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CAVEAT LECTOR
Section of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190
5. MOLECULES IMPLICATED IN SPERM-OOCYTE INTERACTION
Because fertilization essentially involves cell-to-cell fusion of a sperm with the oocyte, it is believed that a number of integrin molecules may be involved in this process. Integrins mediating adhesion of cells are present in an active or inactive conformation, and in addition to adhesive events, they transfer signals to the cell inducing changes in gene expression (21-23, 41). Mammalian sperm membrane-oolemma fusion results in tyrosine phosphorylation of egg proteins, increased production of phosphoinositol lipids, and substantial cytoskeletal reorganization thereby suggesting receptor-mediated signal transduction across the egg plasma membrane (42-43).
A number of recent studies also suggest a causal relationship between the complement (C) system and reproduction, particularly the expression of cell membrane-associated C regulatory proteins, C3b receptors and C inhibitors (34, 44-45). C3 fragments and C3 receptors are involved in cell-cell adhesion reactions in both natural and inducible immunity and in membrane apposition before fusion and entry of eukaryotic cells by bacteria, virus, protozoa, and yeast cells (46). Not surprisingly, a role for C3 fragments and C3 receptors in sperm-oocyte apposition and fusion has been invoked (44).
At least three cell surface C-binding regulatory proteins are expressed on human sperm: Membrane cofactor protein (MCP or CD46), decay accelerating factor (DAF or CD55), and membrane inhibitor of reactive lysis (MIRL or CD59) have been demonstrated by biochemical and immunolocalization studies (47-53). Both CD55 and CD59 are also expressed on the plasma membrane, whereas, CD46 is only expressed as an unusual alternatively spliced product on the inner acrosomal membrane (49-50). In addition to their probable role in local C binding and inactivation, these proteins have also been implicated in human reproductive events (34, 45). In somatic cells, CD46, CD55, and CD59 in addition to their primary role in protecting cells from C-mediated damage, also participate in cell adhesion and both CD55 and CD59 contribute to cell signaling (54-56). In vitro gamete interaction studies using a panel of mAbs has led to the hypothesis that these C proteins may also participate in sperm-oocyte interaction or in signaling induction and protect sperm from C-mediated damage in the reproductive tract.
5.1. Adhesion molecules on the sperm
Members of a recently discovered gene family called ADAMs have been strongly implicated in integrin-mediated sperm-oocyte binding and fusion in the guinea-pig and mouse model systems (8-9, 13). Fertilins possess both a potential adhesion domain and a potential protease domain (57). The PH-30, a transmembrane protein, isolated from the guinea-pig sperm plasma membrane by Primakoff and colleagues appears to have sperm-oolemma fusion, and metalloprotease properties (58) and may be considered as a potential oocyte receptor ligand. The complex is expressed on the posterior surface of the mature sperm head, at the appropriate location for molecules involved in oocyte fusion (59). mAbs to PH-30 disrupts the fusion of acrosome-reacted guinea-pig sperm with the vitelline membrane of the oocyte (59). Antibody to PH-30 precipitates two tightly coupled immunologically distinct subunits ( and ) that behave as a single integral membrane protein (8). Both and subunits are generated as large precursor proteins that seem to be modified by proteolytic cleavage during testicular differentiation and subsequent epididymal maturation of the sperm (60).
The mature PH-30 alpha/ß complex, being involved in sperm-oocyte fusion, resembles certain viral fusion proteins in membrane topology and predicted binding and fusion functions (8). Cloning of PH-30 and sequence analysis suggests that it may be responsible for both recognition and fusion with the guinea-pig oolemma. The alpha subunit is composed of a peptide core of 289 amino acids, with a single membrane-spanning domain towards the C terminus, a large extracellular domain and a short cytoplasmic tail. The putative fusion peptide has been identified on the alpha subunit, and it appears to be somewhat similar in sequence to a potential fusion peptide on the rubella virus (61). A feature of the fusion protein motif on PH-30 alpha is its relative hydrophobicity which, in conjunction with the transmembrane anchoring segment on the same subunit, is thought to permit the molecule to interact simultaneously with the plasma membranes of both sperm and the oocyte (62).
