Hum. Reprod. Advance Access originally published online on January 20, 2006
Human Reproduction 2006 21(4):1002-1008; doi:10.1093/humrep/dei435
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Tyrosine phosphorylation on capacitated human sperm tail detected by immunofluorescence correlates strongly with sperm–zona pellucida (ZP) binding but not with the ZP-induced acrosome reaction
1 Department of Obstetrics and Gynaecology, University of Melbourne, 2 Andrology Laboratory and 3 Reproductive Services, Royal Women’s Hospital, 4 Melbourne IVF, Melbourne, Australia
5 To whom correspondence should be addressed. E-mail: dyl{at}unimelb.edu.au
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Abstract |
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BACKGROUND: Protein tyrosine phosphorylation (TP) of human sperm is related to sperm capacitation and zona pellucida (ZP) binding. The aim of this study was to determine whether the TP of capacitated sperm is a useful marker for the ability of sperm to bind to the ZP and undergo the ZP-induced acrosome reaction (AR). METHODS: Semen samples were obtained from 115 subfertile men with sperm count
20 x 106/ml, motility 25% and variable morphology. Motile sperm (2 x 106/ml) selected by swim-up were incubated with four oocytes for 2 h, and the number of sperm bound to the ZP and the ZP-induced AR was examined. TP of sperm tail was assessed by immunofluorescence (IF) with anti-phosphotyrosine monoclonal antibody. The time course and effects of dibutyryl cyclic adenosine monophosphate (dbcAMP) and phorbol myristate acetate (PMA) on TP were also studied. RESULTS: TP was stimulated more by dbcAMP (P < 0.001) and less by PMA (P < 0.05). TP increased significantly with time of incubation of sperm. TP was not detectable on the surface of unfixed live sperm by either Dynabeads or IF. Sperm TP at 2, 4 and 20 h incubation was all significantly correlated with sperm–ZP binding but not with the ZP-induced AR. CONCLUSION: Sperm TP detected by IF correlates strongly with sperm–ZP binding capacity but not with the ZP-induced AR. This simple IF assay of TP may be a clinically useful test of sperm function that is predictive of normal sperm ZP-binding capacity.
Key words: immunofluorescence/sperm–zona binding/tyrosine phosphorylation/zona-induced acrosome reaction
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Introduction |
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Human sperm require capacitation before they are able to interact with oocytes during the process of fertilization (Yanagimachi, 1994
). Protein tyrosine phosphorylation (TP) occurs during capacitation of human spermatozoa. Although the mechanism of sperm capacitation is currently not fully understood, it is known to involve a complex of molecular and biochemical events such as membrane cholesterol efflux, calcium influx, increase in bicarbonate uptake, increased membrane fluidity, hyperactivated motility and activation of cyclic adenosine monophosphate (cAMP) pathways resulting in protein TP (Leclerc et al., 1996; Galantino-Homer et al., 1997; Visconti and Kopf, 1998; Nassar et al., 1999; Urner and Sakkas, 2003). The TP is regulated by protein kinase A (PKA), and incubation of sperm with dibutyryl cAMP (dbcAMP) in vitro significantly promotes TP (Visconti and Kopf, 1998). With immunofluorescence (IF), human sperm TP was mainly identified in the principal piece of the tail (Sakkas et al., 2003; Buffone et al., 2005). In the mouse, early studies showed that only a small proportion of sperm (<10%) displayed TP in the acrosome region, and this did not change with capacitation (Leyton and Saling, 1989; Leyton et al., 1992; Urner et al., 2001). Furthermore, most mouse sperm bound to the zona pellucida (ZP) had no TP detected by IF in the acrosome region. More recently, Asquith et al. (2004) showed that almost all sperm bound to the mouse ZP had TP in the acrosome region, and they believe that TP may play a crucial role in assembly of the ZP receptors on the sperm surface via activation of chaperone proteins. Buffone et al. (2005) reported that reduced TP in capacitated sperm was associated with the poor motility in asthenozoospermic men. Sakkas et al. (2003) showed that human sperm TP assessed by IF was related to sperm–ZP binding and the ability of sperm to fertilize oocytes in vitro. Brewis et al. (1998) reported that TP plays an important role in human acrosome reaction (AR) induced by recombinant human ZP3. Therefore, it is possible that assessment of TP in sperm using simple IF in vitro may provide an assay highly predictive of the ability of sperm to bind to the ZP and undergo the ZP-induced AR—processes critical for human fertilization.
