A sperm's perspective of fertilization1
D. J. Miller2
Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana 61801
Abstract
Recent advances in mammalian fertilization are summarized in this review. Because the most information exists about mouse
fertilization, mouse data are considered in the most detail, but research on swine gametes is also reviewed. These two species are
contrasted to illustrate which steps in fertilization may be conserved between mammals and which may be divergent. In all mammals, for a sperm to fertilize an oocyte it must be transported to the site of fertilization and then bind to the mammalian egg coat,
known as the zona pellucida. Binding is a receptor-mediated event and, although it was demonstrated in 1980 that one zona
pellucida glycoprotein, ZP3, binds sperm, its receptor is still the subject of debate. In mice, the most-studied ZP3 receptor is an
enzyme called β1,4-galactosyltransferase. First identified in the Golgi, sperm have this enzyme on their surface. Rather than
acting as an enzyme, on the cell surface β1,4-galactosyltransferase acts as a lectin and binds oligosaccharides of ZP3. It also
activates intracellular signaling, leading to the release of the sperm acrosome, an exocytotic step necessary for penetration
through the zona pellucida. Either through direct or indirect coupling to G proteins, β1,4-galactosyltransferase triggers signaling
within cells, leading to exocytosis. Additional zona pellucida receptors on porcine sperm seem to be involved in gamete binding.
Although porcine sperm β1,4-galactosyltransferase binds to the zona pellucida, it is neither necessary nor sufficient to bind
sperm to oocytes. Identifying the receptors and signaling mechanisms they trigger in sperm is important for diagnosing and successfully treating male infertility. Using a more repeatable competitive fertilization assay, studies are underway to determine
steps in which subfertile males are most often deficient. With this information, rational molecular screening of sperm can be
performed to diagnose infertility problems.
Key Words: Spermatozoa, Acrosome, Zona Pellucida, Oocytes, Eggs, Fertility
Introduction
Sperm Transport
The ultimate destination of the fertilizing sperm is only
reached after a long journey from the testis through the male
and female reproductive tracts. Those sperm that survive this
journey have an even more difficult task ahead. The fertilizing sperm must bind and penetrate the tough coat around the
oocyte, known in mammals as the zona pellucida (Figure 1).
Sperm bind to the zona pellucida using specific adhesion
molecules. At least one of these adhesion molecules must be
a receptor for the zona pellucida that triggers signals within
sperm activating the release of the acrosome. Gamete receptors must maintain sperm adhesion to the zona pellucida
while exocytosis of the acrosome allows sperm to penetrate
through the zona. Once sperm move inside the zona pellucida
and enter the perivitelline space, sperm bind to and fuse with
the oocyte plasma membrane. Another set of gamete receptors is engaged to accomplish this step. Either binding or
fusion of the gametes reawakens the quiescent oocyte, allowing completion of meiosis, the release of cortical granules,
and a host of other changes collectively referred to as egg
activation. Contained in this review is an update of recent
data elucidating the key molecules that allow gametes to bind
and fuse together.
At ejaculation, sperm are deposited into the female reproductive tract. Although most sperm are lost through retrograde sperm transport, some sperm are carried to the site of
fertilization, most often the ampulla of the oviduct. During
this passage, sperm bind to the oviduct epithelial cells
(Hunter, 1981; Hunter and Nichol, 1983). In fact, the lower
portion of the oviduct (isthmus) serves as a reservoir for
sperm (Hunter et al., 1987; Hunter et al., 1991). From the
isthmus, groups of sperm are released that move to the ampulla to provide a relatively constant supply of sperm able to
fertilize oocytes (Hunter, 1984; Smith, 1991). Binding of
sperm to the epithelial cells of the isthmus seems to be quite
strong, because repeated flushing of the oviduct is required to
release adhering sperm (Smith and Yanagimachi, 1990).
Strong attachment and timely release from the reservoir help
ensure that the appropriate number of sperm arrive at the site
of fertilization, ready to fertilize oocytes.
In addition to providing a sperm reservoir, attachment to
the oviduct extends the lifespan of sperm in vitro (Ellington
et al., 1991; Dobrinski et al., 1997; Suarez et al., 1998).
Some evidence suggests that, as sperm develop the ability to
fertilize oocytes (capacitation) and hyperactivated motility,
they are released from the oviductal epithelium, freeing them
to ascend to the ampulla to fertilize oocytes (Fazeli et al.,
1999). Therefore, the surface changes that occur as sperm are
capacitated may reduce their ability to bind to the oviduct.
