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BOR - Papers in Press, published online ahead of print October 14, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.007856
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BIOLOGY OF REPRODUCTION 68, 1–9 (2003)
DOI: 10.1095/biolreprod.102.007856
© 2003 by the Society for the Study of Reproduction, Inc.

Minireview

Cell Adhesion and Fertilization: Steps in Oocyte Transport, Sperm-Zona Pellucida Interactions, and Sperm-Egg Fusion1

Prudence Talbota, Barry D. Shurb, and Diana G. Myles2,c

a Department of Neuroscience, University of California-Riverside, Riverside, California 92521 b Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia 30322 c Section of Molecular and Cell Biology, University of California-Davis, Davis, California 95616


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 ROLE OF THE CUMULUS...
 SPERM RECOGNITION OF THE...
 SPERM ADHESION TO (AND...
 SUMMARY
 REFERENCES
 
Fertilization in mammals requires the successful completion of many steps, starting with the transport of gametes in the reproductive tract and ending with sperm-egg membrane fusion. In this minireview, we focus on three adhesion steps in this multistep process. The first is oocyte "pick-up," in which the degree of adhesion between the extracellular matrix of the cumulus cells and oviductal epithelial cells controls the successful pick-up of the oocyte-cumulus complex and its subsequent transfer into the oviduct. The second part of this review is concerned with the interaction between the sperm and the zona pellucida of the egg. Evidence is discussed that a plasma membrane form of galactosyltransferase on the surface of mouse sperm binds to ZP3 in the zona pellucida and initiates an acrosome reaction. Additional evidence raises the possibility that initial sperm binding to the zona pellucida is independent of ZP3. Last, we address the relationship between sperm adhesion to the egg plasma membrane and membrane fusion, especially the role of ADAM family proteins on the sperm surface and egg integrins.

acrosome reaction, fertilization, oviduct, ovum pick-up/transport


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
ROLE OF THE CUMULUS...
 SPERM RECOGNITION OF THE...
 SPERM ADHESION TO (AND...
 SUMMARY
 REFERENCES
 
The bringing together of two gametes in mammals is a complex process that starts with cells moving through the reproductive tracts of both the male and female organisms until they arrive close to each other in the female reproductive tract. The subsequent interaction between the two gametes requires several steps that result in gamete fusion to produce a zygote. Cell adhesion is a major part of these unique processes. Cell adhesion is involved in the transport of both egg and sperm, and it is also integral to the steps in sperm-egg interaction. In this review, we examine three separate cell adhesion phenomena: 1) adhesion between the oviduct epithelial cells and the extracellular matrix of the oocyte-cumulus complex (OCC), 2) adhesion/signaling between the sperm membrane and the extracellular coat of the egg, and 3) adhesion/fusion between egg and sperm membranes. In each case, we discuss recent studies that investigate the molecular basis of these adhesion events.


   ROLE OF THE CUMULUS MATRIX IN OOCYTE PICK-UP
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 ABSTRACT
 INTRODUCTION
 ROLE OF THE CUMULUS...
SPERM RECOGNITION OF THE...
 SPERM ADHESION TO (AND...
 SUMMARY
 REFERENCES
 
The OCCs, consisting of an oocyte surrounded by a zona pellucida and cumulus layer, are ovulated from mammalian ovarian follicles into the peritoneal or bursal cavity, where they are picked up by the infundibulum of the oviduct [1–3]. Pick-up, which is brought about by cilia covering the outer surface of the infundibulum, is a vital process in reproduction, and its failure to occur can lead to ectopic implantation [2].

The cumulus layer of the OCC contains numerous cumulus cells plus an extracellular matrix that binds the cells together. The cumulus cells and their matrix are probably involved in several reproductive processes (summarized in [4]), including pick-up of the OCC by the oviduct [4, 5]. Cilia that cover the exterior surface of the infundibulum beat in the direction of the ostium and are important in moving the OCC into the oviduct. The matrix of the OCC is rich in hyaluronan [6–10], and numerous proteins are reported to be present in the matrix, including inter-{alpha}-trypsin inhibitor [11], a dermatin sulfate proteoglycan, and a 46-kDa protein [12]. Microscopic studies first suggested that the proteins and hyaluronan are linked together to form a meshwork comprised of granules and filaments between cumulus cells [3, 13, 14]. Inter-{alpha}-trypsin inhibitor, which is present throughout the matrix, was subsequently shown to become covalently linked to chains of hyaluronan during cumulus expansion [11], thereby stabilizing the cumulus matrix and retaining it in the complex.

Studies regarding the process of OCC pick-up have been facilitated by development of an in vitro procedure, using hamsters as a model species, that allows video microscopy to be performed during pick-up [15]. In addition, ciliary beat frequency, muscle contraction rate, adhesion between the OCC and infundibulum, and OCC pick-up rate can be measured quantitatively in the same preparations [4, 16–18]. Video microscopy has shown that OCC pick-up involves adhesion of the OCC to the surface of the infundibulum [15]. The OCC, which is relatively large, adheres to the oviductal cilia and slides over the surface of the infundibulum during pick-up. When the OCC reaches the ostium, the pick-up process slows while the OCC, which is too large to pass directly through the opening, churns for several minutes in the ostium. During this churning process, the cumulus matrix becomes compacted, making the diameter of the OCC smaller so that it can move into the lumen of the infundibulum [15]. QuickTime movies showing pick-up and churning can be viewed on the Internet (http://www.molbiolcell.org/cgi/content/full/10/1/5). Electron microscopy shows that adhesion occurs between the tips of the cilia (specifically, the ciliary crown) and the cumulus matrix of the OCC [4]. In electron micrographs, both protein and hyaluronan appear to adhere to the ciliary crowns. Adhesion between the cilia and the cumulus matrix is specific, because OCCs do not adhere to other intact epithelial surfaces, including the ciliated cells of the trachea and the ependyma.

