Structural change of the endoplasmic reticulum during fertilization: evidence for loss of membrane continuity using the green fluorescent protein.

Green fluorescent protein (GFP) was targeted to the lumen of the endoplasmic reticulum (ER) of starfish eggs by injecting mRNA coding for a chimeric protein containing a signal sequence and the KDEL ER retention sequence. By confocal microscopy, the GFP chimeric protein was localized in intracellular cisternae (membrane sheets) and the nuclear envelope, showing that it had been successfully targeted to the ER. The labeling pattern closely resembled that produced by the fluorescent dicarbocyanine DiI, which has been used previously to label the ER (Jaffe and Terasaki, Dev. Biol. 164, 579-587, 1994). Eggs expressing the GFP chimera were used to examine whether there is a loss of ER continuity at fertilization. The time required for recovery of fluorescence after photobleaching for both the GFP chimera and DiI was much longer in eggs at 1 min postfertilization than in unfertilized eggs or in 20-min-postfertilized eggs. This result provides strong evidence for a transient loss of continuity of the ER associated with Ca release at fertilization.