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Sperm Capacitation
As sperm are moved to the site of fertilization, the female
reproductive tract capacitates them (Visconti and Kopf,
1998). During capacitation, proteins that coat sperm are
removed and modified, sperm develop hyperactivated motility, and the plasma membrane loses cholesterol (Suarez,
1996; Cross, 1998; Visconti and Kopf, 1998). The active
agents in the tract may be serum albumin, high-density lipoproteins, glycosaminoglycans, and(or) other unidentified
components (Parrish et al., 1989a,b; Cross, 1998). Although
the molecular details of capacitation are not completely clear,
recent data suggest that cholesterol removal leads to a protein
kinase A-dependent increase in phosphorylation of many
proteins on tyrosine residues, a process correlated with capacitation (Visconti et al., 1995a,b; Visconti and Kopf,
1998). Apparently, activation of protein kinase A leads to
stimulation of tyrosine kinase activity through an unidentified
mechanism. The functional importance of tyrosine phosphorylation to capacitation is unknown.
A recent observation in the study of capacitation is that
incubation with serum albumin under capacitating conditions
results in membrane hyperpolarization, whereas incubation in
noncapacitating conditions does not change sperm membrane
potential (Arnoult et al., 1999). Sperm that underwent membrane hyperpolarization could respond to zona pellucida
proteins by increasing intracellular calcium and releasing the
acrosome. Data suggest that capacitation allows sperm lowvoltage-activated calcium channels to progress from an inactivated state to a closed state so that they can be opened to
trigger the acrosome reaction in response to zona pellucida
binding (Arnoult et al., 1999).
Murine Sperm-Egg Binding and the Acrosome Reaction
More studies have been performed on fertilization in
mice than in any other mammal. From work with mice, useful
themes have been developed that apply to other mammals,
although there seem to be some differences between species.
The following sections review important findings from murine research and then porcine research.
Sperm Binding to the Zona Pellucida
The extracellular coat of the mouse oocyte is a fairly simple structure composed of three glycoproteins, termed ZP1,
ZP2, and ZP3, in descending order of molecular weight
(Wassarman, 1988). The molecular weights of the three zona
pellucida glycoproteins are 200, 120, and 83 kDa, respectively. Although the molecular mass of each zona pellucida
glycoprotein is heterogeneous, each zona glycoprotein can be
clearly separated from the others by SDS-PAGE. This has
made the study of individual zona glycoproteins of the mouse
much easier than zona glycoproteins from other species, in
which the molecular masses overlap. The intact zona pellucida seems to be formed by ZP2-ZP3 heterodimers that are
connected to form filaments. These filaments are crosslinked
Proceedings of the American Society of Animal Science, 1999
occasionally by ZP1, and the entire complex yields a thick
resilient extracellular matrix.
Pioneering studies of the function of individual zona pellucida proteins were carried out with preparative SDSPAGE. The individual soluble zona glycoproteins were
added to sperm to determine which would bind to sperm and
inhibit binding of sperm to intact oocytes, a competition
assay (Bleil and Wassarman, 1980). Using this assay, it was
demonstrated that ZP3 inhibited binding of sperm to intact
oocytes but other zona glycoproteins did not. Later studies
demonstrated that oligosaccharides on ZP3 were responsible
for binding sperm (Florman and Wassarman, 1985). In addition to binding sperm, ZP3 also induces the acrosome reaction, an effect requiring the protein core in addition to oligosaccharides (Bleil and Wassarman, 1983; Florman et al.,
1984; Leyton and Saling, 1989b).
The Acrosome Reaction of the Fertilizing Sperm
Is Triggered by ZP3
The sperm acrosome is a membrane-bound organelle lying underneath the plasma membrane at the anterior portion
of the sperm head. The acrosome reaction, which can be
considered as a specialized type of exocytosis, proceeds as
the plasma membrane undergoes a series of point fusions
with the underlying outer acrosomal membrane. These fusions eventually form hybrid vesicles that are released, exposing the contents of the acrosome. The acrosome reaction
is required for sperm to penetrate the zona pellucida.
Most evidence indicates that the acrosome reaction of the
fertilizing sperm occurs on the zona pellucida. Observations
of sperm moving through the cumulus mass surrounding the
zona pellucida show only acrosome-intact sperm penetrating
the cumulus matrix (Cherr et al., 1986). Prematurely acrosome-reacted sperm do not penetrate the cumulus mass, and
blocking the zona-induced acrosome reaction specifically
inhibits fertilization (Florman and Storey, 1982; Yanagimachi, 1994). In addition to ZP3, there is evidence that a soluble hormone induces the acrosome reaction. Progesterone
can induce the acrosome reaction, apparently by binding to a
plasma membrane receptor (Blackmore et al., 1991; Meizel
et al., 1997; Baldi et al., 1999). Because relatively high
progesterone concentrations are required, it is not certain that
the progesterone concentration is adequate near the oocyte to
induce the acrosome reaction independently. One attractive
hypothesis is that progesterone synergizes with the zona
pellucida to induce higher frequencies of the acrosome reaction, but studies do not support this hypothesis (Melendrez et
al., 1994; Roldan et al., 1994; Murase and Roldan, 1996).