The sequence data on the ß subunit indicate that it contains a 353-amino acid peptide core, with two potential sites for N-glycosylation and a single membrane spanning domain. The ß subunit contains a putative oocyte recognition domain characteristic of a family of small, soluble (about 50 amino acids) integrin-binding peptide ligands called disintegrins (57-58). Disintegrins, which are present in many snake venoms, contain RGD, or related sequences, in the context of extended loop structure (63). They recognize sites on integrins that normally interact with the RGD-based cell recognition sequences in many matrix-associated glycoproteins (e.g., fibronectin, fibrinogen). These soluble disintegrins block platelet aggregation and promote uncontrolled bleeding by competitively inhibiting the function of the platelet integrin GPIIb/IIIa (alphaIIbß3). In the case of the sperm, the disintegrin domain is immobilized as part of a larger transmembrane glycoprotein and therefore it has the potential to act as a cell surface-bound ligand (counter receptor) to promote a cell-cell adhesion event (64).
The fusogenic properties of sperm protein have been investigated using liposomes. A synthetic peptide representing the putative fusion domain of PH-30 binds to vesicles composed of both neutral and acidic lipids (64). In the intervesicular lipid mixing assay, the synthetic peptide undergoes conformational transition to a ß-structure and induces fusion of large unilamellar vesicles supporting the hypothesis that PH-30 mediates sperm fusion with oocyte (64). The role of the sperm surface protein PH-30 in sperm-oocyte fusion has been further tested by using peptide analogues of a potential integrin binding site in the fertilin ß subunit. Peptide analogues that induce a QDE sequence (Thr-Asp-Glu) from the disintegrin region of fertilin ß have been shown to bind to the oolemma and strongly inhibit sperm-oocyte fusion (58). Even though, the disintegrin motif of PH-30 ß does not contain an RGD sequence, there are ample examples of integrin ligands in which this particular tripeptide is absent.
A mouse analogue of PH-30, has recently been identified on mouse sperm (65). Mouse sperm fertilin has a QDE tripeptide (instead of RGD) in its cell recognition region and synthetic peptides containing QDE inhibit sperm-oocyte fusion in the mouse bioassay.
A human fertilin ß has recently been identified using cDNA cloning, deduced amino acid sequence analysis, Northern blot analysis, and chromosomal localization (66). Human fertilin ß is encoded by a 2205 nucleotide (735 amino acids) open reading frame; its deduced amino acid sequence contains pro-metalloproteinase-like, disintegrin-like, and cysteine-rich domains, which are structurally homologous to P-III snake venom metalloproteinases. Human fertilin ß shares a 90% amino acid identity to monkey, 56% to guinea pig, and 55% to mouse fertilins. A FEE binding tripeptide is located within the disintegrin loop of human fertilin ß and could be the site competing for recognition by integrin and other receptors on the oolemma. Northern blot analysis of poly (A)+ RNA from 16 human tissues revealed human fertilin ß's 2.9 kb message only in the testis (66).
Presence of disintegrin domain on guinea pig, mouse, and human sperm suggests that an oocyte integrin might serve as a receptor for sperm. Integrins, through their property to recognize the RGD tripeptide sequence, also mediate cell-cell and cell-extracellular matrix recognition and adhesion in reproductive tissues. The recent discovery of ß1-integrins on human sperm (67), human endometrium (68), and human trophoblast (69) has expanded the role of these molecules in the biology of reproduction. Indirect evidence suggest that ß1 integrins are involved in sperm membrane-oolemma recognition and fusion as well. The most relevent receptors are the family of integrins which bind to fibronectin, vitronectin, and laminin. ß1-integrins are represented by the very-late-activation antigen (VLA) molecules. Each VLA molecule contains a common ß1 subunit in association with one of at least nine different alpha subunits (70). Glandner and Schaller described the binding patterns of ß1-integrins (alpha3, alpha4, alpha5, alpha6) and their matrix proteins, fibronectin and laminin, on human sperm by flow cytometry (67, 71). They compared the expression of ß1 integrin with the fertilizing ability of sperm from men of couples who underwent IVF for either unexplained, tubal or male factor infertility. When compared with normal sperm, sperm from patients with teratozoospermia showed a significant decrease in expression of these adhesion molecules. Compared with semen samples from men with unexplained or male factor infertility patients, samples from patients with tubal infertility had significantly higher percentage of sperm expressing ß1 integrins. Also, higher IVF success rate and pregnancy rates were obtained when compared with couples suffering from male or unexplained infertility (71). Immunolocalization techniques using a panel of mAbs against the ß1-integrin cell adhesion molecules indicated that the alpha chains 4, 5, and 6 are expressed by a small percentage of sperm from fertile individuals (67, 71). After the loss of the acrosome a significantly higher expression of alpha4, alpha5, and alpha6 chains of integrins was detected in the fertile semen group (72). These findings suggest that certain ß1 integrin cell adhesion molecules, through their ability to recognize the RGD sequence, may be involved in the early stages of sperm-oocyte recognition and interaction.
