Diana G. Myles

The ADAM gene family: surface proteins with adhesion and protease activity

An ADAM is a transmembrane protein that contains a disintegrin and metalloprotease domain and, therefore, it potentially has both cell adhesion and protease activities. Currently, the ADAM gene family has 29 members, although the function of most ADAM gene products is unknown. We discuss the ADAM gene products with known functions that act in a highly diverse set of biological processes, including fertilization, neurogenesis, myogenesis, embryonic TGF-alpha release and the inflammatory response.

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

Abstract 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.

A map of the guinea pig sperm surface constructed with monoclonal antibodies

The surface of mammalian sperm is known to exhibit regional heterogeneity. Using monoclonal antibodies, we have analyzed the nature of this regional variation at the molecular level. A map of the surface of guinea pig sperm has been constructed that defines a number of regions in which surface antigens are localized and analyzes the diversity of antigens found in each region. In an initial screening of 117 hybridoma antibodies to a sperm membrane preparation, a remarkable result was obtained: all the antibodies bound to a localized region of the cell. From the initial hybrid lines, we established a collection of 56 stable hybridomas producing antibodies to surface antigens. These antibodies detect antigens localized in five surface regions: anterior head (AH), posterior head (PH), whole head (WH), posterior tail (PT), and whole tail (WT). At least 12 distinct surface antigens are recognized that bind antibodies in one of the localized regions (five AH antigens, three PH, two WH, one PT, and one WT). Some of the recognized antigens have been identified as proteins, comprised of either one or several 125I-labeled polypeptides. The identified AH antigens have labeled polypeptides of molecular weights (Mr) 52,000 (52K); 70K, 62K, 46K, 25K, and 18K; 62K, 52K, and 38K; 16K; and 38K. Identified PH antigens have polypeptides of Mr 60K; 66K, 48K, and 41K; and 58K and 48K. Identified WH antigens have polypeptides of Mr 89K and 45K; and 42K. We conclude that the sperm cell can maintain contiguous membrane domains which have quite different compositions. Its surface is a mosaic consisting of multiple regions and each region can contain several localized antigens.

None of the integrins known to be present on the mouse egg or to be ADAM receptors are essential for sperm-egg binding and fusion.

Antibody inhibition and alpha6beta1 ligand binding experiments indicate that the egg integrin alpha6beta1 functions as a receptor for sperm during gamete fusion; yet, eggs null for the alpha6 integrin exhibit normal fertilization. Alternative integrins may be involved in sperm-egg binding and fusion and could compensate for the absence of alpha6beta1. Various beta1 integrins and alphav integrins are present on mouse eggs. Some of these integrins are also reported to be receptors for ADAMs, which are expressed on sperm. Using alpha3 integrin null eggs, we found that the alpha3beta1 integrin was not essential for sperm-egg binding and fusion. Oocyte-specific, beta1 integrin conditional knockout mice allowed us to obtain mature eggs lacking all beta1 integrins. We found that the beta1 integrin null eggs were fully functional in fertilization both in vivo and in vitro. Furthermore, neither anti-mouse beta3 integrin function-blocking monoclonal antibody (mAb) nor alphav integrin function-blocking mAb inhibited sperm binding to or fusion with beta1 integrin null eggs. Thus, function of beta3 or alphav integrins does not seem to be involved in compensating for the absence of beta1 integrins. These results indicate that none of the integrins known to be present on mouse eggs or to be ADAM receptors are essential for sperm-egg binding/fusion, and thus, egg integrins may not play the role in gamete fusion previously attributed to them.

Infertility in female mice with an oocyte-specific knockout of GPI-anchored proteins

Glycosylphosphatidylinositol-anchored proteins on the egg surface have been proposed to play a role in gamete fusion on the basis of in vitro experiments. We tested this hypothesis by asking if oocyte GPI-anchored proteins are required for fertilization in vivo. Oocyte-specific knockout mice were created using the Cre/loxP system to delete a portion of the Pig-a gene, which encodes an enzyme involved in GPI anchor biosynthesis. Conditional Pig-a-knockout females are infertile, and eggs recovered from the females after mating are unfertilized. In in vitro assays, the knockout eggs are severely deficient in their ability to fuse with sperm. These results demonstrate that GPI-anchored proteins are required for gamete fusion. Loss of the GPI-anchored complement of plasma membrane proteins could prevent fusion by altering the organization and function of GPI-anchored protein-containing lipid domains. Alternatively, a single GPI-anchored protein may be required in the fusion process. To distinguish between these possibilities, we have begun to identify the GPI-anchored proteins on the egg surface. We have identified one egg GPI-anchored protein as CD55, an ∼70 kDa complement regulatory protein. It has previously been found that CD55-knockout mice are fertile, demonstrating that CD55 is not essential for fertilization. This finding also means that the presence of the full complement of egg GPI-anchored proteins is not necessary for gamete fusion. Other egg GPI-anchored proteins acting in the fusion process can now be investigated, with the goal of understanding the mechanism of their function in sperm-egg fusion.

