REVIEW ARTICLE

Molecular Reproduction & Development 76:933–941 (2009)

Zona Pellucida Glycoprotein ZP3 and

Fertilization in Mammals
EVELINE S. LITSCHER, ZEV WILLIAMS,† AND PAUL M. WASSARMAN*

Department of Developmental and Regenerative Biology, Mount Sinai School of

Medicine, New York, New York

SUMMARY
An early step in mammalian fertilization is species-restricted binding of sperm to the
egg’s zona pellucida (ZP), a thick extracellular coat that surrounds eggs. Sperm bind . . .[the] polypeptide encoded
to the ZP of unfertilized eggs, but not to the ZP of fertilized eggs. Shortly after binding to by ZP3 exon-7 is both
the unfertilized egg ZP, sperm undergo the acrosome reaction, a form of cellular
necessary and sufficient for
exocytosis that enables sperm to penetrate the ZP. Three glycoproteins, mZP1-3,
binding of sperm
constitute the mouse egg’s ZP and participate in the process of fertilization. For
example, sperm exposed to unfertilized egg mZP3 at nanomolar concentrations are
inhibited from binding to eggs and undergo the acrosome reaction. Neither mZP1 nor
mZP2 has an effect on sperm binding or the acrosome reaction. Furthermore, mZP3 * Corresponding author:
from fertilized eggs has no effect on sperm binding and is unable to induce the 1 Gustave L. Levy Place, NY 10029.
acrosome reaction. These and other properties of mZP3 suggest that it is a receptor E-mail: [Link]@[Link]
for sperm and inducer of the acrosome reaction. Mapping of the mZP3 combining-site
for sperm suggests that it is located near the C-terminus of the polypeptide, just †
Current address:
downstream of the ZP domain, in a region encoded by exon-7 of the mZP3 gene. This Center for Reproductive Medicine and
region of mZP3 is a site of positive Darwinian selection. When mZP3 exon-7 is fused to Infertility
Weill-Cornell Medical Center
the Fc fragment of human IgG and sperm exposed to the chimeric protein, sperm are New York, NY 10021.
inhibited from binding to eggs. However, the chimeric protein does not induce the
acrosome reaction. Therefore, polypeptide encoded by mZP3 exon-7 is necessary
and sufficient for binding of mouse sperm.

Mol. Reprod. Dev. 76: 933–941, 2009. ß 2009 Wiley-Liss, Inc.

Published online 5 June 2009 in Wiley InterScience
([Link]).
Received 3 March 2009; Accepted 2 April 2009 DOI 10.1002/mrd.21046

PROLOGUE on fertilization in mammals to a symposium honoring the life

The subject of fertilization is certainly appropriate for a of E.E. Just.
symposium dedicated to Dr. Ernest Everett Just. E.E. Just’s
Ph.D. thesis at the University of Chicago (1916) concerned
research on fertilization in marine invertebrates. His thesis MAMMALIAN FERTILIZATION AND THE
supervisor, Frank R. Lillie, was together with Jacques Loeb, ZONA PELLUCIDA
one of the most influential practitioners of fertilization re-
A primary characteristic of living organisms is their ability
search in the first part of the 20th century. E.E. Just pub-
to reproduce. Of the nearly 14 million species on Earth,
lished several important papers in 1919 and 1920 on the
approximately 99% of them reproduce sexually. Many
‘‘fertilization-reaction’’ in Echinarachnius parma and these
papers had considerable impact on the field. Consequently,
E.E. Just’s international reputation as an outstanding
Abbreviations: ZP, zona pellucida; mZP3, mouse ZP3; hZP3, hamster ZP3;
research scientist and scholar was established early on EC, embryonal carcinoma; CHO, Chinese hamster ovary; CFCS, consensus furin
(Manning, 1983). It is a great pleasure to contribute a paper cleavage-site; TM, transmembrane; CT, cytoplasmic tail.

ß 2009 WILEY-LISS, INC.

 10982795, 2009, 10, Downloaded from [Link] by Cochrane Mexico, Wiley Online Library on [31/03/2023]. See the Terms and Conditions ([Link] on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Molecular Reproduction & Development LITSCHER ET AL.

Figure 1. Light and electron micrographs of the mouse ZP. A: Light micrograph (Nomarski differential
interference contrast) of sperm bound to the egg ZP. B: Scanning electron micrograph of the egg ZP taken
from Familiari et al. (2006), with permission. Note the extensive network of ZP fibrils.

aspects of the fertilization process in mammals have been in addition to serving as a structural protein, mZP3 serves as
investigated extensively and resulted in an enhanced ap- a receptor for sperm and inducer of the acrosome reaction.
preciation of egg and sperm molecules and mechanisms Shortly after fertilization mZP3 is modified, probably by
involved in fertilization. cortical granule enzymes, such that it is no longer recog-
All mammalian eggs are surrounded by a thick extracel- nized by sperm. Modification of mZP2 and mZP3 following
lular coat, the zona pellucida (ZP) (Fig. 1). The mouse egg fertilization contributes to the prevention of polyspermy,
ZP is
6.5 mm thick, contains
3.5 ng protein, and consists typically a lethal event (Yanagimachi, 1994; Florman and
of three glycoproteins, mZP1-3, that are synthesized exclu- Ducibella, 2006).
sively by growing oocytes and assembled into an extensive Mouse embryonal carcinoma (EC) cells stably trans-
network of crosslinked fibrils (Wassarman, 1988, 2008). fected with mZP3 synthesize and secrete biologically active
Several features of nascent ZP polypeptides control their
processing, secretion, and assembly by oocytes (Jovine
et al., 2007). mZP2 and mZP3 are present in equimolar
amounts in the ZP and are located every 15 nm or so along
ZP fibrils; the fibrils are noncovalently crosslinked by
mZP1. Each ZP glycoprotein has a characteristic region
(
260 amino acids) known as the ZP domain that also is
present in proteins related to ZP1-3 that make up the
extracellular coat (vitelline envelope) of fish, frog, and bird
eggs (Jovine et al., 2005; Litscher and Wassarman, 2007).
The ZP domain is a bipartite structure and the N-terminal
half of the polypeptide serves as a polymerization module
(Jovine et al., 2004, 2006; Monne  et al., 2008). ZP and
vitelline envelope proteins have been conserved for more
than 600 million years. It should be noted that the ZP domain
is not restricted to egg-coat proteins, but is present in
hundreds of extracellular proteins found in virtually all
multicellular organisms.
A large body of evidence strongly suggests that capaci-
tated mouse sperm recognize and bind to mZP3 during Figure 2. Multiple steps along the path to fertilization in mammals.
fertilization and, as a result, undergo the acrosome reaction, Capacitation enables sperm to bind to the egg ZP and undergo the
a form of cellular exocytosis that enables sperm to acrosome reaction. Acrosome-intact sperm bind to mZP3 and undergo
penetrate the ZP (Yanagimachi, 1994; Wassarman, 1999, the acrosome reaction. Acrosome-reacted sperm penetrate the egg ZP
and use plasma membrane remaining at the posterior of the sperm
2005; Wassarman et al., 2001; Florman and Ducibella, head to fuse with egg plasma membrane, thereby restoring the egg to a
2006; Wassarman and Litscher, 2008) (Fig. 2). Therefore, diploid state and activating it.

