In this project, Drs. Chandler and Bieber will focus on the role of the egg extracellular matrix (ECM) in controlling sperm and egg interactions during vertebrate fertilization. These interactions are essential for successful fertilization in animals ranging from invertebrates such as echinoderms to vertebrates such as amphibians and mammals. In many phyla animal eggs are surrounded by an inner ECM layer, such as the vitelline envelope or zona pellucida and outer ECM coats such as the egg jelly layers of amphibian eggs and the cumulus mass of mammalian oocytes. The goal of the current project is to use an amphibian model system (X. laevis) to evaluate the biological role of the three jelly layers sperm must pass through in order to penetrate and fertilize the egg. Although the egg jelly layers are poorly studied in comparison to any of the inner ECM layers, these outer layers are known to be essential for fertilization in many amphibian species. Despite this essential role, to date biological activities have not been reported for any egg jelly macromolecule in vertebrates.
Appropriate assays have been developed to demonstrate that at least two biological activities can be attributed to egg jelly macromolecules - a factor that acts as a sperm chemotactic agent and a factor that acts to promote fertilization at the moment of sperm-egg binding. First, using these bioassays, Drs. Chandler and Bieber intend to isolate the biologically active proteins by a combination of molecular sieve chromatography, ion-exchange chromatography and HPLC. Second, these proteins will be characterized by mass spectroscopy, capillary electrophoresis and amino acid sequencing. Third, the physiological actions of the fertilization promoting factor on sperm will be studied in detail. A working hypothesis is that this factor has a direct effect on one of four steps in the fertilization process: 1) enhancement of sperm motility, 2) initiation of an acrosome reaction or surface remodeling of the sperm, 3) enhancement of sperm-vitelline envelope binding, or 4) enhancement of sperm-egg plasma membrane fusion and sperm penetration. Protocols for evaluating each of these physiological steps in the fertilization process have been developed. Fourth, sperm-egg jelly interactions in situ will be characterized by a combination of confocal microscopic, video microscopic and quick-freeze, deep-etch electron microscopic technologies in order to correlate the distribution of these biologically active factors with the physiological status of the sperm as it traverses the jelly layers.
The longer term goals of this research are a) to use PCR amplification techniques to amplify and sequence the genes coding for these proteins and b) to construct primers and use PCR technology to identify homologous genes from mammals that may code for similar proteins essential to fertilization.
