Modulation of Calcium signalling by F-actin during maturation and fertilization of starfish oocytes

The Calcium ion (Ca2+) is a universal intracellular messenger that is implicated in the regulation of a great variety of intracellular processes. Among them, there is the meiotic maturation of oocytes from several species and their fertilization. The cell model system chosen for the work of this thesis has been the starfish oocytes. They constitute an exceptional model to study the properties of the dynamic release of Ca2+. First, the cells are large and nearly transparent, making them suitable for imaging experiments after microinjection of fluorescent markers. Immature oocytes extracted from the gonad are arrested at the prophase stage of the first meiotic division. Overcoming the meiotic arrest (maturation), is induced by the application of the maturing hormone 1-Methyladenine (1-MA), whose action involves intracellular Ca2+ transients. During the maturation process, the oocytes develop their ability to be successfully activated by a fertilizing spermatozoon by liberating higher levels of calcium. 1-MA also induces dramatic actin cytoskeleton rearrangements. In the present work, the relevance of the actin cytoskeleton in the modulation of the Ca2+ signals generated by hormonal stimulation and by the sperm has been studied. For this purpose, actin-specific disrupting agents and a specific antibody targeting the actin binding protein depactin have been delivered into living cells by needle microinjection, to study their effects using confocal laser fluorescent microscopy. Thus, the Ca2+ increases induced either by 1-MA, by the fertilizing sperm or by the injection of exogenous InsP3. which is the canonical Ca2+ mobilizer second messenger, have been monitored with video fluorescent microscopy. The results described indicate that actin filaments play direct and indirect roles in modulating the intracellular Ca2+ mobilization induced by the maturation process triggered by 1-MA as well as in the Ca2+ responses induced by the sperm