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From the * Department of Natural Sciences,
Fayetteville State University, Fayetteville, North Carolina; and the
Department of Biology, Chemistry and
Environmental Health Science, Benedict College, Columbia, South
Carolina.
| Correspondence to: Dr Subir K. Nagdas, Department of Natural Sciences, Fayetteville State University, 1200 Murchison Rd, Fayetteville, NC 28301 (e-mail: snagdas{at}uncfsu.edu). |
| Received for publication November 17, 2008; accepted for publication May 18, 2009. |
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Abstract |
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Key words: Sperm, acrosome, acrosomal hydrolases, protein-protein interaction
Acrosin, a trypsinlike endoprotease derived from the enzymatically inactive precursor proacrosin, functions both in sperm binding to and in penetration of the zona pellucida (Meizel, 1984; Jones and Brown, 1987). Natural and synthetic inhibitors of acrosin prevent fertilization both in vivo and in vitro (Stambaugh et al, 1969; Bhattacharyya et al, 1979). Acrosin-null mice penetrate the zona pellucida and fertile homologous zona-intact egg at a lower rate than wild types (Baba et al, 1994). However, Moreno et al (2002) showed that the in vitro fertilization of mouse zona-intact oocytes was inhibited in the presence of a polyclonal antibody raised against a unique 18-amino-acid domain in the polysulfate-binding domain of proacrosin/acrosin. This study clearly suggests that proacrosin/acrosin has a key role during sperm penetration through the zona pellucida. The release of acrosomal proteins from the acrosin-null sperm was significantly delayed during the acrosome reaction induced by calcium ionophore or by solubilized zona pellucida, suggesting the role of acrosin to accelerate the release of acrosomal components during acrosome reaction (Yamagata et al, 1998). The decreased ability of acrosin-null mouse sperm to disperse acrosomal proteins could explain the delayed sperm penetration of zona pellucida. Mouse sperm N-acetylglucosaminidase (NAGA), one of the major acrosomal glycohydrolases, exhibits a 20-fold higher activity than other acrosomal glycohydrolases (Miller et al, 1993). NAGA is released during the acrosome reaction (Akruk et al, 1979; Miller et al, 1993; Takada et al, 1994), and a specific NAGA inhibitor blocks the penetration of mouse sperm through the zona pellucida (Miller et al, 1993). It has been proposed that, in the boar, NAGA functions before sperm interaction with the zona pellucida, facilitating sperm passage through the cumulus cell layer (Takada et al, 1994). Thus, several studies suggest that mammalian fertilization is indeed a complicated process in which multiple hydrolases are involved. In several mammalian species, the acrosomal contents are segregated into spatially distinct domains of differing ultrastructural appearance (Phillips, 1972; Fawcett, 1975; Westbrook-Case et al, 1995). In the guinea pig, specific acrosomal matrix polypeptides localized to restricted domains of the apical segment have been identified and characterized (Noland et al, 1994; Reid and Blobel, 1994; Westbrook-Case et al, 1994, 1995; Foster et al, 1997). This suggests that regionally localized matrix assemblies may partition the acrosomal interior into distinct functional domains. It has also been proposed that binding interactions between the acrosomal matrix and specific hydrolases may account for the differences between hydrolases in their solubility and release rates during the acrosome reaction (Green, 1978; DiCarlantonio and Talbot, 1988; Olson et al, 1988; Nuzzo et al, 1990; Hardy et al, 1991; NagDas et al, 1996a). For example, although some acrosomal proteins such as hyaluronidase, dipeptidyl peptidase I, and AA 1 do not appear to be associated with the acrosomal matrix and are freely soluble and rapidly released upon cell disruption or during the acrosome reaction, other components such as acrosin are associated with a particular sperm function and are released more slowly during the acrosome reaction (Srivastava et al, 1974; Noland et al, 1989; Hardy et al, 1991). We have previously isolated and characterized a localized, stable acrosomal matrix assembly from the bovine acrosome termed the outer acrosomal membrane–associated matrix complex (OMC; Olson et al, 1985; NagDas et al, 1996a). This stable matrix assembly exhibits specific binding activity for acrosin (NagDas et al, 1996a) and is restricted to the apical and principal segments of the acrosome, where it is associated with the luminal surface of the outer acrosomal membrane (Olson et al, 1985; NagDas et al, 1996a). Similarly, the potential interaction of other glycohydrolases with the acrosomal matrix has not been explored. In the study reported here, we investigated the binding competency of OMC to NAGA, one of the major acrosomal glycohydrolases (Miller et al, 1993; NagDas et al, 1996b). This stable acrosomal fraction provides a model system for investigating protein-protein interactions between the acrosomal matrix and acrosomal hydrolases. The present study is focused to identify the matrix proteins that specifically interact/bind to acrosomal hydrolases.
