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The forms and locations of centrosomes in mouse oocytes and in sea urchin eggs were followed through the whole course of fertilization and first cleavage by immunofluorescence microscopy. Centrosomes were identified with an autoimmune antiserum to centrosomal material. Staining of the same preparations with tubulin antibody and with the DNA dye Hoechst 33258 allowed the correlation of the forms of the centrosomes with the microtubule structures that they generate and with the stages of meiosis, syngamy, and mitosis. The results with sea urchin eggs conform to Boveri's view on the paternal origin of the functional centrosomes. Centrosomes are seen in spermatozoa and enter the egg at fertilization. Initially, the centrosomes are compact, but as the eggs enter the mitotic cycle the forms of the centrosomes go through a cycle in which they spread during interphase, apparently divide, and condense into two compact poles by metaphase. In anaphase, they spread to form flat poles. In telophase and during reconstitution of the daughter nuclei, the centrosomal material is disposed as hemispherical caps around the poleward surfaces of the nuclei. Mouse sperm lack centrosomal antigen. In the unfertilized mouse oocyte, the meiotic spindle poles are displayed as broad-beaded centrosomes. In addition, centrosomal material is detected in the cytoplasm as particles, about 16 in number, which are foci of small aster-like arrays of microtubules. The length and number of astral microtubules correlate with the size of the centrosomal foci. After sperm incorporation, as the pronuclei develop and more cytoplasmic microtubules assemble, a few of the foci associate with the peripheries of the nuclei. The number of foci multiplies during the first cell cycle. At the end of interphase, all of the centrosomal foci have concentrated on the nuclear peripheries and the cytoplasmic microtubules have disappeared. At prophase, the centrosomes are seen as two irregular clusters, marking the poles which, at metaphase and anaphase, appear as rough bands with foci, and the spindle is typically barrel-shaped. At telophase, the centrosomes are seen as arcs that lie on the nuclear peripheries after cleavage. The ordering of microtubules in all the stages reflects the shapes of the centrosomes. The findings on the sea urchin confirm the classical theory of the paternal origin of centrosomes and contrast with observations tracing the mitotic poles of the mouse egg to maternal centrosomal material. This evidence strengthens the conclusion that mouse centrosomes derive from the oocyte.

Centrosomes undergo cell cycle-dependent changes in shape and separations, changes that govern the organization of the cytoskeleton. The cytoskeleton is largely organized by the centrosome; however, this investigation explores the importance of cytoskeletal elements in directing centrosome shape. Since the sea urchin egg during fertilization and mitosis displays dramatic and synchronous changes in centrosome shape, the effects of cytoskeletal inhibitors on centrosome compaction, expansion, and separation were explored by the use of anticentrosome immunofluorescence microscopy. Centrosome expansion and separation was studied during two phases: the transition after sperm incorporation, when the compact sperm centrosome enlarges and the sperm aster develops, and from prometaphase to telophase, when the compact spindle poles enlarge. Compaction was investigated when the dispersed centrosome at interphase condenses into the two spindle poles at prometaphase. Although centrosome expansion and separation typically occur concurrently, beta-mercaptoethanol results in centrosome separation independent of expansion. Microtubule inhibitors prevent centrosome expansion and separation, and expanded centrosomes collapse. Since pronuclear union is arrested by microtubule inhibitors, this treatment also affords the opportunity to explore the relative attractiveness of the male and female pronuclei for these centrosomal antigens. Both pronuclei acquire centrosomal material; though only the male centrosome is capable of organizing a functional bipolar mitotic apparatus at first division, the female centrosome nucleates a monaster. Microfilament inhibition (cytochalasin D) prevents centrosome separation but not expansion or compaction. These results demonstrate that as the centrosome shapes the cytoskeleton, the cytoskeleton alters centrosome shape.