To determine if the cumulus matrix is necessary for pick-up, unexpanded and expanded hamster OCCs were examined using an in vitro assay for OCC pick-up [4]. Expanded OCCs were picked up at the normal rate, as observed previously [17]. In contrast, unexpanded OCCs, which had cumulus cells but not cumulus matrix, failed to adhere to the infundibulum and were not picked up at all. Interestingly, when unexpanded OCCs were placed in the ostium, they remained in place and were not picked up even though cilia were beating normally. This experiment demonstrates that the adhesion occurring between the cumulus matrix and the ciliary crown is necessary for pick-up to occur in hamsters, and it further illustrates the importance of the cumulus matrix in reproduction. In the absence of such matrix (i.e., unexpanded OCCs), the OCC does not adhere to the infundibulum. In addition, pick-up does not occur, because the OCC cannot pass through the ostium. In contrast to the situation for hamsters, the cumulus layer of shrews does not expand before ovulation, and sperm are able to penetrate unexpanded shrew cumulus complexes in the oviduct, indicating that cumulus matrix is not needed for pick-up in Insectivora, a more primitive group of eutherian mammals [19, 20].

An assay was developed to measure adhesion between the OCC and oviduct with the hamster model [4]. Using this assay, it was determined that adhesion between the OCC and cilia is very consistent from measurement to measurement both within and between females. To determine if modulating adhesion alters the OCC pick-up rate, adhesion was measured using experimental conditions chosen to either increase or decrease adhesion. When adhesion was decreased by compressing the cumulus matrix, the OCC pick-up rate tended to decrease. In addition, OCCs could not enter the ostium, because they did not adhere well to the infundibulum. When adhesion was increased, such as by preincubating the infundibulum in the lectin WGA (wheat germ agglutinin) or in poly-L-lysine, the OCC pick-up rate slowed or completely stopped. These results show that slight changes in adhesion prevent proper pick-up and emphasize the importance of the correct degree of adhesion for pick-up to occur normally.

Finally the effects of cigarette smoke solutions on OCC pick-up and adhesion were examined with the in vitro assay. Both mainstream and sidestream cigarette smoke solutions, made as described previously [21, 22], inhibited OCC pick-up rate, and in most cases, this inhibition was accompanied by an increase in adhesion. Because increased adhesion was shown previously to slow the OCC pick-up rate [4], the smoke solutions likely were decreasing pick-up rate by increasing adhesion. These observations may explain why women who smoke have higher rates of ectopic pregnancy than women who do not smoke [23].

Taken together, these data show that adhesion between the cumulus matrix and ciliary crown is necessary for pick-up of the OCC by the infundibulum in hamsters, that the degree of adhesion is important for proper pick-up, and that altering adhesion experimentally or via environmental factors can preclude proper pick-up and may explain some cases of ectopic pregnancy.


   SPERM RECOGNITION OF THE EGG COAT, THE ZONA PELLUCIDA
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 INTRODUCTION
 ROLE OF THE CUMULUS...
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Sperm penetration through the cumulus matrix is facilitated by a membrane-bound hyaluronidase [24]. Eventually, the sperm encounters the zona pellucida, the extracellular coat of the egg where species-specific gamete recognition is believed to occur. In the mouse, the zona is composed of three glycoproteins, referred to as ZP1, ZP2, and ZP3, with the sperm-binding activity residing on ser/thr-linked oligosaccharide chains (i.e., O-linked oligosaccharides) of the ZP3 glycoprotein [25]. Much of the research on the zona and sperm recognition of the zona has been carried out using the mouse as a model system. Unless otherwise indicated, the work reviewed below used mouse gametes.

Structure of the Sperm-Binding Oligosaccharide on ZP3 Remains Undefined

Although O-linked oligosaccharide chains on ZP3 act as a binding epitope for sperm, the molecular composition of the sperm-binding oligosaccharide remains obscure. Competition assays have implicated at least four different monosaccharide residues as being critical for initial sperm binding: 1) {alpha}-galactose, 2) ß-N-acetylglucosamine (GlcNAc), 3) fucose, and 4) mannose [26–29]. Insight regarding this apparent paradox comes from two recent observations. First, immunocytochemical analysis at the ultrastructural level reveals that the sugar composition of the zona is heterogeneous [30, 31], with some sugars being confined to the inner portions of the zona pellucida whereas others are dispersed uniformly throughout the zona. Thus, competition assays using soluble glycosides suffer from not knowing whether the inhibitory sugar being assayed is available to the sperm at initial binding, during later aspects of zona penetration, or not at all. In this regard, {alpha}-galactosyl residues are confined to the inner zona matrix and are not accessible to sperm on the zona surface; consequently, {alpha}-galactose residues are unlikely to participate in initial gamete recognition. Consistent with this, eggs from females bearing null mutations in {alpha}-galactosyltransferase are fertilized normally [32], at least in vivo.