934 Mol Reprod Dev 76:933–941 (2009)

 10982795, 2009, 10, Downloaded from [Link] by Cochrane Mexico, Wiley Online Library on [31/03/2023]. See the Terms and Conditions ([Link] on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
ZP GLYCOPROTEIN ZP3 AND FERTILIZATION

mZP3 (EC-mZP3). Like egg mZP3, EC-mZP3 inhibits bind-

ing of sperm to eggs and induces the acrosome reaction
(Kinloch et al., 1991). Certain oligosaccharides also can
inhibit binding of sperm to eggs, however, the nature of the
terminal sugar, anomeric linkage, and branching sugar can
dramatically affect the extent of inhibition (Florman and
Wassarman, 1985; Litscher et al., 1995; Tulsiani et al.,
1997; Hanna et al., 2004; Kerr et al., 2004). Despite exten-
sive evidence in support of an essential role for oligosac-
charides in binding of sperm to mZP3, recent attempts
to identify and characterize the specific oligosaccharides
involved have been relatively unsuccessful (Clark and Dell,
2006). By themselves, the oligosaccharides do not induce
the acrosome reaction. Figure 3. Schematic representation of the overall organization of
ZP3 polypeptides from different mammals are highly mZP3 and hZP3 polypeptides encoded by cDNA constructs trans-
conserved, but differentially glycosylated. For example, fected into EC cells. Shown are polypeptides designated as mZP3
(8 exons; 424 amino acids) (A) and hZP3 (8 exons; 422 amino acids)
mZP3 and hamster ZP3 (hZP3) polypeptides are
86% (B) with an N-terminal signal peptide (amino acids 1–22 for mZP3 and
identical and purified egg hZP3 inhibits binding of mouse hZP3 [yellow]), a ZP domain (amino acids 45–304 for mZP3 and
or hamster sperm to mouse eggs (Moller et al., 1990). hZP3 45–302 for hZP3 [red]), a CFCS (amino acids 350–353 for mZP3 and
synthesized by oocytes from transgenic mice carrying the 348–351 for hZP3 [yellow]), a C-terminal TM domain (amino acids
hZP3 gene also inhibits binding of mouse sperm to mouse 387–409 for mZP3 and 385–407 for hZP3 [yellow]), and a short CT
(amino acids 410–424 for mZP3 and 408–422 for hZP3). The
eggs to the same extent as mZP3 (Kinloch et al., 1992). On positions of polypeptide encoded by exons 1–6 are indicated, as are
the other hand, EC- or Chinese hamster ovary (CHO-) cells exons 7 and 8 of mZP3 [blue] and hZP3 [cyan].
stably transfected with hZP3 synthesize EC- or CHO-hZP3
that is inhibitory with hamster, but not with mouse gametes
(Litscher and Wassarman, 1996a). However, swapping the has an almost identical domain organization to that of mZP3
last three exons of hZP3, exons 6–8, for the corresponding (Kinloch et al., 1990) (Fig. 3).
exons from mZP3 imparts inhibitory activity with mouse
gametes to EC-hZP3 (Kinloch et al., 1995). This suggests
that one or more of the last three exons of mZP3 can restore Production of Proteins EC-hZP3/m6, /m7, and /m8
the ability of EC- or CHO-hZP3 to recognize mouse sperm. In our constructs EC-hZP3/m6, /m7, and /m8, we ex-
Here, we review our extended exon-swapping experi- changed exons 6/7/8 of hZP3 with the corresponding exons
ments on mZP3 and hZP3 carried out to further assess the of mZP3 (Williams et al., 2006) (Fig. 4). EC cells were stably
location of the sperm combining-site of mZP3. We deter- transfected by electroporation with each of the three con-
mined which of the last three exons of mZP3 is responsible structs and cultured in D-MEM supplemented with 10% fetal
for imparting inhibitory activity to EC-hZP3 and assessed
whether this activity is retained independent of other regions
of mZP3 polypeptide (Williams et al., 2006). The latter
was analyzed by using a chimeric protein consisting of a
portion of mZP3 polypeptide fused to a foreign protein, the
Fc portion of human IgG. It is concluded that polypeptide
encoded by mZP3 exon-7 is both necessary and sufficient
for binding of sperm. It is likely that differential glycosylation
of the polypeptide could be responsible for the changes
in sperm binding specificity observed with EC- and
CHO-hZP3.

