The Questions of Developmental Biology

acetylglucosamine residues are removed at fertilization, ZP3 will no longer serve as a substrate
for the binding of other sperm. ZP2 is clipped by the cortical granule proteases and loses its
ability to bind sperm as well (Moller and Wassarman 1989). Thus, once a sperm has entered the
egg, other sperm can no longer initiate or maintain their binding to the zona pellucida and are
rapidly shed.
Calcium as the initiator of the cortical granule reaction.
The mechanism of the cortical granule reaction is similar to that of the acrosomal
reaction. Upon fertilization, the intracellular calcium ion concentration of the egg increases
greatly. In this high-calcium environment, the cortical granule membranes fuse with the egg
plasma membrane, releasing their contents (see Figure 7.24). Once the fusion of the cortical
granules begins near the point of sperm entry, a wave of cortical granule exocytosis propagates
around the cortex to the opposite side of the egg.
In sea urchins and mammals, the rise in calcium concentration responsible for the cortical
granule reaction is not due to an influx of calcium into the egg, but rather comes from within the
egg itself. The release of calcium from intracellular storage can be monitored visually using
calcium-activated luminescent dyes such as aequorin (isolated from luminescent jellyfish) or
fluorescent dyes such as fura-2. These dyes emit light when they bind free calcium ions. When a
sea urchin egg is injected with dye and then fertilized, a striking wave of calcium release
propagates across the egg (Figure 7.25). Starting at the point of sperm entry, a band of light
traverses the cell (Steinhardt et al. 1977; Gilkey et al. 1978; Hafner et al. 1988). The calcium ions
do not merely diffuse across the egg from the point of sperm entry. Rather, the release of calcium
ions starts at one end of the cell
and proceeds actively to the other
end. The entire release of calcium
ions is complete in roughly 30
seconds in sea urchin eggs, and the
free calcium ions are resequestered
shortly after they are released.
If two sperm enter the egg
cytoplasm, calcium ion release can
be seen starting at the two separate
points of entry on the cell surface
(Hafner et al. 1988).
Several experiments have demonstrated that calcium ions are directly responsible for
propagating the cortical granule reaction, and that these calcium ions are stored within the egg
itself. The drug A23187 is a calcium ionophore (a compound that transports free calcium ions
across lipid membranes, allowing these cations to traverse otherwise impermeable barriers).
Placing unfertilized sea urchin eggs into seawater containing A23187 causes the cortical granule
reaction and the elevation of the fertilization envelope. Moreover, this reaction occurs in the
absence of any calcium ions in the surrounding water. Therefore, A23187 must be causing the
release of calcium ions already sequestered in organelles within the egg (Chambers et al. 1974;
Steinhardt and Epel 1974).