Terminal GlcNAc residues have been implicated in sperm-zona binding by enzymatic modification of either intact zona pellucida or of purified ZP3 [27]. Immunocytochemical studies illustrate GlcNAc residues dispersed throughout the zona matrix, and one class of O-linked oligosaccharides on ZP3 has been characterized as a small trisaccharide with a terminal GlcNAc [33]. Although these data further implicate GlcNAc as a participant in sperm binding, whether this specific trisaccharide has sperm-binding activity is not yet known. Furthermore, structural analysis of the major classes of O-linked sugars in the mouse zona pellucida have failed to detect this GlcNAc-terminating trisaccharide [34], suggesting that it is present in very small quantities or is degraded during analysis.

Finally, Lewis X-type fucosyl residues are able to competitively inhibit sperm-egg binding, suggesting that these glycoside structures mimic a sperm-binding oligosaccharide on the zona [28]. However, these structures are not detectable immunocytochemically in the zona pellucida of mice or rats, making it unlikely that they participate in sperm-egg interactions [30]. All these results emphasize the need to interpret competition assays in light of the carbohydrate composition and heterogeneity that characterize the zona matrix.

A second recent observation that affects our understanding of sperm-binding macromolecules is that at least two distinct binding events occur during the early stages of sperm-egg interaction: a ZP3-dependent and a ZP3-independent event (unpublished observations). Thus, which, if any, binding event is being perturbed by the addition of soluble competitive glycosides is not known. Similarly, that the zona matrix is modified after ovulation by the adsorption of oviduct-secreted glycoproteins [35, 36] is noteworthy. This highlights the possibility that the sperm-binding glycans exposed on the zona surface are distinct from those assayed when zona pellucida is isolated from ovarian homogenates.

A Wide Variety of Sperm Surface Components Are Implicated in Zona Binding

Sperm proteins thought to participate in zona binding have been identified by a range of approaches, including 1) analysis of mutations that affect fertilizing ability [37], 2) development of inhibitory monoclonal antibodies (MAbs) [38], 3) identification of sperm proteins that bind zona pellucida affinity columns [39, 40], 4) identification of sperm proteins that can be photoaffinity cross-linked to the zona matrix [41], 5) use of radiolabeled zona glycoproteins to overlay blots of sperm lysates [42], 6) use of competitive glycoside substrates [43, 44], and 7) indirect implication that they require the chaperone, calmegin, for proper folding and expression [45]. Still other approaches have been used as well [46]. Some of these sperm proteins have been specifically implicated as primary sperm receptors for ZP3 [27, 41, 42], whereas others are thought to function as generalized adhesive proteins for the zona matrix [39, 40]. Thus far, among the sperm proteins thought to bind ZP3 oligosaccharides in particular, sperm surface ß1,4-galactosyltransferase-I (GalT) satisfies virtually all the criteria expected of a ZP3 receptor.

Found on the dorsal, anterior aspect of the sperm head, GalT behaves as an integral membrane protein [47]. On cauda epididymal sperm, GalT is masked by epididymally secreted glycoconjugates that are shed from the sperm surface during capacitation, thus making GalT available to bind its oligosaccharide ligand in the zona [48]. The importance of GalT in sperm-zona binding has been demonstrated using a series of reagents that block GalT or the GalT-recognition site on the zona pellucida, all of which inhibit sperm-zona binding [47–50]. Similarly, blocking or removing the GalT-binding site on the zona destroys sperm-binding activity. Sperm GalT selectively binds O-linked oligosaccharides on ZP3, and the interaction between sperm GalT and ZP3 oligosaccharides is necessary for sperm-zona binding, as shown by two observations [27]. First, when GalT-recognition sites in soluble ZP3 are blocked by glycosylation, ZP3 can no longer bind GalT and loses its sperm-binding activity. Second, removal of the GalT-recognition site on ZP3 by digestion with GlcNAc results in a loss of ZP3 sperm-binding activity.

The binding of ZP3 to sperm activates a range of intracellular signal cascades that culminate in fusion of the plasma membrane and underlying outer acrosomal membrane (i.e., the acrosome reaction) [51]. The best-studied among these signal cascades involve 1) a pertussis toxin (PTx)-sensitive, heterotrimeric G-protein [52, 53] and 2) a voltage-independent cation channel that regulates membrane potential, which, in turn, regulates a voltage-dependent cation channel [54, 55]. These cascades, among others, result in elevation in [Ca2+]i and pHi, which are required for acrosomal exocytosis [54]. The ability of ZP3 to activate PTx-sensitive G-proteins in sperm appears to involve ZP3-induced aggregation of GalT, because GalT aggregation, in the absence of ZP3 binding, induces PTx-sensitive G-protein activation [56]. Zona binding also activates a sperm glycine receptor/Cl- channel that may contribute to the control of voltage-sensitive calcium influx [57].