Although the initial signal cascade activated by ZP3 and
progesterone is divergent, both agonists eventually lead to
increases in cytoplasmic calcium concentrations and the
acrosome reaction (Meizel et al., 1997).
The ZP3 Receptor: An Enigma
Although ZP3 was identified in 1980 in mice as the zona
glycoprotein that bound sperm (Bleil and Wassarman, 1980),
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its corresponding receptor on sperm has been the subject of
much controversy. Different methods of isolating the receptor have yielded several different molecules. Of the putative
receptors studied, the most information exists about a bifunctional enzyme called β1,4-galactosyltransferase (GalTase).
Named for its ability to add galactose to glycoproteins and
glycolipids during their biosynthesis in the Golgi, GalTase is
also found on the plasma membrane of some cells (Shur,
1993). Because the galactose donor, uridine diphosphogalactose (UDP-Gal), is present in the lumen of the Golgi apparatus, GalTase adds galactose to the galactose acceptor (i.e.,
glycoproteins). UDP-Gal has not been found on the cell surface, so GalTase seems to bind the galactose acceptor in a
stable complex without completing the catalytic reaction,
much like a lectin binds an oligosaccharide (Shur, 1993).
This model is supported by experiments in which addition of
UDP-Gal allows completion of the catalytic reaction and
disrupts cell adhesion due to GalTase (Shur and Hall, 1982;
Lopez et al., 1985).
Cloning GalTase cDNA revealed that GalTase has a
transmembrane domain. Finding two translation initiation
sites in GalTase mRNA suggested a possible mechanism for
its dual localization (Shaper et al., 1988; Russo et al., 1990).
A longer form of GalTase is translated from an upstream
translation initiation site, and it has 13 additional N-terminal
amino acids compared to the short form. Within that Nterminal extension is apparently a sequence that overrides the
Golgi retention signal, carrying the longer form to the plasma
membrane (Youakim et al., 1994). Very recently, additional
GalTase-related genes have been identified (Amado et al.,
1999). None seems to have two translation start sites, so the
protein products of these genes may be not be located on the
cell surface. At least one of the newly identified GalTase
isomers is expressed in the testis, although it is not clear
whether it is expressed in the germ cells (Sato et al., 1998).
Some of the newly identified GalTases prefer glycolipid
substrates to glycoproteins, so they would not be expected to
bind ZP3 even if they were on the sperm plasma membrane
(Schwientek et al., 1998). All studies of GalTase function
during fertilization have been performed on the first GalTase
identified, dubbed GalTase-I.
GalTase-I on the sperm surface can bind to ZP3 but not
to other zona pellucida glycoproteins (Miller et al., 1992).
Interestingly, although all three mouse zona pellucida glycoproteins have terminal N-acetylglucosamine residues, GalTase-I only binds to ZP3 (Miller et al., 1992). Thus, sperm
GalTase-I recognizes N-acetylglucosamine residues only in
the context of a specific oligosaccharide. The biological
importance of GalTase-I was demonstrated using a variety of
GalTase inhibitors (Shur and Hall, 1982; Lopez et al., 1985;
Miller et al., 1992). Antibodies, substrate modifier proteins,
and soluble purified GalTase-I (as a competitor) all inhibit
sperm-egg binding. Furthermore, blocking or removing the
GalTase-I binding sites from ZP3 that have terminal Nacetylglucosamine residues prevents ZP3 from binding sperm
(Figure 2).
Other ZP3 receptors have been proposed. One is a sperm
protein that, after SDS-PAGE and transfer to nitrocellulose,
Proceedings of the American Society of Animal Science, 1999
binds labeled ZP3 (Leyton and Saling, 1989a). There is evidence in mice that this is an unusual form of hexokinase
(Kalab et al., 1994; Visconti et al., 1996). Saling and colleagues suggested that its human orthologue had been cloned,
but what was cloned instead seems to be c-mer (Burks et al.,
1995; Bork, 1996; Tsai and Silver, 1996). Another potential
ZP3 receptor called sp56 was identified by ZP3 crosslinking
experiments and by ZP3 affinity chromatography (Bleil and
Wassarman, 1990; Cheng et al., 1994; Bookbinder et al.,
1995). However, recent studies report that, rather than being
a plasma membrane protein, sp56 is found inside the acrosome, where it could not bind sperm until the acrosomal
contents are exposed (Foster et al., 1997). Other potential
ZP3 receptors have been described, and the function of these
putative ZP3 receptors requires further study (Cornwall et al.,
1991; Tanphaichitr et al., 1993; Gao and Garbers, 1998).
