My laboratory is interested in the molecular basis of cellular interactions during fertilization and development. We rely upon a multidisciplinary approach using diverse model systems, ranging from tissue culture cells to mice and zebrafish, to identify novel cell surface receptors that mediate a wide variety of cell-cell and cell-matrix interactions.

Whereas much is known regarding the receptors that bind protein ligands, little is known about the cell surface proteins that recognize complex carbohydrate ligands. The glycosyltransferases are the intracellular enzymes that synthesize complex carbohydrates, and it is now clear that some glycosyltransferases are also expressed on the cell surface. In this location, glycosyltransferases can function as signal transducing receptors by binding their glycoside substrates on adjacent cell surfaces or in the matrix. Most efforts have focused on the role of ß1,4-galactosyltrnasferase (GalT), in particular.

Mammalian Fertilization

One of the best studied, and most important illustrations of how cells recognize and communicate with one another in a highly regulated fashion is during mammalian fertilization. Previous studies have shown that the sperm surface protein, GalT, binds to a specific oligosaccharide structure within the egg coat glycoprotein, ZP3. Multiple ZP3 oligosaccharide chains leads to GalT aggregation, dephosphorylation of its cytoplasmic domain, and activation of a heterotrimeric G-protein cascade that induces the acrosome reaction (Miller, D.J., Macek, M.B., and Shur, B.D. 1992. Nature 357: 589-593; Gong, X., Dubois, D. H., Miller, D.J. and Shur, B.D. 1995. Science 269: 1718-1721). After fertilization, the egg releases a specific glycosidase that removes the GalT-binding oligosaccharide from the egg coat, thus preventing polyspermy (Nixon, B., Lu, Q., Wassler, M.J., Foote, C.I., Ensslin, M.A., and Shur, B.D. 2001. Cells Tissues Organs. 168: 46-57).

Eliminating GalT by homologous recombination leads to sperm that are unable to bind ZP3 nor are they able to undergo the acrosome reaction or penetrate the egg coat. Unexpectedly, however, GalT-null sperm are still able to bind the egg coat (Lu, Q. and Shur, B.D. 1997. Development 124: 4121-4131). This indicates the existence of two independent, and necessary, receptor-ligand complexes that are responsible for sperm-egg binding. The first mediates initial sperm-egg adhesion, and involves a novel sperm protein that binds to an oviduct-secreted glycoprotein associated with the egg coat surface. This initial gamete binding brings the sperm and egg coat sufficiently close to engage the second receptor-ligand interaction involving sperm GalT binding to ZP3, which subsequently induces of the acrosome reaction.

The realization that sperm-egg binding requires two distinct receptor-ligand interactions represents a major advancement in our understanding of mammalian fertilization. The identification of these novel receptors is underway in my laboratory.

SED1: SED1 is homologous to a small group of Secreted cell-matrix adhesive proteins that contain Notch-like EGF repeats and discoidin/F5/8 type C domains. SED1 is expressed in the Golgi of spermatogenic cells and is secreted by the initial segment of the caput epididymis, resulting in SED1 localization on the sperm plasma membrane overlying the acrosome. SED1 binds specifically to the zona pellucida of unfertilized oocytes, but not to the zona of fertilized eggs. Recombinant SED1 and anti-SED1 antibodies competitively inhibit sperm-egg binding, as do truncated SED1 proteins containing a discoidin/C domain. SED1-null males are subfertile and their sperm are unable to bind to the egg coat in vitro. These studies illustrate that Notch-like EGF and discoidin/C domains, protein motifs that facilitate a wide range of cellular interactions, participate in gamete recognition as well (Ensslin, M.A. and Shur, B.D. 2003. Cell 114:405-417). SED1 is also expressed by a number of epithelia, including the mammary ductal tree, where it plays critical roles during epithelial morphogenesis.

Oviduct-derived ligand: We recently identified a novel ZP3- and GalT I-independent mechanism for sperm adhesion to the egg coat (Rodeheffer, C. and Shur, B.D. 2004. Development 131:503-512). Results show that the ovulated zona pellucida contains at least two distinct ligands for sperm binding: a ZP3-independent ligand that is peripherally associated with the egg coat and which facilitates gamete adhesion, and a ZP3-dependent ligand that is present in the insoluble zona matrix and which is recognized by sperm GalT I to facilitate acrosomal exocytosis. The ZP3-independent ligand is not a result of contamination by egg cortical granules, nor is it the mouse homolog of Oviduct-specific Glycoprotein. It behaves as a 250 kDa, WGA-reactive glycoprotein with a basic isoelectric point, distinguishing it from the acidic glycoproteins that form the insoluble matrix of the egg coat. When eluted from isoelectric focusing gels, the acidic matrix glycoproteins possess sperm-binding activity for wild-type sperm, but not for GalT I-null sperm; whereas, the basic glycoprotein retains sperm-binding activity for both wild-type and GalT I-null sperm. Thus, GalT I-null sperm are able to resolve gamete recognition into at least two distinct binding events, leading to the characterization of a novel, peripherally-associated, sperm-binding ligand on the ovulated zona pellucida. The identity of this novel ligand is being identified by microsequencing.

 

Glycosyltransferase Function During Embryonic Development

It has also been know for many years that early embryonic development is dependent upon the expression and function of complex carbohydrates; however, their mode of action remains obscure. In some instances, a precise role for specific cell surface glycosyltransferase receptors and extracellular glycoside ligands can be defined. For example, in vitro studies have shown that GalT is expressed on the leading edge of migrating cells, where it associates with the cytoskeleton and facilitates lamellipodial formation on basal lamina (Wassler, M.J. and Shur, B.D. 2000. J. Cell Sci. 113: 237-245). Furthermore, manipulating GalT expression leads to developmental abnormalities, including defective mammary gland development when GalT is over-expressed (Hathaway, H.J. and Shur, B.D. 1996. Development 122: 2859-2872), and neonatal lethality when GalT is deleted by targeted mutagenesis (Lu, Q., Hasty, P. and Shur, B.D. 1997. Dev. Biol. 181: 257-267). However, the complexity of the glycosyltransferase superfamily makes their analysis extremely difficult using the mouse model system.

Consequently, we have adapted the zebrafish system to analyze the expression and function of each glycosyltransferase gene product during early vertebrate development. Zebrafish are ideal for this analysis since the genome sequence is complete enabling the rapid identification of fish glycosyltransferase families. Their expression patterns are readily determined by whole mount in situ hybridization, and their developmental function can be analyzed through the use of morpholino-oligonucleotides that eliminate translation of a functional glycosyltransferase protein. In this way, we are compiling the first catalogue describing the expression and function of specific glycosyltransferase families during early vertebrate development.