Project Summary The first step to initiate development of a new organism is to convert the terminally-differentiated mature oocyte to a totipotent cell capable of undertaking development upon fertilization. This change in cell state, called egg activation, includes the resumption of meiosis from its arrest in the mature oocyte and the translation of some stored maternal mRNAs and the degradation of others. Although these events are critical for fertility, what induces egg activation is not understood, except that a rise in oocyte calcium level is required. Since the transition initiates with no new transcription or translation, we hypothesized that post- translational switches mediate the transition from oocyte to activated egg by turning on or off the activity of proteins present in the egg. Consistent with this hypothesis, we found that the phospho-states of many proteins change during egg activation in Drosophila; this has since been seen in three other taxa. We hypothesize that calcium activates enzymes that regulate the phospho-state of critical proteins in the oocyte, allowing these proteins to then mediate critical egg activation events. We will test this hypothesis using Drosophila, whose large oocytes, excellent genetics, ‘omics tools, and high conservation of biomedically-relevant genes provide an optimal system for answering these questions. The first aim stems from our findings that a conserved mechanically-gated cation channel (TRPM) initiates a calcium rise in the oocyte and that the calcium-regulated phosphatase calcineurin, which is necessary for egg activation, is required for a significant fraction of the phosphoproteome changes. Molecules other than TRPM can then cause a slower, important, rise in calcium. We will determine their nature and contributions (Aim 1a). Because phosphorylation of some proteins increases upon egg activation, we will determine whether the calcium-regulated kinase CaMKII is also needed for egg activation in Drosophila, as it is in mouse (Aim 1b). In Aim 2, we will dissect the events downstream of those studied in Aim 1. By manipulating kinase activity using optogenetics, we will determine the effect on egg activation of altering the proteome’s phospho-state (Aim 2a). We will then focus on selected phospho-regulated proteins, particularly those involved in cell cycle control and translation initiation, testing whether the phospho-modifications that occur during egg activation alter their activity in ways that facilitate egg activation processes (Aim 2b). Given the conservation of egg activation, the mechanisms we define should reveal the basis of some pre- implantation-stage infertilities in humans (including identifying cases that would not be treatable with IVF) and for assessing the efficacy of IVF conditions. Our results will also help to understand the mechanisms by which calcium regulates myriad cell processes and their transitions.

Narra$ve While essen)al for development, the transi)on from mature oocyte to embryo is poorly understood at the molecular level, largely due to technical limita)ons with studying this process in mammals. We will use Drosophila to dissect the nature and consequences of the evolu)onarily conserved calcium increase and protein-modifica)on changes that mediate this transi)on, exploi)ng the fruit fly’s large eggs and excellent gene)c tools. In addi)on to contribu)ng to our basic understanding of how calcium changes cell fates, the results of this research will shed light on infer)li)es in people, including assessing who will or will not benefit from IVF.