LOCATING THE SPERM COMBINING-SITE OF ZP3

Eight exons encode mZP3 (Wassarman, 2008) (Fig. 3). A
signal peptide and the N-terminal region of the ZP domain is
encoded by exon-1 and the bulk of the ZP domain are
encoded by exons 2–6 which accounts for more than
80% of the polypeptide. A consensus furin cleavage-site
(CFCS) that is utilized during secretion of nascent mZP3 is Figure 4. Schematic representation of the overall organization of
encoded by exon-7. A propeptide that includes a C-terminal hybrid polypeptides generated by swapping exon-6, -7, and -8 from
hZP3 (cyan) for the corresponding exons from mZP3 (blue), producing
transmembrane (TM) domain and a short cytoplasmic tail hZP3/m6 (A), hZP3/m7 (B), and hZP3/m8 (C). The positions of the
(CT), encoded by exon-8, is lost upon secretion due to N-terminal signal sequence, CFCS, and C-terminal TM domain are
proteolysis at the CFCS. hZP3, also encoded by 8 exons, indicated [yellow].

Mol Reprod Dev 76:933–941 (2009) 935

 10982795, 2009, 10, Downloaded from [Link] by Cochrane Mexico, Wiley Online Library on [31/03/2023]. See the Terms and Conditions ([Link] on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Molecular Reproduction & Development LITSCHER ET AL.

bovine serum. Supernatant was collected in serum-free

D-MEM, concentrated, and purified. Aliquots of purified
supernatant containing EC-hZP3/m6, /m7, and /m8, as
well as EC-mZP3 and EC-hZP3 (control proteins), were
analyzed by SDS–PAGE and Western immunoblotting.
On SDS–PAGE, egg mZP3 (
83 kDa) migrated more
slowly than EC-mZP3 (
69 kDa), and egg hZP3 (
55 kDa)
migrated more slowly than EC-hZP3 (
51 kDa) suggesting
that although EC cells glycosylate nascent mZP3 and
hZP3 polypeptides (
37 kDa), EC-mZP3 and EC-hZP3 are
glycosylated to a lesser extent than their native counter-
parts. EC-hZP3/m6, /m7, and /m8 have a Mr of
51,
65,
and
51 kDa, respectively, and are also glycosylated. There
are six potential N-linked glycosylation sites (-Asn-X-Ser/
Thr-) in mZP3 polypeptide with exon-7 containing two sites,
and four potential N-linked glycosylation sites in hZP3 poly-
petide with only one site in exon-7. Thus, the exchange
of exon-7 in EC-hZP3 (EC-hZP3/m7) that gives a Mr
more similar to that of EC-mZP3 than to that of EC-hZP3/ Figure 5. Effect of recombinant ZP3 on the binding of sperm to eggs.
m6 and /m8 can be explained by the introduction of Shown are the number of capacitated sperm bound/egg  SD and
an additional N-linked glycosylation site. EC-hZP3/m8 the average % inhibition of sperm binding to eggs for EC-mZP3 [blue],
EC-hZP3 [red], and hybrid forms of EC-hZP3 [pink]. In the control
(
51 kDa) is the same size as EC-hZP3 (
51 kDa) and sample, sperm were pre-incubated in the presence of M199-M alone
suggests that polypeptide encoded by exon-8 is cleaved [black]. In the remaining samples, recombinant glycoproteins were
at the CFCS upon secretion. Partial deglycosylation of present at
5 ng/ml. The values represent the average of three to four
EC-hZP3/m7 and egg hZP3 with N-glycanase shifted their separate experiments with each sample. See Williams et al. (2006)
for further details.
Mr from
65 and
55 kDa, respectively, to a relatively broad
band with a Mr of
42 kDa as shown by SDS–PAGE.
was collected, purified by IgG-affinity chromatography, and
samples were analyzed by SDS–PAGE and Western im-
Effect of Proteins on Sperm Binding to Eggs munoblotting. In order to visualize column-bound huIgG(Fc),
As shown previously (Kinloch et al., 1995), EC-hZP3/ immunoblots were incubated with anti-IgG(Fc)-HRP and
m6-8 inhibits binding of mouse sperm to mouse eggs as well developed by ECL [note: polypeptide encoded by exon-8
as egg mZP3, egg hZP3 and EC-mZP3, all at nanomolar was removed by excision at the CFCS by EC cells; see
concentrations. EC-hZP3 is not inhibitory with mouse ga- below]. On SDS–PAGE under reducing conditions secreted
metes. These data indicate that the sperm binding-site of EC-IgG(Fc)/mZP3(7) migrated at
55 kDa Mr, while under
mZP3 is located in the C-terminal portion of mZP3 that is nonreducing conditions it migrated at
125 kDa Mr, suggest-
encoded by exon 6, 7, and 8. ing that it was secreted as a dimer. It always showed a broad
We found that mouse sperm binding to mouse eggs was smear on SDS–PAGE even after hydrolysis with N-glyca-
significantly inhibited (
30–65%) when sperm were expos- nase (removal of N-linked carbohydrates) which reduced the
ed to egg mZP3, EC-mZP3, or EC-hZP3/m7 at
5 ng/ml. Mr by
6 kDa. To visualize the mZP3 portion of EC-IgG(Fc)/
Sperm incubated with EC-hZP3, EC-hZP3/m6, or EC-hZP3/ mZP3(7), immunoblots were stained with anti-mZP3 specif-
m8 had little (<10%), if any, effect (Fig. 5). Small differences ic for mZP3(7) (polyclonal rabbit antibody against an mZP3
in estimating the concentrations of purified recombinant EC- peptide encompassing amino acids 328–344 encoded by
mZP3 and EC-hZP3 constructs versus purified egg mZP3 exon-7). To determine whether the portion of mZP3 encoded
may account for the lesser inhibition seen with EC-mZP3 by exon-8 was present in secreted EC-IgG(Fc)/mZP3(7),
and EC-hZP3/m7. Sperm binding is inhibited four to nine
times more with EC-mZP3 and EC-hZP3/m7 than with EC-
hZP3, EC-hZP3/m6, or EC-hZP3/m8. These data suggest
that the inhibitory activity of EC-hZP3/m7 with mouse ga-
metes resides in mZP3 exon-7.