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Materials and Methods |
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500 x
106 spermatozoa) was washed twice by resuspension in Hanks balanced
saline solution followed by centrifugation as described above. The final
pellet was resuspended in a Tris-saline-protease inhibitor solution (TNI)
composed of 150 mM NaCl, 25 mM Tris-HCl (pH 7.5), 2 mM benzamidine, 1 µg/mL
leupeptin, 1 µg/mL pepstatin, and 0.05% sodium azide, and was centrifuged
at 1500 x g for 10 minutes at 4°C. The pellet was washed 1
additional time with TNI as described above.
Isolation of Sperm Heads and Tails
The washed sperm pellet was resuspended into 35 mL of TNI and sonicated for
60-second intervals 5 times with Misonix Miroson Ultrasonic Cell Disruptor XL
Sonifier (Misonix, Formingdale, New York). The samples were examined by
phase-contrast microscopy to assure that more than 90% of the sperm were
decapitated. The sonicated sperm suspension was centrifuged at 1500 x
g for 10 minutes at 4°C. The supernatant was discarded, and the
pellet was washed 3 times by resuspension in TNI followed by centrifugation at
1500 x g for 10 minutes at 4°C. The final pellet was
resuspended with 20 mL of TNI, and 3.3-mL aliquots of this suspension were
layered on discontinuous sucrose gradients composed of 3.3 mL 55% sucrose, 1.7
mL 70% sucrose, and 1.7 mL 75% sucrose. All sucrose solutions contained 25 mM
Tris-HCl, pH 7.5. The gradients were centrifuged at 100 000 x g
for 60 minutes in a Beckman SW40 rotor (Beckman Instruments, Palo Alto,
California). Heads were present in the pellet and tails banded at the 55%/70%
interface. Both fractions were collected, diluted with 20 mL TNI, and pelleted
by centrifugation at 15 000 x g for 10 minutes at 4°C
(NagDas et al, 1996a).
Isolation of OMC
The sperm head pellet was resuspended into 20 mL of TNI containing 0.6%
Triton X-100 and extracted overnight at 4°C with constant agitation. To
detach the detergent-insoluble OMC from the heads, the suspension was
homogenized with 40 strokes of a glass-Teflon homogenizer (Knotes, Vineland,
New Jersey). The acrosomal components and heads were separated by
centrifugation on a Percoll gradient
(NagDas et al, 1996a). The
gradient mixture consisted of 20 mL sperm head suspension and 80 mL of a
solution of 50% Percoll, 0.25 M sucrose, and 0.05 M Tris-HCl, pH 7.5. This
mixture was then centrifuged at 60 000 x g for 35 minutes in a
Beckman 70Ti rotor. The outer acrosomal membrane–matrix fraction banded
near the top and was collected. The fraction was diluted in a 1:1 suspension
with TNI and pelleted in a Beckman SW40 rotor by centrifugation at 100 000
x g for 1 hour.
Hydrolase Assays
For the assay of hydrolases, a cauda sperm pellet, after washing in Hanks
balanced saline solution, was resuspended in 150 mM NaCl, 25 mM Tris-HCl, pH
7.5 (TN), and centrifuged at 1500 x g for 10 minutes at
4°C. The pellet was washed 1 additional time with TN as described above.
The pellet was then extracted into a high-salt Triton X-100 solution (500 mM
NaCl, 0.1% Triton X-100, 25 mM Tris-HCl, pH 7.5) for 1 hour at 4°C and
centrifuged at 15 000 x g for 10 minutes. The supernatant was
to be used as a source of hydrolases. Acrosin activity was measured
spectrophotometrically at 410 nm in 0.05 M Tris-HCl buffer (pH 8.0), 0.1%
Triton X-100, 0.05 M CaCl2, and 0.1 mM
N-p-Tosyl-Gly-Pro-Arg-p-nitroanilide (Sigma
Chemical Co, St Louis, Missouri;
Lottenberg et al, 1981). One
unit of acrosin is defined as the quantity of enzyme required to hydrolyze 1
µM of N-p-Tosyl-Gly-Pro-Arg-p-nitroanilide per
minute. β-NAGA activity was also measured spectrophotometrically at 410
nm using 2 mM p-nitrophenyl N-acetyl-β-D-glucosaminide as a
substrate (Miller et al,
1993). The assay buffer was 60 mM trisodium citrate, 40 mM
NaH2PO4, 1 mM CaCl2, 1 mM MgCl2, 1
mM KCl, and 1 mM benzamidine, pH 5.0. One unit of NAGA is expressed in mM
p-nitrophenol released per hour at 37°C. Protein was estimated by the
procedure of Bradford
(1976).
