JCS ePress online publication date 4 August 2009
Research Article
3153
Functional consequences of cleavage, dissociation
and exocytotic release of ZP3R, a C4BP-related
protein, from the mouse sperm acrosomal matrix
Mariano G. Buffone1,*, Kye-Seong Kim2,*, Birgit J. Doak1, Esmeralda Rodriguez-Miranda3 and
George L. Gerton1,‡
1
Center for Research on Reproduction and Womenʼs Health, Department of Obstetrics and Gynecology, University of Pennsylvania
School of Medicine, Philadelphia, PA 19104, USA
2
Department of Anatomy and Cell Biology, Hanyang University College of Medicine, Seoul 135-081, Korea
3
Instituto Tecnológico Superior de Irapuato (ITESI), Colonia El Copal, Irapuato 36821, Guanajuato, México
*These authors contributed equally to this work
‡
Author for correspondence ([email protected])
Journal of Cell Science
Accepted 16 June 2009
Journal of Cell Science 122, 3153-3160 Published by The Company of Biologists 2009
doi:10.1242/jcs.052977
Summary
The acrosome is an exocytotic vesicle located on the apical tip
of the sperm head. In addition to having different morphological
regions, two biochemically distinct compartments can be defined
within the acrosome: a particulate acrosomal matrix and a
soluble partition. The domains within the acrosome participate
in the release of acrosomal proteins from the sperm during
exocytosis, depending on whether the proteins partition into
either the soluble or matrix compartments of the acrosome. We
have examined the mechanism of differential release by
evaluating the solubilization of acrosomal matrix protein ZP3R
(sp56) from mouse sperm during the course of spontaneous
acrosomal exocytosis. Using indirect immunofluorescence and
immunoblotting, we found that the ZP3R monomer is processed
from 67,000 Mr to 43,000 Mr by proteases coincident with release
Introduction
The acrosome is a membrane-bound organelle located at the
anterior tip of the sperm head. It is essential for fertilization in
mammals; humans and mice whose sperm lack acrosomes are
infertile (Dam et al., 2007; Schill, 1991; Sotomayor and Handel,
1986). Structurally and functionally, the acrosome consists of soluble
and particulate compartments. Some of the proteins described as
constituents of the soluble compartment are hyaluronidase,
dipeptidyl peptidase, and CRISP2 (also known as autoantigen 1 or
Tpx1) (Foster and Gerton, 1996; Hardy et al., 1991). The proteins
of the particulate compartment or acrosomal matrix have been
studied most extensively in the guinea pig and include proacrosin,
AM50 (also known as p50, apexin, Narp, and neuronal pentraxin
II), AM67 (the orthologue of mouse sperm protein ZP3R; previously
known as sp56), and proacrosin-binding protein (Baba et al.,
1994b; Honda et al., 2002; Noland et al., 1994).
The acrosome and its contents have been implicated in spermZP interactions (Yanagimachi, 1994). During mammalian
fertilization, sperm bind to and then penetrate the zona pellucida
(ZP), the extracellular matrix that surrounds the oocyte. It has been
proposed that the initial interaction with ZP macromolecules
triggers several molecular processes in sperm that lead to acrosomal
exocytosis (AE). AE allows the sperm to penetrate the ZP in a
process that also requires hyperactive sperm motility. Subsequently,
from the acrosome. Sperm require a maturational step, termed
capacitation, before they are competent for acrosomal exocytosis
and the processing of ZP3R is dramatically reduced under noncapacitating conditions. The cleavage probably takes place in
complement control protein domain (CCP) 6 or the bridge
region between CCP6 and CCP7, which is not present in the
guinea pig orthologue AM67. The cleaved form of ZP3R does
not bind to unfertilized eggs. We have incorporated these
structural considerations into a model to explain the functional
consequences of acrosomal exocytosis on sperm-zona
interactions.
Key words: ZP3R (sp56), Acrosomal matrix, Mouse fertilization,
Sperm
the fertilizing sperm interacts and fuses with the egg plasma
membrane to form the zygote.
Although there are only a few ZP sulfoglycoproteins involved
in these binding events (ZP1, ZP2 and ZP3 in the mouse), several
sperm molecules have been proposed as ZP-binding proteins or
signaling receptors on mammalian sperm, based on their affinity
for the ZP (Tanphaichitr et al., 2007). Among these molecules are
β-1,4 galactosyltransferase, zonadhesin, SED1, spermadhesins, and
ZP3R (Bleil and Wassarman, 1990; Ensslin and Shur, 2003; Hardy
and Garbers, 1995; Miller et al., 1992; Sanz et al., 1992). Without
precluding a role for any candidates in sperm-ZP interactions, the
binding to the ZP appears to be a redundant process since the
targeted deletion of SED1, β-1,4 galactosyltransferase, or proacrosin
does not result in complete male infertility (Baba et al., 1994a;
Ensslin and Shur, 2003; Lu and Shur, 1997). Rather, some degree
of subfertility or disregulation of acrosomal exocytosis is seen.
