Mitotic apparatus formation and cleavage induction by micromanipulation of the nucleus and centrosome: The centrosome forms a spindle together with only the chromosomes at a short distance

We micromanipulated the nucleus and centrosomes in the zygote of the starfish, Asterina pectinifera, in order to investigate their roles in mitotic apparatus formation and cleavage induction. The zygote cleaved without spindle formation when its nucleus was removed. When one or two centrosomes were transplanted, they formed asters in the recipient cell, which cleaved into three or four blastomeres so that each blastomere might contain one centrosome or aster. When one centrosome was removed, a half-spindle formed in the manipulated cell, which did not cleave until the other centrosome was duplicated. When both centrosomes were removed, no microtubular structures such as the spindle and the aster appeared in the manipulated cell, which failed to cleave. These results indicate that two centrosomes or more in the cell induce cleavage with or without the nucleus and that one centrosome or less does not induce cleavage. It is also concluded that the centrosome(s) together with the nucleus forms a half-spindle or bipolar spindle. However, from the experiments of nucleus transplantation and displacement, spindle formation is found to depend on the distance between chromosomes and centrosomes. The half-spindle formed when the distance from the centrosome to the chromosomes was shorter than 22 microns; on the other hand, when the distance was longer than 22 microns, the nucleus remained apart from the aster, which means that the functional range of the astral microtubule's ability to engage chromosomes was 22 microns from the centrosome.     

Germinal vesicle components are not required for the cell-cycle oscillator of the early starfish embryo

We show that certain events of the cell cycle can still occur in starfish oocytes or fertilized eggs from which the germinal vesicle (the prominent nucleus of prophase-arrested oocytes) has been removed before the induction of meiotic maturation. Two meiotic asters develop following hormonal induction of meiotic maturation in these enucleated oocytes. The asters then divide to form a transient tetrapolar figure. When enucleated oocytes are fertilized, the sperm centrosome duplicates at the times corresponding to each cleavage in control nucleated embryos. Periodic changes in the organization of the asters and in the morphology of the cell surface also occur in synchrony with controls. Decondensation of the sperm nucleus, spindle formation, and cleavage do not occur when enucleated oocytes are fertilized. Ultimately the number of asters increases to approximately 520 (about 2(9] before the pseudo-embryo arrests and cytolyzes. Fertilized eggs from which both pronuclei but not the sperm aster have been removed undergo nine cleavages and then cease cell division. The cessation of division may be related to the events that cause the midblastula transition after seven cleavages in normal nucleated embryos.     

Spindle-to-cortex communication in cleaving,polyspermic Xenopus eggs

Mitotic spindles specify cleavage planes in early embryos by communicating their position and orientation to the cell cortex using microtubule asters that grow out from the spindle poles during anaphase. Chromatin also plays a poorly understood role. Polyspermic fertilization provides a natural experiment in which aster pairs from the same spindle (sister asters) have chromatin between them, whereas asters pairs from different spindles (nonsisters) do not. In frogs, only sister aster pairs induce furrows. We found that only sister asters recruited two conserved furrow-inducing signaling complexes, chromosome passenger complex (CPC) and Centralspindlin, to a plane between them. This explains why only sister pairs induce furrows. We then investigated factors that influenced CPC recruitment to microtubule bundles in intact eggs and a cytokinesis extract system. We found that microtubule stabilization, optimal starting distance between asters, and proximity to chromatin all favored CPC recruitment. We propose a model in which proximity to chromatin biases initial CPC recruitment to microtubule bundles between asters from the same spindle. Next a positive feedback between CPC recruitment and microtubule stabilization promotes lateral growth of a plane of CPC-positive microtubule bundles out to the cortex to position the furrow.     

Poor centrosomal function of cat testicular spermatozoa impairs embryo development in vitro after intracytoplasmic sperm injection