Fractionation of OMC Polypeptides
The OMC fraction was extracted overnight at 4°C with 100 mM
3-[cyclohexylamino]-1-propanesulfonic acid (CAPS) buffer (pH 10.5; Sigma) and
then centrifuged for 30 minutes at 100 000 x g in a Beckman
SW40 rotor (Olson et al,
1997b). Sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) revealed that the 38–19-kd polypeptide family (termed rpf
family) was solubilized by high-pH extraction, whereas 54-, 50-, and 45-kd
polypeptides were present in the particulate fraction. The 32-kd polypeptide
from the high-pH soluble fraction was purified following the method of Olson
et al (1997b) and the
purification of the 45-kd polypeptide (OMC45) from the high pH insoluble
fraction was performed by continuous-elution SDS-PAGE on 7.5% acrylamide gels
using a Model 491 Prep Cell (Bio-Rad Laboratories, Hercules, California). The
32-kd (OMC32) and the 45-kd (OMC45) polypeptides were used for subsequent
studies. Polyclonal antibodies of OMC45 polypeptide were raised in 2 rabbits.
The primary injection consisted of OMC45 emulsified in Freund complete
adjuvant and was followed by 2 booster injections at 2-week intervals of OMC45
emulsified in Freund incomplete adjuvant. Two weeks after the final booster
injection, animals were given a lethal dose of Nembutal (Sigma Chemical Co.),
blood was collected by cardiac puncture, and a serum fraction was prepared.
The 2 antisera gave identical results by immunoblotting and
immunocytochemistry.
Gel Electrophoresis and Western Blotting
SDS-PAGE was performed on 12% or 7.5% continuous or 7.5% to 15% gradient
polyacrylamide gels (Laemmli,
1970). Polypeptides were then electrophoretically transferred to
polyvinylidene difluoride membranes
(Towbin et al, 1979) or
stained with Coomassie blue (Fairbanks et
al, 1971) or silver (Wray et
al, 1981). Two-dimensional polyacrylamide gel electrophoresis
(PAGE) was performed using a Bio-Rad precast immobilized pH (3–10)
gradient gel–ready strip (7 cm) for isoelectric focusing (IEF). IEF was
done in a Bio-Rad Protean IEF cell following the conditions described in the
Bio-Rad manual.
Immunoblots were blocked in phosphate-buffered saline (PBS) blocking buffer containing 5% heat-inactivated normal goat serum (NGS), 2.5% bovine serum albumin, 0.1% Tween-20, and 5% nonfat dry milk for 1 hour at room temperature and then washed in PBS containing 0.1% Tween-20 (PBS-TW) 3 times. To detect the OMC45 polypeptide, membranes were incubated with anti-OMC45 in PBS containing 0.1% Tween-20 and 1% goat serum (PBS-TW-GS) respectively for 1 hour at room temperature; typically, 1:2500 dilution of immune or preimmune sera was utilized. Following 3 washes in PBS-TW at room temperature, membranes were incubated with affinity-purified horseradish peroxidase–conjugated secondary antibody (1:5000; KPL Inc, Gaithersburg, Maryland) diluted in PBS-TW-GS for 1 hour at room temperature. Membranes were washed 3 times in PBS-TW. Immunoreactive bands were visualized using H2O2 and diaminobenzidine (Sigma) for color development.
Immunofluorescence Microscopy
Spermatozoa were fixed in 4% formalin in 0.1 M PBS, pH 7.4, at 4°C for
30 minutes and plated on poly-L-lysine–coated coverslips.
Cells were then washed in PBS and permeabilized in PBS containing 0.1% Triton
X-100 for 30 minutes at 4°C. After 3 rinses in PBS, samples were blocked
with 1% NGS in PBS (PBS-NGS). Coverslips were then incubated with equal
dilutions of immune or preimmune serum in PBS-NGS for 1 hour, and washed 3
times with PBS. Cells were then incubated with fluorescein
isothiocyanate–conjugated goat anti-rabbit IgG (KPL) in PBS-NGS for 1
hour. Following incubation, cells were then washed 3 times with PBS to remove
unbound antibody molecules. Coverslips were examined by phase contrast and
epifluorescence microscopy.