Production of Chimeric Protein EC-IgG(Fc)/ZP3(7)

We designed a chimeric protein construct which involved
an IgG fusion strategy that facilitated protein purification as
well as characterization (Williams et al., 2006). The cDNA Figure 6. Schematic representation of the overall organization of
encodes the Fc portion of human IgG and is fused to mZP3 chimeric polypeptide huIgG(Fc)/mZP3(7) in which the Fc portion of
human IgG (amino acids 1–269 [green]) was joined to the C-terminal
exons 7 and 8 (Fig. 6). A stably transfected EC cell line, end (exon-7 and -8) of mZP3 (amino acids 309–424 [blue]). The
called IgG(Fc)/mZP3(7,8), was produced by electroporation. positions of the N-terminal signal sequence, CFCS, and C-terminal TM
Again, serum-free supernatant from transfected EC-cells domain are indicated [yellow].

936 Mol Reprod Dev 76:933–941 (2009)

 10982795, 2009, 10, Downloaded from [Link] by Cochrane Mexico, Wiley Online Library on [31/03/2023]. See the Terms and Conditions ([Link] on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
ZP GLYCOPROTEIN ZP3 AND FERTILIZATION

immunoblots were also stained with anti-mZP3 specific binding of sperm to homomeric fibrils of mZP3 that form
for mZP3(8) (polyclonal rabbit antibody against an mZP3 under nondenaturing conditions (Litscher et al., 2008).
peptide encompassing amino acids 369–388 encoded by Several reports have concluded that the C-terminal re-
exon-8). As reported earlier (Litscher et al., 1999; Qi et al., gion of mZP3 polypeptide is responsible for the ability of
2002; Zhao et al., 2002), polypeptide encoded by exon-8 of mZP3 to inhibit binding of sperm to eggs and to induce the
mZP3 is removed by proteolysis during secretion of nascent AR (Rosiere and Wassarman, 1992; Kinloch et al., 1995;
native mZP3. Similarly, we did not detect any immuno- Litscher and Wassarman, 1996b; Li et al., 2007). In this
positive stain for the C-terminal portion of mZP3, suggesting context, Millar et al. (1989) reported that a short peptide
that the same is true for EC-IgG(Fc)/mZP3(7). (16 amino acids) from the C-terminal region of mZP3 pro-
duced antibodies in mice to mZP3. These antibodies recog-
nized a 7 amino acid epitope on mZP3 in vivo which resulted
Visualization of Chimeric Protein Bound to Sperm in long-lasting infertility. In a similar experiment, Rosiere and
Mouse sperm samples were incubated with EC-IgG(Fc)/ Wassarman (1992) exposed eggs to polyclonal antibodies
mZP3(7) or with egg mZP3 followed by a rabbit anti-mZP3 against the same region of mZP3 which also inhibited sperm
antibody (positive control), or with M199-M alone (negative binding to eggs in vitro. It is of interest that ZP3 is among the
control) followed by anti-human IgG-gold conjugate (goat 10% most divergent proteins in mammals, that the region of
anti-human IgG-gold) and silver enhancement. Sperm were polypeptide encoded by mZP3 exon-7 has undergone a
screened by light microscopy for colloidal gold-protein com- significant number of changes during evolution compared
plexes on sperm heads and the number of complexes per to other regions, and that exon-7 is a site of positive Darwini-
head were counted. [Note: Sequence homology between an selection (Swanson et al., 2001). These features may be
human and rabbit IgG(g) (>75%) allowed us to gold-label related to the proposed role of ZP3 in species-restricted
rabbit anti-mouse mZP3 bound to egg mZP3 with antibody fertilization (Wassarman, 1990, 1999, 2005).
specific for human IgG]. mZP1-3 null mutant mice have been produced by homol-
Gold-labeling on sperm heads incubated with EC-IgG- ogous recombination in embryonic stem cells. Mutant fe-
(Fc)/mZP3(7) or egg mZP3 was present specifically over the males heterozygous for mZP3 are fertile, although their egg
sperm head and not over any portion of the mid-piece or tail ZP is approximately one-half the thickness (2.7  1.2 mm) of
(Fig. 7). By counting the gold particles per sperm head, there the wild-type ZP (6.2  1.9 mm) (Wassarman et al., 1997).
were on average 21.4  8.9 particles (range ¼ 11–39) for EC However, homozygous mutant females missing either
-IgG(Fc)/mZP3(7) and 22.9  10.2 particles (range ¼ 9–44) mZP2 or mZP3 fail to lay down a ZP during oocyte growth,
for egg mZP3. All sperm heads labeled with gold had an possess oocytes and eggs that lack a ZP, and are complete-
intact acrosome; that is, a ridge typically associated with the ly infertile (Liu et al., 1996; Rankin et al., 1996, 2001). Failure
acrosome. Sperm treated with M199-M alone averaged only to synthesize either ZP glycoprotein prevents assembly of
2.6  2.7 particles (range ¼ 0–10) per sperm head. the synthesized ZP glycoprotein into a ZP. mZP2/ and
mZP3/ females possess fewer growing oocytes, Graafian
follicles, and ovulated eggs than wild-type females
Effect of Chimeric Protein on Sperm (Wassarman et al., 1998). On the other hand, oocytes and
Binding to Eggs eggs from mZP1/ females have a ZP, but are not as fertile
To determine the extent of inhibition of sperm binding as wild-type mice (Rankin et al., 1999). Although mZP2 and
to eggs, we incubated mouse sperm with purified EC-IgG- mZP3 assemble into a ZP in mZP1/ females, it exhibits
(Fc)/mZP3(7), egg mZP3, and human IgG(Fc) (control large pores due to insufficient crosslinking of ZP fibrils.
glycoprotein) (Fig. 8). Sperm binding was inhibited by Human ZP2 and ZP3 can replace mZP2 and mZP3 and
65  23% (
10 ng/ml), 71  9% (
10 ng/ml), and 1  1% restore a ZP to oocytes in homozygous null female mice.
(
25 ng/ml), for EC-IgG(Fc)/mZP3(7), egg mZP3, and Although the mosaic ZP permits binding of mouse sperm, it
human IgG(Fc), respectively, as compared to media alone does not permit binding of human sperm (Dean, 2007). This
(M199-M). These results indicate that EC-IgG(Fc)/mZP3(7) difference in sperm binding may be related to the glycans
inhibits sperm binding to eggs like egg mZP3. However, carried by human ZP3 that are synthesized by oocytes of
unlike mZP3, the chimeric protein does not induce sperm to transgenic mice (Dell et al., 2003; discussed below).
undergo the acrosome reaction (Fig. 9). As noted above, EC-hZP3 does not inhibit mouse sperm
binding to mouse eggs, but if exons 6–8 of hZP3 are
replaced with the equivalent exons of mZP3 (EC-hZP3/m6
-8), it does inhibit sperm binding to eggs. Purified native
RELEVANT OBSERVATIONS AND IMPLICATIONS hZP3 from hamster eggs and hZP3 from transgenic
The mouse ZP consists of 3 glycoproteins, however, mice carrying the hZP3 transgene inhibits binding of mouse
only purified mZP3 from unfertilized eggs inhibits binding sperm to mouse eggs (Kinloch et al., 1995). Results pre-
of mouse sperm to eggs at nanomolar concentrations (Bleil sented here indicate that inclusion of mZP3 exon-7, not exon
and Wassarman, 1980; Wassarman, 2005) and induces -6 or -8, enables EC-hZP3 to become inhibitory with mouse
sperm to undergo the acrosome reaction (Bleil and Wassar- gametes (Figs. 4 and 5) (Williams et al., 2006). Radiolabeled
man, 1983; Arnoult et al., 1996). Purified mZP1 and mZP2 and gold-labeled mZP3 bind preferentially to plasma mem-
have no effect on sperm binding or the acrosome reaction. It brane overlying mouse sperm heads (Bleil and Wassarman,
appears likely that the biological effects of mZP3 are due to 1986; Mortillo and Wassarman, 1991). Such behavior is