In the domestic cat, morula-blastocyst formation in vitro is compromised after intracytoplasmic sperm injection (ICSI) with testicular compared to ejaculated spermatozoa. The aim of this study was to determine the cellular basis of the lower developmental potential of testicular spermatozoa. Specifically, we examined the influence of sperm DNA fragmentation (evaluated by TUNEL assay) and centrosomal function (assessed by sperm aster formation after ICSI) on first-cleavage timing, developmental rate, and morula-blastocyst formation. Because the incidences of DNA fragmentation were not different between testicular and ejaculated sperm suspensions, DNA integrity was not the origin of the reduced developmental potential of testicular spermatozoa. After ICSI, proportions of fertilized and cleaved oocytes were similar and not influenced by sperm source. However, observations made at 5 h postactivation clearly demonstrated that 1) zygotes generally contained a large sperm aster after ICSI with ejaculated spermatozoa, a phenomenon never observed with testicular spermatozoa, and 2) proportions of zygotes with short or absent sperm asters were higher after ICSI with testicular spermatozoa than using ejaculated spermatozoa. The poor pattern of aster formation arose from the testicular sperm centrosome, which contributed to a delayed first cleavage, a slower developmental rate, and a reduced formation of morulae and blastocysts compared to ejaculated spermatozoa. When a testicular sperm centrosome was replaced by a centrosome from an ejaculated spermatozoon, kinetics of first cell cycle as well as embryo development quality significantly improved and were comparable to data from ejaculated spermatozoa. Results demonstrate for the first time in mammals that maturity of the cat sperm centrosome (likely via epididymal transit) contributes to an enhanced ability of the spermatozoon to produce embryos that develop normally to the morula and blastocyst stages.     

Induced formation of asters and cleavage furrows in oocytes of Xenopus laevis during in vitro maturation

We have previously reported that injection of purified basal bodies or sperm into unfertilized eggs of Xenopus laevis induced the formation of asters and irregular cleavage furrows. Fully grown oocytes were found to be unable to form asters or cleavage furrows. In this paper we show that the oocyte acquires the ability to form asters upon basal body injection at the time of germinal vesicle breakdown during in vitro maturation. Our evidence indicates that aster formation requires progesterone-stimulated changes in the oocyte and mixing of cytoplasm and germinal vesicle plasm. The ability of the oocyte to form cleavage furrows arises six to eight hours after germinal vesicle breakdown. We infer that some maturational change in the cell cortex occurs to enable the egg surface to furrow. Experiments on the relationship of aster formation to furrow initiation indicates that asters stimulate furrow formation. However, some furrowing could be induced without aster formation in mature oocytes and unfertilized eggs by an activation stimulus, showing that asters are not essential for cleavage initiation. The significance of these observations are discussed in the light of our current understanding of meiotic maturation, cell cleavage and aster growth.     

Protein synthesis and the cell cycle: centrosome reproduction in sea urchin eggs is not under translational control

The reproduction, or duplication, of the centrosome is an important event in a cell's preparation for mitosis. We sought to determine if centrosome reproduction is regulated by the synthesis and accumulation of cyclin proteins and/or the synthesis of centrosome-specific proteins at each cell cycle. We continuously treat sea urchin eggs, starting before fertilization, with a combination of emetine and anisomycin, drugs that have separate targets in the protein synthetic pathway. These drugs inhibit the postfertilization incorporation of [35S]methionine into precipitable material by 97.3-100%. Autoradiography of SDS-PAGE gels of drug-treated zygotes reveals that [35S]methionine incorporates exclusively into material that does not enter the gel and material that runs at the dye front; no other labeled bands are detected. Fertilization events and syngamy are normal in drug-treated zygotes, but the cell cycle arrests before first mitosis. The sperm aster doubles once in all zygotes to yield two asters. In a variable but significant percentage of zygotes, the asters continue to double. This continued doubling is slower than normal, asynchronous between zygotes, and sometimes asynchronous within individual zygotes. High voltage electron microscopy of serial semithick sections from drug-treated zygotes reveals that 90% of the daughter centrosomes contain two centrioles of normal appearance. From these results, we conclude that centrosome reproduction in sea urchin zygotes is not controlled by the accumulation of cyclin proteins or the synthesis of centrosome-specific proteins at each cell cycle. New centrosomes are assembled from preexisting pools of ready-to-use subunits. Furthermore, our results indicate that centrosomal and nuclear events are regulated by separate pathways.     

Aster Formation in Sea Urchin Eggs Induced by the injection of Enzyme-Treated Sperm Centrioles

To determine the responsible components of isolated sperm centrioles for the aster induction in sea urchin eggs, the sperm centriolar fraction was treated with various enzymes and was injected into the unfertilized eggs, then the aster formation in first division was observed after fertilization.
Treatment with 1 μg/ml or higher concentration of trypsin inhibited the centriolar activity for aster induction, whereas the treatment with 50 μg/ml of DNase 1, 80 μg/ml of RNase A, 40 μg/ml of RNase T1, or 0.1 μg/ml of trypsin had no inhibitory effect to induce asters. Injection of 0.5 μg/ml of RNase A or 1 mUg/ml of RNase T1 into the egg caused the detention of mitosis at the streak stage. To examine the temperature effect for aster induction, the centriolar fraction was pre-treated with boiling temperature, and it was found that the fraction became incapable to induce any aster.
Results obtained suggest that the effective components of the sperm centriolar fraction to induce asters in the fertilized sea urchin eggs are the proteins but not the nucleic acids. The aster inducing activity is destroyed by heat treatment.     