OMC-NAGA Binding Assay
A centrifugation assay (NagDas et al,
1996a) was employed to determine whether OMC possessed specific
binding sites for NAGA. The OMC fraction and the sperm tail fraction were
extracted with 0.5 M NaCl, 25 mM Tris-HCl (pH 7.5), 2 mM benzamidine, 1 µg
/mL leupeptin, and 1 µg/mL pepstatin (HST) for 1 hour at 4°C. The high
salt extraction depleted residual NAGA. The OMC and the tails were pelleted
after extraction by centrifugation at 100 000 x g for 10
minutes, then washed once with TN (25 mM Tris-HCl, pH 7.5, 150 mM NaCl) and
resuspended into fresh TN. The high-salt extract (HST supernatant) of cauda
sperm, prepared as described above, was used as a source of NAGA. The HST
supernatant was dialyzed against binding buffer containing 25 mM Tris-HCl (pH
7.5) and 150 mM NaCl and then centrifuged at 15 000 x g for 10
minutes to remove particulate material. Aliquots of the supernatant were
incubated with variable amounts of HST-extracted OMC (20–60 µg) in a
total volume of 0.2 mL for 1 hour at 4°C. As a control for binding
specificity, incubations were performed in which equal amounts of tails were
substituted for OMC. Samples were then centrifuged at 100 000 x
g for 10 minutes. The supernatant fractions were collected and the
pellets were resuspended in 0.2 mL TN. Supernatant and pellet fractions were
assayed for NAGA.
Identification of hydrolases binding polypeptides of OMC complex
Both the OMC32 and OMC45 polypeptides were isolated from high-pH soluble
and insoluble fractions of OMC complex, respectively, by continuous-elution
SDS-PAGE, and subsequently coupled to an AminoLink Plus resin at pH 10.0
following the procedure provided by the manufacturer (Pierce Chemical Co,
Rockford, Illinois). A centrifugation assay was performed to determine the
binding specificity of hydrolases to OMC32 and OMC45 polypeptides. The
high-salt extract of cauda sperm was dialyzed against binding buffer (25 mM
Tris-HCl, pH 7.5, and 150 mM NaCl) and centrifuged, and the supernatant was
used as a source of acrosin and NAGA. Aliquots of the supernatants were
incubated with variable amounts of OMC32 and OMC45 polypeptides (10–70
µg proteins) coupled to AminoLink beads in a total volume of 0.2 mL for an
hour at 4°C. As a control, for binding specificity, parallel incubations
were performed in which equal amounts of the tails' protein were extracted at
pH 11.0, coupled to AminoLink Plus beads. Samples were then centrifuged at 15
000 x g for 10 minutes and the pellets were suspended in a
volume of binding buffer equal to the volume of supernatant fraction (0.2 mL).
Pellets and supernatants were assayed for acrosin and NAGA.
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Results |
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Experiments were next performed to determine whether the OMC45 polypeptide is associated with a particulate sperm fraction. Sperm were disrupted by sonication; the sperm suspension was centrifuged, and the pellet and supernatant fractions were analyzed by immunoblot analysis with anti-OMC45. The pattern of immunoreactive polypeptide observed in the purified OMC fraction (Figure 3, lane 1) was identical to that seen in the pellet fraction of sonicated (lane 2) spermatozoa. No OMC45 polypeptide was detected in the supernatant fraction of sonicated spermatozoa (Figure 3, lane 3). These studies demonstrate that the OMC45 polypeptide is associated with a particular cellular fraction in mature spermatozoa.
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To determine whether OMC45 was comprised of charge variant isoforms, OMC fraction was separated by 2-dimensional PAGE and subjected to immunoblot analysis. Blots stained with anti-OMC45 exhibited a charge train of 5–6 spots, with isoelectric points ranging between pH 5.5 and 7.2 (Figure 4). No stained bands were seen with preimmune serum (data not shown).
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Identification of Hydrolase (Acrosin and NAGA)-Binding Polypeptide(s) of OMC Complex
To identify matrix-specific hydrolase-binding polypeptides, a
centrifugation assay was utilized. Both OMC32 and OMC45 polypeptides were
isolated from the OMC complex and coupled to AminoLink Plus beads. Soluble
acrosomal hydrolases were incubated with OMC32- and OMC45-conjugated beads for
1 hour at 4°C. After centrifugation, both the pellet and the supernatant
fractions were assayed for acrosin and NAGA. Both acrosin
(Figure 7A-I) and NAGA
(Figure 7B-I) specifically
bound the OMC32 polypeptide in a concentration-dependent fashion, as evidenced
by the increased fraction of sedimentable acrosin and NAGA activities. In
contrast, the OMC45 polypeptide depleted acrosin activity
(Figure 7A-II) from the
supernatant in a concentration-dependent manner, whereas the interaction
between OMC45 polypeptide and NAGA activity was not significant
(Figure 7B-II), indicating a
strong binding interaction between acrosin and OMC45 polypeptide. Parallel
control incubation that substituted equal amounts of high-pH extracted sperm
tail proteins for the OMC32 and OMC45 polypeptides exhibited no significant
binding of either hydrolase (Figure 7A-III
and 7B-III). These experiments demonstrate that the OMC32
polypeptide possesses specific binding sites for both acrosin and NAGA,
whereas the OMC45 polypeptide exhibited significant interaction to acrosin.