Mol Reprod Dev 76:933–941 (2009) 937

 10982795, 2009, 10, Downloaded from [Link] by Cochrane Mexico, Wiley Online Library on [31/03/2023]. See the Terms and Conditions ([Link] on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Molecular Reproduction & Development LITSCHER ET AL.

Figure 7. Binding of gold-labeled glycoproteins to mouse sperm. Shown are light micrographs of
individual capacitated mouse sperm incubated in the presence of EC-huIgG(Fc)/mZP3(7) (
10 ng/ml)
(A,B; [red]), egg mZP3 (
10 ng/ml) (C,D; [blue]) followed by rabbit anti-mZP3, or M199-M alone
(E,F; [purple]). All samples were exposed to goat anti-human IgG-gold conjugate (5 nm gold) followed by
silver enhancement. It should be noted that EC-huIgG(Fc)/mZP3(7) and egg mZP3 are specifically
associated with heads of acrosome-intact sperm. See Williams et al. (2006) for further details.

consistent with the ability of mZP3 to inhibit binding of sperm provides further support for the suggestion that mZP3 exon-
to eggs. We also found that the chimeric protein EC-IgG(Fc)/ 7 encodes the region of mZP3 polypeptide that serves as a
mZP3(7), but not IgG(Fc), binds specifically to sperm sperm combining-site and other regions are not required
heads and inhibits sperm binding to eggs as effectively for sperm binding (Fig. 10). It also suggests that, if carbohy-
as intact egg mZP3 (Figs. 6–8) (Williams et al., 2006). This drate serves as the ligand for sperm binding to EC-IgG(Fc)/

Figure 8. Effect of chimeric ZP3 on the binding of sperm to eggs. Figure 9. Effect of chimeric ZP3 on the acrosome reaction. Capaci-
Shown are the number of capacitated sperm bound/egg  SD and the tated sperm were incubated for 1 h with M199-M alone (control),
average % inhibition  SD of sperm binding to eggs for IgG(Fc), egg ionophore A23187 (10 mM), or EC-IgG(Fc)/mZP3(7) (
25 and
mZP3, and EC-IgG(Fc)/mZP3(7). In control samples, sperm were
50 ng/ml). At the end of the incubation, sperm were fixed, stained
pre-incubated in the presence of M199-M alone [black] and in the with Coomassie blue to reveal the status of the acrosome, and were
presence of IgG(Fc) (
25 ng/ml; [green]). In the remaining samples, examined by bright-field microscopy. Shown are the number of sperm
egg mZP3 (
10 ng/ml; [blue]) and EC-IgG(Fc)/mZP3(7) (
10 ng/ml; remaining acrosome-intact (SD; [blue]) and the number of sperm
[red]) were present. The values represent the average of two to four acrosome-reacted (SD; [red]). For each experimental group
separate experiments with each sample. See Williams et al. (2006) for
300 sperm were scored for the presence or absence of an intact
further details. acrosome.