Taxol blocks the migrations of the sperm and egg nuclei in fertilized eggs and induces asters in unfertilized eggs of the sea urchins Lytechinus variegatus and Arbacia punctulata. Video recordings of eggs inseminated in 10 microM taxol demonstrate that sperm incorporation and sperm tail motility are unaffected, that the sperm aster formed is unusually pronounced, and that the migration of the egg nucleus and pronuclear centration are inhibited. The huge monopolar aster persists for at least 6 h; cleavage attempts and nuclear cycles are observed. Colcemid (10 microM) disassembles both the large taxol-stabilized sperm aster in fertilized eggs and the numerous asters induced in unfertilized eggs. Antitubulin immunofluorescence microscopy demonstrates that in fertilized eggs all microtubules are within the prominent sperm aster. Within 15 min of treatment with 10 microM taxol, unfertilized eggs develop numerous (greater than 25) asters de novo. Transmission electron microscopy of unfertilized eggs reveals the presence of microtubule bundles that do not emanate from centrioles but rather from osmiophilic foci or, at times, the nuclear envelope. Taxol-treated eggs are not activated as judged by the lack of DNA synthesis, nuclear or chromosome cycles, and the cortical reaction. These results indicate that: (a) taxol prevents the normal cycles of microtubule assembly and disassembly observed during development; (b) microtubule disassembly is required for the nuclear movements during fertilization; (c) taxol induces microtubules in unfertilized eggs; and (d) nucleation centers other than centrioles and kinetochores exist within unfertilized eggs; these presumptive microtubule organizing centers appear idle in the presence of the sperm centrioles.

Griseofulvin (4-6 X10-5 and 1 X 10-4 M) prevents the formation of any microtubule-based structures of sea urchin (Strongylocentrotus purpuratus, Lytechinus variegatus, Arbacia punctulata) eggs at fertilization. Sperm incorporation occurs, though the migrations of the pronuclei, dependent on the formation of the sperm aster, are arrested. Similarly in "streak" and the mitotic apparatus fail to assemble. Cycles of DNA synthesis, chromosome activity, nuclear breakdown and reconstitution, and even cleavage attempts occur on schedule in the absence of any mitotic movements. The action of griseofulvin, unlike that of colchicine, is readily reversible by the removal of the drug. Microtubules are formed, and the chromosome are separated. At 1 X 10-6 M, diminutive microtubule-based structures (e.g. sperm aster, mitotic apparatus) are observed though syngamy and division are arrested. These results demonstrate an independence of the cycle of microtubule-mediated events from other cyclical processes during the first cell cycles.

Latrunculin A, a marine toxin from a Red Sea sponge, is a potent inhibitor of the microfilament-mediated processes of fertilization and early development in sea urchins and in mice. Sperm from sea urchins, but not those from Limulus or mice, were affected by latrunculin, and fertilization in both sea urchins and in mice was arrested but at different stages. Sea urchin sperm treated with 2.6 microM latrunculin are unable to assemble acrosomal processes and their ability to fertilize eggs is impaired. The unwinding of the Limulus sperm acrosomal process occurs in the presence of latrunculin. Treated mouse sperm are able to fertilize mouse oocytes in vitro, suggesting that microfilaments may not be required in this mammalian sperm. In sea urchin eggs, sperm incorporation, microvillar elongation and cytokinesis are inhibited. Microtubule-mediated motility occurs normally. 20 nM latrunculin prevents the morphogenetic movements during gastrulation. It reduces the viscosity of actin gels from sea urchin egg homogenates. In unfertilized mouse oocytes, it prevents the colcemid-induced dispersion of the meiotic chromosomes; accumulations of cortical actin are noted adjacent to the scattered chromosomes. Sperm incorporation during mouse fertilization in vitro is unaffected suggesting that sperm entry may occur independent of microfilament activity in mammals. However, the apposition of the pronuclei at the center of the egg cytoplasm does not occur, providing evidence that cytoplasmic microfilaments may be required for the motions leading to pronuclear union during mouse fertilization. It inhibits the second polar body formation and cytokinesis. These results indicate that latrunculin is a potent inhibitor of microfilament-mediated processes in sperm, eggs and embryos, and that it may prove to be a powerful new drug for exploring the cellular behavior of microfilaments in the maintenance of cell shape and during motility.