Chromatin and microtubule morphology during the first cell cycle in bovine zygotes

Chromatin and microtubule configurations during the first cell cycle of bovine zygotes were analyzed by DNA staining and microtubule immunolocalization using an IVM/IVF system and oocytes matured and fertilized in vivo, in order to investigate the origin of the active centrosome and to characterize the nuclear and the cytoplasmic changes following bovine fertilization. Our results suggest that the paternal centrosome is active during early zygotic development, forming a conspicuous sperm aster soon after fertilization. We also report that polyspermy in bovine eggs, leads to the formation of numerous sperm asters with different degrees of association with the chromatin. The maternal structures in both monospermic and polyspermic zygotes can be lost or degenerate. Consequently, these cells may resume the first cell cycle as androgenotes, very often with several types of mitotic activity taking place in different regions of the cell cytoplasm at the same time. As indicated by a comparison of monospermic and polyspermic fertilization rates to rates of development, it is possible that some androgenetic embryos cleave and develop to the blastocyst stage. © 1993 Wiley-Liss, Inc.     

SOME STRUCTURAL AND FUNCTIONAL ASPECTS OF THE MITOTIC APPARATUS IN SEA URCHIN EMBRYOS

The mitotic figures in dividing cells of sea urchin embryos, from first division to the onset of cilia formation, were studied with regard to the filament system and its relation to kinetochores, chromosomes, and poles, as well as to fixation conditions which would best preserve these structures. With regard to fixation, variations in the salt concentration and pH of the fixative indicated that an extraction effect on the chromosomes noted in earlier work was probably due to a combination of neutral pH and salt concentration equivalent to sea water. The presence of the 15 mµ filaments depended on the presence of either of two stabilizing conditions: pH 6.1 or presence of the salts of sea water, presumably the divalent cations of Ca and Mg. Kinetochores and centrioles were unaffected by the fixative variations. The 15 mµ filaments, reported earlier in the central spindle, are also found in great numbers in the asters of early cleavage divisions. However, with successive divisions and reduction in cell size, the aster disappears at about the 32 to 64 cell stage, and the 15 mµ filaments are entirely associated with the central spindle. This disappearance of the aster suggests that it may be, in fact, merely a specialization of large cells for cytokinesis.     

The first two cleavages in Tubifex involve distinct mechanisms to generate asymmetry in mitotic apparatus

We have investigated factors which determine inequality of the first two cleavages in Tubifex hattai. A mitotic spindle for the first cleavage, which is located at the center of the egg, possesses an aster at one pole, but not at the other pole. Inequality of the first cleavage is determined by the asymmetric organization of the spindle poles, rather than by the spindle position in the egg. A centrosome which appears as a dot stained with an anti--tubulin antibody is found at one pole (at the center of the aster) of the spindle, but not at the other pole. This centrosome appears to be maternal in origin. In contrast to the first cleavage, the poles of the second cleavage spindle are not different from each other either in their ability to form asters or in -tubulin distribution. As a result of an interaction of one of the spindle poles with the cell cortex, however, an asymmetric spindle is formed in the cell CD, giving rise to unequal division in this cell. Thus, factors generating asymmetry in spindle organization are intrinsic to the mitotic spindle in the first cleavage, but not in the second cleavage.     

Distribution of tubulin-containing structures in the egg of the sea urchin Strongylocentrotus purpuratus from fertilization through first cleavage

Eggs of the sea urchin Strongylocentrotus purpuratus were examined by indirect immunofluorescence microscopy for tubulin-containing structures at intervals from fertilization through first cleavage. The staining revealed that the monaster is made up not only of the sperm aster but also of tubulin-staining fibers originating elsewhere in the egg. The monaster does not divide directly but is broken down first before the amphiaster or interphase asters begin to form. The interphase asters reach a peak of development at the streak stage and are in turn broken down before the formation of the mitotic apparatus. The breakdown of the monaster, interphase asters, as well as the asters of the mitotic apparatus proceeds from the cell center or aster centers to the periphery of the cell and is followed by growth of new asters, also proceeding outward from the aster centers. The pattern suggests a transient wavelike movement of some condition, or factor, which favors microtubule depolymerization.     

