbcAMP. The test was repeated on two of the five men with enhanced sperm–ZP binding using sperm from a different ejaculate, and similar results were obtained. However, in all other cases, PMA and dbcAMP had no significant effect on sperm–ZP binding. In all 15 DSZPB patients with normal semen analysis, dbcAMP significantly enhanced TP whereas PMA did not. This dbcAMP-induced enhancement of TP appeared to have no correlation with sperm–ZP binding enhancement (Spearman r = 0.069, P > 0.05).
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In the 15 DSZPB patients with severe teratozoospermia, incubation of sperm with 2 mM dbcAMP or 15 µM PMA did not significantly increase the number of sperm bound to the ZP in any individual. Again, dbcAMP significantly enhanced TP uniformly for all the samples, but PMA had no effect on TP (Table II, Fig. 4).
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Neither dbcAMP nor PMA had any significant effect on the acrosomal status of capacitated sperm. This was true for both men with normal semen analysis and those with severe teratozoospermia (Table II).
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Discussion |
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This study shows that activation of PKA and PKC signal transduction pathways by dbcAMP and PMA significantly enhances sperm–ZP binding capacity in some DSZPB infertile men with normal semen analysis. Sperm ZP–binding capacity was increased to normal levels in 5 of 15 men by PMA and in two of these five men by dbcAMP (Fig. 3). This result suggests that the cause of DSZPB in the five men may include specific defects in pathways upstream of PKA or PKC. However, the majority of normospermic infertile men with DSZPB failed to respond, indicating they may have defects downstream of PKA or PKC pathways, or different defects unrelated to these pathways such as absence of receptors for the ZP on the plasma membrane. This requires further investigation. In contrast, neither dbcAMP nor PMA were able to enhance sperm–ZP binding in any of the 15 DSZPB patients with severe teratozoospermia (NSM
5%). DSZPB in these men may be related to major structural defects particularly in the acrosome region as a structurally normal acrosome is critical for sperm binding to the ZP. Sperm without an acrosome (globozoospermia, small round-headed acrosomeless sperm) are unable to bind to the ZP (Bourne et al., 1995). However, none of the DSZPB men included in this study had this condition. The size of the acrosome in severely teratozoospermic sperm is usually smaller than normal. However, only 30% of men with severe teratozoospermia have DSZPB whereas 70% have normal sperm–ZP binding, but there remains a high frequency (65%) of defective ZP-induced AR in this group (Liu et al., 2007). Thus, the cause of DSZPB may not only be related to acrosomal morphology but may involve other defects, including defective receptors, calcium influx, actin polymerization and signal transduction pathways (Yanagimachi, 1994; Thaler and Cardullo, 1996; Wassarman, 1999; Barratt and Publicover, 2001; Cohen et al., 2004; Liu et al., 2004). Walsh et al. (2008) have recently identified Shaperonin 10, a heat shock protein 1 on the head of capacitated acrosome-intact mouse sperm, and this protein may be involved in mouse sperm–ZP interaction.
For normozoospermic samples with normal sperm–ZP binding, both dbcAMP and PMA significantly enhanced sperm–ZP binding in a dose-responsive manner. Although dbcAMP was also able to enhance TP, PMA was not, suggesting that enhancement of sperm–ZP binding by PMA or dbcAMP may not be due to increased TP. It is known that activation of either or both PKA and PKC pathways can enhance sperm capacitation and subsequently increase the ability of sperm to bind to the ZP. Similar enhancement of sperm–ZP binding capacity by PMA was reported previously in macaque sperm (Tollner et al., 1995). In bovine sperm, it is reported that both PKA and PKC pathways can crosstalk, and activation of either or both pathways can lead to increased intracellular calcium, actin polymerization and activation of phospholipase D (Cohen et al., 2004). However, it remains uncertain how these agents increase sperm–ZP binding.
In human sperm, we found that PMA-induced acrosomal ruffling probably via an increase in actin polymerization and this was associated with exposure of actin on the sperm head (Liu et al., 2002b, 2006). Furthermore, the proportion of sperm with actin detectable on the surface was highly correlated with sperm–ZP binding capacity (Liu et al., 2006). Thus, actin polymerization appears to be an important part of the capacitation events and may be a useful indicator of sperm–ZP binding capacity. However, actin exposure on sperm is unlikely to be one of the co-receptors directly involved in sperm binding to the ZP, as neither an anti-actin monoclonal antibody nor actin polymerization inhibitors (cytochalasin B or D) block sperm binding to the ZP (Liu et al., 2002a).
