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Calcium waves are well-known hallmarks of egg activation that trigger resumption of the cell cycle and development of the embryo. These waves rapidly and efficiently assure that activation signals are transmitted to all regions of the egg. Although the mechanism by which the calcium wave propagates across an egg as large as that of Xenopus is not known, two models prevail. One model is a wave of calcium-induced calcium release (CICR) and the other is propagation by inositol-induced calcium release (IICR). IICR requires a wave of phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis, generating two second messengers, IP3, which then releases calcium and DAG, which activates protein kinase C (PKC). We show here that a wave of PKC-green fluorescent protein travels across the egg immediately following, and at the same velocity as, the calcium wave. This is the first example of a PKC wave in a vertebrate egg and supports the IICR model of wave propagation.
Fig. 1 Calcium and PKC waves at Xenopus egg activation. The
increase in intracellular free calcium is detected by the dye calcium
crimson (left column) as it travels from the site of activation (right)
across the egg; a wave of PKC is seen crossing the egg using PKCGFP
(center column). The merged image shows that the PKC wave
follows the calcium wave (right column).
Fig. 2 Relative change in levels of intracellular free calcium after
egg activation compared with those of PKC-GFP. Calcium levels
reach a maximum within 2 min after artificial egg activation then
slowly decrease toward baseline; PKC levels begin to increase with
a lag of 30 s after the calcium rise and steadily continue to rise.
Fig. 3 Wave of PKC-GFP in egg cortex. The increase in PKC-GFP
is seen in one region of the cell periphery at high magnification
(60 lens) before activation (A) and at 30 s intervals thereafter
(B–I). PKC is slowly seen as it encircles cortical granules (1–3 mm
diameter secretory granules), moving from the right to left of each
frame and slowly increasing in intensity.
Fig. 4 Egg cortex labeled with anti-PKC antibodies. (A) PKC can
be seen surrounding the cortical granules in the unactivated egg. (B)
Activated egg showing that PKC labels granules in the egg
periphery at 15 min post-activation.
Fig. 5 PKC-labeled secretory granules after egg activation. Shortly
after egg activation, PKC-labeled granules (white circles) are
detected as they are propelled from the egg surface to the interior
of the egg by actin comet tails. (A, B) Eggs fixed 5 min after
activation. (C) Eggs fixed 10 min after activation. (D) Egg fixed 15
min after activation.