Fodrin, a spectrin-like protein, is localized in gametes, zygotes, and embryos from sea urchins and mice. Mammalian fodrin comprises two polypeptides with molecular weights of approximately 240 kDa (alpha) and 235 kDa (beta). An antibody specific for mammalian alpha-fodrin cross-reacted with a 240-kDa polypeptide from sea urchin egg extracts. This indicates that sea urchins contain a protein of similar electrophoretic mobility and immunological properties to mammalian alpha-fodrin. When this antibody was used to stain the sea urchin gametes with indirect immunofluorescence, fodrin-specific fluorescence was localized to the acrosome of the sperm and was distributed over the entire egg near the surface in a punctate pattern similar to the distribution of polymeric actin. During sperm incorporation, the fodrin-specific fluorescence is found at the site of sperm incorporation, in the fertilization cone. After fertilization, the intensity of fodrin fluorescence increases. During mitosis and cytokinesis in sea urchins, the entire surface of the egg remains stained; the cleavage furrow also was stained but no more intensely than was the rest of the egg surface. Antibody labeling with colloidal gold followed by electron microscopy showed that fodrin was loated in the cytoplasm immediately beneath the plasma membrane. In unfertilized mouse oocytes, both actin and fodrin were stained most intensely beneath the membrane adjacent to the meiotic spindle. After insemination, the cell surfaces of the pronucleate egg and the second polar body were stained; however, the actin matrix surrounding the apposed pronuclei did not bind the fodrin antibody. During cytokinesis in the mouse, the cleavage furrow stained more intensely than did the rest of the egg cortex, and in embryos the cell borders were delineated. These results indicate that organisms as unrelated to mammals as sea urchins have fodrin-like proteins; the rearrangements of such proteins suggest that they participate in the actin-mediated events at the cell surface during fertilization and early development in both mice and sea urchins.

alpha-Tubulin in the microtubules of mouse oocytes and embryos is acetylated in a specific spatial and temporal sequence. In the unfertilized oocyte, a monoclonal antibody to the acetylated form of alpha-tubulin is bound predominantly at the poles of the arrested metaphase meiotic spindle. The labeling intensity of the spindle microtubules is weaker as observed by immunofluorescence using oocytes double-labeled for total tubulin and acetylated alpha-tubulin, and as measured by immuno high-voltage electron microscopy (immunoHVEM) with colloidal gold; cytasters are not acetylated. At meiotic anaphase, the spindle becomes labeled, and by telophase and during second polar body formation only the meiotic midbody is acetylated. The sperm axoneme retains its acetylation after incorporation though the interphase microtubules are not detected. First mitosis follows a pattern similar to that observed at the second meiosis and during interphase only the mitotic midbodies are acetylated. After treatment with cold, colcemid, or griseofulvin, the remaining stable microtubules are acetylated, but immunoHVEM observations suggest that these fibers might not have been acetylated prior to microtubule disruption. Taxol stabilization does not alter acetylation patterns. Acetylated microtubules are not necessarily old microtubules since acetylated fibers are observed at 30 sec after cold recovery. These results show the presence of acetylated microtubules during meiosis and mitosis and demonstrate a cell-cycle-specific pattern of acetylation, with acetylated microtubules found at the centrosomes at metaphase, an increase in spindle labeling at anaphase, and the selective deacetylation of all but midbody microtubules at telophase.

