MECHANISM AND CONSEQUENCES OF CALCIUM INFLUX DURING DROSOPHILA EGG ACTIVATION

Abstract

At the end of oogenesis, the mature oocyte is arrested in meiosis. It needs to be “activated” to transition to embryonic development. The egg activation process is largely conserved across organisms. It is accompanied by a rise of intracellular calcium in almost all species studied to date. This calcium rise is believed to trigger a series of downstream events in preparation for embryogenesis, including cell cycle resumption, maternal protein and mRNA processing, cytoskeleton rearrangement, egg covering modification and releases of intracellular zinc. In vertebrates, echinoderms, and C. elegans, egg activation is triggered by fertilization. In arthropods, however, egg activation is uncoupled from fertilization. Drosophila egg activation is triggered by mechanical pressure and oocyte-swelling during ovulation. Despite the different triggers, the presence of a calcium level rise and many downstream events are conserved. The calcium rise during Drosophila egg activation initiates through influx of external calcium. This influx nucleates a calcium wave that starts from the oocyte pole(s) and sweeps across the oocyte. The propagation of this calcium wave requires release of calcium from internal stores through IP3R channels. My research uncovered several aspects of Drosophila egg activation. I discovered that the TRP family channel, Trpm, mediates the calcium influx required for calcium wave initiation; I proposed a model for how Trpm is activated. I also showed that Plc21c is required for calcium wave propagation, possibly through the IP3 pathway. Downstream of the calcium wave, I detected a wave of F-actin reorganization that is interdependent with the calcium wave. I also found that zinc levels increase during oocyte maturation and decrease upon egg activation. These phenomena are analogous to those observed in echinoderms or mammals; their mechanisms have not been elucidated. My findings not only suggest conservation of multiple events during egg activation, but also demonstrate that Drosophila is an ideal model with which to dissect the molecular mechanisms of these events. In addition, I carried out experiments to optimize a germline-specific CRISPR/Cas9-mediated genome editing protocol in Drosophila that will facilitate future studies of Drosophila reproduction and germ cells.