The formation and migration of the sperm aster, and the migration of male and female pronuclei during fertilization were investigated in the eggs of the sand dollar, CZypeaster japonicus using the Colcemid-UV method. When an egg in Colcemid sea water was irradiated locally with UV light (about 365 nm wavelength) at a limited region containing sperm head, a sperm aster formed in this region, and migrated to the center of the UV-irradiated region during its formation. When the UV-irradiated region was displaced or its shape was changed after the formation of the sperm aster, the aster migrated to the center of the new UV-irradiated region. The direction of the migration of the sperm aster coincided with the direction of the longest astral rays. Direct contact between astral rays and the egg surface was not essential for sperm aster migration. When a region containing both the sperm centrosome and the female pronucleus was irradiated with UV light, the female pronucleus migrated toward the center of the sperm aster after they were connected by astral rays. The migration was suppressed when UV light was shaded over the region between the aster and the female pronucleus. These results suggest that the female pronucleus migrates to the sperm aster by attractive force between them.Previous studies on the fertilization process in sea urchins by light and electron microsocpy (for reviews see ref. I and 2) have shown that the sperm aster migrates to the center of the egg carrying the sperm pronucleus and that the female pronucleus is carried to the center of the aster along astral rays. Anti-tubulin immunofluorescence microscopy (3, 4) and fluorescent analogue cytochemistry (5) have elegantly visualized microtubule structures which form in sea urchin eggs during fertilization. Results of previous investigators using microtubule inhibitors (6, 7, 8, 9, 10) have demonstrated that motile systems responsible for the pronuclear migration are Colcemid-sensitive, suggesting that the microtubules in fertilized eggs play an important role in these systems.Colcemid is known to bind specifically to tubulin and thereby to prevent it from polymerizing into microbutules (11) and to be inactivated by ultraviolet light (12, 13, 14). Recently we reported a method by which microtubule structures could be destroyed or allowed to form in a limited region of a cell by combining Colcemid treatment and local irradiation with ultraviolet light (15). In the present study, we report the results of some experiments on the formation and migration of the sperm aster and on the migration of female and male pronuclei during fertilization using the Colcemid-UV method and some observations of the pronuclear migration in abnormal eggs with two female pronuclei and in polyspermic eggs. In eggs locally irradiated with UV light, the sperm aster migrated toward the egg surface under certain specific conditions. These examples are noticeably different from migration in untreated control eggs. In normal 143

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