The sea urchin egg at fertilization is an ideal model in which to study actin-mediated surface activity. Electron microscopy of unfertilized eggs demonstrates the presence of thousands of well-arrayed short microvilli, which appear supported by cytochalasin-sensitive actin oligomers as detected with rhodamine-labeled phalloidin staining of permeabilized eggs. At insemination, the previously short microvilli elongate and cluster around the successful sperm during incorporation. Phalloidin staining demonstrates a tremendous recruitement of polymerized actin into the site of sperm incorporation, resulting in the formation of the fertilization cone. Fertilization of cytochalasin-treated eggs results in the normal activation of the metabolic and bioelectric events, but sperm incorporation does not occur since the localized actin assembly required for fertilization cone formation is precluded. After sperm incorporation, the entire fertilized surface is restructured, as a result of a massive polymerization of actin to produce a burst in microvillar elongation. Addition of cytochalasin to eggs immediately following sperm incorporation demonstrates the recruitment of actin assembly for the proper progression through the first cell cycle. During normal cell division, the egg surface retains the long microvilli. The furrow which forms at cytokinesis does not appear as a unique new structure, but rather as a reorganization of the cortical microfilaments. Quantitative fluorescence microscopy argues against an increase in microfilaments during early cytokinesis. At the latest stages of cytokinesis, a thickening of the cortical actin is noted, which could possibly be interpreted as a contractile ring. A minor basal level of actin assembly with numerous nucleation sites in unfertilized eggs and a tremendous but localized assembly of microfilaments surrounding the sperm during incorporation, followed by a massive global microfilament assembly event to elongate the fertilized egg microvilli resulting later in the reorganization of these microfilaments to produce the forces necessary for cytokinesis, highlight the utility of the study of sea urchin eggs at fertilization for understanding actin-membrane interactions.

The regulation of the microtubule-mediated motions within eggs during fertilization was investigated in relation to the shift in intracellular pH (pHi) that occurs during the ionic sequence of egg activation in the sea urchins Lytechinus variegatus and Arbacia punctulata. Microtubule assembly during formation of the sperm aster and mitotic apparatus was detected by anti-tubulin immunofluorescence microscopy, and the microtubule-mediated migrations of the sperm and egg nuclei were studied with time-lapse video differential interference contrast microscopy. Manipulations of intracellular pH were verified by fluorimetric analyses of cytoplasmic fluorescein incorporated as fluorescein diacetate. The ionic sequence of egg activation was manipulated i) to block the pHi shift at fertilization or reduce the pHi of fertilized eggs to unfertilized values, ii) to elevate artificially the pHi of unfertilized eggs to fertilized values, and iii) to elevate artificially or permit the normal pHi shift in fertilized eggs in which the pHi shift at fertilization was previously prevented. Fertilized eggs in which the pHi shift was suppressed did not assemble microtubules or undergo the normal microtubule-mediated motions. In fertilized eggs in which the pHi was reduced to unfertilized levels after the assembly of the sperm aster, no motions were detected. If the intracellular pH was later permitted to rise, normal motile events leading to division and development occurred, delayed by the time during which the pH elevation was blocked. Microtubule-mediated events occurred in eggs in which the intracellular pH was elevated, even in unfertilized eggs in which the pH was artificially increased. These results indicate that the formation and normal functioning of the egg microtubules is initiated, either directly or indirectly, by the shift in intracellular pH that occurs during fertilization.

