1: Biophys J  1995 Mar;68(3):739-48 

Spatiotemporal relationships among early events of fertilization in sea urchin
eggs revealed by multiview microscopy.

Suzuki K, Tanaka Y, Nakajima Y, Hirano K, Itoh H, Miyata H, Hayakawa T, Kinosita
K Jr.

Department of Physics, Faculty of Science and Technology, Keio University,
Yokohama, Japan.

Four early events of egg fertilization, changes in intracellular calcium
concentration and intracellular pH, reorientation of the surface membrane, and
the elevation of the fertilization envelope, were imaged in real time and in
pairs in single sea urchin eggs. The paired imaging allowed the correlation of
the four events spatially and temporally. Three of them propagated as waves
starting at the sperm entry site. The earliest was the calcium wave, visualized
with fluorescent indicator dyes. After a delay of 10 s there followed a large
decrease in the fluorescence polarization of membrane-bound dyes, which we
interpret as arising from membrane reorientation as a result of cortical granule
exocytosis and microvillar elongation. With a further delay of 15 s the
fertilization envelope was seen to rise in transmitted light. All three waves
propagated with similar velocities of approximately 10 microns/s, supporting the
view that calcium triggers the latter two events. The fluorescence polarization
changed in two steps with a clear pause of 10-20 s in between. The second step,
which also propagated as wave, reflects either further elongation of microvilli
or straightening of irregular microvilli. This second step was abolished by
cytochalasin B and was coincident with an increase in cytoplasmic pH, suggesting
that pH-induced actin reorganization may play a role. The cytoplasmic
alkalinization, imaged with a fluorescent probe, was quite different from the
other events in that it took place homogeneously throughout the egg and slowly
(over 100 s). Apparently, the alkalinization is not on a direct downstream
pathway of calcium origin. An opposing possibility, that the alkalinization may
in fact be triggered by the traveling calcium wave, is also discussed.

PMID: 7756541 [PubMed - indexed for MEDLINE]