The focus of this study, mouse sperm ZP-binding protein ZP3R
is closely related to the alpha chain of complement 4-binding protein
(C4BP) (Foster et al., 1997) and both proteins form high order
oligomers. Each monomer of these proteins contains complement
control protein domains (CCP). In the case of ZP3R, there are seven
complete and one partial CCP (numbered consecutively as CCP1CCP8); in the mouse C4BP alpha chain, there are six CCPs followed
by a C-terminal extension. Ligand-binding regions of human C4BP
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have been mapped to the various regions of the N-terminal domain
and include Factor I, C3b, C4b, heparin and bacterial M proteins.
The CCP domains of C4BP interact with proteases in the
complement cascade (i.e. as a cofactor of Factor I in the proteolytic
cleavage of C4b and in preventing the assembly of C3-convertase)
(Blom et al., 2004).
ZP3R was initially reported to be on the surface of capacitated
sperm (Bleil and Wassarman, 1990; Suzuki-Toyota et al., 1995).
However, results from our laboratory demonstrated that ZP3R is a
component of the acrosomal matrix since it could not be detected
on the plasma membrane of non-capacitated sperm by indirect
immunofluorescence and conventional immunoelectron microscopy
(Foster et al., 1997); rather, it localizes to the particulate
compartment of the acrosome (Kim et al., 2001a). One probable
reason for the discrepancy in the localization of ZP3R in mouse
sperm is the state of sperm capacitation; the protein cannot be
detected on the sperm surface of uncapacitated sperm (Foster et al.,
1997), but the protein appears to be on the surface of capacitated
sperm (Bleil and Wassarman, 1990; Suzuki-Toyota et al., 1995).
The association of ZP3R with the acrosome matrix may allow the
protein to be revealed on the sperm surface after exposure to the
external environment during the course of sperm capacitation.
Although this has been described as a movement from the acrosome
to the surface of the sperm (Wassarman, 2009), we believe it is
more probable that intact ZP3R remains associated with the
acrosomal matrix and is exposed to the external milieu through
limited fusion of the outer acrosomal and plasma membranes in
preparation for the more complete fusion of these membranes during
the course of ZP-stimulated acrosomal exocytosis (Buffone et al.,
2008a). As a consequence of AE, the acrosomal matrix gradually
dissolves, releasing ZP3R into the surrounding environment.
Following our finding that ZP3R is part of the acrosomal matrix,
we have begun to reexamine the events of AE and the role of the
acrosome in sperm-ZP interactions. We support the paradigm that
AE occurs prior to or coincident with sperm-ZP binding, a process
involving the acrosomal matrix (Kim and Gerton, 2003). At the
same time, however, a mechanism must exist to enable the sperm
to release from their point of attachment and move through the ZP
without losing contact with the egg’s extracellular matrix.
Because ZP3R is of interest in sperm-ZP interactions, we
investigated its properties during the progression of AE. In this study
we found that, coincident with its release from the acrosome during
the course of AE, the ZP3R monomer was cleaved to a 43,000 Mr
form by proteases. The process was dramatically reduced under
non-capacitating conditions. The cleavage took place in complement
control protein domain (CCP) 6 or the bridge region between CCP6
and CCP7, which is not present in the guinea pig orthologue AM67.
The cleaved form of ZP3R did not bind to unfertilized eggs,
suggesting that the function of this protein was modified during
AE.
acrosomal exocytosis. In this study, we used immunofluorescence
and immunoblotting of both anti-CPT and anti-VYK (an antibody
directed against a peptide in the C-terminal CCP8) to expand these
previous observations. We found that, after 120 minutes of
incubation in capacitating conditions, ZP3R was barely detectable
in sperm, consistent with our previous results (Figs 1 and 2). When
the suspension of sperm undergoing spontaneous acrosomal
exocytosis was centrifuged, the recovery of ZP3R in the supernatant
correlated with its loss from the sperm heads (Fig. 2). In addition,
we discovered that during the course of spontaneous acrosomal
exocytosis, most of the 67,000 Mr form of mouse ZP3R was
converted to a 43,000 Mr form (ZP3RAE) coincident with its release
from the sperm (Fig. 2A). The band corresponding to ZP3R in intact
sperm was barely observed in the supernatant.
Results
Fig. 1. ZP3R is released to the supernatant during the course of spontaneous
acrosomal exocytosis. Mouse sperm were incubated under capacitating
conditions for up to 120 minutes. At the indicated times, sperm were
immunostained with anti-ZP3R antibody (anti-CPT) and observed under the
microscope to determine the presence of ZP3R in the acrosome. After 120
minutes of incubation most of the sperm lost the ZP3R protein. (A) Percentage
of sperm with ZP3R staining in the acrosome. (B) Representative images
showing sperm stained with anti-ZP3R (anti-CPT) antibody at 0 and 120
minutes of incubation in capacitating conditions (left and middle panels
respectively) with their respective bright-field images. The right panels show a
the negative control (rabbit IgG). Results are expressed as the mean ± standard
deviation (n=4).
ZP3R is processed coincident with acrosomal exocytosis and
its release into the medium
Our laboratory previously demonstrated that there is a temporal
relationship among several phases of acrosomal exocytosis,
suggesting that these patterns represent different transitional stages
leading to the eventual, complete release of acrosomal components
(Kim and Gerton, 2003). Using FluoSphere beads coated with antiCPT (an antibody directed against a peptide in CCP6), we showed
that most of ZP3R is lost during the first 2 hours of spontaneous
The cleavage of ZP3R during spontaneous acrosomal
exocytosis takes place in CCP6 or the bridge region between
CCP6 and CCP7
We next set out to determine at what point in the polypeptide
backbone the cleavage of ZP3R takes place. This modification was
apparently due to a proteolytic cleavage between CCP6 and the
Journal of Cell Science
Acrosomal exocytosis and ZP3R
Fig. 2. ZP3R is processed to a lower molecular weight form as it is released
into the supernatant during the course of spontaneous acrosomal exocytosis.