As sperm undergo the acrosome reaction, they must remain transiently attached to the zona before initiation of zona penetration. The binding of acrosome-reacted sperm to the zona may depend on ZP2, because acrosome-reacted sperm lose affinity for ZP3 and gain affinity for ZP2 [58]. Recent developments suggest that the acrosome reaction may sequentially release and/or expose specific components of the acrosomal matrix, which may stabilize sperm adhesion to the zona matrix [59]. Candidates include sp56 [60], zonadhesin [61], and other zona-binding proteins associated with the acrosomal matrix.

After egg activation, cortical granules are exocytosed at the egg plasma membrane. These vesicles contain various hydrolases that act on the zona pellucida such that it loses its ability to bind sperm, thereby blocking subsequent fertilization by other sperm. The loss of sperm-binding activity is due, at least in part, to the release of ß-N-acetylhexosaminidase from the egg cortical granules, which destroys the GalT-binding sites on ZP3 [62].

Altering the Expression of Sperm GalT Affects the Efficacy of Sperm-Egg Binding

The consequences of either elevating or eliminating GalT expression have been examined regarding sperm-fertilizing ability. Elevating GalT expression on the sperm surface in appropriate transgenic animals leads to increased binding of ZP3, increased G-protein activation, and increased sensitivity to zona-induced acrosome reactions [63]. In contrast, eliminating GalT from the sperm surface through homologous recombination leads to reduced ZP3 binding and to sperm that are refractory to zona-induced acrosome reactions [64].

The ability of GalT to function as a signal-transducing receptor for ZP3 is shown by the heterologous expression of GalT on the surface of Xenopus oocytes, which acquire the ability to bind ZP3 but not other zona glycoproteins [65]. The addition of ZP3 or anti-GalT antibodies to GalT-expressing Xenopus oocytes elicits cortical contraction, envelope elevation, and cortical granule exocytosis. This results from GalT-dependent G-protein activation, as shown by the ability of ZP3 or anti-GalT antibodies to increase both GTP{gamma}35S binding and GTPase activity as well as by the ability of PTx to inhibit GalT-induced egg activation. Finally, mutagenesis of a putative G-protein activation motif within the GalT cytoplasmic domain eliminates G-protein activation in response to ZP3 or anti-GalT antibodies. These results strongly suggest that GalT functions as a ZP3 receptor and that ZP3-induced aggregation activates PTx-sensitive G-proteins [65]. Thus, GalT appears to be a member of the growing family of single-pass membrane receptors that activate, directly or indirectly, heterotrimeric G-proteins [66, 67].

Sperm from GalT-Null Males Still Bind to the Zona Pellucida, and Galt-Null Males Remain Fertile

Although GalT-null sperm are unable to bind ZP3 or undergo a ZP3-dependent acrosome reaction, GalT-null males remain fertile. Similarly, males with sperm made null for the acrosomal protease, acrosin, are also fertile [68]. These observations suggest that acrosomal exocytosis specifically induced by ZP3-dependent GalT aggregation is not critical for fertilization. However, the rate of zona penetration by either GalT-null sperm or acrosin-null sperm is severely compromised relative to wild-type sperm [64, 68, 69], indicating that the degree of acrosomal exocytosis influences the efficacy of fertilization. By whatever mechanism, the fertilizing sperm must be acrosome-reacted to bind and fuse with the egg plasma membrane.

Importantly, GalT-null sperm still bind to the coat of ovulated oocytes, although they do not bind ZP3 [64]. This implies that GalT binds ZP3 oligosaccharides in the context of other sperm surface components that are responsible for the initial docking of sperm to ZP3-independent ligands in the egg coat. Conceptually, this paradigm is similar to the concerted action of selectins and integrins in lymphocyte adhesion to the endothelium [70]. Preliminary studies are consistent with the egg coat containing at least two distinct sperm-binding ligands (unpublished observations): 1) one peripherally associated with the egg coat and responsible for sperm-egg adhesion and 2) ZP3, a structural component of the egg coat responsible for inducing the acrosome reaction. That mouse sperm still bind to eggs in which murine ZP3 has been replaced with human ZP3 [71] is consistent with the possibility, among others, that sperm adhesion requires a ZP3-independent ligand on the egg coat.

With this in mind, it is of interest to determine if other sperm surface components thought to facilitate adhesion to the zona are responsible for the binding of GalT-null sperm. Sperm p47 is a particularly attractive candidate for mediating initial sperm-egg adhesion [40]. A peripherally associated sperm protein, p47 was identified by affinity chromatography of solubilized sperm plasma membrane proteins bound to immobilized porcine zona pellucida glycoproteins [40]. It contains two amino-terminal, tandemly arranged, epidermal growth factor-like domains followed by regions similar to the C1 and C2 domains of blood clotting factors V and VIII. The second EGF-like domain contains an RGD sequence, a motif often found in integrin ligands. The current biochemical evidence strongly favors a role for p47 during initial gamete adhesion [40], but a more complete understanding of p47 function must await the analysis of mice bearing null mutations in p47.