Intracellular Signaling to Induce the Acrosome Reaction
Because the acrosome reaction is vital for zona penetration, elucidating the way in which zona binding induces the
acrosome reaction is integral to improving reproductive rates.
In addition to binding sperm, ZP3 also induces the acrosome
reaction. Some investigators have proposed that a second
ligand on ZP3 binds to a unique receptor to trigger the acrosome reaction (Ward and Kopf, 1993). Another hypothesis is
that the same receptor that binds ZP3 also induces the acrosome reaction. In agreement with this model, studies using
GalTase-I antibodies have shown that some GalTase-I antibodies could elicit the acrosome reaction from mouse sperm
(Macek et al., 1991). In fact, some antibodies activated acrosome reactions at the same frequency as solubilized zona
pellucida proteins. Most interesting was the observation that
pertussis toxin, which inhibits the Gi class of G proteins,
inhibits the GalTase-I antibody-induced acrosome reaction
(Gong et al., 1995). Studies from Kopf's laboratory demonstrated that pertussis toxin can block the zona-induced acrosome reaction (Endo et al., 1987, 1988). Binding of GalTaseI by antibodies also activated G proteins (Figure 3), as assessed by increased guanosine triphosphate (GTP) binding
(Gong et al., 1995). Therefore, in addition to acting in gamete adhesion, GalTase-I may also activate signal transduction, leading to the acrosome reaction. Additional studies of
the similarity of the GalTase-I antibody-induced acrosome
reaction and the ZP3-induced acrosome reaction are required
to determine whether GalTase-I can account for the characteristics expected of a ZP3 receptor that activates the acrosome reaction.
In agreement with the hypothesis that GalTase-I induces
the acrosome reaction, mice with a targeted deletion of the
GalTase-I gene produce sperm that are severely compromised in their ability to acrosome-react and penetrate the
zona pellucida (Lu and Shur, 1997). Sperm from these males
had a 93% reduction in zona penetration rates. This confirms
that GalTase is important for inducing the acrosome reaction
in response to the zona pellucida. Surprisingly, there were no
obvious fertility defects in these males during natural mating
(Asano et al., 1997; Lu and Shur, 1997; Lu et al., 1997).
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However, preliminary data suggest that, when GalTase-I null
sperm were mixed with an equal number of sperm from wild
type males, sperm from the wild type males had an advantage
in fertilization rates (B. D. Shur, unpublished observations).
Interestingly, sperm from the GalTase-I knockout mice
still had some residual GalTase activity remaining on their
sperm, amounting to 5% of the total found on sperm from
wild type males (Asano et al., 1997; Lu et al., 1997). Both
groups that produced knockouts reported this residual activity, and this observation led to identification of other GalTase
genes, as described above. It is not known whether the other
GalTase genes are expressed on sperm or have any function
during fertilization. As reviewed above, there are also other
putative ZP3 receptors on sperm. Therefore, to study GalTase-I function in the absence of other putative ZP3 receptors, GalTase-I was expressed in heterologous cells. This
approach also enables one to study specific mutations of
putative ZP3 receptors.
Expression of a ZP3 receptor in heterologous cells would
be predicted to enhance ZP3 binding. Xenopus laevis oocytes
were used to express mouse GalTase-I and study its function.
Xenopus oocytes expressing GalTase-I bound ZP3 but not
other zona pellucida glycoproteins (our unpublished observations). Another reason for selecting Xenopus oocytes to express GalTase-I is that previous studies demonstrated that
activating exogenous G protein coupled receptors expressed
in oocytes would trigger cortical granule exocytosis (Kline et
al., 1988, 1991; Shilling et al., 1994). Envisioning that cortical granule exocytosis may be related to acrosomal exocytosis, we found that when mature oocytes expressing mouse
GalTase-I were incubated with GalTase-I agonistic antibodies or ZP3, the cortical granules were exocytosed (our unpublished results). In sperm, liganded GalTase-I triggered an
increase in GTP binding, indicative of G protein activation
(Gong et al., 1995). Membranes from oocytes expressing
GalTase-I also responded to GalTase-I ligands with an increase in GTP binding. Therefore, signaling induced by GalTase-I was similar in sperm and Xenopus oocytes, suggesting
that this is a useful expression system for studying GalTase-I
function.
The observation that GalTase-I activates G proteins was
quite unexpected. To confirm this, the GalTase cytoplasmic
domain that would be predicted to activate G proteins was
incubated with sperm lysate and those proteins that formed a
complex with the cytoplasmic domain were precipitated.