938 Mol Reprod Dev 76:933–941 (2009)

 10982795, 2009, 10, Downloaded from [Link] by Cochrane Mexico, Wiley Online Library on [31/03/2023]. See the Terms and Conditions ([Link] on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
ZP GLYCOPROTEIN ZP3 AND FERTILIZATION

porcine and bovine sperm (Yonezawa et al., 2005). It is likely

that mouse oocytes modify hZP3 polypeptide with oligosac-
charides recognized by mouse sperm, whereas EC and
CHO cells do not. In this context, it is of interest that mouse
and human ZP3, the latter derived from eggs of transgenic
mice in which human ZP3 replaced mZP3 and to which
Figure 10. Polypeptides encoded by exon-7 of mZP3 and hZP3. mouse, but not human sperm bind (Rankin et al., 1998),
Shown are several features of the polypeptides, including the positions possess identical O-glycans (Dell et al., 2003). The constel-
of 4 conserved Cys residues (yellow box), 2 Pro residues (orange), lation and distribution of glycosylation enzymes in mouse
and consensus furin cleavage-sites (majenta box). Potential sites for
O-linked (Ser/Thr) and N-linked (Asn-X-Ser/Thr) glycosylation are
oocytes probably differ somewhat from human oocytes and
indicated by small (black) and large (cyan) arrows, respectively. such a difference could account for species-restricted bind-
Positively and negatively charged amino acids are indicated, as is the ing of mouse and human sperm to eggs. It is relevant to note
polar (p) or apolar (a) nature of the amino acids. It should be noted that that binding of bacteria, animal viruses, parasites, and other
the portion of polypeptide downstream of the fourth Cys residue is pathogens to their cellular hosts, binding of bacteria to
composed largely (
70%) of polar amino acids (Kinloch et al., 1988,
1990). The arrangement of the 4 Cys residues is conserved in ZP3-like plants, binding of pollen to the plant stigma, sexual aggluti-
proteins from mammals, amphibians, birds, and fish (Wassarman and nation in yeast, and binding of amphibian and marine sperm
Litscher, 2008). to eggs are all considered to be carbohydrate-mediated
events (Varki et al., 1999).
The ZP domain serves as a structural domain for
mZP3(7), then polypeptide encoded by mZP3 exon-7 alone protein–protein interactions and is found in hundreds of
provides all of the information required for appropriate gly- extracellular proteins in a highly diverse group of animals
cosylation by EC cells. In this connection, it has recently (Jovine et al., 2005). It is therefore not too surprising that the
been reported that the C-terminal region of human ZP3 sperm combining-site is encoded by mZP3 exon-7 since the
is able to induce capacitated human sperm to undergo ZP domain is encoded by mZP3 exon 1–6 and the C-terminal
the acrosome reaction and that this ability is dependent peptide missing from mature mZP3 is encoded by mZP3
on proper glycosylation of the polypeptide (Bansal et al., exon-8. Perhaps this class of ZP domain-proteins arose by
in press). combining a common structural building block with added
Interestingly, although EC-IgG(Fc)/mZP3(7) binds to functional and specific sequences (e.g., a sperm combining-
acrosome-intact sperm and inhibits their binding to eggs, it site) into a mosaic architecture; an arrangement that may
does not induce sperm to undergo the acrosome reaction account for the many diverse functions observed with ZP
(Fig. 9). This is reminiscent of the behavior of small mZP3 domain proteins.
glycopeptides (Florman et al., 1984), mZP3 oligosacchar-
ides (Florman and Wassarman, 1985), and synthetic oligo-
saccharides (Litscher et al., 1995) that bind to sperm and
ACKNOWLEDGMENTS
inhibit their binding to eggs, but fail to induce the acrosome
reaction. Perhaps the acrosome reaction occurs only in the We thank Dr. Malcolm Byrnes for his kind invitation to participate
presence of mZP3 homomeric polymers that have been in the Ernest Everett Just Symposium at Howard University in 2008.
Our research was supported in part by the NIH, most recently by
shown to form under nondenaturing conditions (i.e., under grant HD-35105.
conditions used in in vitro assays of sperm binding) (Litscher
et al., 2008). Results of recent experiments suggest that
EC-IgG(Fc)/mZP3(7) does not form polymers and migrates
as a single broad band during electrophoresis under non- REFERENCES
denaturing conditions (E.S. Litscher and P.M. Wassarman, Arnoult C, Zeng Y, Florman HM. 1996. ZP3-dependent activation of
unpublished data). sperm cation channels regulates acrosome secretion during
We suggest that mZP3 binding to sperm is mediated mammalian fertilization. J Cell Biol 134:637–645.
by carbohydrate(s) (Tulsiani et al., 1997; Clark and Dell, Bansal P, Chakrabarti K, Gupta SK. In press. Functional activity
2006), evidence which is consistent with its inhibitory activity of human ZP3-primary sperm receptor resides towards its
even after exposure to high or low temperatures and pH, C-terminus. Biol Reprod.
denaturants and detergents, fixatives, or proteases Bleil JD, Wassarman PM. 1980. Mammalian sperm-egg inter-
(Wassarman, 1988). Similarly, either mZP3 oligosacchar- action: Identification of a glycoprotein in mouse egg zonae
ides (Florman and Wassarman, 1985) or synthetic oligo- pellucidae possessing receptor activity for sperm. Cell 20:873–
saccharides (Litscher et al., 1995) inhibit binding of sperm 882.
to eggs and several putative carbohydrate-specific egg- Bleil JD, Wassarman PM. 1983. Mammalian sperm-egg interac-
binding proteins on sperm have been identified (Miller tion: Sequence of events and induction of the acrosome reaction
et al., 1992; Cheng et al., 1994). Differences in glycosylation by a zona pellucida glycoprotein. Dev Biol 95:317–324.
patterns may be responsible for these different behaviors Bleil JD, Wassarman PM. 1986. Autoradiographic visualization of
with mouse gametes as seen with hZP3 from transgenic the mouse egg’s sperm receptor bound to sperm. J Cell Biol
mice (inhibitory) versus EC- and CHO-hZP3 (not inhibitory). 102:1363–1371.
A similar situation has been reported for native and recom- Cheng A, Le T, Palacios M, Bookbinder LH, Wassarman PM,
binant porcine ZP glycoproteins and their interaction with Suzuki F, Bleil JD. 1994. Sperm-egg recognition in the mouse:

Mol Reprod Dev 76:933–941 (2009) 939

 10982795, 2009, 10, Downloaded from [Link] by Cochrane Mexico, Wiley Online Library on [31/03/2023]. See the Terms and Conditions ([Link] on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Molecular Reproduction & Development LITSCHER ET AL.