This binding specificity indicates that acrosomal matrix polypeptides are
involved in the release of hydrolases during the acrosome reaction.
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Discussion |
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A highly purified OMC fraction, isolated from Triton X-100–extracted bovine sperm heads, is comprised of 3 major (54, 50, and 45 kd) and several minor (38–19 kd) polypeptides (NagDas et al, 1996a; Olson et al, 1997b). In the present study, we purified the 45-kd polypeptide from the high-pH insoluble fraction by continuous-elution SDS-PAGE, and the polyclonal antibody was raised for biochemical and morphological studies. Our immunofluorescence and biochemical data clearly demonstrate that the bovine OMC45 polypeptide is specifically localized to the OMC complex. The presence of OMC45 in a stable acrosomal matrix assembly, which is adherent to the fusigenic domains of the outer acrosomal membrane, suggests that this membrane-associated assembly maintains the structural integrity of the hybrid membrane complex generated by the membrane fusion process of the acrosome reaction.
Two-dimensional PAGE reveals a complex charge-variant pattern of OMC45 polypeptide with 5–6 isoforms; the presence of several isoforms could be due to the posttranslational modification of OMC45 polypeptide. Previously, 2-dimensional PAGE of the OMC complex demonstrated that the 38–19-kd set of OMC polypeptides (OMCrpf) were structurally related, because they migrated a set of charge/size variants (Olson et al, 1985). Whether posttranslational modifications such as glycosylation, sulfation, phosphorylation, and others could contribute to the charge heterogeneity of OMC polypeptides remains to be investigated. Several studies suggest that posttesticular changes of the acrosome occur during epididymal transit that include remodeling of acrosomal shape, development of distinct matrix domains, and the processing of specific acrosomal glycoproteins (Arboleda and Gerton, 1988; O'Brien et al, 1988; Anakwe and Gerton, 1990; Tulsiani et al, 1995; Olson et al, 1997a, 2003, 2004; Yoshinaga et al, 2001) and hydrolases such as proacrosin/acrosin (Anakwe et al, 1991; NagDas et al, 1992). The posttranslational modification(s) of acrosomal matrix polypeptides during epididymal transit could be one of the important biochemical events to produce functionally mature spermatozoa.
We have demonstrated that the OMC possesses proacrosin-binding activity (NagDas et al, 1996a). Our current data reveals that the bovine OMC complex specifically binds NAGA. Several other glycohydrolases that were present in the particulate fraction of detergent-permeabilized bovine spermatozoa may also bind the acrosomal matrix, but this possibility requires experimental verification. In the current study, we identified acrosomal matrix polypeptides that specifically bind to bovine sperm acrosin and N-acetylglucosaminidase. Acrosin and NAGA bound the OMC32 polypeptide in a concentration-dependent manner; in contrast, the OMC45 polypeptide exhibited stronger affinity to acrosin than NAGA. Alternatively, it may be possible that the interaction between OMC 32 and OMC45 polypeptides with acrosin and NAGA is mediated by other protein(s). Future studies will address this issue. The binding specificity of acrosomal matrix proteins to hydrolases strongly suggests that the matrix polypeptides play an important role in the regulation of release of hydrolases during the acrosome reaction and in maintaining a hydrolase pool at the site of sperm-zona interaction. This interaction could also provide one mechanism for segregating and targeting specific hydrolases within the acrosome interior. Because the OMC binds both proacrosin/acrosin and glycohydrolases (NagDas et al, 1996a,b), and acrosin remains associated with the hybrid membrane complex (Richardson et al, 1991), this stable acrosomal complex may also function to maintain a hydrolase pool at the localized site of sperm-zona binding. Moreover, the binding of proacrosin/acrosin to both the OMC (NagDas et al, 1996a) and the zona pellucida (Jones and Brown, 1987) could represent the structural basis for maintaining the attachment of acrosome-reacting spermatozoa to the surface of the zona pellucida. It is also possible that the role of matrix proteins is not limited to regulating the differential release of acrosomal hydrolases; they could also function in the membrane fusion events in sperm-zona interactions. These potential functions, as well as the specific association of acrosomal matrix assembly with the outer acrosomal membrane, will be addressed in future studies.
