THE 2 INTRACELLULAR CA2-TRISPHOSPHATE RECEPTOR, PLAY DIFFERENT ROLES DURING FERTILIZATION IN ASCIDIANS( RELEASE CHANNELS, RYANODINE RECEPTOR AND INOSITOL 1,4,5)

Fertilization in the ascidians triggers an activation wave of calcium release followed by intracellular calcium oscillations synchronous wit h periodic membrane potential excursions during the completion of the meiotic cell cycle. fertilization also causes a fast decrease in the e gg plasma membrane depolarization-activated calcium current and a larg e increase in capacitance thought to represent membrane addition to th e egg surface. We have analyzed the temporal and causal relationships between these changes in the eggs of Phallusia mammillata using whole- cell patch-clamp recording while simultaneously imaging calcium with f ura-2 dextran. We have defined the role of ryanodine receptor (RyR) an d InsP(3) receptor (InsP(3)R) during fertilization and meiosis by look ing at the effects of InsP(3), cyclic ADP ribose (cADPR), and ryanodin e in perfused oocytes. We show that InsP(3) (10 mu M perfused through the patch pipette) is able to trigger sustained oscillations in intrac ellular calcium concentration in unfertilized oocytes, resembling thos e recorded in fertilized egg completing meiosis. In addition the susta ined oscillations resulting from InsP(3) perfusion in unfertilized ooc ytes are sufficient to cause the emission of both polar bodies. In con trast, ryanodine or cADPR never trigger detectable calcium signal in p erfused oocytes. Instead, nanomolar concentrations of ryanodine or cAD PR cause a capacitance change, implying a net insertion of membrane to the oocyte surface, and trigger a fast decrease in the depolarization -activated calcium current. Both changes are similar to the changes in conductance and capacitance naturally observed following fertilizatio n. These effects, although not associated with measurable calcium sign als, are abolished by coperfusion of the calcium chelator BAPTA. In co ntrast to ryanodine or cADPR, sustained perfusion of the oocyte with n anomolar concentrations of InsP(3) causes no capacitance change and a slow and moderate decrease in calcium current. Our observations on ins eminated patch-clamped eggs further indicate that membrane insertion, which starts 15-20 sec after the onset of the membrane conductance cha nge at fertilization, can be altered by interfering with the RyR. Our results imply that, in ascidians, as in some mammals, RyR and InsP(3)R play distinct roles during fertilization. (C) 1997 Academic Press.