1 Department of Obstetrics and Gynecology, University of Melbourne, 2 Reproductive Services and 3 Andrology Laboratory, Royal Women's Hospital and 4 Melbourne IVF, Melbourne, Australia
5 To whom correspondence should be addressed. Email: dyl{at}unimelb.edu.au
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Abstract |
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Key words: actin/capacitation/morphology motility/spermZP binding
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Introduction |
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Actin is a major cytoskeletal protein present in all mammalian cells which plays an important role in regulating cell shape, migration and cellular interaction with extracellular matrices through reversible transformations between monomeric G-actin and filamentous F-actin (Cooper, 1991; Aderem, 1992
). In human sperm, actin has been identified in the acrosome, post-acrosomal area, neck and principal piece of the tail (Clarke et al., 1982
; Virtanen et al., 1984
; Ochs and Wolf, 1985
; Fouquet and Kann, 1992
). Castellani-Ceresa et al. (1993)
showed that actin is mainly in the monomeric form in uncapacitated boar sperm and that it polymerizes to filamentous actin after capacitation. In several other mammalian species including humans, actin polymerization in sperm plays an important role during capacitation and the acrosome reaction (AR) (Brener et al., 2003
; Cabello-Agueros et al., 2003
). Inhibition of actin polymerization with cytochalasin D blocks guinea-pig and human sperm penetration into zona-free hamster oocytes (Rogers et al., 1989
). Castellani-Ceresa et al. (1993)
also found that cytochalasin D inhibits boar fertilization in vitro.
In endocrine and secretory cells, the actin network plays an important role in the regulation of exocytosis (Koffer et al., 1990; Burgoyne et al., 1991
; Dudani and Ganz, 1996
). Our previous work using dual-fluorescent stains for actin and acrosomal contents on the same human sperm showed that actin was abundant in the acrosomal region and that it was lost following the AR (Liu et al., 1999
). Blocking actin polymerization in human sperm with cytochalasin B or D or an anti-actin monoclonal antibody (A-mAb) inhibited the ZP-induced AR (Liu et al., 1999
, 2002
). From such evidence it is suggested that actin plays an important role in the human acrosome reaction (Spungin et al., 1995
; Liu et al., 1999
).
In a previous study of 17 sperm samples we found that 10% of motile sperm had exposed actin detectable by second antibody-coated beads binding to an A-mAb after 2 h incubation in vitro and that there was wide variation between individual sperm samples (Liu et al., 2002
). Immunofluorescence localization of the A-mAb in live capacitated sperm was in the equatorial segment and also, to a lesser extent, the anterior part of the acrosome. The A-mAb does not affect spermZP binding but significantly reduces the ZP-induced AR after binding to the ZP. It is possible that exposure of actin on the surface of the sperm head is an integral part of the process of membrane modification during sperm capacitation or initiation of the AR. Therefore, assessment of the proportion of motile sperm exposing actin during in vitro culture may be a useful marker for sperm capacitation or other aspects of sperm fertilizing ability. In this study, we have used A-mAb and Dynabeads coated with a second anti-IgG antibody to determine the proportion of sperm exposing actin during in vitro culture and have related this to other sperm characteristics and spermZP binding ability. We also varied conditions known to affect capacitation. We tested sperm in semen which were uncapacitated, or after washing and exposure to low bicarbonate to inhibit capacitation (Boatman and Robbins, 1991
) or addition of phorbol myristate acetate to increase capacitation (Furuya et al., 1993
; Zhang and Baker, 1997).
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Materials and methods |
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Human oocytes
Oocytes were used that showed no evidence of two or three pronuclei or cleavage at 4860 h after insemination in conventional IVF. 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 small glass pipette with an inner diameter (120 µm) slightly smaller than the oocyte diameter. Only morphologically normal and unfertilized oocytes with <10 sperm penetrating the ZP (from IVF insemination) were used for the tests. Degenerated, morphologically abnormal and spontaneously activated oocytes were not used.
Sperm samples and preparation
Semen was obtained by masturbation after 23 days of abstinence from 79 men who attended the infertility clinic for fertility assessment and had a sperm concentration >20 x 106/ml. Semen analysis was performed according to World Health Organization (1999) criteria using strict sperm morphology (Kruger et al., 1988
). Motile sperm were selected by a swim-up technique with 1 h incubation as described previously (Liu and Baker, 1996a
,b
). The motile sperm were washed with 2 ml fresh HTFBSA medium by centrifugation and then the sperm pellet was resuspended in fresh HTFBSA medium to a sperm concentration of 2 x 106/ml for subsequent experiments.
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.
SpermZP interaction test
Motile sperm (2 x 106 in 1 ml medium) selected by swim-up were incubated with four oocytes in 4-well culture plates (Nunc, Denmark) for 2 h at 37°C in 5% CO2 in air. After this 2 h incubation period, each group of four oocytes was transferred to phosphate-buffered saline (PBS) containing 2 mg/ml BSA. The oocytes were then washed by aspiration with a wide-bore glass pipette (inside diameter 250 µm) to dislodge sperm loosely adherent to the surface of the ZP. The number of sperm bound tightly to the ZP was counted and recorded. Oocytes with >100 sperm bound were recorded as 100 since it is impossible to count the number accurately when it is >100. Binding of >100 sperm per ZP indicates normal ZP binding. The average number of sperm bound to four oocytes per test was used for statistical analysis.