Characterization of sp56, a sperm protein having specific affinity Kinloch RA, Sakai Y, Wassarman PM. 1995. Mapping the mouse
for ZP3. J Cell Biol 125:867–878. ZP3 combining site for sperm by exon-swapping and site-
Clark GF, Dell A. 2006. Molecular models for murine sperm-egg directed mutagenesis. Proc Natl Acad Sci USA 92:263–267.
binding. J Biol Chem 281:13853–13856. Li D, Cao S, Xu C. 2007. Polypeptide backbone derived from
Dean J. 2007. The enigma of sperm-egg recognition in mice. In: carboxyl terminal of mouse ZP3 inhibits sperm-zona binding.
Gupta SK, Koyama K, Murray JF, editors. Gamete biology. Mol Reprod Dev 74:1327–1336.
UK:Nottingham University Press, pp 359–365. Litscher ES, Wassarman PM. 1996a. Recombinant hamster sperm
Dell A, Chalabi S, Easton RL, Haslam SM, Sutton-Smith M, Pa- receptors that exhibit species-specific binding to sperm. Zygote
tankar MS, Lattanzio F, Panico M, Morris HR, Clark GF. 2003. 4:229–236.
Murine and human zona pellucida 3 derived from mouse eggs Litscher ES, Wassarman PM. 1996b. Characterization of a mouse
express identical O-glycans. Proc Natl Acad Sci USA 100: ZP3-derived glycopeptide, gp55, that exhibits sperm receptor
15631–15636. and acrosome reaction-inducing activity in vitro. Biochemistry
Familiari G, Relucenti M, Heyn R, Micara G, Correr S. 2006. Three- 35:3980–3987.
dimensional structure of the zona pellucida at ovulation. Microsc Litscher ES, Wassarman PM. 2007. Egg extracellular coat proteins:
Res Tech 69:415–426. From fish to mammals. Histol Histopathol 22:337–347.
Florman HM, Ducibella T. 2006. Fertilization in mammals. In: Neill Litscher ES, Juntunen K, Seppo A, Niemala R, Pentilla L, Renkonen
JD, editor. The physiology of reproduction 1. San Diego:Elsevier O, Wassarman PM. 1995. Oligosaccharide constructs with de-
Press, pp 55–122. fined structures that inhibit binding of mouse sperm to unfertilized
Florman HM, Wassarman PM. 1985. O-Linked oligosaccharides eggs in vitro. Biochemistry 34:4662–4669.
of mouse egg ZP3 account for its sperm receptor activity. Cell Litscher ES, Qi H, Wassarman PM. 1999. Mouse zona pellucida
41:313–324. glycoproteins mZP2 and mZP3 undergo carboxy-terminal
Florman HM, Bechtol KB, Wassarman PM. 1984. Enzymatic dis- proteolytic processing in growing oocytes. Biochemistry 38:
section of the functions of the mouse egg’s receptor for sperm. 12280–12287.
Dev Biol 106:243–255. Litscher ES, Janssen WG, Darie CC, Wassarman PM. 2008.
Hanna WF, Kerr CL, Shaper JH, Wright WW. 2004. Lewis X- Purified mouse egg zona pellucida glycoproteins polymerize into
containing neoglycoproteins mimic the intrinsic ability of zona homomeric fibrils under non-denaturing conditions. J Cell Physiol
pellucida glycoprotein ZP3 to induce the acrosome reaction in 214:153–157.
capacitated mouse sperm. Biol Reprod 71:778–789. Liu C, Litscher ES, Mortillo S, Sakai Y, Kinloch RA, Stewart CL,
Jovine L, Qi H, Williams Z, Litscher ES, Wassarman PM. 2004. Wassarman PM. 1996. Targeted disruption of the mZP3 gene
A duplicated motif controls assembly of zona pellucida domain results in production of eggs lacking a zona pellucida and infertili-
proteins. Proc Natl Acad Sci USA 101:5922–5927. ty in female mice. Proc Natl Acad Sci USA 93:5431–5436.
Jovine L, Darie CC, Litscher ES, Wassarman PM. 2005. Zona Manning KR. 1983. Black Apollo of science—The Life of Ernest
pellucida domain proteins. Annu Rev Biochem 74:83–114. Everett Just. NY:Oxford University Press.
Jovine L, Janssen WG, Litscher ES, Wassarman PM. 2006. The Millar SE, Chamow SM, Baur AW, Oliver C, Robey F, Dean J. 1989.
PLAC1-homology region of the ZP domain is sufficient for protein Vaccination with a synthetic zona pellucida peptide produces
polymerisation. BMC Biochem 7:11–19. long-term contraception for female mice. Science 246:935–938.
Jovine L, Qi H, Williams Z, Litscher ES, Wassarman PM. 2007. Miller DJ, Macek MB, Shur BD. 1992. Complementarity between
Features that affect secretion and assembly of zona pellucida sperm surface b-1,4-galactosyltransferase and egg coat ZP3
glycoproteins during mammalian oogenesis. In: Gupta SK, mediates sperm-egg binding. Nature 357:589–593.
Koyama K, Murray JF, editors. Gamete biology. Nottingham, Moller CC, Bleil JD, Kinloch RA, Wassarman PM. 1990. Structural
UK:Nottingham University Press, United Kingdom, pp 187–201. and functional relationships between mouse and hamster zona
Kerr CL, Hanna WF, Shaper JH, Wright WW. 2004. Lewis pellucida glycoproteins. Dev Biol 137:276–286.
X-containing glycans are specific and potent competitive inhibi- Monne  M, Han L, Schwend T, Burendahl S, Jovine L. 2008. Crystal
tors of the binding of ZP3 to complementary sites on capacitated, structure of the ZP-N domain of ZP3 reveals the core fold of
acrosome-intact mouse sperm. Biol Reprod 71:770–777. animal egg coats. Nature 456:653–657.
Kinloch RA, Roller RJ, Fimiani CM, Wassarman DA, Wassarman Mortillo S, Wassarman PM. 1991. Differential binding of gold-
PM. 1988. Primary structure of the mouse sperm receptor’s labeled zona pellucida glycoproteins mZP2 and mZP3 to mouse
polypeptide chain determined by genomic cloning. Proc Natl sperm membrane compartments. Development 113:141–149.
Acad Sci USA 85:6409–6413. Qi H, Williams Z, Wassarman PM. 2002. Secretion and assembly of
Kinloch RA, Ruiz-Seiler B, Wassarman PM. 1990. Genomic orga- zona pellucida glycoproteins by growing mouse oocytes micro-
nization and polypeptide primary structure of zona pellucida injected with epitope-tagged cDNAs for mZP2 and mZP3. Mol
glycoprotein hZP3, the hamster sperm receptor. Dev Biol 142: Biol Cell 13:530–541.
414–421. Rankin T, Familiari M, Lee E, Ginsburg A, Dwyer N, Blanchette-
Kinloch RA, Mortillo S, Stewart CL, Wassarman PM. 1991. Embry- Mackie J, Drago J, Westphal H, Dean J. 1996. Mice homozygous
onal carcinoma cells transfected with ZP3 genes differentially for an insertional mutation in the Zp3 gene lack a zona pellucida
glycosylate similar polypeptides and secrete active mouse sperm and are infertile. Development 122:2903–2910.
receptor. J Cell Biol 115:655–664. Rankin TL, Tong ZB, Castle PE, Lee E, Gore-Langton R, Nelson
Kinloch RA, Mortillo S, Wassarman PM. 1992. Transgenic mouse LM, Dean J. 1998. Human ZP3 restores fertility in Zp3 null mice
eggs with functional hamster sperm receptors in their zona without affecting order-specific sperm binding. Development
pellucida. Development 115:937–946. 125:2415–2424.