   SPERM ADHESION TO (AND FUSION WITH) THE EGG PLASMA MEMBRANE
TOP
 ABSTRACT
 INTRODUCTION
 ROLE OF THE CUMULUS...
 SPERM RECOGNITION OF THE...
 SPERM ADHESION TO (AND...
SUMMARY
 REFERENCES
 
After sperm bind to the egg zona pellucida and acrosome react, they penetrate the zona and enter the perivitelline space, the extracellular region between the zona and the egg plasma membrane. There, the final adhesion in the fertilization process occurs (i.e., adhesion of the sperm plasma membrane to the egg plasma membrane). It is thought that this adhesion normally precedes, and is required for, sperm-egg membrane fusion [72]. Most of the studies discussed below used the mouse as the experimental system; use of other mammalian species is indicated.

Adhesion between the sperm and the egg plasma membranes can be observed in vitro most easily with zona-free eggs, but observations have also been made with zona-intact eggs. In most cases, the initial contact is between the tip of the sperm and the egg plasma membrane, followed by a lateral attachment between the side of the sperm and the egg. Membrane fusion may occur with the sperm in either position (perpendicular or parallel to the egg surface), but fusion is thought to always use the central region of the sperm plasma membrane (near or at the equatorial region) [72].

Sperm-Egg Plasma Membrane Adhesion Assay

Measurements of sperm-egg adhesion are made by counting the number of sperm bound per zona-free egg. Because the assay conditions used for counting the number of bound sperm vary widely, comparisons are best made between samples in a single assay. Even then, however, the physiological relevance of the number of sperm bound to a zona-free egg is questionable. The presumption is that sperm are bound to the egg plasma membrane by a mechanism that can result in sperm-egg membrane fusion, such as the "tethering" or "docking" of membranes observed before fusion of other types of membranes. Several different sperm populations, however, may be included in the counts of bound sperm. Some of the sperm may be bound via interactions that will not lead to fusion. For example, acrosome-intact sperm can bind to zona-free eggs, but they do not fuse [73]. Also, most counts are performed after an extended period of sperm-egg coincubation. There could be considerable difference between the adhesion properties of sperm that bind early (i.e., before sperm-egg fusion) and those of sperm that bind late (i.e., after at least one sperm has fused with the egg), because fusion may change the ability of the egg to bind additional sperm. Fertilized mouse eggs are still able to bind many sperm even after they lose their ability to fuse with sperm (within 1 h after their initial fusion) [74]. However, this "late" sperm binding may be of a different type. The question of different sperm avidities in the sperm-binding assays has recently been addressed, and it has been suggested that the assay could be improved by standardizing the washing techniques to select for sperm binding of higher avidity [75]. Although this partially standardizes the assay, a direct correlation between avidity and fusion capability has not been demonstrated. Any of the in vitro assays may include a heterogeneous population of bound sperm, including nonphysiologically bound sperm, along with a specific population of physiologically relevant sperm that are tethered or docked before fusion. If the first (nonspecific) category is large compared to the second (specific) category, changes in the number of physiologically relevant bound sperm may not be measured. Therefore, when sperm binding is measured by counting the total number of sperm bound to the egg plasma membrane in vitro, the relevance of this measured binding to the fusion of sperm and eggs is unknown.

Lack of Correlation Between Assays of Sperm Binding and Sperm Fusion

Recent evidence indicates that counts of the total number of sperm bound to the egg do not directly correlate with the level of sperm fusion. Null mutations in either or both of two different sperm surface proteins (fertilin ß and cyritestin) resulted in sperm that lost much of their ability to bind to the egg plasma membrane but that were still capable of fusion [76, 77]. By following sperm-egg interactions in insemination drops in vitro, it was observed that almost 100% of the wild-type sperm contacting the zona-free eggs remain bound, whereas only approximately 3% of the fertilin ß knockout sperm form a lasting attachment to the zona-free eggs (unpublished observations). This is in spite of the fact that fusion by fertilin ß knockout sperm is only reduced by 50%. It is also possible to prevent fusion without a measurable effect on sperm binding. For example, CD9-null eggs bind normal or excessive numbers of sperm, but sperm-egg fusion is severely limited or does not occur [78–80]. In addition, inhibition of metalloprotease activity at the time of gamete interaction can result in normal levels of bound sperm but no fusion [81].

The basis for this lack of correlation between the total number of sperm bound to the egg plasma membrane and the ability of sperm to fuse with the egg has not yet been resolved. In vitro assays possibly lead to an artifactual situation in which most of the sperm bound to the egg are in a holding pattern that allows them to remain bound, but these sperm are not in a pathway that will lead to fusion. Although some of these sperm may eventually enter the fusion pathway, others may remain bound without that capability. Also, it is possible that this type of irrelevant sperm binding does not normally occur in vivo. Physiological binding, defined as an adhesion of sperm and egg leading to fusion, may be a step that normally precedes fusion and that makes it more efficient by providing a pool of sperm in the "right position" for fusion. However, the existence of physiologically relevant sperm binding, perhaps analogous to the "docking" step that occurs during intracellular membrane fusion, has not been proven, and in fact, it may not be an absolute requirement for sperm-egg fusion.

