Molecular basis of cellular interactions during fertilization and development.
My laboratory is interested in the molecular basis of cellular interactions during fertilization and development. It is well accepted that cellular interactions are governed by specific cell surface receptors that bind their complementary protein or carbohydrate ligands on adjacent cells or in the ECM. 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.
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. After fertilization, the egg releases a specific glycosidase that removes the GalT-binding oligosaccharide from the egg coat, thus preventing polyspermy.
However, eliminating GalT by homologous recombination leads to sperm that are still able to bind the egg coat, although they do not bind ZP3 nor are they able to undergo the acrosome reaction or penetrate the egg coat. 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. One excellent candidate for the sperm surface protein is p47. We have recently produced p47-null mice and are examining the resultant phenotype, focusing on fertility. The complementary ligand on the egg coat is being identified by microscale 2D gel analysis and microsequencing.
It has also been know for many years that early embryonic development is dependent upon the expression and function of complex carbohydrate ligands, 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. However, the complexity of the glycosyltransferase superfamily makes their analysis in viv