Human sperm binding to the ZP was one of the most important indicators for sperm fertilizing ability (Liu and Baker, 1992). In conventional IVF, defective sperm–ZP interaction was a major cause of failure of fertilization in patients with 0 or <25% fertilization rates (Liu and Baker, 2000). Although some patients with defective sperm–ZP interaction have obvious sperm abnormalities such as severe oligozoospermia, asthenozoospermia or teratozoospermia alone or in combination (Liu et al., 2004), others have normal semen analysis with either abnormal sperm–ZP binding or normal sperm–ZP binding, but defective ZP-induced AR which then leads to failure of sperm–ZP penetration (Liu and Baker, 1994). In the human ejaculate with an average of about 300 x 106 sperm with 50% motility, only about 14% of motile sperm are capable of interacting with the ZP of human oocytes in vitro (Liu et al., 2003). This means that the majority of motile sperm in normal semen do not have the capacity to fertilize, as they are unable to interact with the oocyte.
Currently, assessment of sperm–ZP binding requires human oocytes since no substitute for the human ZP is available. Recombinant human ZP3 protein has been produced by several independent groups, but it has insufficient biological activity for either binding sperm or stimulation of the human AR (van Duin et al., 1994; Brewis et al., 1996; Whitmarsh et al., 1996; Dong et al., 2001; Martic et al., 2004). While human oocytes remain essential for testing sperm–ZP interactions, they are very limited in supply and not available for routine clinical tests. Therefore, an alternative test for prediction of these aspects of sperm function would have great advantages. In this study, we investigated the relationship of sperm TP assessed by IF and both sperm–ZP binding and the ZP-induced AR in large number of subfertile men to evaluate the clinical potential of human sperm TP assessment.
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Chemicals and culture medium
Human tubal fluid (HTF, Irvine Scientific, Irvine, CA, USA) supplemented with 10% (v/v) inactivated human serum was used as culture medium for all the experiments. Human serum and anti-phosphotyrosine monoclonal antibody [PY20, mouse immunoglobulin (Ig)G] labelled with fluorescein isothiocyanate (FITC) were obtained from ICN Pharmaceuticals (Costa Mesa, CA, USA). Dynabeads coated with anti-mouse IgG (anti-mIgG) were purchased from Dynal (Oslo, Norway). Phorbol 12-myristate 13-acetate (PMA), dbcAMP, dimethylsulphoxide, phosphotyrosine antibody inhibitor, a solution of O-phospho-L-tyrosine conjugated to bovine serum albumin (BSA) (specific inhibitor of the reactivity of anti-phosphotyrosine antibody) and Pisum sativum agglutinin conjugated with FITC (PSA–FITC) were purchased from Sigma Chemical Company (St Louis, MO, USA).
Human oocytes
Oocytes for the ZP interaction tests were obtained from the clinical IVF program that showed no evidence of two or three pronuclei or cleavage at 48–60 h after insemination or were immature (with germinal vesicle). Oocytes with any remaining cumulus and corona cells or sperm bound to the ZP from the IVF insemination had these removed by repeated aspiration of the oocyte using a fine glass pipette with an inner diameter (120 µm) slightly smaller than the oocyte diameter (Liu and Baker, 1994, 1996). Degenerate, activated or morphologically abnormal oocytes as well as oocytes with more than 10 sperm penetrating the ZP were not used for the test. All the oocytes were obtained on day 3 of insemination, and the oocytes were pooled from several patients and used for the test on the same day or kept in the 5% CO2 incubator and used within 3 days.