Molecular Basis of Sperm-Egg Membrane Adhesion/Binding

During the last 15 years, a model for sperm-egg plasma membrane binding has developed that suggests sperm-egg binding results from the adhesion between an integrin on the egg and an integrin ligand (i.e., the disintegrin domain of a member of the ADAM family of proteins) on the sperm. The initial step in the development of this model was a screen (using an in vitro fusion assay) of a library of MAbs directed against the guinea pig sperm plasma membrane, which identified a sperm surface protein that is potentially required for sperm-egg fusion [82]. One MAb (PH-30 MAb) that recognized this protein (fertilin/PH-30) was able to inhibit fusion with half-maximal inhibition at 140 µg/ml, whereas a control antibody (nonfunction blocking, PH-1 MAb) showed no inhibition even at 400 µg/ml. The ability of sperm to bind to the egg plasma membrane was not measured in this initial screen. The PH-30 MAb immunoprecipitated a heterodimeric protein composed of two subunits, now named fertilin {alpha} (ADAM 1) and fertilin ß (ADAM 2) [82]. Sequencing of these two subunits revealed three regions of particular interest: 1) a metalloprotease domain (that has a consensus sequence indicating enzyme activity in the fertilin {alpha} subunit but not in the fertilin ß subunit), 2) a disintegrin domain in both subunits (previously shown to be an integrin ligand in snake venom peptides), and 3) a region in the fertilin {alpha} subunit that could be modeled as an amphipathic alpha helix and was therefore reminiscent of the fusion peptides of some viruses [83, 84].

Both subunits were subsequently identified in other mammalian species [85–88], although some species may lack the fertilin {alpha} subunit [89]. The identification and sequencing of fertilin ß and fertilin {alpha} subunits in the mouse also led to the discovery of many other members in this new family of proteins (named the ADAM family because of the presence of a disintegrin and a metalloprotease domain) [90]. As new proteins have been identified, additional functions have been determined for members of this family, and it has been found that domains other than the disintegrin and metalloprotease domains of the protein may be functional [91].

The finding that fertilin and, subsequently, other members of the ADAM family were present on the surface of mature sperm led to the hypothesis that sperm bind via the disintegrin domain of a sperm ADAM to an egg integrin. The putative binding site was determined by alignment with snake disintegrin peptides that were shown to have a short loop formed by disulfide bonds at the base that contained an RGD (a common sequence for many integrin ligands) at its tip [91]. Homologous regions of the ADAM family do not contain RGD; many, but not all, members of the ADAM family have an ECD in this position. Peptides were synthesized using the sequence derived from the putative binding site of the disintegrin domain of guinea pig fertilin ß and were tested by in vitro assays; the results showed a dramatic reduction in sperm-egg fusion with both zona-intact and zona-free eggs [92]. In subsequent studies using the mouse, peptides derived from the fertilin ß sequence blocked sperm-egg plasma membrane binding and fusion, and peptides from cyritestin (ADAM 3) were even more effective. Peptides from other proteins (fertilin {alpha}, ADAM 4, and ADAM 5) had little or no effect [93]. Different peptide and recombinant proteins have subsequently been tested in a variety of species [94]. Inhibition of fusion and binding by the peptides/proteins has been attributed to peptide/protein binding to an egg receptor (predicted to be an integrin), thus blocking specific binding steps required for fusion.

To better assess the requirement for specific members of the ADAM family in binding (and possibly fusion), knockout mice were constructed that were null for fertilin ß, cyritestin, or both (double knockout). Sperm from each of these mouse lines showed dramatically (~90%) reduced binding to zona-free oocytes [76, 77]. The most straightforward interpretation of these results is that each of these proteins is required to mediate sperm-egg plasma membrane binding. However, knocking out either fertilin ß or cyritestin resulted in mature sperm with levels of fertilin {alpha} too low to detect with the available antibodies. In addition, knocking out cyritestin resulted in a reduction in fertilin ß on mature sperm to 60% of wild-type levels, and knocking out fertilin ß resulted in a loss of cyritestin to 11% of the level detected in wild-type sperm [77]. Other sperm surface proteins, GalT, PH-20, and testase 1 (ADAM 24), continued to be expressed at wild-type levels. Because of this loss of additional sperm surface proteins in the knockout mice, possibly other proteins could be lost from the knockout sperm, including unknown proteins responsible for the binding of sperm to the egg plasma membrane.

On the egg side, the initial evidence that a disintegrin might be responsible for sperm-egg membrane adhesion suggested that the egg adhesion molecule might be an integrin. Later studies, in fact, demonstrated that a fertilin ß peptide could be cross-linked to the {alpha}6ß1 integrin on the egg plasma membrane [95]. An early screen of antibodies against egg integrins indicated that GoH3, a MAb that recognizes the {alpha}6 integrin subunit, could inhibit sperm-egg binding, pointing to {alpha}6ß1 as a receptor for sperm on the egg plasma membrane [96]. These assays were carried out with eggs from which the zona had been removed with chymotrypsin. Subsequent in vitro assays using the GoH3 antibody, however, including assays in which the zona was removed from oocytes by an alternative method of acid treatment, did not result in the same inhibition of sperm binding [97]. Also, no inhibition of the fusion step was observed when the zona was left intact [98] or in assays using cumulus-intact eggs [99].

