1. Introduction
The penetration of the oocyte zona pellucida is a crucial step during fertilization. The zona pellucida is the extracellular network-like matrix enveloping the egg that protects
the growing egg and the preimplantation embryo against physical damages. Sperm that are unable to recognize and bind to the zona pellucida glycoproteins, as well as sperm,
upon binding, that are unable to respond with it by undergoing the acrosome reaction fail to fertilize the egg. After entering the Fallopian tube sperm are stored in the isthmic
Ž part of the oviduct by binding to the ciliated cell lining the oviductal epithelium Suarez,
. 1999 . Sperm are stored under conditions that maintain sperm viability and synchronize
sperm transport and the capacitating process. This enables sperm to interact with the oocyte in the appropriate manner.
Reaching the site of fertilization sperm recognize the egg by carbohydrate–protein interactions. It is well accepted that the corresponding carbohydrate-binding proteins of
the sperm surface bind defined oligosaccharide ligands of the zona pellucida. Upon Ž
. binding the signaling cascade leading to the acrosomal exocytosis acrosome reaction is
activated. This allows sperm to penetrate the zona pellucida. After penetrating the zona pellucida sperm interact and fuse with egg vitelline membrane and trigger thus the
Ž .
embryonic development program Yanagimachi, 1994 . The current perspective focuses on the molecules involved in the complex hierarchy
of interactions between sperm and egg in pig and other domestic animals.
2. Molecules involved in gamete recognition
Close to the time of ovulation sperm are released from the oviductal epithelium and swim to the site of fertilization to meet the egg. The hyperactivated motility that sperm
develop during the capacitating process not only enables the sperm to reach the oocyte but also may be necessary for the collision with the egg assisting the manifestation of
Ž .
the adhesion between sperm and egg Thaler and Cardullo, 1996 . The adhesion between both gametes is a complex sequence of binding events implicating low and high affinity
Ž .
binding sites Thaler and Cardullo, 1996 . The fundamental mechanism of gamete recognition appears to be conserved throughout the evolution from marine invertebrates
to eutherian mammals and is based on carbohydrate–protein interactions between the sperm and the oocyte envelope. Oligosaccharides that are presented with a certain
arrangement within the supramolecular architecture of the egg envelope are recognized by complementary carbohydrate receptors of the sperm, thereby mediating gamete
recognition and coordinating sperm functions to warrant fertilization.
3. Zona pellucida
The mammalian zona pellucida is composed of three glycoproteins that are products of the three zona pellucida genes named according to their length: ZPA, ZPB and ZPC
Ž .
genes Harris et al., 1994 . In rodents zp glycoproteins are exclusively synthesized by the oocyte whereas, in domestic animals possessing a thick zona pellucida, e.g. in pig
and cow, during follicular development the surrounding follicle cells contribute to the Ž
. formation of the extra cellular matrix for review see Sinowatz et al., 1999 . The three
proteins build a typical fibrogranular structure by noncovalent interactions presenting a Ž .
complex and highly heterogeneous mixture of asparagine N - and serinerthreonine Ž .
O -linked oligosaccharide side chains. It has been proved that in different mammalian species amino acid sequence of the zp glycoproteins is highly conserved between
different mammalian species. However, variable posttranslational glycosylation and processing of the polypeptide chains as well as the variable assembling of the supra-
Ž .
molecular structure of the zp matrix possibly due to differing biosynthetic pathways Ž
. lead to substantial differences of zp structure and function between rodents mouse and
Ž .
Ž domestic animals pig . In mouse the zona pellucida protein 3 mZP3 encoded by the
. ZPC gene carrying the sperm receptor activity and the acrosome reaction-inducing
Ž .
potency forms together with zona pellucida protein 2 mZP2 encoded by the ZPA gene long periodically heterodimeric filaments that are randomly interconnected with the zona
Ž . Ž
. pellucida protein 1 mZP1 encoded by the ZPB gene
Wassarman and Mortillo, 1991 . Ž
In contrast, in pig the biological active zp protein has been found to be ZPB the mZP1 .
Ž .
homologue that tends to aggregate with ZPC the mZP3 homologue thereby increasing Ž
. the sperm binding capacity Yurewicz et al., 1998 . Murine zp proteins can be separated
Ž .