Sperm samples and preparation
Semen samples were obtained by masturbation after 3–5 days abstinence from men who had sperm count 20 x 106/ml, motility 25% with progressive motility >20% and variable normal sperm morphology. Sperm count and motility were performed after liquefaction within 1 h of collection of semen according to World Health Organization (1999). Per cent normal sperm morphology 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 swim-up technique as follows: 0.3 ml of semen was carefully added to the bottom of a test tube (12 x 75 mm) containing 0.7 ml HTF supplemented with 10% human serum. Care was taken to avoid bubbles and not disturb the interface between semen and the medium. After incubation for 1 h, 0.5 ml of the top layer of the medium containing motile sperm was aspirated. The motile sperm suspension was then centrifuged at 1000g for 5 min, the supernatant removed and the sperm pellet washed again with 1 ml fresh HTF by centrifugation at 1000g for 5 min. The washed sperm pellet was re-suspended with serum-supplemented standard HTF to a motile sperm concentration of 20 x 106/ml for subsequent experiments. Because sperm samples were from men with normal sperm concentration and reasonable motility, progressive motility of sperm after swim-up preparation was >90% for all the samples used in this study.
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.
Sperm–ZP binding
Motile sperm (2 x 106 in 1 ml medium) selected by swim-up were incubated with four oocytes in 4-well culture plates (Nunc, Roskilde, Denmark) for 2 h at 37°C in 5% CO2 in air. After 2 h incubation, each group of four oocytes was transferred to phosphate-buffered saline (PBS) containing 2 mg/ml BSA (Commonwealth Serum Laboratory, Parkville, Victoria, Australia). 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 PBS with 0.2% BSA. The number of sperm bound to each of four oocytes was counted using an inverted contrast microscope, and an average number of sperm bound per ZP was used as end-point. Under this experimental condition, an average number of sperm bound 
40/ZP was considered as poor sperm–ZP binding since sperm from fertile men could have >100 sperm bound per ZP (Liu et al., 2001).
The ZP-induced AR
For sperm samples with normal ZP-binding (average >40 sperm/ZP), all sperm bound to surface of the four ZPs were removed by repeated vigorous aspiration with a narrow gauge pipette with an inner diameter (approximately 120 µm) slightly smaller than the oocyte (Liu and Baker, 1994, 1996). This was performed on a glass slide with about 5 µl PBS containing 0.2% BSA, and the removed ZP-bound sperm were smeared in a limited area (about 16 mm2) that was marked on the back of the slides with a glass pen to help find the sperm under the microscope. This pipetting procedure for removing sperm from the surface of ZP does not affect sperm motility, morphology and acrosome status (Liu and Baker, 1996).
Assessment of acrosome status of sperm bound to the ZP
The AR was assessed using PSA–FITC as described previously (Cross et al., 1986; Liu and Baker, 1996). Sperm smears were fixed in 95% ethanol for 30 min after air-drying and then stained using 25 µg/ml PSA–FITC in PBS for 2 h at 4°C. The slides were washed and mounted with distilled water, and 200 sperm per sample were counted with a fluorescence microscope using excitation wavelengths of 450–490 nm and a magnification of x400. When more than half the head of a sperm was brightly and uniformly fluorescing, the acrosome was considered intact. Sperm with a fluorescing band at the equatorial segment or without fluorescence in acrosome region were considered reacted.
Localization of TP of human sperm
Motile sperm (2 x 106 in 1 ml medium) selected by swim-up were incubated for 2 h (some samples were also incubated for 4 and 20 h) at 37°C in 5% CO2 in air. After incubation, sperm were washed with 10 ml normal saline, and the sperm pellet was re-suspended in about 20 µl saline and 3 µl smeared on a glass slide. After drying in air, the smear was fixed in 90% ethanol for 30 min to permeabilize the plasma membrane. The fixed slides were stored in a box at 4°C for no more than 5 days. TP of sperm was assessed by direct IF using the monoclonal antibody PY20 (ICN Pharmaceuticals) labelled with FITC. The antibody is diluted 1 : 300 in PBS containing 10 mg/ml BSA. Each smear (approximately 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 principal piece of the tail. The percentage of sperm with positive stain (tyrosine phosphorylated sperm) was determined by scoring 400 sperm for each sample using a combination of light and fluorescent microscopy (Figure 1). No fluorescent labelling was detected on the sperm heads with the anti-tyrosine monoclonal antibody (PY20).