Mouse sperm were incubated under capacitating conditions for up to 240
minutes. At the indicated times, the incubation mixture was separated into a
pellet and supernatant and analyzed by 10% SDS-PAGE and immunoblotting
with anti-ZP3R (anti-CPT, A; anti-VYK, B). Each lane contained 1 μg of
protein. Anti-VYK antibody detected a band at ~24,000 Mr but not at ~43,000
Mr.
C-terminal end of the ZP3R monomer, since anti-VYK (an antipeptide antibody generated against the C-terminal region of the
protein) no longer recognized the same band detected by anti-CPT,
an antibody that binds to a more central region of the polypeptide
backbone (Fig. 2B). Instead, the anti-VYK detected an ~24,000 Mr
form in the supernatant resulting from the cleavage of the ZP3R
monomer. Fig. 3A, is a diagram of the ZP3R monomer, illustrating
the relative positions of the peptides used to generate anti-CPT and
anti-VYK antibodies.
The guinea pig orthologue of mouse ZP3R, AM67, is not
released as a cleaved form as a consequence of acrosomal
exocytosis
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Fig. 3. (A) Diagram of mouse sperm ZP3R and the relative positions of the
peptides used to generate the anti-ZP3R antibodies (anti-CPT: residues 350367; anti-VYK: residues 542-558). (B) A comparison of the region comprising
domains CCP6 and CCP7 of guinea pig AM67 (protein accession number
O08569) and mouse ZP3R (protein accession number NM_009581). The
bridge region comprising residues 414-453 of mouse ZP3R has no
corresponding counterpart in guinea pig AM67.
and a cholesterol acceptor such as BSA or cyclodextrin in the
medium for capacitation to occur in vitro (Visconti et al., 1995).
We omitted the cholesterol acceptors from the medium to determine
whether they were required for release and cleavage of ZP3R. The
processing of ZP3R under non-capacitating conditions was greatly
impeded (Fig. 5A) compared with parallel capacitating incubations
with either BSA (Fig. 5B) or cyclodextrin (Fig. 5C), suggesting
that the capacitation process is essential for the cleavage and release
into the surrounding medium. We did not observe any difference
between BSA and cyclodextrin in promoting the ZP3R cleavage
and release of ZP3RAE, indicating that the cholesterol efflux is
critical for creating conditions conducive to ZP3R processing.
Processing of ZP3R to ZP3RAE alters the oligomer stability
We recently demonstrated that recombinant ZP3R monomers form
a high order oligomeric protein containing six to eight disulfidelinked monomers (Buffone et al., 2008b). Native ZP3R was also
found in a high order oligomer (Fig. 6A). After treating ZP3R with
Although guinea pig AM67 is most closely related to mouse ZP3R
(Foster et al., 1997), it does not contain a region corresponding to
the bridge between CCP6 and CCP7 of mouse ZP3R (Fig. 3B;
residues 414-453). Since the guinea pig protein lacks the bridge
region, we hypothesized that AM67 would not undergo proteolytic
cleavage during the course of acrosomal exocytosis similar to mouse
ZP3R. To test this hypothesis, we incubated guinea pig sperm under
capacitating conditions for 1 hour, separated the sperm suspension
into supernatant and pellet by centrifugation, and analyzed the
fractions by immunoblotting with anti-VYK antibodies. As shown
previously with the anti-CPT antibody, AM67 was released into
the supernatant as a consequence of acrosomal exocytosis but not
as a proteolytically processed form (Fig. 4).
Cleavage of ZP3R to ZP3RAE requires capacitating conditions
and acrosomal proteases
Sperm require a maturational step termed capacitation before they
are competent for acrosomal exocytosis. Several studies have
demonstrated that mouse sperm need calcium, glucose, bicarbonate
Fig. 4. AM67, the guinea pig orthologue of ZP3R, is not processed to a lower
molecular weight form but it is released into the supernatant during the course
of spontaneous acrosomal exocytosis. Guinea pig sperm were incubated under
capacitating conditions for up to 60 minutes. The incubation mixture was
separated into a supernatant and pellet and analyzed by 10% SDS-PAGE and
immunoblotting with anti-ZP3R (anti-VYK). Each lane contained 5 μg of
protein.
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Journal of Cell Science
Fig. 6. Oligomerization of ZP3R before and after acrosomal exocytosis is
dependent upon disulfide bonds. Mouse sperm were incubated under
capacitating conditions and, at the indicated times, the incubation mixture was
separated into a pellet (sperm) and supernatant (medium) and analyzed by 7%
SDS-PAGE with amounts of DTT ranging from 0 to 5 mM in the sample
buffer. (A) ZP3R from the sperm pellet before the incubation. (B) ZP3RAE
recovered from the medium after the capacitating incubation. Each lane
contained 1 μg of protein (n=3).
