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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The change in intracellular pH (pHi) upon fertilization and the effects of changing the pH, by microinjection of pH buffers were investigated in the eggs of the sand dollar, Clypeaster japonicus. The pH, was determined by the tint of a pH indicator, phenol red, microinjected into eggs. The pHi ranged from 6.5 to 6.75 in unfertilized eggs and it rose by 0.4 to 0.5 unit within 3 min upon fertilization. The elevated pHi ranging from 7.0 to 7.25 was maintained at least until the first cleavage. As reported in eggs of other species of sea urchin (I+, development of fertilized eggs which had been transferred to Na-free sea water immediately after insemination was arrested and the pHi did not rise remaining at the level of unfertilized eggs. Development was initiated in eggs arrested in Na-free sea water when the pHi was elevated up to the level of fertilized eggs, i.e. 7.0 to 7.25, by microinjecting 1 M HEPES (N-2-hydroxyethylpiperazine-N '-2-ethanesulfonic acid)-KOH buffer at pH 8.0. By microinjection of pH 7.5 buffer, some eggs started development though none of them underwent cleavage. By microinjection of pH 7.0 or pH 6.5 buffer, development was not initiated. The initiation of development depended on the pH value of microinjected pH buffer, and in consequence, on the final pHi. The elongation of microvilli which had been arrested in eggs in Na-free sea water was also induced by microinjection of pH 8.0 or 7.5 buffer.It has been reported that intracellular pH (pH,) increases following a transient increase in intracellular free calcium concentration upon fertilization in sea urchin eggs (5-8). The role of the increase in pH, in fertilization was emphasized by the observation that the increases in K conductance and biosynthesis induced upon fertilization are mimicked by exposing unfertilized eggs to the weak base NH,, thereby raising pHi (3, 9, 10). On the other hand, it has been reported that development of sea urchin eggs is arrested when they are put into Na-free sea water immediately after insemination (1) and that external Na ions are necessary for the rise in the intracellular pH after fertilization (2-4).In the present study, changes in pHi from fertilization to the first cleavage were determined by the tint of pH indicator microinjected into the cell, using very transparent eggs of the sand dollar, Clypeaster japonicus. The effects of changing pH, of fertilized eggs in sea water or Na-free sea water and the relation between pHi and morphological events during early stages of development were investigated by microinjecting pH buffers into eggs.. MATERIALS AND METHODS Experimental mediaArtificial sea water (ASW) and Ca-free artificial sea water (CFSW) purchased from Jamarin Laboratory (Osaka, Japan) were used. Na-free sea water (NFSW) had the composition: 420 mM choline 443

The observations of the fertilization process in the heart-urchin, Clyprastrr .japonicus with a differential interference microscope indicate that the sperm pronucleus is carried to the center of the egg by the growth of the sperm aster as stated by CHAMBERS (3, and that the egg pronucleus is carried to the center of the aster by a filamentous structure formed between them. The curved path of egg pronucleus in the fertilized egg is interpreted as the combination of the movement of the center of the aster and the movement of the egg pronucleus toward the center of the aster. The movement and the rotation of the sperm head result from pushing by the tail being engulfed in the egg.

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