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Antibody specificity was confirmed by control tests demonstrating that phosphotyrosine antibody inhibitor (10 µg/ml) could block the binding of PY20–FITC to the sperm tails. In addition, mIgG–FITC used as an extra control for non-specific binding did not show fluorescence on any of the sperm.
Detection of TP on the surface of live motile sperm
Both IF and Dynabeads coated with anti-mIgG (the second antibody) were used to probe for the binding of the primary anti-tyrosine antibody (PY20) to live sperm to determine whether phosphotyrosine was detectable on the surface of unfixed motile sperm after incubation. For this, sperm were not fixed in 90% ethanol to permeabilize the plasma membrane and allow the anti-tyrosine antibody access to internal TP. The Dynabeads were washed twice with 5 ml HTF–BSA medium and re-suspended in the same medium at a concentration of about 4 x 108/ml before use.
To increase the chances of detection of TP on the surface of motile sperm, sperm were pre-incubated for 2 h in medium with 1 mM dbcAMP to increase the proportion of TP sperm, and then sperm were further incubated for another 1 h after addition of 10 µl (1 : 100) PY20 (for Dynabead test) or PY20–FITC antibody (for IF test). The sperm pellets were then recovered and washed three times with 10 ml HTF–BSA medium to remove excessive primary antibody. The sperm pellet was re-suspended to about 40 x 106/ml with HTF–BSA medium. For the Dynabead test, 5 µl sperm suspension was mixed with 5 µl of Dynabeads and covered with a coverslip (22 x 22 mm). The percentage of motile sperm with one or more beads bound to the head or tail was counted by scoring 400 motile sperm using a phase-contrast microscope at magnification of x400. For the IF test, 5 µl of sperm suspension was added on a glass slide and covered with a coverslip (22 x 22 mm). The proportion of sperm reacting with PY20–FITC was assessed by scoring 400 sperm using fluorescence microscopy.
To compare the results of Dynabead and IF on unfixed live sperm with the results of IF on fixed sperm, the same sperm suspension was smeared on a glass slide and then fixed in 90% ethanol for 30 min to permeabilize plasma membrane, and then direct IF was performed using PY20–FITC as described above.
Effect of incubation time, dbcAMP and PMA on sperm TP
To determine whether direct IF is sensitive enough to detect changes of TP, motile sperm (1 x 106) were incubated for various times (0, 2, 4, 6 and 20 h) or with various concentrations of either PMA (0–15 µM) or dbcAMP (0–2 mM) for 2 h. After incubation, sperm were washed in 10 ml normal saline, smeared on a slide, fixed, and TP was assessed by IF as described above.
Statistical analysis
The significance of differences in the percentage TP sperm at various times of incubation, or concentrations of dbcAMP and PMA, was examined by nonparametric analysis of variance (ANOVA) (Friedman) test. Correlations between the proportion of motile sperm with TP and other sperm characteristics were examined by the nonparametric (Spearman) test. Regression analysis was performed to determine the significance of relationships between sperm test results and percentages of sperm with TP.
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Sperm test results
Semen test results are summarized in Table I. There was a wide range of results for all the tests including TP. Sperm TP was detected by IF only on the principal piece of the tail of sperm fixed in 90% ethanol to permeabilize the plasma membranes. No surface TP was detected on motile sperm by either Dynabead (0%) or IF (0%) even after incubation of sperm with 1 mM dbcAMP to enhance TP. In contrast, IF on fixed sperm showed 45 ± 13% (mean ± SD) (range 31–68%) were positive as assessed in 10 sperm samples from different men with normal semen analysis and normal sperm–ZP binding.
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Effect of incubation time, dbcAMP and PMA on sperm TP
Prolonged incubation of sperm significantly enhanced TP (Figure 2). The dbcAMP significantly increased TP of sperm in a dose–responsive manner (Figure 3). In contrast, PMA had relatively little effect on TP (Figure 3).