Within this GalTase-I complex, a Gi heterotrimer was found
(Gong et al., 1995). Scrambled peptides or a peptide corresponding to the short version of GalTase-I did not precipitate
Gi; therefore, sequences specifically in the long form of GalTase-I are necessary for G protein interaction.
Because most G protein-coupled receptors have seven
transmembrane domains and GalTase-I has only one, further
studies of GalTase activation of G proteins are warranted.
There are examples of single transmembrane receptors that
interact with G proteins (Okamoto et al., 1990; Nishimoto,
1993; Sun et al., 1997). Mastoparan, a short basic peptide,
also activates G proteins (Okamoto et al., 1990; Ward et al.,
1992; Nishimoto et al., 1993). In these peptides, as well as in
Proceedings of the American Society of Animal Science, 1999
some seven transmembrane domain receptors, clusters of
basic amino acid residues are necessary to activate G proteins. Importantly, mutation of a cluster of basic amino acid
residues in the cytoplasmic domain of long GalTase-I eliminates its ability to activate G proteins and trigger cortical
granule exocytosis in Xenopus oocytes (our unpublished
observations). This supports the hypothesis that GalTase-I
activation of G proteins is necessary for exocytosis.
Zona Pellucida Penetration
As the sperm acrosome reaction occurs, sperm must remain adherent to the zona pellucida and start their movement
through a developing slit in the zona pellucida. Adherence
during the acrosome reaction may be mediated by ZP3 binding to sp56, a protein with affinity for ZP3 that is found in
the sperm acrosome (Bleil and Wassarman, 1990; Bookbinder et al., 1995; Foster et al., 1997). Following completion of the acrosome reaction, mouse sperm lose their affinity
for ZP3 but gain an affinity for ZP2 (Bleil and Wassarman,
1986; Bleil et al., 1988). A protein called PH-20 with hyaluronidase activity on acrosome-reacted sperm seems to be
important for maintaining sperm adherence to the zona pellucida (Primakoff et al., 1985; Hunnicutt et al., 1996; Myles
and Primakoff, 1997). Prior to the acrosome reaction, PH-20
is found on the sperm plasma membrane, and it digests the
hyaluronic acid matrix between cumulus cells in animals that
retain the cumulus matrix on oocytes until fertilization
(Hunnicutt et al., 1996; Myles and Primakoff, 1997). Curiously, some PH-20 is relocalized during the acrosome reaction, moving from the postacrosomal region to the inner
acrosomal membrane (Cowan et al., 1991). At this location,
it apparently binds to an unidentified zona glycoprotein.
Studies of the fate of GalTase-I following the acrosome
reaction have been intriguing. On acrosome-intact sperm,
GalTase is located on the plasma membrane overlying the
acrosome (Lopez and Shur, 1987). During the acrosome
reaction, this membrane is released from sperm, but, surprisingly, GalTase-I is redistributed and retained on sperm. Enzyme assays indicate that 90% of total GalTase activity is
salvaged on mouse sperm after the acrosome reaction (Lopez
and Shur, 1987). GalTase-I moves laterally on the sperm
head and seems to be found on the inner acrosomal membrane. Although GalTase-I is present on acrosome-reacted
sperm and is enzymatically active toward simple substrates, it
does not seem to bind any zona pellucida proteins (Miller et
al., 1992). To date, the function of GalTase on acrosomereacted sperm remains unresolved.
One hypothesis was that binding of sperm to ZP2 is mediated by proacrosin, the proenzyme form of the acrosomal
serine protease acrosin (Bleil et al., 1988; Benau et al., 1990;
Williams and Jones, 1993). However, sperm from mice with
a targeted deletion of proacrosin are fertile (Baba et al.,
1994). ZP2 probably binds to undetermined sperm proteins
to maintain binding of sperm after the acrosome reaction. A
ZP2 receptor would have to maintain sperm on the zona
pellucida so that these sperm could start to move into the
zona matrix. This receptor may also retain sperm within the
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matrix so that a fertilizing sperm could not "back out" of a
penetration slit. One can envision a ratchet-like mechanism
with which a receptor would hold sperm in place and then a
combination of motility and formation of a penetration slit
would allow sperm to advance through the zona pellucida.
Formal testing of this model of sperm penetration has not
been reported.