Fig. 5. ZP3R is processed only in conditions supporting capacitation. Mouse
sperm were incubated for up to 240 minutes under non-capacitating condition
(A) and capacitating conditions in the presence of 3 mg/ml BSA (B) or 5 mM
2-OH-propyl-β-cyclodextrin (C). At the indicated times, the incubation
mixture was separated and the supernatant analyzed by 10% SDS-PAGE and
immunoblotting with anti-ZP3R (anti-CPT). Each lane contains 1 μg of
protein. When the sperm were incubated for 30 minutes in the presence of
calcium ionophore (1 μM ionomycin a potent stimulator of the acrosomal
exocytosis), the maximum amount of released ZP3R was observed (B, last
lane).
increasing concentrations of dithiothreitol (DTT), the complex was
broken down into a series of smaller oligomers, suggesting that the
complete oligomer consisted of at least six monomers (Fig. 6A).
By contrast, oligomeric ZP3RAE displayed a series of oligomeric
bands in the absence of DTT (Fig. 6B). In addition, the ZP3RAE
complex was dissociated into monomers using a fourfold more dilute
concentration of DTT (250 μM, Fig. 6B) than is required for ZP3R
(1 mM).
Benzamidine blocks the proteolysis of ZP3R but not its release
The inclusion of a protease inhibitor mixture in the medium
decreased the processing of the ZP3R monomer as well as the
release of ZP3RAE from the sperm relative to controls (Fig. 7A),
suggesting that acrosomal protease activity may inactivate a
tethering protein and cause the release of ZP3RAE from the
acrosomal matrix (Fig. 7B). We suspected that this cleavage may
be mediated by acrosin, and cleavage of ZP3R was indeed inhibited
when a single competitive inhibitor of trypsin and trypsin-like
proteases such as acrosin (1 mM benzamidine) was included in the
capacitation medium (Fig. 7C). Benzamidine did not prevent the
release of the uncleaved ZP3R to the surrounding medium,
indicating that other proteases are involved in the disassembly of
the acrosomal matrix and that proteolysis of ZP3R is not a
prerequisite to its release from the acrosomal matrix. Even after
incubating the sperm in the presence of the protease inhibitor
mixture for 120 minutes, the amount of ZP3RAE did not increase
as occurs in the absence of the inhibitors (Fig. 7B). We confirmed
this observation by immunofluorescence, showing that ZP3R
remains associated with the sperm in the presence of the protease
inhibitor mixture but is released to a greater extent when the only
included protease inhibitor is benzamidine (Fig. 7D).
Proteolysis decreases the stability of ZP3R oligomers
To determine whether the processing of ZP3R to ZP3RAE has an
impact on oligomerization, proteins extracted from sperm or recovered
from the supernatant around sperm incubated for 2 hours under
capacitating conditions were analyzed by immunoblotting under nonreducing conditions. As shown in Fig. 8A, protein extracted from
sperm ran as a high order oligomer with a molecular weight much
greater than 250,000. After incubation for 120 minutes under
capacitating conditions, this protein was essentially depleted from
the sperm (Fig. 8A, lane 2). As shown above (Fig. 7C), uncleaved
ZP3R was released into the supernatant when 1 mM benzamidine
was included in the incubation medium; however, the protein
maintained a similar oligomerization state as the protein extracted
from non-capacitated sperm (Fig. 8B, left lane). In the supernatant
recovered from sperm incubated in the absence of benzamidine, a
ladder of different sized oligomers was detectable when analyzed by
non-reducing SDS-PAGE (Fig. 8B, right lane). The high order
molecular weight complex released into the medium under
capacitating conditions, contains both the N-terminal fragment (Fig.
8B, right lane) as well as the C-terminal fragment of the monomer
(Fig. 8C) as detected by anti-CPT and anti-VYK antibodies,
respectively. For reference purposes, the SDS-PAGE gel of the same
samples run under reducing conditions is shown in Fig. 8D. We
conclude that processing of ZP3R to ZP3RAE decreases the ability
of the monomers to maintain the original oligomeric structure.
ZP3RAE does not bind to the ZP of unfertilized eggs
We hypothesized that cleavage of ZP3R and release of ZP3RAE
from the sperm during the course of acrosomal exocytosis correlates
Journal of Cell Science
Acrosomal exocytosis and ZP3R
Fig. 7. In the presence of benzamidine, ZP3R is not processed to a lower
molecular weight form but it is released into the supernatant during the course
of spontaneous acrosomal exocytosis. Mouse sperm were incubated under
capacitating conditions (A), capacitating conditions in the presence of EDTAfree protease inhibitors (B) or capacitating conditions with 1 mM benzamidine
(C). (D) At the indicated times, the incubation mixture was separated into a
pellet and supernatant (1 μg of protein per lane) and analyzed by 10% SDSPAGE and immunoblotting or immunofluorescence with anti-ZP3R (antiCPT) to determine the presence of ZP3R in the acrosome. Black bars and gray
bars represent the average number of sperm with staining after 0 or 120
minutes of incubation, respectively. Results are expressed as the mean ±
standard deviation (n=4).
