*** 9553031 Foltz NSF Presidential Faculty Fellowship ABSTRACT Research in our laboratory centers around two major questions. First, how is it that the sperm and egg specifically interact? That is, why is fertilization species-specific? And second, once the sperm binds an egg, how is the egg "activated" to begin development? These two interesting but rather broad questions are related at a molecular level. Using the sea urchin as a model system, we have helped identify and characterize a transmembrane protein on the egg surface which binds sperm species-specifically. This same protein (designated the "sperm receptor") is also rapidly tyrosine phosphorylated by an egg kinase at the time of sperm binding, suggesting that it may be involved in events that signal egg activation. One of our long-term goals is to identify the structural aspects of the receptor protein which are responsible for species-specific interactions. To this end, we are identifying and characterizing receptor homologs in other species for comparison with that of the purple sea urchin (Strongylocentrotus purpuratus). In addition, we are assessing variation in the receptor among individuals of the sarne species. This is to test a relatively new idea (put forth primarily by Dr. Steve Palumbi at the University of Hawaii and Dr. Vic Vacquier at Scripps Institute of Oceanography) that gamete recognition molecules such as the sperm receptor will have high intraspecific allelic variability that serves to promote diversity-enhancing selection. We also hope to be able to obtain crystals and eventually three-dimensional structural data for the sperm receptor. We are able to purify large quantities of recombinant receptor protein that binds sperm spccies-specifically~ which iS a major first-step towards this goal. If successful, we will combine mutational analysis and species-specific domain swapping experiments to get at the molecular basis of gamete recognition. The other aspect of the research in the laboratory addresse s the mechanism of egg activation. There are two basic hypotheses for how this occurs. One model is that the sperm delivers an "activating factor" at the time of fusion that triggers egg signaling pathways to initiate development. Another model is that sperm binding triggers a receptor-mediated signal transduction event. Despite years of investigation, neither hypothesis has been adequately tested. In fact, the two mechanisms are not necessarily mutually exclusive; so many changes occur during "egg activation" that independent pathways could exist. Now that we have identified a candidate receptor on the egg surface, we plan to test the hypothesis that this receptor mediates a signal transduction event at the time of sperm binding. We have already established that the receptor interacts with a src-like tyrosine kinase and is phosphorylated within seconds of sperm binding. Our task now is to investigate whether or not this is necessary for egg activation. We will employ several strategies, including a straightforward search for signaling proteins that interact with the receptor and the role they may play in activation. But the real test will require that we "knockout" the ability of the receptor to signal and then ask if activation occurs. In order to accomplish this, we will express the sea urchin sperm receptor in a starfish oocyte. If sea urchin sperm now bind to this oocyte and activate it, then we are in a position to test the hypothesis. We will then express a sea urchin sperm receptor that has been mutated such that it is missing the cytoplasmic signaling domain or has point mutations in that domain. If sperm bind to the oocytes expressing the mutated receptor but no activation occurs, this would support the receptor-mediated activation hypothesis. If this does not appear to be the case, we will begin to assess the alternative hy pothesis in greater detail. Either way, having the ability to deal with molecular details of recognition and activation puts us in the exciting position of being able to investigate these basic questions about how development starts. ***
