The transition from egg to embryo is a critical time in development. Since this process is essential for fertility, a better understanding of its mechanisms is important in developmental biology and will also help to optimize ART procedures. It has thus far been very difficult to identify the critical proteins needed for this transition, in part because it is too rapid to involve changes in gene transcription. Instead, numerous lines of evidence argue that this rapid and comprehensive change in cell state is mostly accomplished by post-translational modification of existing proteins. We are focusing on proteins whose phosphorylation state is modulated during egg activation, reasoning that these proteins constitute an enriched pool of molecules important for the egg-to-embryo transition. We propose to use germline-specific RNAi to screen among 258 conserved proteins that our proteomics screens have found to be phospho-modulated during egg activation in Drosophila. By straightforward phenotypic screening that takes advantage of the latest strains and methods for this model organism, we expect to identify the genes that regulate the transition from a mature oocyte to an "activated" egg that can begin development, as well as those genes that initiate its embryogenesis. We will prioritize the order of screening, giving precedence to genes likely to encode upstream regulators of this transition, and to one class of downstream effectors: those likely to regulate the cell cycle as it transitions from meiosis to mitosis. Once we have identified these new players, we will use Western blotting and phosph-proteomics to begin to order their actions. The goal of the proposed research is to discover the critical genes that carry out the important developmental transition from egg to embryo. These genes will permit future studies to dissect in detail the molecular interactions and pathways that carry out egg activation and initiate embryogenesis.
To understand fertility, as well as to design optimal methods for infertility treatments such as IVF and ICSI, we need to know the molecules that activate an egg to develop immediately after fertilization. Few of these molecules are known because of technical difficulties in previous methods to identify proteins that act during this narrow window of time. Taking a new approach, we propose to identify such molecules by screening genetically among a defined set of proteins chosen because their chemical modification state changes when eggs are triggered to activate development in the genetic model system, Drosophila.