with the loss of the ability of the protein to bind to ZP. To test this
hypothesis, the biological activities of ZP3R and ZP3RAE were
assessed by evaluating their respective abilities to bind to
unfertilized eggs. Since the native proteins are difficult to purify
from mouse sperm, we used an immunoadsorption procedure to
isolate ZP3R and ZP3RAE from complex protein mixtures prepared
from sperm. The starting material for uncleaved ZP3R was an acid
extract of sperm, and as the source for ZP3RAE we used the
supernatant collected from around sperm that had been incubated
for 120 minutes under capacitating conditions. In the binding assay,
beads coated with the uncleaved form of ZP3R bound to the ZP of
unfertilized eggs (Fig. 9A). Consistent with our hypothesis, beads
coated with ZP3RAE did not bind the ZP. To confirm that the beads
effectively bound ZP3R or ZP3RAE, the protein-coated FluoSpheres
were treated with SDS-PAGE sample buffer and the released
proteins were analyzed by immunoblotting. In each case, the
appropriate form of ZP3R was found in the extracts (Fig. 9B). This
finding indicates that proteolysis of ZP3R coincident with acrosomal
exocytosis modifies its function as a ZP-binding protein.
Discussion
The status of the acrosome in fertilization and the role of the
acrosomal matrix
Although the acrosome has long been recognized to be important
for fertilization, the role of this exocytotic organelle in sperm-zona
pellucida interactions is not completely understood. Based on studies
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Fig. 8. Processing of ZP3R to ZP3RAE decreases the ability of the monomers
to maintain the original oligomeric structure. Mouse sperm were incubated
under capacitating conditions with or without 1 mM benzamidine under
reducing and non-reducing conditions. At the indicated times, the incubation
mixture was separated into a pellet (sperm) and supernatant (medium) and
analyzed by 7% SDS-PAGE and immunoblotting with anti-CPT (A, B and D)
or anti-VYK antibodies (C). In C, prior to blocking with 5% nonfat dry milk,
the PDVF membrane was incubated with TBS containing 200 mM DTT for 1
hour at room temperature to reduce the oligomers and enable detection by the
anti-VYK antibody, which does not react with unreduced ZP3R. The
membrane was then washed three times in TBS for 5 minutes, blocked and
probed with anti-VYK antibody. Each lane contains 1 μg of protein. The
samples were separated under non-reducing conditions for A, B and C, and in
the presence of 50 mM DTT for D (n=4).
by Saling et al. (Saling et al., 1979; Saling and Storey, 1979), many
investigators believe that the outer acrosomal and plasma membrane
must be intact for sperm to recognize and bind to the ZP. Following
what has been referred to as primary binding, the Acrosome
Reaction Model posits that the outer acrosomal membrane (OAM)
and plasma membrane (PM) fuse, releasing the contents of the
acrosome (Yanagimachi, 1994). It is then believed that the inner
acrosomal membrane mediates secondary binding of the sperm to
the ZP. Although the idea that sperm must be ‘acrosome-intact’ to
bind to the ZP has become a predominant model for sperm-egg
interactions, we believe that it is virtually impossible to identify
the fertilizing sperm at the instant of sperm-zona interaction and to
define the nature of its acrosome at that specific point. Furthermore,
there are several reports in the literature of mammalian sperm that
are capable of binding to the ZP and/or fertilizing an egg following
acrosomal exocytosis (Huang et al., 1981; Kuzan et al., 1984;
Morales et al., 1989).
Taking these points into consideration, our laboratory supports
an alternative view that we call the Acrosomal Exocytosis Model
(Buffone et al., 2008a; Gerton, 2002). In this model, sperm
capacitation leads to a metastable state of plasma membrane and
outer acrosomal membrane fusion whereby acrosomal matrix
components may be transiently exposed to the external milieu. In
this model, initial fusion of the OAM and PM may occur without
the complete loss of acrosomal components because, while some
proteins are relatively soluble, other materials are found within the
particulate acrosomal matrix. In support of this concept, specific
acrosomal components of guinea pig sperm undergoing acrosomal
exocytosis in response to calcium ionophore A23187 are
differentially released depending on whether they are components
of the soluble or particulate compartments (Hardy et al., 1991; Kim
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is proteolytically processed to AM50AR as a consequence of its
release from guinea pig sperm, consistent with the results of other
studies (Westbrook-Case et al., 1994). As we have shown here, the
monomer that comprises the ZP3R multimer is cleaved to a lower
molecular weight form upon release from mouse sperm undergoing
acrosomal exocytosis. This proteolytic event may be mediated, in
part, by acrosin which appears to function in the dispersion of the
matrix during acrosomal exocytosis in the mouse (Yamagata et al.,
1998).
Journal of Cell Science
Consequences of the structural homologies of ZP3R and
C4BP
Fig. 9. Mouse cleaved ZP3R protein does not bind to unfertilized eggs.
(A) Sperm medium containing the cleaved form of ZP3R after acrosomal
exocytosis was incubated with fluorescent beads (1 μm) conjugated with antiCPT antibody. The ZP3R-antibody-complex-conjugated beads were then
added to droplets containing ovulated metaphase-II arrested eggs and
incubated for 1 hour. Unbound beads were removed by washing. Beads coated
with the cleaved form of ZP3R do not bind to the zona of unfertilized oocytes
(4). The binding of anti-ZP3R-coated beads that were incubated with acidextract of sperm protein (containing the uncleaved acrosomal ZP3R) served as
a positive control (5). As a negative control, BSA-coated FluoSpheres were
used (6). 1, 2 and 3 are the phase contrast images of 4, 5 and 6, respectively.
