Analysis of the Role of Astral Rays in Pronuclear Migration in Sand Dollar Eggs by the Colcemid‐UV Method

Abstract

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

Centrosome positioning and directionality of cell movements

“…The cellular basis of such a stabilizing effect is not understood, but it seems plausible that the microtubules extending from the centrosome are required, presumably by maintaining the centrosome in a near-centroid position (27). Directional changes initiated (and executed) in the cell periphery may lead to a gradual repositioning of the microtubule-organizing center by virtue of dynamic length changes of microtubules, as suggested by the elegant experiments of Hamaguchi and Hiramoto (37). Thus microtubules may be considered as sensors of directional changes that help maintain the centrosome in a position near the cell center where the microtubule system can respond quickly to rapid and sometimes gross changes in cell morphology in the course of cell movement.…”

Intracellular organelles mediate cytoplasmic pulling force for centrosome centration in the Caenorhabditis elegans early embryo

“…In this case, the pulling force per microtubule increases as the length of the microtubule increases because the longer the microtubule, the more contact with cytoplasmic dynein (6). This length-dependent pulling mechanism was initially proposed by Hamaguchi and Hiramoto in sand dollar eggs (25). In a computer simulation assuming that the centrosome centration anchors are located throughout the cytoplasm, the in vivo profile of centrosome centration in C. elegans was reproduced (16).…”

“…However, these cytoplasmic dyneins at the cortex may not be required for centrosome centration. In sand dollar eggs, centrosome centration occurs in a cortex-independent manner (25). In large amphibian eggs, the plus ends of microtubules do not reach the cell cortex in the traveling direction during centrosome centration (20).…”

Mechanisms of spindle positioning: cortical force generators in the limelight

“…Laser microsurgery experiments in Fusarium solani or C. elegans suggested that this link corresponds to astral microtubules connecting the spindle poles with the cell cortex [6,7,8,9,10 ]. Although not the focus of this review, there are instances where pulling forces are exerted along the length of astral microtubules instead of at their plusend located at the cell cortex [11,12,13]. Work in several systems in recent years has increased our understanding of the basic principles governing spindle positioning and identified core molecular players and aspects of their mechanism of action.…”