Fig. 2. Series I and series II physiological calcium wave pacemakers (PM1 and PM2) are associated with two distinct ER-rich domains. (A) Variations of [Ca²⁺]c displayed as normalized ratio CG/TR fluorescence (DF_CG/TR/F_CG/TR(0)) (1 image every 14 seconds). The [Ca²⁺]c scale on the right corresponds to earlier quantitative measurements of [Ca²⁺]c using aequorin (see Speksnijder et al., 1990a; Speksnijder et al., 1990b; Roegiers et al., 1999). Fertilization elicits series I calcium oscillations (PM1 boxed in blue) and the extrusion of the first polar body (pb1). Series II calcium oscillations (PM2 boxed in pink) can be observed after the extrusion of pb1, in the last 15-20 minutes, and end just before the extrusion of the second polar body (pb2). (B) Only the red lines, which represent the ER network, and the blue chromosomes are depicted. The red arrows show the origin and direction of calcium waves initiated by the different pacemakers. (Left) PM1-confocal ratio imaging of cytosolic calcium (CG/TR) (1 image every 9 seconds). The ratios are shown as pseudocoloured images: higher [Ca²⁺]c concentrations are in red and yellow; lower [Ca²⁺]c in blue. The CG/TR ratio image sequence shows the initiation and propagation of the second calcium wave triggered by the moving series I pacemaker (PM1, white arrowhead at 4 minutes 0 seconds (4'00'')). All calcium waves in the series start in a peripheral ER-rich domain corresponding to the sperm aster (sa; arrowhead in TR image), which moves with the cortical contraction. The TR image shown (6'08'') corresponds to the third calcium wave initiated by pacemaker PM1. (Right) PM2-ratio CG/TR sequence of a series II calcium oscillation (1 image every 14 seconds). All waves are initiated from the same vegetal cortical region of the egg (contraction pole area; white arrowhead at t=12'15''). The sperm aster (sa, arrowhead in TR image) has enlarged and has moved into the interior of the egg away from the calcium wave pacemaker (PM2) located in the contraction pole which, in this case, is below the imaged confocal plane.