(B) The conjugated beads were analyzed by 10% SDS-PAGE and
immunoblotting with anti-CPT antibody.
et al., 2001b). Proteins such as CRISP2 (autoantigen 1) are readily
released from sperm undergoing acrosomal exocytosis whereas
acrosomal matrix protein AM50 remains associated with the sperm
for a longer period of time. Another acrosomal matrix protein,
AM67, is also released more slowly than CRISP2 but is more readily
solubilized than AM50 (in the supernatant fluid 10 minutes after
ionophore treatment). In mouse sperm, there is a temporal
relationship among several stages of acrosomal exocytosis and we
conclude that these patterns represent different transitional stages
leading to the essentially complete release of acrosomal components
from the sperm (Kim and Gerton, 2003). These stages could be
defined morphologically using green fluorescent protein-tagged
acrosomes and beads coated with antibodies to ZP3R.
In this study we have extended our knowledge of the mechanism
whereby AM components are secreted into the surrounding milieu.
For some components such as ZP3R, the release of acrosomal matrix
proteins from the sperm is correlated with post-translational
modifications. Another example is proacrosin, which can be
detected in immunoblots of intact guinea pig sperm, but it is not
detectable in either the pellet or supernatant of A23187-treated
sperm, suggesting that the protein is modified during acrosomal
exocytosis (Kim et al., 2001b). Related to this conclusion, protease
activity consistent with the activation of proacrosin to lower
molecular weight forms of acrosin are detected in the soluble
acrosomal components released from guinea pig sperm stimulated
to undergo acrosomal exocytosis with A23187. Furthermore, AM50
The proteolytic cleavage of ZP3R has profound implications for
the stability of the mouse sperm acrosomal matrix. ZP3R is a
member of the complement regulatory superfamily. Other than
guinea pig AM67 and rat ZP3R, mouse ZP3R is most closely related
to the alpha chain of complement 4-binding protein (Foster et al.,
1997; He et al., 2003). Both of these proteins form high order
oligomers (six to eight monomers of ZP3R per protein complex
and seven alpha and one beta subunits in human C4BP) that contain
both intra- and inter-chain disulfides (Kask et al., 2002). The
monomers of each protein contain CCP domains. In the case of
ZP3R, there are seven complete and one partial CCP; in the mouse
C4BP alpha chain, there are six CCPs followed by a C-terminal
extension and these are held together by non-covalent (presumably
hydrophobic) interactions (Kaidoh et al., 1981; Kristensen et al.,
1987). As shown for human C4BP, these regions contain two
cysteine residues each and an amphipathic α-helix region, which
is required to polymerize the molecule within the endoplasmic
reticulum (Kask et al., 2002). Ligand-binding regions of human
C4BP have been mapped to the various regions of the N-terminal
domain (Blom et al., 2004).
Based on its oligomeric structure and the sequence homology of
the ZP3R monomer to the alpha subunit of C4BP (Bookbinder et
al., 1995; Foster et al., 1997), we propose that ZP3R forms a similar
spider-like structure with the C-termini oriented toward the center
of the complex (Fig. 10). Coupling this model with our results using
two peptide-specific antibodies, anti-CPT and anti-VYK, which
target CCP6 and CCP8, respectively, and our analysis of the products
released by acrosomal exocytosis, we conclude that proteolysis of
ZP3R occurs in the latter part of CCP6 or in the bridge region
between CCP6 and CCP7 (Fig. 10) because anti-VYK no longer
recognized the same band detected by anti-CPT. A corresponding
bridge region is not present in guinea pig AM67, the orthologue of
mouse ZP3R, which is interesting since our analysis of the material
released from guinea pig sperm during acrosomal exocytosis
established that AM67 is released but is not cleaved (Kim et al.,
2001b) (Fig. 4). Thus, cleavage of AM67 is not a prerequisite for
its release from guinea pig sperm during the course of acrosomal
exocytosis.
Capacitation, processing and release of ZP3R from sperm
As is shown in Fig. 7, the cleavage of mouse ZP3R was inhibited
in the presence of benzamidine, a competitive inhibitor of trypsin
and trypsin-like enzymes, such as acrosin. This inhibitor did not
prevent the release of mouse ZP3R to the surrounding medium.
Therefore, as was found previously for guinea pig AM67,
proteolysis of mouse ZP3R is not a prerequisite for its release from
sperm undergoing acrosomal exocytosis. However, when we used
a broad-range protease inhibitor mixture, both the cleavage and
release of mouse ZP3R were reduced, suggesting the active
Acrosomal exocytosis and ZP3R
3159
Materials and Methods
Preparation of culture media and spermatozoa
Journal of Cell Science
Fig. 10. Schematic representation of ZP3R before and as a consequence of
acrosomal exocytosis. The 67,000 Mr monomer is composed of eight
contiguous complement control protein domains (CCP, red). CCPs 1-6 are
linked to CCPs 7 and 8 by a ‘bridge’ domain (blue). The monomers form a
high order oligomeric protein containing six to eight monomers linked by
disulfide bonds (green). During the course of spontaneous acrosomal
exocytosis, the 67,000 Mr form of mouse ZP3R is converted to a 43,000 Mr
form coincident with its release into the surrounding medium. Note: the data
support the presence of at least one disulfide in the N-terminal domain and one
in the C-terminal region; the positions of the disulfide bonds in the figure have
been arbitrarily assigned.
participation of non-benzamidine-inhibited acrosomal proteases or
processes in the processing of ZP3R. In addition, the processing of
ZP3R in mouse sperm was reduced under conditions that do not
support capacitation (Fig. 5). Consequently, we hypothesize that
some of the events associated with capacitation, such as the rise in
intracellular pH, lead to the activation of acrosomal proteases or
processes involved in cleaving and/or releasing ZP3R and other
matrix proteins.
