In the Egg of the Ascidian Phallusia mammillata, Removal of External Ca2+ Modifies the Fertilization Potential, Induces Polyspermy, and Blocks the Resumption of Meiosis

Abstract

The fertilization potential of the Phallusia mammillata egg has been found to consist, in natural or standard artificial sea water (NSW and ASW), of an initial sperm-triggered rapid depolarization comprising two distinct successive components, followed by a phase of membrane depolarization comprising a plateau and two series of membrane potential oscillations. These oscillations occur during meiotic divisions and they end just before formation of the second polar body. In the present work, the effects of removing external Ca2+ on the fertilization potential time course on the rate of polyspermy and on the resumption of meiosis of the P. mammillata egg were explored. Eggs inseminated in Ca2+-free ASW usually responded by several successive sperm-induced electrical responses strikingly different from that obtained in standard ASW: removing external Ca2+ during insemination and the initial depolarizing shift (free (Ca2+), 5-11 μM in Ca2+-free ASW) and during the subsequent period of the electrical responses (free (Ca2+), 0.2 μM in Ca2+-free ASW), completely suppressed both the second component of the initial depolarizing shift, the plateau, and membrane oscillations of the depolarization phase, did not impair sperm entry, but always blocked the resumption of meiosis. Under these conditions eggs were polyspermic (91%, n = 22, c.l. 69-99%). Lastly, only a short period of contact (∼20 sec at minimum) with a physiological concentration of external Ca2+ ( 12 mM) at the onset of the fertilization potential was necessary for the egg to resume meiosis and then to undergo complete embryonic development. These results indicate that in the P. mammillata egg, the second component of the initial depolarizing shift involves voltage-gated Ca2+ channels. The possibility is discussed that external Ca2+ ions might contribute to the activation of the P. mammillata egg by providing a source for an increase of (Ca2+)i and/or for the reloading of the Ca2+ internal stores.