Aster formation in eggs of Xenopus laevis. Induction by isolated basal bodies

We have assayed various materials for their ability to induce aster formation by microinjection into unfertilized eggs of Xenopus laevis. We have found that purified basal bodies from Chlamydomonas reinhardtii and Tetrahymena pyriformis induce the formation of asters and irregular cleavage furrows within 1 h after injection. Other microtubule structures such as flagella, flagellar axonemes, cilia, and brain microtubules are completely ineffective at inducing asters or cleavage furrows in unfertilized eggs. When known amounts of sonicated Tetrahymena and Chlamydomonas preparations are injected into unfertilized eggs, 50% of the injected eggs show a furrowing response at approximately 3 cell equvalents for Chlamydomonas and 0.1 cell equivalent for Tetrahymena. These results are close to those expected if basal bodies were the effective astral-inducing agent in these cells. Other materials effective at inducing asters in unfertilized eggs, such as crude brain nuclei, sperm, and a particulate fraction from brain known to induce parthenogenesis in eggs of Rana pipiens, probably contain centrioles as the effective agent. Our experiments provide the first functional assay to indicate that centrioles play an active role in aster initiation. None of the injected materials effective in unfertilized eggs produced any observable response in fully grown oocytes. Oocytes and eggs were found to have equal tubulin pools as judged by colchicine-binding activity. Therefore, the inability of oocytes to form asters cannot be due to a lack of an organizing center or to a lack of tubulin. Experiments in which D2O was found to stimulate aster-like fibrous areas in eggs but not oocytes suggest that the inability of oocytes to form asters may be due to an inability of tubulin in oocytes to assemble.     

High incidence of multiple aster formation in vitrified-warmed bovine oocytes after in vitro fertilization

In vitro-matured bovine oocytes do not tolerate vitrification as well as mature murine or human oocytes. Delayed first cleavage in vitrified and in vitro-fertilized bovine oocytes may be responsible for the decreased yield of blastocysts in vitro. Because formation of sperm-aster and the subsequent assembly of microtubule network play an important role for migration and fusion of both pronuclei, aster formation in vitrified-warmed oocytes was analyzed by confocal laser-scanning microscopy. At 10 h post-insemination (hpi), proportions of oocytes fertilized normally were comparable between the vitrified and fresh control groups (67 and 70%, respectively). Proportions of oocytes that exhibited microtubule assembly were similar between the two groups (95% each), but the proportion of oocytes with multiple asters was higher in the vitrified group when compared with the fresh control group (68 vs 29%, P < 0.05). Both migration and development of two pronuclei were adversely affected by multiple aster formation. In the next experiment, multiple asters observed in 5.5 vs 8 hpi pronuclear zygotes were located near the male pronucleus, suggesting that those multiple asters were not the cytoplasmic asters of maternal origin. In conclusion, multiple aster formation frequently observed in vitrified-warmed bovine oocytes may be related to loss of ooplasmic function responsible for normal microtubule assembly from the sperm-aster.     

Cytoskeleton and chromatin reorganization in horse oocytes following intracytoplasmic sperm injection: patterns associated with normal and defective fertilization

Intracytoplasmic sperm injection (ICSI) is the method of choice for fertilizing horse oocytes in vitro. Nevertheless, for reasons that are not yet clear, embryo development rates are low. The aims of this study were to examine cytoskeletal and chromatin reorganization in horse oocytes fertilized by ICSI or activated parthenogenetically. Additional oocytes were injected with a sperm labeled with a mitochondrion-specific vital dye to help identify the contribution of the sperm to zygotic structures, in particular the centrosome. Oocytes were fixed at set intervals after sperm injection and examined by confocal laser scanning microscopy. In unfertilized oocytes, microtubules were present only in the metaphase-arrested second meiotic spindle and the first polar body. After sperm injection, an aster of microtubules formed adjacent to the sperm head and subsequently enlarged such that at the time of pronucleus migration and apposition it filled the entire cytoplasm. During syngamy, the microtubule matrix reorganized to form a mitotic spindle on which the chromatin of both parents aligned. Finally, after nuclear and cellular cleavage were complete, the microtubule asters dispersed into the interphase daughter cells. Sham injection induced parthenogenetic activation of 76% of oocytes, marked by the formation of multiple cytoplasmic microtubular foci that later developed into a dense microtubule network surrounding the female pronucleus. The finding that a parthenote alone can produce a microtubule aster, whereas the aster invariably forms at the base of the sperm head during normal fertilization, indicates that both gametes contribute to the formation of the zygotic centrosome in the horse. Finally, 25% of sperm-injected oocytes failed to complete fertilization, mostly due to absence of oocyte activation (65%), which was often accompanied by failure of sperm decondensation. In conclusion, this study demonstrated that union of the parental genomes in horse zygotes is accompanied by a series of integrated cytoskeleton-mediated events, failure of which results in developmental arrest.     

