The goal of this project is to increase the number of minorities involved in research addressing the biomedical problems of fertility and fertilization, signaling across membrane, and intracellular movement of organelles via studies of activated ascidian sperm at the cellular and biochemical levels. Thirty percent of the students who have worked in my' lab over the past ten years have been minorities. Many of these students have gone into health professions, but few have continued in biomedical research. The participation of students in my lab in the MBRS program would increase their exposure to biomedical research as a viable career option. These students would be studying the early events of fertilization include sperm-egg binding, sperm activation and, subsequently, sperm penetration of egg vestments, processes which ultimately lead to fusion of sperm and egg plasma membranes. The cellular mechanisms of sperm activation and penetration, as triggered by sperm-egg binding, are the foci of this project. It is hypothesized that signaling events which cross the membrane must connect binding to activation and that rearrangement of force-producing cytoskeletal elements must connect activation to mitochondrial migration. Investigating the transmembrane signaling connection requires loading of cells with activators or blockers to indirectly test for the presence of the components of a polyphosphainositide-linked mechanism. Direct evidence will be gathered by biochemical analysis of some of the protein members of the pathway, by measuring the appearance of the products of their actions, and by studying their behavior in the presence of antibodies which either identify or interfere with them. This polyphosphainositide-dependent regulatory pathway was chosen because, in some cells, it is known to elevate intracellular pH and calcium, both of which occur as part of ascidian sperm activation. Studying mitochondrial movement requires probing the biochemical nature and cellular location of participating cytoskeletal proteins. The presence of specific cytoskeletal proteins and motility motors will be established by polyacrylamide gel electrophoresis and immunoblotting. The cellular location of these proteins will be studied by indirect immunofluorescence and immunoelectron microscopy.