The acrosome reaction is initiated when receptors in the periacrosomal plasma membrane bind ZP3 of the zona pellucida (Wassarman, 1992). In the spermatozoon, this interaction initiates signaling pathways regulated by G proteins, alters protein phosphorylation patterns, and raises intracellular calcium levels (Kopf and Gerton, 1991; Ward and Kopf, 1993). The target proteins for many of theses regulatory processes have not been localized, and thus their role in the membrane fusion and acrosomal matrix dispersion processes is limited. Sukardi et al (2001) demonstrated that 33- and 39-kd polypeptides of the acrosomal membrane fraction of ram ejaculated spermatozoa bind 45Ca2+. Internal amino acid sequence data of 39-kd polypeptide obtained after cyanogen bromide cleavage showed 68% homology with SP-10 protein precursor and 64%–72% homology with various annexins. It could be possible that the OMC is a target structure for the activation and regulatory pathways controlling the acrosome reaction. Future studies will better define the role of OMC polypeptides in mammalian fertilization.
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Footnotes |
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References |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Anakwe OO, Gerton GL. Acrosome biogenesis begins during meiosis: evidence from the synthesis and distribution of an acrosomal glycoprotein, acrogranin, during guinea pig spermatogenesis. Biol Reprod. 1990;42: 317 –328.[Abstract]
Anakwe OO, Sharma S, Hardy DM, Gerton GL. Guinea pig proacrosin is synthesized principally by round spermatids and contains O-linked as well as N-linked oligosaccharide side chains. Mol Reprod Dev. 1991; 29: 172 –179.[CrossRef][Medline]
Arboleda CE, Gerton GL. Proacrosin/acrosin during guinea pig spermatogenesis. Dev Biol. 1988; 125: 217 –225.[CrossRef][Medline]
Baba T, Azuma S, Kashiwabara S-I, Toyoda Y. Sperm from mice
carrying a targeted mutation of the acrosin gene can penetrate the oocyte zona
pellucida and effect fertilization. J Biol Chem. 1994
:269: 31845
–31849.
Barros C, Bedford JM, Franklin LE, Austin CR. Membrane vesiculation
as a feature of the mammalian acrosome reaction. J Cell
Biol. 1967;34: C1
–C5.
Bearer EL, Friend DS. Morphology of mammalian sperm membranes during differentiation, maturation, and capacitation. J Electron Microsc Tech. 1990;16: 281 –297.[CrossRef][Medline]
Beebe SJ, Leyton L, Burks D, Ishikawa M, Furest T, Dean J, Saling PL. Recombinant mouse ZP3 inhibits sperm binding and induces the acrosome reaction. Dev Biol. 1992; 151: 48 –54.[CrossRef][Medline]
Bhattacharyya AK, Goodpasture JC, Zaneveld LJD. Acrosin of mouse spermatozoa. Am J Physiol. 1979; 237: E40 –E44.[Medline]
Bleil JD, Wassarman PM. Autoradiographic visualization of the mouse
egg's sperm receptor bound to sperm. J Cell Biol. 1986; 102: 1363
–1371.
Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976; 72: 248 –254.[CrossRef][Medline]
Byrd W. In vitro capacitation and the chemically induced acrosome reaction in bovine spermatozoa. J Exp Zool. 1981; 215: 35 –46.[CrossRef][Medline]
Cohen-Dayag A, Eisenbach M. Potential assays for sperm capacitation
in mammals. Am J Physiol Cell Physiol. 1994; 267(5): C1167
–C1176.
Cross NL. Role of cholesterol in sperm capacitation.
Biol Reprod. 1998; 59(1): 7
–11.
de Lamirande EP, Leclerc P, Gagnon C. Capacitation as a regulatory
event that primes spermatozoa for the acrosome reaction and fertilization.
Mol Hum Reprod. 1997; 3(3): 175
–194.
DiCarlantonio G, Talbot P. Evidence for sequential deployment of secretory enzymes during the normal acrosome reaction of guinea pig sperm in vitro. Gamete Res. 1988; 21: 425 –438.[CrossRef][Medline]
Eddy EM, O'Brien DA. The spermatozoon. In: Knobil E, Neill JD, eds. The Physiology of Reproduction. New York, NY: Raven Press; 1993: 29–77.
Fairbanks G, Steck TL, Wallach DFH. Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry. 1971; 10: 2606 –2617.[CrossRef][Medline]
Fawcett DW. The mammalian spermatozoon. Dev Biol. 1975;44: 394 –436.[CrossRef][Medline]
Foster JA, Friday, BB, Maulit MT, Blobel C, Winfrey VP, Olson GE,
Kim K-S, Gerton GL. AM67, a secretory component of the guinea pig sperm
acrosomal matrix, is related to mouse sperm protein sp56 and the complement
component 4-binding proteins. J Biol Chem. 1997; 272: 12714
–12722.
Green DPL. The activation of proteolysis in the acrosome reaction
of guinea-pig sperm. J Cell Sci. 1978; 32: 153
–164.