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Relationships between TP and other sperm characteristics
The sperm TP at 2 h incubation was significantly correlated with sperm–ZP binding but not with the ZP-induced AR (Figure 4). In 44 sperm samples, TP was assessed after incubation of sperm for 2, 4 and 20 h, and TP results of all the three incubation times were significantly correlated with sperm–ZP binding (Spearman r = 0.771, 0.738 and 0.519, all P < 0.001). However, TP at 2 and 4 h incubation gave the highest correlations.
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The percentages of sperm with normal morphology in both semen (n = 115, Spearman r = 0.359, P < 0.001) and swim-up preparations (n = 115, Spearman r = 0.298, P = 0.001) were significantly correlated with TP of sperm at 2 h incubation. Also, sperm morphology in semen (n = 79, Spearman r = 0.338, P = 0.003) and swim-up (n = 79, Spearman r = 0.353, P = 0.002) was also significantly correlated with the ZP-induced AR. The other sperm characteristics including sperm count, motility and viability were not significantly correlated with either sperm–ZP binding or sperm TP.
When all the data were analysed by multiple regression, only sperm count (P < 0.05) and TP of sperm at 2 or 4 h (P < 0.001) were significantly related to sperm–ZP binding.
Comparison of TP results between men with normal and abnormal sperm–ZP binding
Sperm samples with normal sperm–ZP binding had significantly higher TP than those with abnormal sperm–ZP binding (11 ± 9 versus 2 ± 3.8%, P < 0.001). Over 92% (49 of 53) of men with TP 5% at 2 h had normal sperm–ZP binding. In contrast, only 66% (41 of 62) of men with TP <5% had normal sperm–ZP binding. Thus, 2 h TP <5% had reasonable sensitivity (84%) and negative predictive value (94%) but low specificity (54%) and positive predictive value (34%) for abnormal sperm–ZP binding capacity.
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The present study shows that TP is highly correlated with sperm–ZP binding ability but not with the ZP-induced AR. These results suggest that TP is a good indicator of the capacity of sperm to bind to the ZP. The majority (>92%) of sperm samples with
5% TP of sperm at 2 h incubation had normal sperm–ZP binding (Figure 4), and most of those with very low TP (0 –1%) had poor sperm–ZP binding 85% (28 of 33). However, TP appears to be less accurate for prediction of abnormal sperm–ZP binding: about half the men with low TP have normal sperm–ZP binding capacity. Assessment of sperm TP by simple IF may be a useful routine test that is predictive of normal sperm–ZP binding ability: men with TP on >5% of sperm tails after 2 h incubation are likely to have normal sperm–ZP binding capacity. The monoclonal antibody PY20–FITC is commercially available, and the procedure is simple, cheap and could be performed in any clinical Andrology Laboratory with a fluorescence microscope. Addition of the TP test to the routine semen analysis could improve clinical diagnosis of sperm defects and management of patients by assisted reproduction techniques. For example, patients with unexplained infertility or mildly abnormal semen analyses with a normal female partner are difficult to advise whether they should be treated by conventional IVF or ICSI. In such patients, additional screening of sperm TP may help predict which patients will have normal sperm–ZP binding who can be treated by IVF. On the other hand, those with low TP need a sperm–ZP binding test to decide whether they should be treated with ICSI. Because human oocytes are difficult to obtain for routine testing, the TP test might allow this limited resource to be better applied.
Sperm normal morphology in semen and insemination medium was significantly related to TP. Sperm morphology is also correlated with the ZP-induced AR. These results confirm that sperm morphology is one of the most important sperm characteristics related to sperm fertilizing ability (Liu et al., 2003). However, prediction of sperm fertilizing ability either in vivo or in vitro by sperm morphology alone is still not very accurate. Testing sperm–ZP binding improves prediction of sperm fertilizing ability (Liu et al., 2004). As we reported previously, defective ZP binding and penetration were the most frequent sperm causes for failure of fertilization with standard IVF (Liu and Baker, 2000).
