Calcium waves occur as Drosophila oocytes activate.

Egg activation is the process by which a mature oocyte becomes capable of supporting embryo development. In vertebrates and echinoderms, activation is induced by fertilization. Molecules introduced into the egg by the sperm trigger progressive release of intracellular calcium stores in the oocyte. Calcium wave(s) spread through the oocyte and induce completion of meiosis, new macromolecular synthesis, and modification of the vitelline envelope to prevent polyspermy. However, arthropod eggs activate without fertilization: in the insects examined, eggs activate as they move through the female's reproductive tract. Here, we show that a calcium wave is, nevertheless, characteristic of egg activation in Drosophila. This calcium rise requires influx of calcium from the external environment and is induced as the egg is ovulated. Pressure on the oocyte (or swelling by the oocyte) can induce a calcium rise through the action of mechanosensitive ion channels. Visualization of calcium fluxes in activating eggs in oviducts shows a wave of increased calcium initiating at one or both oocyte poles and spreading across the oocyte. In vitro, waves also spread inward from oocyte pole(s). Wave propagation requires the IP3 system. Thus, although a fertilizing sperm is not necessary for egg activation in Drosophila, the characteristic of increased cytosolic calcium levels spreading through the egg is conserved. Because many downstream signaling effectors are conserved in Drosophila, this system offers the unique perspective of egg activation events due solely to maternal components.

Efficient measurement of H2O2 resistance in Drosophila using an activity monitor.

We have established a method for the efficient measurement of oxidative stress resistance in Drosophila melanogaster, using a commercially available activity monitor. Conditions under which flies in glass tubes placed in the monitor can survive over one month at 25 degrees C were optimized. The active periods of flies were reduced by administration of H(2)O(2) into the media in a dose-dependent manner. Although we used only eight flies per assay, far fewer individuals than in conventional methods, it was possible to detect the effects of H(2)O(2) at a statistically significant level. Increased levels of H(2)O(2) resistance were confirmed in transgenic flies overexpressing antioxidant enzymes, catalase or Cu/Zn superoxide dismutase. We applied the method to determine oxidative stress resistance in fly lines bearing insertions of a gene misexpression vector. H(2)O(2) resistance in these flies varied considerably depending on the insertion, and positively correlated with previously determined longevity. We identified one insertion that conferred a significantly higher level of resistance to H(2)O(2) compared to controls. Molecular analysis of the insertion revealed that a misexpressed transcript matched an expressed sequence tag, and suggested that its full-length product was overproduced upon GAL4 activation. Our method should be applicable to the systematic screening for genes involved in the antioxidant mechanism in Drosophila.