Hardy DM, Oda MN, Friend DS, Huang TTF. A mechanism for differential release of acrosomal enzymes during the acrosome reaction. Biochem J. 1991; 275: 759 –766.[Medline]
Jones R, Brown CR. Identification of a zona-binding protein from boar spermatozoa as proacrosin. Exp Cell Res. 1987; 171: 503 –508.[CrossRef][Medline]
Kopf GS, Gerton GL. The mammalian sperm acrosome and the acrosome reaction. In: Wassarman PM, ed. Elements of Mammalian Fertilization. Boca Raton, FL: CRC Press; 1991: 153 –203.
Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227: 680 –685.[CrossRef][Medline]
Lottenberg R, Christensen U, Jackson CM, Coleman PL. Assay of coagulation proteases using peptide chromogenic and fluorogenic substrates. Methods Enzymol. 1981; 80: 341 –361.[CrossRef][Medline]
Meizel S. The importance of hydrolytic enzymes to an exocytotic event, the mammalian sperm acrosome reaction. Biol Rev. 1984;59: 125 –157.[Medline]
Miller DJ, Gong X, Shur BD. Sperm require B-N-acetylglucosaminidase
to penetrate through the egg zona pellucida.
Development. 1993; 118: 1279
–1289.
Moreno RD, Bustamante E, Schatten G, Barros., C. Inhibition of mouse in vitro fertilization by an antibody against a unique 18-amino acid domain in the polysulfate-binding domain of proacrosin/acrosin. Fertil Steril. 2002; 77: 812 –817.[CrossRef][Medline]
NagDas SK, Skudlarek MD, Orgebin-Crist M-C, Tulsiani DRP.
Biochemical alterations in the proacrosin-acrosin system during epididymal
maturation of rat spermatozoa. J Androl. 1992; 13: 36
–43.
NagDas SK, Winfrey VP, Olson GE. Proacrosin-acrosomal matrix binding interactions in ejaculated bovine spermatozoa. Biol Reprod. 1996a;54: 111 –121.[Abstract]
NagDas SK, Winfrey VP, Olson GE. Identification of hydrolase binding activities of the acrosomal matrix of hamster spermatozoa. Biol Reprod. 1996b; 55: 1405 –1414.[Abstract]
Noland TD, Davis LS, Olson GE. Regulation of proacrosin conversion
in isolated guinea pig sperm acrosomal apical segments. J Biol
Chem. 1989;264: 13586
–13590.
Noland TD, Friday BB, Maulit MT, Gerton GL. The sperm acrosomal
matrix contains a novel member of the pentaxin family of calcium-dependent
binding proteins. J Biol Chem. 1994; 269: 32607
–32614.
Nuzzo NA, Anderson RA, Zaneveld LJD. Proacrosin activation and acrosin release during the guinea pig acrosome reaction. Mol Reprod Dev. 1990;25: 52 –60.[CrossRef][Medline]
O'Brien DA, Gerton GL, Eddy EM. Acrosomal constituents identified with a monoclonal antibody are modified during late spermiogenesis in the mouse. Biol Reprod. 1988; 38: 955 –967.[Abstract]
Olson GE, Ming Bi WV, Hardy DM, Nagdas SK. Zonadhesin assembly into
the hamster sperm acrosomal matrix occurs by distinct targeting strategies
during spermiogenesis and maturation in the epididymis. Biol
Reprod. 2004;71: 1128
–1134.
Olson GE, Winfrey VP. Structure-function relationships in the sperm acrosome. Ann N Y Acad Sci. 1991; 637: 240 –257.[CrossRef][Medline]
Olson GE, Winfrey VP, Davenport GR. Characterization of matrix domains of the hamster acrosome. Biol Reprod. 1988; 39: 1145 –1158.[Abstract]
Olson GE, Winfrey VP, Garbers DL, Noland TD. Isolation and characterization of a macromolecular complex associated with the outer acrosomal membrane of bovine spermatozoa. Biol Reprod. 1985; 33: 761 –779.[Abstract]
Olson GE, Winfrey VP, NagDas SK. Temporal expression and localization of protein farnesyltransferase during spermiogenesis and posttesticular sperm maturation in the hamster. Mol Reprod Dev. 1997a;48: 71 –76.[CrossRef][Medline]
Olson GE, Winfrey VP, NagDas SK. Structural modification of the hamster sperm acrosome during posttesticular development in the epididymis. Microsc Res Tech. 2003; 61: 46 –55.[CrossRef][Medline]
Olson GE, Winfrey VP, Neff JC, Lukas TJ, NagDas SK. An antigenically related polypeptide family is a major structural constituent of a stable acrosomal matrix assembly in bovine spermatozoa. Biol Reprod. 1997b;57: 325 –334.[Abstract]
Parrish JJ, Susko-Parrish J, Winer MA, First NL. Capacitation of bovine sperm by heparin. Biol Reprod. 1988; 38: 1171 –1180.[Abstract]
Phillips DM. Substructure of the mammalian acrosome. J Ultrastruct Res. 1972;38: 591 –604.[CrossRef][Medline]
Reid MS, Blobel CP. Apexin, an acrosomal pentaxin. J
Biol Chem. 1994;269: 32615
–32620.
