The overall goal of this proposal is to investigate the dynamics of sperm surface zona pellucida receptors (ZPrs) during fertilization. Fluorescently and isotopically labeled zona pellucida glycoproteins and glycopeptides will be used to determine: (1) the distribution of different ZPrs during sperm development, (2) the binding properties of ZPrs during fertilization, and (3) the sequence of cell surface events requisite for fertilization. Rather than using standard biochemical techniques requiring large populations of sperm, state-of-the-art digital imaging techniques will be used to determine the distributions and dynamics of zona pellucida receptors on the sperm surface. Using these methods, the investigator will observe the dynamics of sperm surface ZPrs during epididymal maturation, capacitation, and the acrosome reaction. A number of fluorescent substrates will be used for probing active sites of putative ZPrs on the surface of single sperm in well defined physiological states. Finally, a number of monoclonal and polyclonal antibodies will be used to follow the sequence of various cell surface molecules that are involved in the fertilization pathway. This research will yield important information about the distribution and dynamics ZPrs necessary for sperm-zona adhesion and induction of the acrosome reaction and may have implications both for the development of contraceptives and for understanding infertility problems involving sperm adhesion to the egg. In addition, elucidating the molecular dynamics of sperm adhesion to the zona pellucida will lead to a model of ligand-induced, receptor-mediated events important in many areas of cell biology.
| De Los Santos, Carla; Chang, Ching-Wei; Mycek, Mary-Ann et al. (2015) FRAP, FLIM, and FRET: Detection and analysis of cellular dynamics on a molecular scale using fluorescence microscopy. Mol Reprod Dev 82:587-604 |