This study concerned changes in the motional properties of cellular water during the first cell cycle of fertilized sea urchin eggs (Lytechinus variegatus). There was a significant decrease in proton NMR T1 relaxation time and in cytoplasmic ice crystal growth during mitosis and a significant increase in T1 time and cytoplasmic ice crystal size during cleavage. This was not caused by egg water content changes as reflected by egg volume measurements. Removal of both the fertilization membrane and the hyaline layer shortly after fertilization did not alter the pattern of T1 time changes at mitosis and cleavage as compared to whole eggs; thus, the pattern of T1 time changes was attributed to intracellular events. Treatment of fertilized eggs with cytochalasin B, an inhibitor of actin polymerization, did not block the fall in T1 time at mitosis, but did block cytokinesis and the increase in T1 time, which normally occurred at cleavage. A significant pattern of actin disassembly and reassembly at mitosis and cytokinesis was found by studies on the total amount of monomeric actin (G actin) using the DNase I assay. This led to the hypothesis that the observed changes in T1 time and ice crystal size during the first cell cycle were due to the depolymerization and polymerization of cytoplasmic actin. To test this, the effect of the in vitro polymerization of purified actin on the T1 time and on ice crystal growth was examined. It was concluded that changes in the T1 time and ice crystal growth upon polymerization of actin in vitro resembled the changes seen in vivo. These results suggest that changes in the motional properties of cytoplasmic water during the first cell cycle are due, at least in part, to the state of polymerization of cytoplasmic actin.

Motility and the behavior and inheritance of centrosomes are investigated during mouse and sea urchin fertilization. Sperm incorporation in sea urchins requires microfilament activity in both sperm and eggs as tested with Latrunculin A, a novel inhibitor of microfilament assembly. In contrast the mouse spermhead is incorporated in the presence of microfilament inhibitors indicating an absence of microfilament activity at this stage. Pronuclear apposition is arrested by microfilament inhibitors in fertilized mouse oocytes. The migrations of the sperm and egg nuclei during sea urchin fertilization are dependent on microtubules organized into a radial monastral array, the sperm aster. Microtubule activity is also required during pronuclear apposition in the mouse egg, but they are organized by numerous egg cytoplasmic sites. By the use of an autoimmune antibody to centrosomal material, centrosomes are detected in sea urchin sperm but not in unfertilized eggs. The sea urchin centrosome expands and duplicates during first interphase and condenses to form the mitotic poles during division. Remarkably mouse sperm do not appear to have the centrosomal antigen and instead centrosomes are found in the unfertilized oocyte. These results indicate that both microfilaments and microtubules are required for the successful completion of fertilization in both sea urchins and mice, but at different stages. Furthermore they demonstrate that centrosomes are contributed by the sperm during sea urchin fertilization, but they might be maternally inherited in mammals.

Nuclear structural changes during fertilization and embryogenesis in mice and in sea urchins have been followed by using antibodies against the nuclear lamins A/C and B and against antigens at the periphery of nuclei and chromosomes. Lamins are found on all pronuclei and nuclei during mouse fertilization, but with a diminished intensity on the second polar body nucleus. On sperm in both systems, lamins are reduced and detected only at the acrosomal and centriolar fossae. In sea urchin eggs, lamins are found on both pronuclei. Unlike in other dividing cells, the mitotic chromosomes of sea urchin eggs and embryos retain an association with lamins. The peripheral antibodies delineate each chromosome and nucleus except the mature mouse sperm nucleus. A dramatic change from the expected lamin distribution occurs during early development. In mouse morulae or blastocysts, lamins A/C are no longer recognized, although lamin B remains. In sea urchins both lamins A/C and lamin B, as detected with polyclonal antibodies, are lost after the blastula stage, although a different lamin A/C epitope emerges as recognized by a monoclonal antibody. These results demonstrate that pronucleus formation in both systems involves a new association or exposure of lamins, that the polar body nucleus is largely restricted from the cytoplasmic pool of lamins, and that mitotic chromosomes in the rapidly proliferating sea urchin egg retain associated lamins. They also suggest that changes in the expression or exposure of different lamins are a common feature of embryogenesis.