Binding of both acrosome-intact and acrosome-reacted guinea pig sperm to the zona pellucida during in vitro fertilization

Mammalian sperm-egg adhesion occurs when sperm bind to the zona pellucida of the egg. In this study with guinea pig gametes, we have asked if sperm can initiate binding to the zona before and after the acrosome reaction and if the sperm surface protein PH-20 is involved in the binding at these two stages. Sperm binding to the zona was examined under a variety of conditions. Sperm were suspended in 0.9% NaCl or capacitated by two different methods. Eggs were immobilized on lectin-coated coverslips, compressed between a coverslip and a glass slide, or free in tissue culture dishes. The sperm-egg interaction was recorded on videotape or assessed after fixation of the eggs with bound sperm. Under all conditions studied, both acrosome-intact and acrosome-reacted sperm initiated binding to the zona. The binding was persistent and not transitory. In particular, acrosome-intact sperm that bound the zona were observed to remain bound for up to 80 min. One acrosome-intact sperm, bound to the zona, was videotaped while it acrosome-reacted. When mixed sperm populations (on the average 24% acrosome-intact and 76% acrosome-reacted) were incubated with eggs for 30 min, an average of 10% of the bound sperm were acrosome-intact. The PH-20 monoclonal antibody has previously been shown to inhibit zona binding by guinea pig sperm of undetermined acrosomal status (P. Primakoff, H. Hyatt, and D. G. Myles (1985), J. Cell Biol. 101, 2239-2244). In this study, when the two populations of sperm were counted separately, PH-20 inhibited the binding of acrosome-reacted but not acrosome-intact sperm. Our results show that both acrosome-intact and acrosome-reacted guinea pig sperm can initiate binding to the zona; however, the binding in the two cases may not occur by the same mechanism.

Izumo is part of a multiprotein family whose members form large complexes on mammalian sperm

Izumo, a sperm membrane protein, is essential for gamete fusion in the mouse. It has an Immunoglobulin (Ig) domain and an N‐terminal domain for which neither the functions nor homologous sequences are known. In the present work we identified three novel proteins showing an N‐terminal domain with significant homology to the N‐terminal domain of Izumo. We named this region “Izumo domain,” and the novel proteins “Izumo 2,” “Izumo 3,” and “Izumo 4,” retaining “Izumo 1” for the first described member of the family. Izumo 1–3 are transmembrane proteins expressed specifically in the testis, and Izumo 4 is a soluble protein expressed in the testis and in other tissues. Electrophoresis under mildly denaturing conditions, followed by Western blot analysis, showed that Izumo 1, 3, and 4 formed protein complexes on sperm, Izumo 1 forming several larger complexes and Izumo 3 and 4 forming a single larger complex. Studies using different recombinant Izumo constructs suggested the Izumo domain possesses the ability to form dimers, whereas the transmembrane domain or the cytoplasmic domain or both of Izumo 1 are required for the formation of multimers of higher order. Co‐immunoprecipitation studies showed the presence of other sperm proteins associated with Izumo 1, suggesting Izumo 1 forms a multiprotein membrane complex. Our results raise the possibility that Izumo 1 might be involved in organizing or stabilizing a multiprotein complex essential for the function of the membrane fusion machinery. Mol. Reprod. Dev. 76: 1188–1199, 2009.

Sperm-egg fusion: events at the plasma membrane

Sperm-egg fusion is a cell-cell membrane fusion event essential for the propagation of sexually reproducing organisms. In gamete fusion, as in other fusion events, such as virus-cell and intracellular vesicle fusion, membrane fusion is a two-step process. Attachment of two membranes through cell-surface molecules is followed by the physical merger of the plasma membrane lipids. Recent progress has demonstrated an essential role for an oocyte tetraspanin, CD9, in mouse sperm-egg fusion, and a specific molecular site crucial for CD9 function has been identified. Absence of glycosylphosphatidylinositol-anchored proteins on the oocyte surface also results in loss of oocyte fusion competence in this gamete. These discoveries provide a strong starting point for the identification of additional proteins that have roles in sperm-egg fusion.

The guinea pig sperm plasma membrane protein, PH-20, reaches the surface via two transport pathways and becomes localized to a domain after an initial uniform distribution

The PH-20 protein is first detected in the Golgi complex at the start of differentiation of round spermatids into a polarized cell (spermiogenesis), and next appears in the membrane of the developing secretory granule (the acrosome). Thereafter, a second population of PH-20 is inserted directly into the plasma membrane. Initially, both the acrosomal membrane (PH-20AM) and the plasma membrane (PH-20PM) populations are uniformly distributed in each membrane. Subsequently, PH-20AM is restricted to the inner acrosomal membrane, and during epididymal passage PH-20PM becomes localized to the posterior head surface domain. Therefore, the PH-20 protein does not become localized to either domain by intracellular sorting and insertion into a localized domain, but by restriction following uniform insertion. When the sperm undergoes Ca2+-regulated exocytosis (the acrosome reaction), the inner acrosomal membrane becomes confluent with the plasma membrane. Consequently, the population of PH-20AM is now inserted into the plasma membrane. The PH-20 protein isolated from developing testicular cells contains a major form, approximately 66 kDa, and a minor form, approximately equal to 56 kDa, but it remains to be determined if each form enters only one or both pathways. The developmental control of surface expression of PH-20 during spermiogenesis in the guinea pig may reflect the regulation of a protein involved in sperm-egg adhesion. (Primakoff, P., Hyatt, H., and Myles, D. g. (1985), J. Cell. Biol. 101, 2239-2244).

Cell-cell membrane fusion during mammalian fertilization.

The mechanism of sperm–egg fusion in mammals is a research area that has greatly benefited from the use of gene deletion technology. Because fertilization is internal in mammals and the gametes (particularly the eggs) are sparse in number, in vitro studies have considerable limitations. Using gene deletions, a few cell surface proteins in both gametes have been identified as essential for gamete fusion. Ongoing studies are directed at analysis of the function of these proteins and the search for additional proteins that may be involved in this process. So far, no mammalian proteins have been found that also function in sperm–egg fusion of non‐mammalian species or in other types of cell–cell fusion.