Structural alterations may have functional consequences
Once attached, how might a sperm pass through the zona? ZP3R
is a zona pellucida-binding protein (Bleil and Wassarman, 1990;
Buffone et al., 2008b). Once a sperm attaches to the zona, there
must be a mechanism that enables the sperm to move through the
zona without losing its grip. The fact that the cleaved form of mouse
ZP3R does not bind to unfertilized eggs (Fig. 9) leads us to the
following model. We envision that a capacitated sperm encountering
an egg binds to the zona via transiently exposed acrosomal matrix
proteins, not a plasma membrane ZP-binding protein.
Simultaneously, signaling molecules on the sperm surface interact
with ligands in the ZP, stimulating signal transduction and leading
to an accelerated rate of OAM and PM fusion (ZP-stimulated
acrosomal exocytosis) compared with spontaneous acrosomal
exocytosis. The newly exposed, particulate acrosomal matrix
components assist the penetration of the spermatozoa through the
ZP by the restricted disassembly of this egg vestment, either
enzymatically or stoichiometrically. At the periphery of the exposed
acrosomal matrix, ZP3R is processed and loses its ability to bind
to the ZP. The tethering of the sperm to the zona is transiently lost
until fresh, underlying (but intact) ZP3R oligomers are exposed on
the surface of the sperm and come into contact with the zona.
Release of the peripheral matrix proteins might be mediated by
proteolysis. Processing of acrosomal matrix proteins could result
from proteolytic cleavage by acrosin or other trypsin-like proteases.
As a consequence of the cyclical binding, proteolysis coupled with
loss of binding followed by renewed binding mediated by freshly
exposed ZP3R, the sperm, propelled by its flagellum, would be able
to ratchet its way through the zona.
The solution used for the preparation and culture of mouse sperm was a modified
Krebs-Ringer bicarbonate medium (HMB-Hepes buffered), as described by Lee and
Storey (Lee and Storey, 1986). This medium was first prepared in the absence of
calcium, bovine serum albumin (BSA), NaHCO3, and pyruvate, sterilized by filtration
(0.2 μm tissue culture filter unit; Nalgene) and frozen at –20°C in aliquots for single
use. The working ‘complete’ medium was prepared by adding CaCl2 (1.7 mM),
pyruvate (1 mM), NaHCO3 (25 mM), and BSA (3 mg/ml), followed by gassing with
5% CO2, 95% air to pH 7.3. Uncapacitated cauda epididymal sperm were collected
from mature (8-week old) CF-1 mice (Charles River, Wilmington, MA) by placing
minced caudae epididymides in 1 ml of HM medium without Ca2+, BSA and
NaHCO3. After 5 minutes, the sperm were washed in 1 ml of the same medium by
centrifugation at 800 g for 10 minutes at room temperature. The sperm were
resuspended to a final concentration of 2⫻107 sperm/ml. For the time-course
experiments, aliquots of sperm were incubated for different periods of time, the
sperm were spun down for 5 minutes at 5,000 g at each time point, and the medium
was collected to evaluate the release of ZP3R. The pellet containing the sperm was
then used for immunoblotting analysis or immunofluorescence. In some experiments,
1 mM benzamidine or EDTA-free protease inhibitors (catalog number 11873580001,
Roche) were added to the medium according to the manufacturer’s instructions for
samples with high proteolytic activity. This mixture inhibits a broad spectrum of
serine and cysteine proteases but not metallo- and aspartic proteases. Guinea pig
spermatozoa were obtained and stimulated to undergo acrosomal exocytosis as
previously described (Kim et al., 2001b).
Antibodies
Antibodies generated against two internal peptide sequences designated as anti-CPT
and anti-VYK (residues 350-367 in CCP6: CPTPDMEKIKIVSERRDF, and residues
542-558 in CCP8: VYKLFLEIERLEHQKEK, respectively) were affinity purified
on chromatographic columns conjugated to each respective peptide and characterized
by immunoblotting using protein extracts from spermatozoa as the source of ZP3R
(Kim et al., 2001a).
Extraction of sperm protein
Cauda epididymal mouse sperm were washed twice by centrifugation for 5 minutes
at 300 g and resuspended in PBS (5⫻108 sperm/ml). After the final wash the sperm
pellet was resuspended with 200 μl of extraction buffer (phosphate-buffered saline
containing 1% Triton X-100, 1% sodium deoxycholate and protease inhibitor mixture
containing 1 mg/ml leupeptin, 1 mg/ml aprotinin and 100 μM p-aminobenzamidine,
pH 7.3). The sperm suspension was placed on a rocking platform for 1 hour at 4°C.