We have used in situ hybridization and cell fractionation methods to follow the distribution of U1 RNA and immunofluorescence microscopy to follow the distribution of snRNP proteins in oocytes, eggs, and embryos of several sea urchin species. U1 RNA and U1-specific snRNP antigens are concentrated in germinal vesicles of oocytes. Both appear to relocate after oocyte maturation because they are found primarily, if not exclusively, in the cytoplasm of mature unfertilized eggs. This cytoplasmic residence is maintained during early cleavage and U1 RNA is first detectable in nuclei of micromeres at the 16-cell stage. Between morula and gastrula stages the steady-state concentrations of both RNA and antigens gradually increase in nuclei and decrease in cytoplasm. Surprisingly, analysis of the distribution of newly synthesized U1 RNA shows that it does not equilibrate with the maternal pool. Instead new transcripts are confined to nuclei, while cytoplasmic U1 RNAs are of maternal origin. This lack of equilibration and the conversion of maternal U1 RNAs from nuclear species in oocytes to cytoplasmic in embryos suggests that these RNPs (or RNAs) are structurally altered when released to the cytoplasm at oocyte maturation.

The events of mammalian fertilization overlap with the completion of meiosis and first mitosis; the pro-nuclei never fuse, instead the parental genomes first intermix at the mitotic spindle equator at metaphase. Since kinetochores are essential for the attachment of chromosomes to spindle microtubules, this study explores their appearance and behavior in mouse oocytes, zygotes and embryos undergoing the completion of meiosis, fertilization and mitoses. Kinetochores are traced with immunofluorescence microscopy using autoimmune sera from patients with CREST (CREST = calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia) scleroderma. These sera cross-react with the 17 kDa centromere protein (CENP-A) and the 80 kDa centromere protein (CENP-B) found at the kinetochores in human cell cultures. The unfertilized oocyte is ovulated arrested at second meiotic metaphase and kinetochores are detectable as paired structures aligned at the spindle equator. At meiotic anaphase, the kinetochores separate and remain aligned at the distal sides of the chromosomes until telophase, when their alignment perpendicular to the spindle axis is lost. The female pronucleus and the second polar body nucleus each receive a detectable complement of kinetochores. Mature sperm have neither detectable centrosomes nor detectable kinetochores, and shortly after sperm incorporation kinetochores become detectable in the decondensing male pronucleus. In pronuclei, the kinetochores are initially distributed randomly and later found in apposition with nucleoli. At mitosis, the kinetochores behave in a pattern similar to that observed at meiosis or mitosis in somatic cells: irregular distribution at prophase, alignment at metaphase, separation at anaphase and redistribution at telophase. They are also detectable in later stage embryos. Colcemid treatment disrupts the meiotic spindle and results in the dispersion of the meiotic chromosomes along the oocyte cortex; the chromosomes remain condensed with detectable kinetochores. Fertilization of Colcemid-treated oocytes results in the incorporation of a sperm which is unable to decondense into a male pronucleus. Remarkably kinetochores become detectable at 5 h post-insemination, suggesting that the emergence of the paternal kinetochores is not strictly dependent on male pronuclear decondensation.

Scanning electron microscopy (SEM) has been successfully employed for the study of several surface-mediated events during fertilization and early development in sea urchins. In addition to basic morphological descriptions of the sperm, the extrusion of the acrosomal process has been documented with SEM. During sperm incorporation, short microvilli are found to elongate around the successful sperm. In eggs denuded of vitelline layers, in which the elevation and hardening of this fertilization coat is prevented, numerous long microvilli have been shown to cluster around and elongate over the entering sperm during sperm incorporation. Following sperm incorporation and the elevation of the fertilization coat, scanning electron microscopy has been utilized to study the bursts of elongation of the previously short egg microvilli. These microvilli appear to undergo two bursts in length due primarily to the new assembly of microfilaments in the egg cortex. Cytokinesis occurs shortly after the second burst of microvillar elongation. The morula stage is characterized by loosely attached cells which become more closely apposed in subsequent cell divisions to result in the hollow blastula. The ciliated blastula hatches from the fertilization coat, whereupon gastrulation occurs, resulting in a free-swimming, feeding larval stage. This paper reviews the surface alterations and the contribution of scanning electron microscopy to the study of these surface alterations, from fertilization through early development.