Ž .
by SDS-PAGE showing molecular masses of 80 kDa ZP3rZPC , 120 kDa ZP2rZPA Ž
Ž . Ž
. and 220 kDa ZP1 ZPB
reviewed by Wassarman, 1999 . In pig the zona pellucida Ž
. Ž
. protein 3 pZP3 has been shown to be a mixture of the so-called pZP3a ZPB and
Ž .
pZP3b ZPB with apparent molecular masses of 55 kDa and in cow the ZP3arb Ž
. Ž
homologues reveal overlapping bands 78–88 kDa after 2-D PAGE Noguchi and .
Nakano, 1992; Topper et al., 1997 . Information on the oligosaccharide structure of zp glycoproteins is almost complete for pig and partially available for mouse and cow
Ž .
Hokke et al., 1994; Nakano et al., 1996; Topfer-Petersen, 1999 . Although the
¨
structures of the oligosaccharide chains are basically the same in these three species, they differ in the percentage and structure of the neutral N-linked carbohydrates.
Ž Whereas, in mouse the N-glycans are almost acidic, the major neutral N-glycans about
. 25 of the porcine zp glycoproteins belong to the bi-antennary fucosylated complex
N-type and is a high mannose-type in cattle. In cattle the major neutral N-glycan has Ž
. been implicated in sperm–egg recognition Nakano et al., 1996 and may bind to still
Ž poorly described mannose-binding proteins of the capacitated sperm Suarez, personal
. communication . In pig sperm receptor activity has been mapped to O- and N-linked
Ž .
glycans of ZPB Yurewicz et al., 1991; Yonezawa et al., 1995 . The trirtetra-antennary Ž
. N-glycans localized in the N-terminal region of the mature ZPB pZP3a mediate the
binding of sperm to the zona pellucida whereas, the structural identical trirtetra-anten- Ž
. nary N-glycans of the ZPC molecule
pZP3b appear to play no role in gamete
Ž .
recognition Kudo et al., 1998, Yonezawa et al., 1999 . The only difference between these oligosaccharides is the C-terminally localized glycosylation site within the ZPC
molecule, possibly leading to a reduced accessibility of the glycan chains, dependent on Ž
. folding of the protein Fig. 1 . This observation emphasizes that the assembling of the zp
glycoproteins and the correct presentation of the biological active oligosaccharides
Fig. 1. Schematic presentation of the known N-glycosylation sites in porcine zp glycoproteins. Of the six potential N-glycosylation sites of ZPA only Asn268 has been identified carrying biantennary N-glycans
Ž .
Ž Topfer-Petersen, unpublished . ZPB and ZPC both possess three glycosylation sites Kudo et al., 1998;
¨
. Ž
. Yonezawa et al., 1999 . The mature ZPB molecule is N-terminally processed Asp 137 and the mature ZPC
Ž .
molecule losses the signal peptide and is N-terminally blocked pyro-Gln23 . Signal peptide is marked in grey.
within the three-dimensional structure are important features to achieve the physio- logical relevant binding between both gametes. In mouse low- and high-affinity binding
sites have been identified on the sperm surface implicating a hierarchy of binding events Ž
. Johnston et al., 1998 . Interestingly, the high-affinity site recognizing fucosylated
oligosaccharides can be occupied with less affinity by other carbohydrate structures. The multivalent presentation of the biological active oligosaccharides may be a necessity to
create high-affinity binding. In mouse the relevant O-linked oligosaccharides are clus- Ž
. tered at the C-terminal sperm-combing sites of mZPC reviewed by Wassarman, 1999 .
In pig the arrangement of the oligosaccharides within the heteromultimeric ZPBrZPC complex may be responsible for the manifestation of high-affinity binding to the sperm
Ž .
Yurewicz et al., 1998 . There is a considerable controversy regarding the structural Ž .
entity of the sperm oligosaccharide ligands in both species suggesting that i different Ž
experimental conditions and the possible binding of uncapacitated and capacitated .
Ž . sperm to the zona; Miranda, 1998 andror ii an allowed structural variety of the
presented carbohydrates at the sperm-binding site of the intact zona pellucida may account for these differences.
4. Zona pellucida binding proteins
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