The sample was then centrifuged at 14,000 g for 15 minutes at 4°C. The supernatant
was separated and stored at –20°C until used. The protein concentration of the extract
was determined by the bicinchoninic acid assay (Pierce).
Fluorescent bead binding assay
Red fluorescent FluoSphere sulfate microspheres, diameter 1.0 μm, were obtained
from Molecular Probes, Inc. Affinity-purified anti-CPT antibody against mouse ZP3R
was dissolved in 1 ml phosphate-buffered saline (PBS: 50 mM phosphate buffer, pH
7.4, containing 0.9% NaCl) at a final concentration of 2 mg/ml, mixed with 2 ml of
2% aqueous suspension of microspheres, and incubated at room temperature for 12
hours. The mixtures were centrifuged to separate the antibody-labeled microsphere
particles from unreacted protein (3,000–5,000 g for 20 minutes). The pellet was
resuspended in PBS by gentle vortexing. The washing step was repeated twice more
(total of three washes). The antibody-conjugated microspheres were resuspended in
3 ml of PBS containing 1% BSA to block the non-specific sites and incubated in the
BSA solution for 1 hour at room temperature. Sodium azide was added to a final
concentration of 2 mM, and the FluoSpheres were stored at 4°C. The anti-CPT-coated
beads were then incubated for 1 hour at room temperature with a sperm protein extract
containing the non-cleaved protein (10 mg/ml in PBS). Alternatively, the beads were
incubated with cleaved ZP3R (10 mg/ml in PBS) recovered after a 2-hour incubation
of mouse sperm under capacitating conditions. The beads were then washed twice
with PBS and resuspended in 100 μl PBS. For the binding assay, mouse metaphaseII-arrested eggs were mixed with 100 μl of protein-conjugated FluoSpheres for 1
hour at 37°C in 5% CO2. The eggs were then washed three times in PBS containing
0.1% BSA, 0.01% polyvinyl alcohol and examined by phase-contrast and fluorescence
microscopy.
Acid extraction of sperm proteins
Cauda epididymal mouse sperm were washed twice by centrifugation for 5 minutes
at 300 g and resuspended in PBS (5⫻108 sperm/ml). After the final wash, the sperm
pellet was resuspended in two volumes of acid extraction buffer (1% glacial acetic
acid with protease inhibitor mixture containing 1 mg/ml leupeptin, 1 mg/ml aprotinin,
and 100 μM p-aminobenzamidine, pH 3.0) to 1 volume of sperm pellet. The sperm
suspension was placed on a rocking platform for 1 hour at 4°C and then centrifuged
at 10,000 g for 30 minutes at 4°C. The supernatant was then dialyzed with several
changes against 1 mM HCl, pH 3.0 at 4°C for 12–24 hours. The protein concentration
of extract was determined by the BCA assay (Pierce).
3160
Journal of Cell Science 122 (17)
Immunoblotting
Sperm proteins were dissolved in Laemmli SDS-PAGE sample buffer containing the
protease inhibitor mixture (Roche). The proteins were separated by 7%, 3-8% or 10%
SDS-PAGE according to the method of Laemmli (Laemmli, 1970) and transferred
to a PVDF membrane by the method of Towbin et al. (Towbin et al., 1979). Blots
were blocked in 5% nonfat dry milk in TBS and incubated for 1 hour at room
temperature with anti-ZP3R (anti-CPT or anti-VYK) antibodies diluted in blocking
buffer (1:1,000). After washing to remove the primary antibodies, the blots were
incubated with alkaline phosphatase-conjugated goat anti-rabbit IgG secondary
antibody diluted 1:2,500 in blocking buffer. Blots were developed using enhanced
chemifluorescence and analyzed on a STORM system (Molecular Dynamics,
Sunnyvale, CA).
Indirect immunofluorescence
Journal of Cell Science
Mouse sperm obtained from caudae epididymides were washed twice by centrifugation
for 5 minutes at 300 g, resuspended in PBS, and placed onto polylysine-coated
coverslips. Sperm were then treated with PBS containing 4% paraformaldehyde for
15 minutes at room temperature and then permeabilized with methanol. After washing
in PBS, coverslips were incubated in PBS containing 10% normal goat serum
(blocking buffer) for 30 minutes at room temperature, then with primary antibody
(200 nM for anti-CPT) diluted in blocking buffer for 1 hour at 37°C. Following a
washing step (three times, 3.5 minutes in PBS), coverslips were incubated with FITCconjugated goat anti-rabbit IgG diluted 1:50 in blocking buffer for 1 hour at 37°C,
washed again, and mounted using Fluoromount G. Slides were examined using an
inverted microscope (Nikon Eclipse TE 2000-U; Nikon Corporation) and
photographed with a Scion Corporation CFW-1610C digital FireWire camera using
the NIH ImageJ Imaging Software (Rasband, 2007).
We are very grateful to Xue-Jun Fan and Moon-Chan Cha for help
with technical issues. We thank Bayard Storey and James Foster for
their insightful comments on the manuscript. This study was supported
by grant HD-41552 (G.L.G.) and the Fogarty International Center
Fellowship Program 5D43TW000671 (M.G.B.) from the US National
Institutes of Health. Deposited in PMC for release after 12 months.
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