Unfertilized and fertilized sea urchin eggs were used for pulsed proton NMR spin-lattice relaxation time (T1) measurements of cellular water. An 81% increase in T1 time at fertilization was largely explained by the accumulation of extracellular water in the perivitelline space. To assess the role of microtubule and actin filament assembly and disassembly, eggs were treated with drugs that are known to change these cytoskeletal elements (i.e., colchicine, taxol and cytochalasin B). Egg volume was also monitored in all studies to rule out the influence of water content changes on the observed T1 relaxation time changes. Neither assembly nor disassembly of microtubules changed the T1 relaxation time. The role of actin polymerization and depolymerization is discussed as a possible explanation for the observed cell cycle dependent water proton T1 relaxation time changes.

Microtubule assembly is required for the formation of the male and female pronuclei during mouse, but not sea urchin, fertilization. In mouse oocytes, 50 microM colcemid prevents the decondensation of the maternal meiotic chromosomes and of the incorporated sperm nucleus during in vitro fertilization. Nuclear lamins do not associate with either of the parental chromatin sets although peripherin, the Pl nuclear peripheral antigen, appears on both. DNA synthesis does not occur in these fertilized, colcemid-arrested oocytes. This effect is limited to the first hours after ovulation, since colcemid added 4-6 hours later no longer prevents pronuclear development, lamin acquisition, or DNA synthesis. Neither microtubule stabilization with 10 microM taxol nor microfilament inhibition with 10 microM cytochalasin D or 2.2 micrograms/ml latrunculin A prevent these pronuclear events; these drugs will inhibit the apposition of the pronuclei at the egg center. In sea urchin eggs, colcemid or griseofulvin treatment does not result in the same effect and the male pronucleus forms with the attendant accumulation of the nuclear lamins. The differences in the requirement for microtubule assembly during pronucleus formation may be related to the cell cycle: In mice the sperm enters a meiotic cytoplasm, whereas in sea urchin eggs it enters an interphase cytoplasm. Refertilization of mitotic sea urchin eggs was performed to test the possibility that this phenomenon is related to whether the sperm enters a meiotic/mitotic cytoplasm or one at interphase; during refertilization at first mitosis, the incorporated sperm nucleus is unable to decondense and acquire lamins. These results indicate a requirement for microtubule assembly for the progression from meiosis to first interphase during mouse fertilization and suggest that the cytoskeleton is required for changes in nuclear architecture necessary during fertilization and the cell cycle.

Microtubules forming within the mouse egg during fertilization are required for the movements leading to the union of the sperm and egg nuclei (male and female pronuclei, respectively). In the unfertilized oocyte, microtubules are predominantly found in the arrested meiotic spindle. At the time for sperm incorporation, a dozen cytoplasmic asters assemble, often associated with the pronuclei. As the pronuclei move to the egg center, these asters enlarge into a dense array. At the end of first interphase, the dense array disassembles and is replaced by sheaths of microtubules surrounding the adjacent pronuclei. Syngamy (pronuclear fusion) is not observed; rather the adjacent paternal and maternal chromosome sets first meet at metaphase. The mitotic apparatus emerges from these perinuclear microtubules and is barrel-shaped and anastral, reminiscent of plant cell spindles; the sperm centriole does not nucleate mitotic microtubules. After cleavage, monasters extend from each blastomere nucleus. The second division mitotic spindles also have broad poles, though by third and later divisions the spindles are typical for higher animals, with narrow mitotic poles and fusiform shapes. Colcemid, griseofulvin, and nocodazole inhibit the microtubule formation and prevent the movements leading to pronuclear union; the meiotic spindle is disassembled, and the maternal chromosomes are scattered throughout the oocyte cortex. These results indicate that microtubules forming within fertilized mouse oocytes are required for the union of the sperm and egg nuclei and raise questions about the paternal inheritance of centrioles in mammals.