In fact, there is debate about whether zona penetration is
a proteolytic process (Bedford, 1998). In the sea abalone, a
sperm protein (lysin) dissolves a hole through the vitelline
envelope by disrupting the structure of the envelope proteins
nonenzymatically (Shaw et al., 1993, 1995). In the same
manner, mammalian acrosomal proteins could bind to zona
pellucida proteins and disrupt the interaction with other zona
proteins, "melting" a hole in the zona pellucida through
which sperm can move. Mice lacking proacrosin, one of the
more abundant acrosomal enzymes, produce sperm that are
able to fertilize oocytes, although fertilization is slowed
(Baba et al., 1994; Yamagata et al., 1999). The time lag may
be due to slowed dispersion of the acrosome contents, a function of acrosin (Yamagata et al., 1999). However, zona penetration may also be an enzymatic process requiring that acrosomal enzymes digest the zona locally to form a penetration
slit (Kohno et al., 1998; Yamagata et al., 1998).
Once inside the zona pellucida, sperm can fuse with the
oocyte plasma membrane. Binding between sperm and the
oocyte membrane seems to be accomplished by a heterodimeric protein on sperm called fertilin or fertilin-related
proteins (Blobel et al., 1992; Almeida et al., 1995; Yuan et
al., 1997). Blocking fertilin with antibodies prevents sperm
from binding to the oocyte membrane, and sperm from fertilin β knockouts are less able to bind oocytes (Myles, 1993;
Myles and Primakoff, 1997; Cho et al., 1998). Curiously,
sperm from fertilin β knockouts are also transported through
the female tract less efficiently and are less able to bind the
zona pellucida. Deletion of this gene causes a variety of
defects in addition to reducing sperm binding to the oocyte
membrane, suggesting that fertilin β has functions during
spermatogenesis and zona pellucida binding in addition to
oocyte plasma membrane binding.
Porcine Fertilization
Although the mouse has been a useful animal for
fertilization studies, it is important to study fertilization in
other species, given the possible differences that might occur
due to divergence and separation of species. Some have
predicted that gamete receptors may be among the most
divergent genes because that divergence would promote the
formation and isolation of new species (Vacquier, 1998).
Alternatively, rather than separate species by means of
divergent versions of the same genes, species could develop
different genes for fertilization. To study this further, many
researchers have used porcine gametes because it is relatively
easy to purify large amounts of zona pellucida protein from
ovaries collected at an abattoir. Unfortunately, the molecular
weights of the porcine versions of mouse ZP1 and ZP3,
overlap so the study of individual zona glycoproteins has
Proceedings of the American Society of Animal Science, 1999
study of individual zona glycoproteins has been problematic.
Several laboratories have succeeded in separating ZP1 and
ZP3 after partial deglycosylation with endo-β-galactosidase
(Yurewicz et al., 1987, 1998). However, this enzyme may
remove oligosaccharides that bind sperm and cause a partial
loss of sperm binding activity, so caution should be exercised.
The naming conventions of the zona pellucida proteins
have been inconsistent, because these proteins were named
before the genes were cloned and orthologues were identified
(Harris et al., 1994). A table of the nomenclature is provided
(Table 1).
Sperm Binding to the Porcine Zona Pellucida
Orthologues of the mouse zona pellucida proteins are
found in the porcine zona pellucida. Initial studies showed
that, after partial deglycosylation and purification, porcine
ZPB (orthologue of mouse ZP1) retained some spermbinding activity (Sacco et al., 1989). Recent studies suggest
that a heterodimer of ZPB and ZPC retains greatest binding
affinity (Yurewicz et al., 1998). These conclusions with partially deglycosylated zona proteins are not consistent with
studies of the mouse zona pellucida. Because the oligosaccharides of these glycoproteins bind sperm, the difference
may be because the active oligosaccharides are added to
different zona glycoproteins during their biosynthesis. A
second possibility is that each species may use different oligosaccharides found on different zona glycoproteins to bind
sperm.
As in mice, the identity of the receptor on boar sperm for
the zona pellucida is not completely clear. To isolate zona
receptors, most researchers have used the intact porcine zona
or soluble zona proteins that have not been separated from
each other. Several peripheral membrane proteins have been
described that bind the porcine zona pellucida (Ensslin et al.,
1998; Topfer-Petersen et al., 1998). Many of these are products of the accessory glands and are added to sperm at ejaculation. Some are lost during capacitation, whereas others are
retained on sperm because of their affinity for phospholipids
(Topfer-Petersen et al., 1998). The binding specificity to the
zona glycoproteins has not been described. Zonadhesin is an
integral membrane protein from cauda epididymal sperm
membranes that was purified based on its species-specific
affinity for the whole porcine zona pellucida (Hardy and
Garbers, 1994, 1995). Zonadhesin is related to von Willebrand Factor, another cell matrix receptor. Results of studies
concerned with zonadhesin's zona protein binding specificity
and functional importance have not been published.