All sperm bound to the surface of the ZP of the four oocytes were then removed by repeated vigorous aspiration with a narrow-bore glass pipette with an inner diameter (120 µm) slightly smaller than that of the oocyte (Liu and Baker, 1996a
,b
). This procedure was performed on a glass slide with
35 µl PBS containing 0.2% BSA and the dislodged sperm were then smeared in a limited area (
4 x 4 mm) of the slide which was marked with a diamond pen to help find the sperm under the microscope for acrosome assessment. After the pipetting there were usually no sperm remaining bound on the surface of the ZP. Our previous studies confirmed that this pipetting procedure does not affect acrosome status or damage the sperm, as most of them remain motile after removal from the ZP (Liu and Baker, 1994
, 1996a
).
In the above spermZP binding experiments, there was no A-mAb added to the insemination medium. Parallel experiments for bead binding on the same sperm samples were performed as described below.
Assessment of acrosome status
The acrosome status of sperm which had been bound to the ZP was determined with fluorescein-labelled Pisum sativum agglutinin (PSA; Sigma Co., USA) as described previously (Cross et al., 1986; Liu and Baker, 1996a
). Sperm smears were fixed in 95% ethanol for 30 min after air-drying and then stained using 25 µg/ml PSA 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 450490 nm and oil immersion at 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 (a rare pattern) were considered acrosome-reacted.
Anti-mouse IgG Dynabead test
Dynabeads coated with anti-mouse IgG were used to detect A-mAb on the surface of sperm in the culture medium and after removal from the ZP. The Dynabeads were washed twice with 5 ml HTFBSA medium and resuspended in the same medium at a concentration of 4 x 108/ml.
To determine the effect of time of incubation on the exposure of actin on the surface of sperm, motile sperm (2 x 106/ml) were incubated with A-mAb (1:100) for 0, 30 min, 2 and 4 h. The sperm pellets were recovered and washed with 10 ml HTFBSA medium three times. The sperm pellet was resuspended to 20 x 106/ml with HTFBSA medium. Finally, 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 was counted by scoring 400 motile sperm using a phase-contrast microscope at magnification of x400 (Figure 1).
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Effect of sperm capacitation conditions on sperm exposing actin
To determine whether capacitation state could affect the proportion of sperm exposing actin on the surface, motile sperm were incubated for 2 h in HTFBSA medium containing 1:100 A-mAB with (test) or without (control) 15 µmol/l PMA (Furuya et al., 1993), or in bicarbonate-free medium (HTFHEPES) to inhibit capacitation (Boatman and Robbins, 1991
). The proportions of sperm with exposed actin were assessed and compared between the test and control groups. Uncapacitated sperm in semen (0.1 ml) were also directly incubated in medium (0.5 ml) containing 1:100 A-mAb for 2 h and the proportion of sperm with exposed actin was assessed.
Statistical analysis
The significance of the differences between sperm with exposed actin under the various conditions was examined by non-parametric (Friedman and Wilcoxon rank sum) tests. Correlations between the proportion of motile sperm with exposed actin and sperm characteristics were examined by the non-parametric Spearman test. Multiple regression analysis was performed to determine which of the sperm test results were most significantly associated with the proportion of sperm with exposed actin.
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Results |
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Time-course of A-mAb binding to human sperm
For all the experiments, motile sperm were preincubated for 1 h during swim-up preparation and also two subsequent washes before exposing sperm to the A-mAb. The time-course study showed that increasing the time of incubation of sperm with the A-mAb up to 2 h increased the percentage of sperm which bound beads. However, prolonging the incubation to 4 h did not result in any further increase in bead binding (Figure 2). Similar results were also obtained when sperm were incubated with A-mAb for 2 h or preincubated for 1.5 h and then incubated with A-mAb for 30 min (n=7, mean±SD, 8.2±5.4 versus 8.1±4.9%, not significant). We used 2 h incubation of sperm with the A-mAb for all subsequent experiments.
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Discussion |
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The beads bind exclusively to the sperm head, particularly the equatorial segment and acrosome and not the neck and tail of motile sperm. Our previous study using immunofluorescent labelling also showed that actin was exposed in the acrosome and equatorial segment on 10% of motile sperm after 2 h incubation in standard culture conditions (Liu et al., 2002
). We suspect that this pattern of exposure of actin on the sperm head is related to capacitation or the initiation of the acrosome reaction because the membrane vesiculation is believed to start immediately anterior to the equatorial segment (Yanagimachi, 1994
). However, sperm with detectable actin exposure on the surface still had an intact acrosome with no structural damage of plasma and acrosomal membranes since these motile sperm did not stain with either Hoechst 33258 or propidium iodide (Liu et al., 2002
, unpublished observation). It has been reported previously that capacitation induces actin polymerization in boar sperm (Castellani-Ceresa et al., 1993
). It is possible that changes in the plasma membrane during capacitation may facilitate exposure of actin epitopes on sperm, as observed in this study. There are several precedents for actin exposure on the surface of the plasma membranes of somatic cells. For example, endothelial cell surface actin serves as a binding site for plasminogen, tissue plasminogen activator and lipoprotein (Dudani and Ganz, 1996
). Also, Owne et al. (1978)
reported the presence of actin on the surface of human B lymphocytes. The exact molecular state of the cell surface-exposed actin requires further study.