Previous studies showed that activation of the PKC pathway by PMA can induce the AR of human sperm in vitro as assessed by a modified triple stain, but effects were usually very small (De Jonge et al., 1991). When we used electron microscopy and PSA-FITC to examine the AR of sperm, we found that treatment with PMA did not significantly induce the AR of sperm in medium in either this or our previous study (Liu et al., 2002b). However, several studies showed that PMA significantly enhanced the ZP-induced AR of sperm in the mouse, macaque monkey and human (Lee et al., 1987; Tollner et al., 1995; Liu and Baker, 1997; Liu et al., 2002b). PMA is very effective at inducing acrosomal ruffling via activation of actin polymerization and the proportion of sperm with PMA-induced acrosomal ruffling is highly correlated with PMA-enhanced ZP-induced AR of human sperm (Liu et al., 2002b). In this study, all the patients had DSZPB, and therefore there were insufficient sperm bound to the ZP to study the effects of PMA or dbcAMP on the ZP-induced AR.
All 30 DSZPB patients with either NSM or abnormal sperm morphology had very low TP under capacitating conditions, but all exhibited significant enhancement of TP by dbcAMP. However, this did not lead to enhanced sperm–ZP binding for any of the men with teratozoospermia, or for most of the men with normal semen. In contrast, PMA did not enhance TP, but enhanced sperm–ZP binding in some cases of DSZPB with NSM. Thus, low levels of TP in DSZPB with either NSM or abnormal sperm morphology may be the consequence of sperm dysfunction, but it is unlikely to be the primary cause of DSZPB. These results may also indicate that TP is unlikely to be directly involved in sperm binding to the ZP, as TP in human sperm is mainly detectable by IF in the principle pieces of the sperm tail and not on the sperm head (Liu et al., 2006). However, the level of TP of capacitated sperm is highly correlated with increased sperm motility and hyperactivation (Visconti and Tezon, 1989; Visconti and Kopf, 1998; Turner et al., 1999; Bajpai and Doncel, 2003) and this may explain why TP of capacitated sperm is significantly correlated with sperm–ZP binding capacity in both humans and mice (Turner et al., 1999; Sakkas et al., 2003; Asquith et al., 2004; Buffone et al., 2005; Liu et al., 2006). TP is regulated by cAMP and is a useful marker for sperm capacitation and normal sperm function (Visconti and Tezon, 1989; Galantino-Homer et al., 1997; Visconti and Kopf, 1998). As protein TP involves several protein kinases, immunofluorescent staining with the PY20 antibody does not discriminate between different proteins, but is limited to displaying TP in general. Recently, the protein tyrosin kinase SRC has been identified in both head and tail of capacitated and acrosome-intact human sperm and it is possible that SRC may be potentially involved in sperm–ZP interaction (Lawson et al., 2008; Mitchell et al., 2008).
This study shows that PMA and dbcAMP can significantly enhance sperm–ZP binding in some patients with DSZPB and normal semen analysis. However, it is unclear if this would be of therapeutic value in clinical ART, as PKC activators, particularly PMA, are not suitable for use in vivo and agents which act on PKA such as caffeine and pentoxifylline may be ineffective. Nevertheless, diagnosis of DSZPB in infertile men before commencing ART treatment would be very useful to assist clinical assignment of patients to ICSI instead of IVF, particularly those with normal semen analysis. Patients with DSZPB will have very poor fertilization results in conventional IVF as their sperm have very low or no chance to penetrate the ZP (Liu et al. 2004, 2007). In unexplained infertile men with normozoospermic semen, 15% are diagnosed with DSZPB as the cause of their infertility (Liu et al., 2007).
In conclusion, the presence of DSZPB in some infertile men with NSM may be due to defective signal transduction pathways upstream of PKA and PKC. However, most DSZPB infertile men with normal semen, and all those associated with severe teratozoospermia, are likely to have downstream disorders, structural defects or absence of sperm receptors for binding the ZP. TP is an indicator of both normal sperm function and capacitation, but it does not play a major role in the process of sperm–ZP binding, as significant enhancement of TP has no impact on sperm–ZP binding capacity in DSZPB men with primary low TP.
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This study was supported by the National Health and Medical Research Council with a grant number 400069.
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The authors thank all the scientists in both Royal Women's Hospital and Melbourne IVF Laboratories for collecting the oocytes, scientists in the Andrology Laboratory for sperm samples and Dr Claire Garrett for her kind assistance in editing the manuscript.
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References |
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Submitted on May 12, 2008; resubmitted on August 8, 2008; accepted on August 12, 2008.
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