During fertilization, the sperm initiates a transient increase in the concentration of free intracellular Ca2+ in the egg, which is critical for activating the egg to begin development. The Ca2+ increase during fertilization is a fundamental process occurring in eggs of all the organisms that have been studied. One of the roles of this Ca2+ increase is to cause cortical granule exocytosis, which acts as a barrier to polyspermy in echinoderms, frogs, and mammals. The increase in Ca2+ may also act to initiate cell cycle progression. However, the molecular mechanism through which sperm-egg interaction leads to Ca2+ release is not completely understood. The experiments outlined in this proposal will assemble the signaling pathway that links the Ca increase to the initiating signal, whether it is a soluble molecule from the sperm or egg cytoplasm or cell surface receptor in the egg (or sperm) plasma membrane. The research objective of this proposal is to investigate the signaling molecules that interact with Src and PLC? to mediate egg activation during fertilization in the starfish. Molecular tools based upon signaling molecules previously cloned from a starfish oocyte cDNA library will be used for the in- depth study of the signaling pathways that are initiated at fertilzation. The starfish has been chosen as the model system for several reasons, including the availability of high quality molecular reagents, publicly-available genome and transcriptome sequences, the ability to obtain large quantities of gametes for use in molecular and biochemical studies, the ease with which cells can be microinjected and assayed for signaling events, and the optical clarity of the egg for microscopy. Specific signaling domains from known signaling molecules (Src1, Src3, and PLC? are in hand) will be expressed as fusion proteins for use in in vivo competition experiments and as affinity reagents for the identification of interacting proteins, including cell surface molecules. The identity of the proteins that recognized to interact specifically with Src1, Src3, or PLC? will be determined by tandem mass spectrometry amino acid sequencing followed by database identification. The identified cDNA for each signaling molecule will be cloned by RT-PCR to produce a library of molecules with potential function at fertilization. The result will be the elucidation of a network of proteins that interact in the echinoderm egg to facilitate egg activation during fertilization. One novel aspect of this proposal is the ability to work 'inside-out' from known signaling molecules to identify egg cell surface proteins that could be involved in sperm- egg interaction or the initiation of egg activation. The ultimate goal is to learn how the fertilization signal translates into activation of the zygotic cell cycle and the initiation of rapid embryonic cell divisions; thus, determining which molecules are present and how they interact is an important first step. This project will rely extensively on graduate and undergraduate research assistants, and is anticipated to provide salary and research material support for two Ph.D. students and 6-9 undergraduates over the grant period, as well integrating primary research into the introductory undergraduate genetics class taken by all Biology majors and many other majors (typical class size is 80 students). The undergraduate students will be individually trained to work with starfish gametes, bioinformatics, and selected molecular biology methods by the PI and Ph.D. students. If their data is used in published manuscripts they will be given coauthorship to credit their work. Finally, project funds will be used to support these students to attend research conferences to present their work.
This project will utilize the starfish (Patiria miniata) as a model system for understanding the molecular signals that function at fertilization. This signaling pathway is absolutely essential for the initiation of embryonic development. This is important for public health because many of the exact same signals are expected to operate during fertilization in humans.