Our previous study showed that both Dynabead labelling and immunofluorescence can be used for detecting actin exposure on motile sperm, and similar results were obtained when the same samples were assessed by these two methods (Liu et al., 2002). We used Dynabead labelling instead of immunofluorescence in this study because the former is simpler and easier to score large numbers of motile sperm for a large number of samples. In this study, maximum binding was observed after 2 h incubation of sperm with A-mAb. Since sperm had been incubated for 1 h during the preparatory swim-up procedure, sperm had a total of 3 h incubation in vitro. During sperm capacitation, the sperm plasma membrane undergoes numerous biochemical and molecular modifications. The major changes include removal of cholesterol from the plasma membrane which alters membrane fluidity and subsequent sequential changes including an increase in bicarbonate uptake, activation of adenyl cyclase and protein kinase A and then an increase in protein tyrosine phosphorylation (Yanagimachi, 1994
; Visconti and Kopf, 1998
). Although various markers have been used for human sperm capacitation, the process has still not been well defined and the exact specificity of the markers is unclear. Capacitation conditions clearly affect the proportion of sperm exposing actin. Incubation of sperm with PMA, which promotes sperm capacitation, increased the proportion of sperm exposing actin. Incubation of sperm in bicarbonate-free medium, which inhibits the acrosome reaction, decreased the proportion of sperm exposing actin. Furthermore, the presence of seminal plasma reduced actin exposure on sperm. All of these observations are consistent with exposure of actin to the surface being related to the state of capacitation.
PMA also induces acrosomal ruffling which is dependent on actin polymerization events but not the PKC pathway. PMA-induced acrosomal ruffling can be inhibited by cytochalasin B or D (inhibitors of actin polymerization). PMA-induced acrosomal ruffling may also be related to PMA-enhanced sperm actin exposure observed in this study. Interestingly, although PMA induces acrosomal ruffling and enhances the ZP-induced AR, it is not capable of inducing the AR (Liu et al., 2002).
In this study we observed in normozoospermic men with unknown fertility that an average of only 10% of motile sperm exposed actin under standard capacitation conditions. Eisenbach (1999) found that only a small fraction of the sperm population (average 10%) was chemotactically responsive and he suggested that this was the subpopulation of capacitated sperm. Recently we showed that an average of 14% of motile sperm from fertile men and 4% from normozoospermic infertile men were capable of binding to the ZP in vitro with standard culture conditions (Liu et al., 2003
). Therefore, capacitated sperm with exposed actin may reflect the small subpopulation of sperm which have the potential for specific interaction with the ZP.
There is a significant correlation between the percentage of motile sperm binding A-mAb after 2 h incubation and sperm motility and morphology in semen and spermZP binding, which are important indicators for sperm fertilizing capacity. These results suggest that exposure of actin on the surface of the sperm head may reflect the functional competence of the sperm. In insemination medium, an average of 9.4% of motile sperm had exposed actin. In contrast, 44% of sperm, which had been attached to the ZP, had exposed actin. The proportion of sperm in insemination medium with exposed actin was highly correlated with the number of sperm bound/ZP. This relationship may indicate that (i) sperm with exposed actin are more capable of binding to the ZP, or (ii) more sperm expose actin during initiation of the ZP-induced AR following binding to the ZP. However, the actin exposed on the surface of sperm is unlikely to be directly involved as a sperm receptor for binding to the ZP since the addition of A-mAb to sperm and oocytes in culture medium does not inhibit spermZP binding but does block the ZP-induced AR after the sperm have bound to the zona (Liu et al., 2002). Actin polymerization appears to be critical for the ZP-induced AR but not for spermZP binding (Liu et al., 2002
). A recent study showed that a newly identified sperm-specific protein, Haprin, plays an important role in the membrane fusion process during the initiation of the human AR via association with actin filaments and components of the SNARE regulatory complex (Kitamura et al., 2003
). Antibodies to SNARE proteins can block the ionomycin-induced AR in bovine sperm in vitro (Ramalho-Santos et al., 2000
).
In conclusion, this study has shown that an average of 9.4% of motile sperm expose actin on the surface of the head in the equatorial region and acrosome after 2 h incubation, and that the percentage is significantly correlated with sperm morphology in semen and the ability of sperm to bind to the ZP. The proportion of sperm with exposed actin is dependent upon capacitation conditions. Thus, the proportion of sperm exposing actin during in vitro culture may reflect the small subpopulation of sperm with fertilizing ability.
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Acknowledgements |
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References |
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Submitted on September 23, 2004; resubmitted on November 23, 2004; accepted on December 7, 2004.
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