Regulation of TP in sperm and changes during sperm capacitation have been extensively studied previously (Visconti and Kopf, 1998; Urner and Sakkas, 2003). Although TP is mainly regulated by protein kinase A, many other factors such as membrane cholesterol efflux, increase in intracellular bicarbonate and calcium and activation of phosphokinase are also involved (Visconti and Kopf, 1998; Nassar et al., 1999; Urner and Sakkas, 2003). The effect of TP on mouse sperm–ZP binding and the ZP-induced AR was examined using kinase inhibitors (Leyton and Saling, 1989). The data obtained by IF assay in the present study provide further support that TP is dependent on time of incubation of sperm and also that TP is highly regulated by PKA which is enhanced by dbcAMP. PMA had less effect on TP suggesting that protein kinase C is not directly involved in the regulation of TP. The experiments with these agents were performed to confirm that the direct IF assay is sensitive enough to detect changes of TP during sperm capacitation. Although the time-course study showed that all the three incubation times (2, 4 and 20 h) were significantly correlated with sperm–ZP binding, the TP of sperm at 2 or 4 h incubation was the most significant. In this study, sperm–ZP binding was performed by co-incubation of sperm with the oocytes for only 2 h; therefore, TP of sperm at 2 h incubation was chosen for the main part of the study. Also a shorter time of incubation is more desirable for a routine clinical test.
In this study, we did not detect any TP on the surface of live sperm after incubation by either Dynabead or IF assays. However, Sakkas et al. (2003) showed TP in the heads of a few sperm. In contrast, in other mammalian sperm such as mice, TP was identified on the surface of the heads of unfixed live sperm and nearly all sperm bound to the ZP of mouse oocytes (Asquith et al., 2004). It is possible that there are species differences or the level of TP on the human sperm head surface may be too low to be detected by IF. In the literature, most studies with IF showed that TP was mainly in the principal piece of the tail of human spermatozoa after fixation to permeabilize the membrane.
In conclusion, the proportion of TP sperm at 2 or 4 h incubation was highly correlated with sperm–ZP binding capacity but not with the ZP-induced AR. A simple IF assay for TP may be a useful clinical test of sperm function. If the tails of >5% of the sperm are positive, the man is likely to have normal sperm–ZP binding capacity. This may assist clinical decision-making as to the use of IVF or ICSI. However, a caution is that the TP test does not predict the ability of sperm to undergo the ZP-induced AR. The clinical value of the TP test and the underlying mechanism of the relationship between TP and sperm–ZP binding require further study.
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The authors thank Mingli Liu for technical assistance, the scientists in IVF Laboratories for collecting the oocytes and the scientists in the Andrology Laboratory for collecting sperm samples.
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Submitted on September 7, 2005; resubmitted on November 13, 2005; accepted on November 16, 2005.
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P.-h. Hung, M. G. Miller, S. A. Meyers, and C. A. VandeVoort Sperm Mitochondrial Integrity Is Not Required for Hyperactivated Motility, Zona Binding, or Acrosome Reaction in the Rhesus Macaque Biol Reprod, August 1, 2008; 79(2): 367 - 375. [Abstract] [Full Text] [PDF]
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Z. Zhang, X. Shen, B. H Jones, B. Xu, J. C Herr, and J. F Strauss III Phosphorylation of Mouse Sperm Axoneme Central Apparatus Protein SPAG16L by a Testis-Specific Kinase, TSSK2 Biol Reprod, July 1, 2008; 79(1): 75 - 83. [Abstract] [Full Text] [PDF]
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 L.A. Mitchell, B. Nixon, and R.J. Aitken Analysis of chaperone proteins associated with human spermatozoa during capacitation Mol. Hum. Reprod., September 1, 2007; 13(9): 605 - 613. [Abstract] [Full Text] [PDF]
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D. Y. Liu, M. L. Liu, C. Garrett, and H.W. G. Baker Comparison of the frequency of defective sperm-zona pellucida (ZP) binding and the ZP-induced acrosome reaction between subfertile men with normal and abnormal semen Hum. Reprod., July 1, 2007; 22(7): 1878 - 1884. [Abstract] [Full Text] [PDF]
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