The question of which, if any, integrins are required for sperm-egg binding (and subsequent fusion) has also been approached using knockout mice. Recently, oocytes from mice null for the {alpha}6 integrin subunit were shown to have no reduction in sperm-egg binding (or fusion), demonstrating that if {alpha}6 integrins do participate in sperm-egg adhesion, they are not an absolute requirement [99].

To summarize, the best candidates for a role in sperm binding to the egg plasma membrane are fertilin ß and cyritestin. Sperm null for either of these proteins show dramatic reductions in the number of total sperm bound. Because other sperm proteins may be lost in these knockouts, this reduction in total sperm binding is possibly caused by loss of an unidentified protein (another ADAM?). The role of integrins in sperm binding is less convincing. The primary candidate, {alpha}6 integrin, has been removed from eggs by knocking out the gene with no consequent reduction in total sperm binding or in the potential subset of physiological binding that leads to fusion (see below).

Molecular Basis of Sperm-Egg Membrane Fusion

Membrane fusion requires a mechanism in which two lipid bilayers are transformed from two separate barriers into a single bilayer. The best-characterized example of membrane fusion events are intracellular membrane fusion, associated with membrane trafficking [100], and fusion of a virus with its host cell [101]. In comparison, the molecular basis of membrane fusion between two eukaryotic cells is poorly understood.

Sperm surface proteins Only a few sperm proteins that are candidates for a role in sperm-egg fusion have been studied in detail. Recent studies in the mouse using peptide/protein competition or antibody inhibition indicate a potential role in fusion for the proteins DE [102] and equatorin [103]. Both antibody-inhibition and peptide-inhibition studies have also indicated that fertilin ß and cyritestin are required for sperm-egg fusion. However, more definitive studies using gene deletions, including a double knockout of fertilin ß and cyritestin [77], show modest (~50%) [76] or no inhibition of sperm-egg fusion [77, 104]. This could reflect redundancy of fertilin ß and cyritestin, or it could mean that neither fertilin ß nor cyritestin have a role in fusion. These results are somewhat surprising in light of the high inhibition of fusion found with disintegrin active-site peptide studies, but this may indicate that the peptides act in an unexpected way. For instance, disintegrin peptides may act through integrins to initiate signaling that blocks fusion. The signaling pathway may be one that is normally quiescent during fertilization (in the absence of peptides) and irrelevant to fusion.

Fertilin {alpha} has also been considered to be a prime candidate for a fusion protein because of the tantalizing finding that guinea pig fertilin {alpha} contains a short sequence that shares structural properties with viral fusion peptides and that could be modeled as an amphipathic alpha helix. However, sperm from knockout mice, in which fertilin {alpha} could not be detected by Western blot analysis, were still capable of fusion [76, 77, 105]

Egg surface proteins To our knowledge, the identification of any egg integrin required for fusion has not yet been accomplished. The same study in which {alpha}6 integrin knockout eggs showed no reduction in the number of bound sperm also found no reduction in sperm-egg fusion [99].

Two studies, however, do give us ideas regarding potential egg membrane proteins that are key players in sperm-egg fusion. First, a glycosyl phosphatidylinositol (GPI)-anchored protein may be required, because removal of egg GPI-anchored proteins with PI-PLC (phosphatidyl-inositol-specific phospholipase C) results in eggs that can be bound by sperm but that are greatly inhibited (>95%) in their ability to fuse with sperm [106]. However, the specific GPI-anchored protein that may be active has not been identified.

A second result is that absence of the tetraspanin protein, CD9, on the eggs of knockout mice, while not affecting the number of bound sperm, resulted in an almost complete loss of fusion ability [78–80]. CD9 was previously implicated as having a role in sperm-egg fusion by antibody-inhibition studies [107]. So far, the mechanism of CD9 function in fusion has not been clarified, but it may act in coordination with other egg surface proteins. In other cells, tetraspanins are reported to associate with different proteins in the same membrane and are thought to regulate their function [108]. Recent results suggest that CD9 functions via its large extracellular loop (EC2) and that a particular amino acid sequence (SFQ) in the EC2 domain is required for CD9 function in sperm-egg fusion [109]. Further evidence indicates that interaction between the EC2 loop of CD9 and another molecule on the egg (not the sperm) surface is responsible for this function [109].

Another intriguing result is the prevention of fusion by the inhibition of Zn2+-dependent metalloprotease activity. Although inhibitor specificity points to a member of the metzincin class of proteases, which includes matrix metalloproteases and ADAM family members, the identity of the metalloprotease, its substrates, or which gamete expresses it is not known [81].

Considering these findings, we have much less than a complete picture regarding the molecular basis of sperm-egg fusion. Recent results point away from the involvement of sperm ADAMs or egg integrins. Instead, we know that egg CD9 function is essential and that a specific GPI-anchored protein will perhaps prove to play a required role. The sperm proteins required for fusion remain to be established.


   SUMMARY
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 ABSTRACT
 INTRODUCTION
 ROLE OF THE CUMULUS...
 SPERM RECOGNITION OF THE...
 SPERM ADHESION TO (AND...
 SUMMARY
REFERENCES
 
Molecular adhesion between two cell surfaces can be either permanent or transitory. In some cases, it serves to keep cells together, but adhesion can also lead to cell signaling. Adhesion between gametes and surrounding cells, extracellular matrices, and egg and sperm plasma membranes are all thought to be key steps required for successful fertilization in mammals. Most of these adhesion steps are by necessity transitory, because gametes must continue to move forward toward each other. The exception is the final adhesion of the two gamete membranes before membrane fusion. This review concerns three separate adhesion steps and focuses on work from our laboratories.