Asters play only a dispensable role in the induction of the cleavage furrow in the blastomeres of early Xenopus embryos

Kuroda et al. (2001) of our laboratory have previously revealed that exposure of early Xenopus embryos to 150 mm urethane results in complete suppression of formation of the asters and the cleavage furrow, as well as significant reduction of the size of the spindle in the blastomeres, allowing only 1 or 2 cycles of mitosis but not cytokinesis. In the course of closer examination of the effect of urethane on the cleavage of blastomeres of early Xenopus embryos, we unexpectedly discovered that exposure of early Xenopus embryos to 75 mm urethane did not prevent cell division at all, though asters were not detected in the blastomeres. Instead, they contained a spindle that appeared rather normal. They also formed the diastema, a thin yolk-free structure, which is considered to play an essential role in the induction of the cleavage furrow. Essentially the same results were obtained in the exposure of embryos to vinblastine, a well-known microtubule inhibitor: exposure of embryos to 20 micro g/mL vinblastine resulted in complete suppression of cleavage of the blastomeres, where formation of both the spindle and asters were perfectly suppressed. By contrast, exposure of embryos to 5 microg/mL vinblastine did not prevent cleavage in the blastomeres though asters were not detected, whereas the rather normal spindle was formed. Thus, there was a close correlation between the formation of the normal spindle, not asters, and that of the cell division furrow and the diastema in the blastomeres of early Xenopus embryos. We suggest that while the spindle plays an essential role, asters are likely to play only a dispensable role in the induction of the cleavage furrow in even very large cells like the blastomeres of early Xenopus embryos.     

Nuclear transplantation in mouse embryos: assessment of recipient cell stage

Enucleated zygotes were compared with enucleated two-cell embryos as recipients for donor nuclei from eight-cell embryos. Only one or two cleavage divisions were observed when eight-cell nuclei were transplanted to enucleated zygotes. Development of enucleated two-cell embryos containing a transplanted eight-cell nucleus was appreciably better with 51% (45/89) of the embryos forming blastocysts in vitro and 42% (25/60) initiating implantation. Of these, eleven implantation sites on Day 10 of gestation were examined histologically and two contained normally developing embryos. No development was observed beyond Day 12 of gestation. These observations indicate that a major transition occurs between the zygote and two-cell stage that results in the two-cell recipient being more compatible with the eight-cell nucleus than with the zygote.     

Microtubules in ascidian eggs during meiosis, fertilization, and mitosis

The sequential changes in the distribution of microtubules during germinal vesicle breakdown (GVBD), fertilization, and mitosis were investigated with antitubulin indirect immunofluorescence microscopy in several species of ascidian eggs (Molgula occidentalis, Ciona savignyi, and Halocynthia roretzi). These alterations in microtubule patterns were also correlated with observed cytoplasmic movements. A cytoplasmic latticework of microtubules was observed throughout meiosis. The unfertilized egg of M. occidentalis had a small meiotic spindle with wide poles; the poles became focused after egg activation. The other two species had more typical meiotic spindles before fertilization. At fertilization, a sperm aster first appeared near the cortex close to the vegetal pole. It enlarged into an unusual asymmetric aster associated with the egg cortex. The sperm aster rapidly grew after the formation of the second polar body, and it was displaced as far as the equatorial region, corresponding to the site of the myoplasmic crescent, the posterior half of the egg. The female pronucleus migrated to the male pronucleus at the center of the sperm aster. The microtubule latticework and the sperm aster disappeared towards the end of first interphase with only a small bipolar structure remaining until first mitosis. At mitosis the asters enlarged tremendously, while the mitotic spindle remained remarkably small. The two daughter nuclei remained near the site of cleavage even after division was complete. These results document the changes in microtubule patterns during maturation in Ascidian oocytes, demonstrate that the sperm contributes the active centrosome at fertilization, and reveal the presence of a mitotic apparatus at first division which has an unusually small spindle and huge asters.     