Richardson RT, Nikolajczyk BS, Abdullan LH, Beavers JC, O'Rand MG. Localization of rabbit sperm acrosome during the acrosome reaction induced by immobilized zona matrix. Biol Reprod. 1991 :45 20 –26.[Abstract]
Roldan ERS, Murase T, Shi Q-X. Exocytosis in spermatozoa in
response to progesterone and zona pellucida. Science. 1994; 266: 1578
–1581.
Srivastava PN, Munnell JF, Yang CH, Foley CW. Sequential release of
acrosomal membranes and acrosomal enzymes of ram spermatozoa. J
Reprod Fertil. 1974;36: 363
–372.
Stambaugh R, Brackett BG, Mastrioanni L. Inhibition of in vitro fertilization of rabbit ova by trypsin inhibitors. Biol Reprod. 1969;1: 223 –227.[Abstract]
Sukardi S, R, Elliott RMA, Withers JO, Fontaine U, Millar JD, Curry MR, Watson PF. Calcium binding proteins from the outer acrosomal membrane of ram spermatozoa: potential candidates for involvement in the acrosome reaction. Reproduction. 2001; 122: 939 –946.[Abstract]
Takada M, Yonezawa N, Yoshizawa M, Noguchi S, Hatanaka Y, Nagai T, Kikuchi K, Aoki H, Nakano M. pH-sensitive dissociation and association of B-N-acetylhexosaminidase from boar sperm acrosome. Biol Reprod. 1994;50: 860 –868.[Abstract]
Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of
proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some
applications. Proc Natl Acad Sci U S A. 1979; 76: 4350
–4354.
Tulsiani DP, NagDas SK, Skudlarek MD, Orgebin-Crist H-C. Rat sperm plasma membrane mannosidase: localization and evidence for proteolytic processing during epididymal maturation. Dev Biol. 1995; 167: 584 –595.[CrossRef][Medline]
Visconti PE, Kopf GS. Regulation of protein phosphorylation during
sperm capacitation. Biol Reprod. 1998; 59(1): 1
–6.
Ward CR, Kopf GS. Molecular events mediating sperm activation. Dev Biol. 1993; 158: 9 –34.[CrossRef][Medline]
Wassarman PM. Profile of a mammalian sperm receptor. Development. 1992; 108: 1 –17.
Wassarman PM. Towards molecular mechanisms for gamete adhesion and fusion during mammalian fertilization. Curr Opin Cell Biol. 1995;7: 6658 –664.
Westbrook-Case VA, Winfrey VP, Olson GE. A domain-specific 50-kilodalton structural protein of the acrosomal matrix is processed and released during the acrosome reaction in guinea pig. Biol Reprod. 1994;51: 1 –13.[Abstract]
Westbrook-Case VA, Winfrey VP, Olson GE. Sorting of the domain-specific acrosomal matrix protein AM50 during spermiogenesis in the guinea pig. Dev Biol. 1995; 167: 338 –349.[CrossRef][Medline]
Wray W, Boulikas T, Wray VP, Hancock R. Silver staining of proteins in polyacrylamide gels. Anal Biochem. 1981; 118: 197 –203.[CrossRef][Medline]
Yamagata K, Murayama K, Okabe M, Toshimori T, Nakanishi T,
Kashiwabara S-I, Baba T. Acrosin accelerates the dispersal of sperm acrosomal
proteins during acrosome reaction. J Biol Chem. 1998
:273 10470
–10474.
Yanagimachi R. Mammalian fertilization. In: Knobil E, Neill JD, eds. The Physiology of Reproduction. New York, NY: Raven Press; 1993: 189 –317.
Yanagimachi R, Suzuki F. A further study of lysolecithin-mediated acrosome reaction of guinea pig spermatozoa. Gamete Res. 1985;11: 29 –40.[CrossRef]
Yoshinaga K, Tanii I, Oh-oka T, Toshimori K. Changes in distribution and molecular weight of the acrosomal protein acrin2 (MC41) during guinea pig spermiogenesis and epididymal maturation. Cell Tissue Res. 2001;303: 253 –261.[CrossRef][Medline]
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