940 Mol Reprod Dev 76:933–941 (2009)

 10982795, 2009, 10, Downloaded from [Link] by Cochrane Mexico, Wiley Online Library on [31/03/2023]. See the Terms and Conditions ([Link] on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
ZP GLYCOPROTEIN ZP3 AND FERTILIZATION

Rankin T, Talbot P, Lee E, Dean J. 1999. Abnormal zonae Wassarman PM. 2008. Zona pellucida glycoproteins. J Biol Chem
pellucidae in mice lacking ZP1 result in early embryonic loss. 283:24285–24289.
Development 126:3847–3855. Wassarman PM, Litscher ES. 2008. Mammalian fertilization: The
Rankin TL, O’Brien M, Lee E, Wigglesworth K, Eppig J, Dean J. egg’s multifunctional zona pellucida. Intl J Dev Biol 52:665–676.
2001. Defective zonae pellucidae in ZP2-null mice disrupt Wassarman PM, Qi H, Litscher ES. 1997. Mutant female mice
folliculogenesis, fertility, and development. Development 128: carrying a single mZP3 allele produce eggs with a thin zona
1119–1126. pellucida, but reproduce normally. Proc Roy Soc Biol Sci 264:
Rosiere TK, Wassarman PM. 1992. Identification of a region of 323–328.
mouse zona pellucida glycoprotein mZP3 that possesses sperm Wassarman PM, Liu C, Chen J, Qi H, Litscher ES. 1998. Ovarian
receptor activity. Dev Biol 154:309–317. development in mice bearing homozygous or heterozygous null
Swanson WJ, Yang Z, Wolfner M, Aquadro CF. 2001. Positive mutations in zona pellucida glycoprotein gene mZP3. Histol
Darwinian selection drives the evolution of several female repro- Histopathol 13:293–300.
ductive proteins in mammals. Proc Natl Acad Sci USA 98: Wassarman PM, Jovine L, Litscher ES. 2001. A profile of fertiliza-
2509–2514. tion in mammals. Nature Cell Biol 3:E59–E64.
Tulsiani DR, Yoshida-Komiya H, Araki Y. 1997. Mammalian Williams Z, Litscher ES, Jovine L, Wassarman PM. 2006. Polypep-
fertilization: A carbohydrate-mediated event. Biol Reprod 57: tide encoded by mouse ZP3 exon-7 is necessary and sufficient
487–494. for binding of mouse sperm in vitro. J Cell Physiol 207:30–39.
Varki A, Cummings R, Esko J, Freeze H, Hart G, Marth J. 1999. Yanagimachi R. 1994. Mammalian fertilization. In: Knobil E,
Essentials of glycobiology. NY: Cold Spring Harbor Laboratory Neill JD, editors. The Physiology of Reproduction. 1. NY: Raven
Press. Press, pp 189–317.
Wassarman PM. 1988. Zona pellucida glycoproteins. Annu Rev Yonezawa N, Kudo K, Terauchi H, Kanai S, Yoda N, Tanokura M,
Biochem 57:415–442. Ito K, Miura K, Katsumata A, Nakano M. 2005. Recombinant
Wassarman PM. 1990. Profile of a mammalian sperm receptor. porcine zona pellucida glycoproteins expressed in Sf9 cells bind
Development 108:1–19. to bovine sperm but not to porcine sperm. J Biol Chem 280:
Wassarman PM. 1999. Mammalian fertilization: Molecular aspects 20189–20196.
of gamete adhesion, exocytosis, and fusion. Cell 96:175–183. Zhao M, Gold L, Ginsberg AM, Liang LF, Dean J. 2002. Conserved
Wassarman PM. 2005. Contribution of mouse egg zona pellucida furin cleavage site not essential for secretion and integration of
glycoproteins to gamete recognition during fertilization. J Cell ZP3 into the extracellular egg coat of transgenic mice. Mol Cell
Physiol 204:388–391. Biol 22:3111–3120.

Mol Reprod Dev 76:933–941 (2009) 941