Similar to its location on mouse sperm, GalTase-I is
found on the plasma membrane overlying the acrosome of
boar sperm. The presence of GalTase-I on sperm seems to be
common in mammals. Localization to the head has been
demonstrated in all mammals examined to date: cattle, humans, rats, guinea pigs, stallions, and rabbits (Larson and
Miller, 1997). With the exception of mice, studies of GalTase function in other species are limited. Recently it was
demonstrated that GalTase on porcine sperm bound to a band
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of pig zona proteins in the ZPB/ZPC molecular mass region
(Rebeiz and Miller, 1999). Although GalTase bound porcine
zona proteins, when UDP-Gal was added to complete the
catalytic reaction, porcine sperm did not dissociate from the
oocytes. Furthermore, removing the GalTase binding site
from the zona pellucida did not affect sperm binding (Rebeiz
and Miller, 1999). These data suggest that GalTase is not
required for porcine sperm-oocyte binding. Therefore, it
seems that porcine sperm GalTase is part of a redundant
receptor system; when it is blocked, other receptors can still
bind sperm to oocytes. Nevertheless, it was still possible that
GalTase, by itself, was sufficient to mediate gamete binding.
To test this possibility, soluble porcine zona proteins that had
been treated with N-acetylglucosaminidase to remove the
GalTase binding site were incubated with sperm. These zona
proteins would bind to and block all putative zona pellucida
receptors on sperm except GalTase, leaving GalTase exposed
to bind oocytes. Sperm treated in this manner did not bind to
oocytes, suggesting that GalTase alone is insufficient to bind
sperm to oocytes (Rebeiz and Miller, 1999).
Because GalTase was not necessary or sufficient for porcine sperm to bind the zona pellucida and there are a number
of other zona receptor candidates whose functional importance is unknown, it is essential to characterize zona binding
to sperm to determine which of these candidate receptors is
most likely to be biologically important. In studies using
mice, ZP3 was radiolabeled directly and incubated with
sperm. Autoradiography revealed binding to the entire head
of acrosome-intact sperm (Bleil and Wassarman, 1986).
After the acrosome reaction, ZP2 bound to the head (Bleil
and Wassarman, 1986). Using colloidal gold-labeled zona
proteins, ZP3 bound to the entire head of acrosome-intact
sperm, but ZP2 bound mostly to the inner acrosomal membrane of acrosome-reacted sperm (Mortillo and Wassarman,
1991). In other species, reports of the region of the sperm
head that binds zona proteins used fixed or permeabilized
sperm (Jones, 1991; Yurewicz et al., 1993). Because fixation
may expose intracellular proteins that bind zona pellucida
proteins, it was important to examine binding of directly
labeled porcine zona proteins to live porcine sperm. On live
sperm the vast majority of zona protein binding was to the
thin apical ridge (Burkin and Miller, 2000). As sperm acrosome reacted or if sperm were fixed or otherwise permeabilized, zona proteins bound to the entire anterior half of the
head matching the location of the acrosome. Therefore, zona
binding proteins are apparently distributed throughout the
acrosome, where they are available to bind the zona pellucida
during and after the acrosome reaction. These data also suggest that zona protein receptors involved in initial binding
should be localized to the apical ridge of live, acrosomeintact porcine sperm.
Identification of Frequent Causes of Male Subfertility
A useful benefit of fertilization research is to be able to
identify and correct defects causing subfertility. Although
some subfertile sperm have obvious defects, others have
normal motility and morphology. To identify these, one
Proceedings of the American Society of Animal Science, 1999
could test for the presence and function of proteins that are
important for successful fertilization. Tests of proteins that
are most commonly defective are expected to be of greatest
value in identifying subfertile males. Currently, it is not
known which proteins most often cause subfertility. In fact, it
is not known at which stage in fertilization defects frequently
occur. Specific stages in progression through fertilization can
be observed using in vitro fertilization, and several laboratories have used this process to determine whether in vitro
fertilization outcome is related to in vivo fertility. Some
publications report a correlation between in vitro fertilization
rate and in vivo fertility, but others do not (Graham, 1996;
Truelson et al., 1996). In some cases, high concentrations of
sperm were used for in vitro fertilization (Siddequey and
Cohen, 1982). In this situation, because only one sperm per
oocyte is sampled, only a very small sample of the total
sperm is assayed. In addition, any variation in the way gametes in different droplets of medium are handled adds variation to the in vitro fertilization rates and reduces the repeatability of the assay. It is likely that these problems have
contributed to inconsistent reports.
To attempt to develop an in vitro fertilization assay that
provided more repeatable results, a competitive assay using
sperm from two samples was developed (Miller et al., 1998).