Reproductive maternal centrosomes are cast off into polar bodies during maturation division in starfish oocytes

Animal egg inherits a maternal centrosome from the meiosis-II spindle and sperm can introduce another centrosome at fertilization. It has been believed that in most animals only the sperm centrosome provides the division poles for mitosis in zygotes. This uniparental (paternal) inheritance of the centrosome must depend on the loss of the maternal centrosome. In starfish, suppression of polar body (PB) extrusion is a prerequisite for induction of parthenogenesis (Washitani-Nemoto et al. (1994) Dev. Biol. 163, 293-301), suggesting that the centrosomes cast off into PBs have reproducing capacity. Due to the absence of centriole duplication in meiosis II of starfish oocytes, each centrosome of a meiosis-II spindle has only one single centriole, whereas in meiosis I each has a pair of centrioles (Sluder et al. (1989) Dev. Biol. 131, 567-579; Kato et al. (1990) Dev. Growth Differ. 32, 41-49). Hence, the first PB (PB1) has two centrioles, whereas the second PB (PB2) and the mature egg have only one centriole, respectively. The present study examined the reproductive capacity of PB centrosomes by transplanting them into artificially activated eggs, and then the recipient egg nucleus with the surrounding cytoplasm was removed. A transplanted PB2 centrosome with a single centriole formed a monopolar spindle at the first mitosis, followed by formation of a bipolar spindle in the next mitosis, leading to actual cleavage and subsequent development. This proves the reproducing capacity of the single centriole in the PB2 centrosome. The behavior of the transplanted PB1 centrosome was exactly the same as in the PB2 centrosome, in spite of the difference in the number of centrioles. These results clearly show that four maternal centrioles are heterogeneous in duplicating capacity, during meiosis only one centriole in each of the centrosomes of a meiosis-I spindle pole retains duplicating capacity, the reproductive centrioles are successively cast off into PBs, and finally a mature egg inheriting a nonreproductive centriole alone is formed, and the presence of a single reproductive centriole is sufficient condition for embryonic development in starfish.     

Evidence for an involvement of actin in the positioning and motility of centrosomes

Cultured human polymorphonuclear leukocytes exposed to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) spread on the substratum and undergo centrosome splitting. The two centrioles may separate by a distance of several micrometers, each being surrounded by an aster of microtubules. Here we show that the centriole/aster complexes are in constant, rapid motion through the cytoplasm, carrying with them some of the cytoplasmic granules while pushing aside others, or deforming and displacing the nucleus. An analysis of this unique motility phenomenon was undertaken. We show that intact microtubules are required for TPA-induced centrosome splitting and aster motility, but not for cell spreading. More importantly, disruption of the actin network inhibits both centrosome splitting and cell spreading, and even reverses splitting (induces convergence and fusion of asters) in polymorphonuclear leukocytes pretreated with TPA alone. These observations indicate the existence of a dynamic relationship between microtubules and actin networks and provide evidence for a role of actin in determining the position of the centrosome by way of interaction with the microtubules radiating from it.     

Microinjected Polystyrene Beads Move Along Astral Rays in Sand Dollar Eggs

Movements of polystyrene beads along astral rays of the sperm aster and the mitotic aster were investigated in eggs of the sand dollars, Clypeaster japonicus and Scaphechinus mirabilis . Polystyrene beads injected into the unfertilized egg were at a standstill in the protoplasm. After fertilization, these beads exhibited movements toward the center of the sperm aster along the rays, and finally gathered around the astral center. They were distributed in blastomeres together with the mitotic centers during successive cleavages. When injected into eggs during mitosis, beads moved to the centers of the mitotic asters along astral rays. The injected beads did not move when the aster was disorganized by treatment with Colcemid, and moved when it formed after UV-irradiation. These results indicate that microtubules of astral rays are essential to the movement of polystyrene beads. The movement of small polystyrene beads (0.2–0.3 μm in diameter) resembled the saltatory movement of endogenous cytoplasmic granules, and the movement of large beads (ca. 1 μm in diameter) resembled the female pronuclear migration. All of these movements observed in fertilized eggs were demonstrated to be microtubule-dependent, perhaps sharing the same basic mechanisms.