An early adhesion step is the binding of the newly ovulated OCC to the ciliary tips of the epithelial cells lining the distal region of the oviduct. The adhesion is critical for proper pick-up of the oocyte off the ovary wall following ovulation. The degree of adhesion controls the pick-up and subsequent transport and compaction of the complex that allows its passage into the oviduct lumen, where it will contact the sperm that are swimming up the oviduct. Altering adhesion experimentally or via environmental factors can preclude proper pick-up and transport into the oviduct lumen and may explain some cases of ectopic pregnancy. The molecular basis of this adhesion step depends on the accessibility of the cumulus matrix, which contains both hyaluronan and protein. The receptor for the matrix on the oviductal epithelial cells is currently unknown.

The second adhesion step discussed is that between the mouse sperm plasma membrane and the zona pellucida. A long history of investigation in the mouse has pointed to a requirement for an interaction between an O-linked oligosaccharide of the zona glycoprotein, ZP3, and an unknown sperm receptor on acrosome-intact sperm. No general agreement exists regarding which specific sugars of the zona are required for this interaction. This question is further complicated by the fact that the zona is heterogeneous in the localization of carbohydrate groups, so that some sugars may not be available to sperm on the outer edge of the zona where sperm first bind.

Identification of the sperm membrane receptor for the zona also remains elusive, even though a wide variety of sperm surface components have been implicated. One sperm surface protein, GalT, was found to satisfy many of the criteria expected of a ZP3 receptor. Furthermore, it was shown, using a heterologous expression system, that GalT functions as a signal-transducing receptor for ZP3. Additional studies of GalT function have examined what happens to the fertilization ability of sperm when sperm surface expression of GalT is either elevated or eliminated. Elevating GalT expression on the sperm surface in transgenic mice results in increased binding of ZP3 and increased sensitivity to zona-induced acrosome reactions. In contrast, eliminating GalT from the sperm surface through homologous recombination leads to reduced ZP3 binding and to sperm that are refractory to zona-induced acrosome reactions. Although GalT-null sperm are unable to bind ZP3 or to undergo a ZP3-dependent acrosome reaction, GalT-null males are still fertile. Surprisingly, GalT-null sperm can bind to the coat of ovulated oocytes, although they do not bind ZP3. This implies that GalT binds ZP3 oligosaccharides in the context of other sperm surface components that are responsible for the initial docking of sperm to ZP3-independent ligands in the egg coat. Identification of these additional ligands and sperm surface components awaits further investigation.

The final adhesion step in fertilization is the binding of sperm to the egg plasma membrane. In vitro assays include binding of supernumerary sperm that may be bound by a mechanism distinct from that of the fusing sperm, and such assays may complicate study of the adhesion required for sperm-egg fusion. Fusion of sperm and eggs is inhibited by the elimination of either GPI-anchored proteins or CD9 from the egg surface. Currently, no clear evidence suggests the participation of egg integrins in either sperm-egg binding or fusion. The ADAM family proteins on the sperm surface may participate in the adhesion of sperm and eggs that precedes fusion, but if so, the sperm ADAMs must be redundant with other proteins (and perhaps even with other sperm ADAMs). Sperm that are null for both fertilin and cyritestin (the two ADAM proteins most studied in conjunction with sperm-egg adhesion) are still capable of fusion with eggs.

Adhesive properties of gametes are key to their changing interactions with various surfaces contacted on the route to fertilization. Understanding the molecular basis of these adhesive events is an active area of research, but many questions remain unanswered. It is apparent, however, that new tools, including the construction of mice with null mutations for specific proteins, are proving useful in studying the cell biology of different steps in gamete adhesion.


   FOOTNOTES
 
1 Supported by grants from the Tobacco Related Disease Research Program of California (P.T.) and from the National Institutes of Health HD 23479 (B.D.S.) and HD 16580, U54 HD 29125 (D.G.M.). This minireview is derived from talks delivered as part of the BioPore Minisymposium: Sperm-Egg Interactions Leading to Fertilization at the 33rd Annual Meeting of the Society for the Study of Reproduction, July 15–18, 2000, at the University of Wisconsin, Madison, WI. The symposium was organized by D.G.M. and included the following presentations: "The Role of the Cumulus Matrix in Oocyte Pick-up" by P.T., "Molecular Aspects of Gamete Recognition in the Mouse" by B.D.S., and "Sperm-Egg Plasma Membrane Binding and Fusion" by D.G.M. Because this article reflects the contents of the symposium talks, it is not meant to be a comprehensive review of the topics discussed but is primarily focused on work from the laboratories of the respective investigators. Back

2 Correspondence: Diana G. Myles, University of California, Section of Molecular and Cell Biology, One Shields Avenue, Davis, CA 95616. FAX: 530 752 3085; dgmyles{at}ucdavis.edu Back

Received: 5 June 2002.

First decision: 21 June 2002.

Accepted: 5 August 2002.


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