The use of two lipophilic dyes that do not affect sperm motility and oocyte binding ability allows identification of mixed
sperm within a droplet of medium. When sperm from two
males or sperm treated in dissimilar ways are stained with
different dyes, they can be identified after mixing and addition to oocytes. The number of sperm bound to or penetrating
the zona from each sample can be counted. Because sperm
are incubated in the same droplet with the same oocytes, the
samples are treated more uniformly.
These lipophilic dyes were tested to be certain they did
not affect sperm binding to the zona pellucida. Sperm from
the same collection were stained with either DiQ (red) or
DiOC16 (green), mixed in various ratios, and added to oocytes. The ratio of sperm bound to the zona pellucida
matched the proportion of red/green sperm in the surrounding droplet of medium. These results demonstrated there was
no preferential effect of either dye on the ability of sperm to
bind the zona pellucida (Miller et al., 1998). Studies to identify the specific stage of fertilization most often defective in
subfertile sperm are underway.
Implications
Despite recent progress, many molecular aspects of ovum
fertilization remain an enigma. Fertilization in most mammals proceeds through mechanisms that are generally similar
to those described in the most commonly studied mammals,
mice. A variety of mammals, amphibians, and birds express
the zona pellucida glycoprotein ZP3. Although ZP3 was
shown to bind mouse sperm in 1980, its receptor on sperm is
still debated. Many mammals have the putative ZP3 receptor,
β1,4galactosyltransferase-I, on sperm, but the protein’s func© 2000 American Society of Animal Science
7
tion may differ among species. Mouse sperm lacking a functional β1,4galactosyltransferase-I gene have limited ability to
acrosome-react and penetrate the zona pellucida. In swine,
β1,4galactosyltransferase-I is a redundant receptor. Redundancy in gamete receptors should not be unexpected, considering the importance of gamete binding. Nevertheless, in
mouse sperm and other cells expressing mouse
β1,4galactosyltransferase-I, this ZP3 receptor activates G
protein-mediated signal transduction necessary for fertilization. A lucid understanding of fertilization and technologies
to increase fertility seems within reach.
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Notes
1. The NSF (IBN 94-18077), NIH (HD38311), the Illinois
Council for Food and Agricultural Research, and the Illinois Agric. Exp. Sta. have supported work in the author’s
laboratory. Suggestions for this manuscript from Charles
Graves, Heather Burkin, and Xudong Shi were greatly appreciated. Heather Burkin and Xudong Shi also contributed many unpublished observations.
2. Correspondence: phone: 217-333-3408; fax: 217-3338286; E-mail: [email protected].
© 2000 American Society of Animal Science
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Table 1. Nomenclature of mouse and porcine zona pellucida (ZP) glycoproteins
Species
Mouse
Swine
Zona pellucida glycoprotein orthologue
ZP1
ZP2
ZP3
ZPB or ZP3α
ZP1 or ZPA
ZP3β or ZPC
Figure 1. Progression of the fertilizing sperm, following a
clockwise movement on the figure. The fertilizing sperm
binds to the zona pellucida and the zona activates the acrosome reaction. During the acrosome reaction, the plasma
membrane fuses with the underlying outer acrosomal
membrane, releasing both membranes. This frees the acrosomal contents and exposes the inner acrosomal membrane. The acrosome-reacted sperm penetrates the zona
pellucida through a slit in the zona pellucida and enters the
perivitelline space. The sperm binds and fuses with the
oocyte plasma membrane, activating the oocyte and triggering the release of the cortical granules. Cortical granules
contain enzymes that modify the zona pellucida so additional sperm cannot penetrate the zona pellucida.
Figure 2. Model illustrating mouse gamete receptors. ZP3,
a zona pellucida glycoprotein, binds to GalTase-I on the
surface of acrosome intact sperm.
Terminal Nacetylglucosamine residues (GlcNAc) within specific oligosaccharides of ZP3 bind to GalTase-I. GalTase-I binding
activates a heterotrimeric G protein, triggering the acrosome reaction. Acrosome-reacted sperm remain on the
zona pellucida by binding to ZP2 and begin migrating
through the zona matrix.
Proceedings of the American Society of Animal Science, 1999
© 2000 American Society of Animal Science
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Figure 3. Activation cycle of heterotrimeric G proteins. When G proteins are activated by a receptor, the
α subunit exchanges GDP for GTP. Once it has
bound GTP, the α subunit dissociates from the βγ
subunit and the α and βγ subunits act on downstream
effectors. After acting on effectors, the endogenous
GTPase activity of the α subunit hydrolyzes the bound
GTP, releasing free phosphate and allowing the heterotrimer to reform, deactivating the G protein.
Therefore, typical G protein-coupled receptors stimulate both GTP binding and GTP hydrolysis, a result
also triggered by GalTase-I.
Proceedings of the American Society of Animal Science, 1999
© 2000 American Society of Animal Science