The 3 integrin complexes detected on human sperm are known to be expressed on several mesenchymal and epithelial cells types where they function as receptors for extracellular matrix proteins such as fibronectin, laminin, and collagens (73). Two ß1 integrins (alpha4/ß1, alpha5/ß1) that bind fibronectin utilize the variable region (CS-1) and RGD binding domains, respectively (73-74). alpha3/ß1 is a receptor for several extracellular matrix proteins including fibronectin and laminin (74). Its binding to fibronectin involves the RGD site while its binding to laminin is RGD-independent. alpha6/ß1 is a receptor for laminin and it does not recognize the RGD sequence (74). In addition to being extracellular matrix receptors, alpha5/ß1 and alpha3/ß1 can be involved in dynamic cell to cell adhesion. In somatic cells, these integrins are localized in areas of cell to cell contact (75). The integrins expressed on sperm might therefore function in cell-cell and/or cell-matrix interaction during sperm-oocyte fusion.
The extracellular matrix proteins, fibronectin and vitronectin have been identified on the plasma membrane of capacitated sperm (76-77). It has been demonstrated that co-incubation of human sperm and zona-free hamster oocytes in the presence of micromolar concentration of RGD-containing oligopeptides results in a significant decrease in the number of oolemma-adherent sperm as well as complete inhibition of fertilization (24, 78). The effect of these peptides is greatly reduced by changing the D residue into an E, a mutation known to abolish recognition by integrins. Beads coated with RGD-containing peptides bind to the oocyte, but not to the sperm surface indicating that the oocyte express RGD-receptors (24). These findings suggest a role for ß1 integrins in fertilization. In contrast, ß2 integrins, the primary mediators of adhesive immune interaction of sperm with phagocytes are not present on the surface of sperm (79). Fibronectin has also been localized in the region where the sperm fuses first with the egg plasma membrane during fertilization (11). Sperm adhesion to the egg can be inhibited by antifibronectin antibodies. The wide variation in the fibronectin concentration in semen obtained from different donors also suggest that defects in fibronectin expression might play a role in sperm dysfunction and infertility (76-77). Fibronectin is localized on the entire sperm surface and on the equatorial segment, laminin is localized solely on the sperm tail and vitronectin is detectable primarily on the equatorial band on the sperm head (76-77, 80). Bronson and associates suggest that vitronectin and fibronectin expressed on the surface of capacitated sperm could act as a ligand for specific receptors on the oocyte, and might play a role in sperm-oolemmal adhesion (77).
ß1 integrins are known to function in a dual capacity as adhesion and signaling molecules (82). Vitronectin is recognized as an adhesive substrate by cells expressing at least one of four known vitronectin receptors: integrins alphav1, alphav3, alphav5, or alphaIIbß3 (82). Thus, antibodies reacting with ß1 integrins can lead to defective sperm-oocyte interaction. This deficiency may occur either by direct interference with the function of the integrins or by integrin cross-linking, leading to alterations in membrane functions. Therefore, integrins may find utility as prognostic markers for clinical outcome or as therapeutic targets in infertility.
The receptors for C3, the primary mediators of phagocyte-microbe interaction (46), have also been shown to facilitate sperm-oocyte interaction during the hamster penetration test (44). Anderson and associates showed that some mAbs specific for C receptor type 1 (CD35) and type 3 (CR3) bind to the human oolemma, indicating that specific C-binding molecules may play a role in the attachment of C3 catabolites to oocytes (44). In their bioassay, subsaturating concentrations of dimeric C3b (<1 µM) promoted penetration of hamster oocytes by human sperm, whereas saturating doses (>10 µM) inhibited this process. In addition, antibodies to both CD46 and C3 significantly inhibited penetration of hamster oocytes by human sperm (44). These data suggest that regulated gamete-induced generation of C3 fragments and the binding of these fragments by selectively expressed receptors on sperm and oocytes may be an initial step in sperm-oocyte interaction, and subsequent membrane fusion and fertilization. The dimeric C3b at low levels is thought to serve as a bridge between sperm (CD46) and oocyte (CR1, CR3) C receptors, facilitating fertilization. At high levels, however, dimeric C3b could saturate all receptor binding sites for C3 fragment and inhibited the apposition of gamete membranes. CR3, a ß2-integrin, which is expressed on oocytes may bind the human sperm homologue of the PH-30 integrin and facilitate membrane fusion by the PH-30 chain homologue (44).
5.1.4. Complement inhibitors (CD35, CD46, CD55, and CD59)
Acrosome intact and acrosome-reacted human sperm express C inhibitors, CD55 and CD59, whereas, CD46 is expressed only on the sperm head region of acrosome-reacted sperm (45, 49-53). The CD46 and CD59 on sperm have C-inhibitory activity in vitro (50-51). However, the low potency of these proteins in protecting antisperm antibody-induced C-damage in vitro suggest that they have functions other than binding and regulation of C activity (49). In fact, mAbs directed against CD46 and CD59 variably inhibit the penetration of hamster oocytes by human sperm, suggesting that CD46 and perhaps C3 play a role in sperm-oocyte interaction (34, 44-45).
CD46 serves as a C3b/C4b inactivating factor for the protection of host cells from autologous C attack (83). Naniche et al.. have recently shown that CD46 acts as a human cellular receptor for measles virus, allowing cell binding, fusion, and viral replication (84). Okada et al. showed that CD46 is a keratinocyte receptor for the M protein of the group A streptococcus (85). On human sperm, CD46 is located on the inner acrosomal membrane (34). Johnson and associates showed that human sperm binding and pronuclear formation in zona-free human oocytes can be significantly inhibited by preincubation of both sperm and oocytes with anti-CD46 mAb (86). This effect was not observed when either of these gametes alone was incubated with mAb. CD46 is also expressed by zona-free human oocytes (86). The expression of CD46 by zona-free oocytes and acrosome-reacted sperm suggest a role for CD46 at the level of apposition of the inner acrsomal membrane and the oolemma.
CD46 consists of 4 short consensus repeat (SCR) regions which are the predominant extracellular structural motifs each of which is a cysteine-rich domain of approximately 60 amino acids. The cofactor activity of CD46 for C inhibition resides in SCR-3/4 while the site for human sperm-oocyte interaction resides on SCR-1, since only mAbs directed to epitopes in SCR-1 inhibit in vitro gamete interaction (86). Also, the sites for measles virus binding and for C3b/C4b inactivation appear to reside on different SCR. Using Chinese hamster ovary cell clones expressing each SCR deletion mutants, Iwata and associates confirmed which of the 4 SCR of CD46 contribute to its function (87). The functional domains of CD46 for the natural ligands C3b, C4b and measles virus were mapped to different SCR domains. They found that SCR1 and SCR2 were mainly involved in measles virus binding and infection and SCR2, SCR3, and SCR4 contribute to protection from C damage (88). Motifs for N-glycosylation on CD46 are present on SCR1, 2, and 4 (88). These N-linked glycans are essential for the recognition of CD46 by measles virus (89), whereas, the presence of nonglycosylated CD46 on acrosome-reacted sperm suggest a protein-protein type gametic interaction.
CD55 blocks the C cascade by accelerating the decay of C3/C5 convertase complexes whereas CD59 interferes with the assembly of the membrane attack complex of C (90-91). Both CD55 and CD59 are glycosyl phosophatidylinositol (GPI)-anchored glycoproteins. Perturbation of GPI-anchored CD55 and CD59 molecules by cross-linking with antibodies causes activation of T cells and neutrophils (54-56). The mediators of signal transduction are likely to be the tyrosine kinases that are tightly associated with GPI-anchored molecules on GPI-rich membrane clusters (55). CD59 acts as a signal-transducing molecule (56). CD59 associates with kinases in membrane clusters and gains Ca signaling capacity (55). Nonlethal C attack induces a similar series of events, including increase in intracellular-free Ca concentration and production of reactive oxygen radicals (92). During sperm-oocyte fusion, there is a rapid membrane depolarization followed by release of internal Ca (42-43). CD59 expressed by acrosome-reacted sperm and oolemma may be involved in these events before sperm-oocyte fusion. CD55 may be involved in acrosome reaction. For example, mAbs to CD55 antigen inhibit acrosome reaction induced by the oocyte-cumulus complex but not by calcium ionophore. Thus, during the physiological sperm acrosome reaction both CD55 and CD59 complement-binding proteins may be part of the signaling machinary. In addition, the binding of sperm to oocyte and activation of ovum by hydrolysis of phosphotidylinositol 4-5-biphosphate may involve a multimolecular complex that includes CD55 and CD59. Inositol 1, 4, 5-triphosphate (IP3)-gated Ca channels and upstream components (including Galphaq/11, PLCß1 and IP3 gated Ca store) of the phosphoinositide signaling system have been identified in the acrosomal region of sperm (93). In fact, the level of IP3 binding in sperm is among the highest observed in mammalian tissues (93).
There is also a report that C1q, a component of the classical C pathway accelerates the human sperm-hamster oocyte adhesion but inhibits fusion (94). C1q is one of the high molecular weight proteins that bind with fibronectin (95). C1q is a component of classical C pathway that can react with Fc fragment of immunoglobulins and with other proteins, such as fibronectin, and laminin. Presence of C1q in the coincubation media resulted in a significant increase in sperm adhesion to oolemma but inhibited penetration of ovum (94). C1q receptor was immunolocalized both on hamster oolemma and human sperm (94). mAb to C1q variably inhibited penetration of ovum.
Immunogold localization of ultrathin sections of human sperm before and after incorporation into hamster oocyte revealed redistribution of calmodulin on the sperm head (96). Following entry of sperm head into egg cytoplasm, post acrosomal calmodulin disappeared whereas the subacrosomal calmodulin was unaltered (96). These data suggest a role for calmodulin in sperm-oocyte fusion and in the initial pulse of intracellular Ca occurring during fertilization.
5.2. Adhesion molecules on the oolemma
Successful fertilization requires communication between the fertilizing sperm and the oocyte. In somatic cells, expression of major histocompatibility complex (MHC) molecules participates in cell-cell interaction (97). Similarly, the expression of IgG Fc receptors provides a class of surface molecules which promote cell-cell interactions (98). Human oocytes lack class I MHC antigen (99). However, all three classes of Fc(gamma) receptors have been demonstrated on unfertilized human egg oolemma but not on sperm (25, 100). Oolemmal Fc receptors promoted the adhesion of antisperm antibody labeled human sperm to zona-free hamster oocytes (100).
The finding that a disintegrin domain-containing molecule may be the sperm ligand for the oolemma implies that the oocyte receptor is an integrin. In fact, integrin subunits alpha2, alpha4, alpha5, alphaL, ß1, ß2, and ß7 have been immunolocalized on human oocytes (25, 99). Unfertilized mouse oocytes also express ß1 integrins both at mRNA and protein levels (101). In these cells, mRNAs for the ß1, alpha5 and alpha6 and corresponding proteins, alpha5ß1 and the alpha6ß1 complexes are present (101). alpha3ß1 was expressed on the oocyte surface (101). Integrin subunits alpha6 and ß1 were differentially distributed on the oocyte surface. alpha6 antigen was mainly confined to the microvilli while ß1 was homogeneously distributed over the whole oolemma (101). Recent reports suggest that mouse oocyte alpha6/ß1 functions as a sperm receptor (9). In addition, studies using human sperm-hamster zona-free oocyte penetration assay suggest that echistatin (a disintegrin known to block the binding of fibronectin and vitronectin to their respective integrin ligands, alpha5/ß1 and alphav/ß1), interacts with gametic fusion events (102). The binding of sperm to oolemma was echistatin-sensitive whereas, the gamete membrane fusion was resistant to echistatin (102). Whether integrins can actually function as human sperm receptors in the human fertilization process, however, has not yet been unequivocally demonstrated.
C inhibitors and signal transducing molecules, CD55, and CD59 are also expressed on human oolemma (103-104). The human C3b/C4b receptor CR1 is not expressed on sperm or oolemma, whereas, CR3, a ß2 integrin is expressed on oolemma (44). It is thought that CR3 binds the human sperm homologue of the PH-30 integrin and facilitates membrane fusion. Anderson and associates suggest that C3 fragments (C3b/iC3b) may serve as bridging ligands between sperm CD46 and oocyte CR3 and facilitate apposition of the sperm inner acrosomal membrane with the oolemma (44).
The various adhesion molecules that have been implicated in human sperm plasma membrane-oolemma adhesion to date are listed in table 1.