Motility and centrosomal organization during sea urchin and mouse fertilization

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.     

Activation of maternal centrosomes in unfertilized sea urchin eggs.

Centrosomes are undetectable in unfertilized sea urchin eggs, and normally the sperm introduces the cell's microtubule-organizing center (MTOC) at fertilization. However, artificial activation or parthenogenesis triggers microtubule assembly in the unfertilized egg, and this study explores the reappearance and behavior of the maternal centrosome. During activation with A23187 or ammonia, microtubules appear first at the cortex; centrosomal antigen is detected diffusely throughout the entire cytoplasm. Later, the centrosome becomes more distinct and organizes a radial microtubule shell, and eventually a compact centrosome at the egg center organizes a monaster. In these activated eggs, centrosomes undergo cycles of compaction and decompaction in synchrony with the chromatin, which also undergoes cycles of condensation and decondensation. Parthenogenetic activation with heavy water (50% D2O) or the microtubule-stabilizing drug taxol (10 microM) induces numerous centrosomal foci in the unfertilized sea urchin egg. Within 15 min after incubation in D2O, numerous fine centrosomal foci are detected, and they organize a connected network of numerous asters which fill the entire egg. Taxol induces over 100 centrosomal foci by 15 min after treatment, which organize a corresponding number of asters. The centrosomal material in either D2O- or taxol-treated eggs aggregates with time to form fewer but denser foci, resulting in fewer and larger asters. Fertilization of eggs pretreated with either D2O or taxol shows that the paternal centrosome is dominant over the maternal centrosome. The centrosomal material gradually becomes associated with the enlarged sperm aster. These experiments demonstrate that maternal centrosomal material is present in the unfertilized egg, likely as dispersed undetectable material, which can be activated without paternal contributions. At fertilization, paternal centrosomes become dominant over the maternal centrosomal material.     

T-1, a mitotic arrester, alters centrosome configurations in fertilized sea urchin eggs

T-1 induces modifications in the shape of the centrosome at division in fertilized eggs of the North American sea urchin, Lytechinus pictus. Phase contrast microscopy observations of mitotic apparatus isolated from T-1-treated (1.7-8.5 microM) eggs at first division shows that the centrosomes already begin to spread or to separate by prophase and that the mitotic spindle is barrel-shaped. When eggs are fertilized with sperm that have been preteated with T-1, the centrosomes become flattened; the spindles are of normal length. Immunofluorescence microscopy using an anti-centrosomal monoclonal antibody reveals that T-1 modifies the structure of the centrosome so that barrel-shaped spindles with broad centrosomes are observed at metaphase, rather than the expected focused poles and fusiform spindle. Higher concentrations of T-1 induce fragmentation of centrosomes, causing abnormal accumulation of microtubules in polar regions. These results indicate that T-1 directly alters centrosomal configuration from a compact structure to a flattened or a spread structure. T-1 can be classified as a new category of mitotic drugs that may prove valuable in dissecting the molecular nature of centrosomes.     

Microtubules are required for centrosome expansion and positioning while microfilaments are required for centrosome separation in sea urchin eggs during fertilization and mitosis

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.     

The centrosome cycle in the mitotic cycle of sea urchin eggs

When sea urchin eggs entering mitosis are exposed to an appropriate concentration of mercaptoethanol, the chromosome cycle is restrained while the centrosome cycle advances. The two poles of the mitotic apparatus separate into four poles, while the chromosomes remain in their metaphase arrangements until released by the removal of the mercaptoethanol. We follow the centrosomes through the stages of the generation of two poles by each original pole. In electron microscopic studies, the osmiophilic component of the centrosomes serves as an indicator of their changing forms as each pole generates two poles. In light microscopic studies, including observations of birefringence, the shapes of the polar ends of the spindles are taken as indicators of the shapes of the centrosomes. The successive stages of the centrosome cycle are (1) compact spherical centrosomes at the time of formation of the mitotic apparatus; (2) expansion and flattening of the centrosomes, leading to (3) formation of thin flat plates, perpendicular to the spindle axis. Corresponding to the extended flat shape of the centrosomes, the spindle poles are flat; microtubules 'point' to the centrosomal plate and not the centrioles. The centrioles are separated in the flattening of the centrosomes. (4) The flat plate divides into two and each of the two halves becomes more compact, defining two separate poles. Our findings resurrect and update Boveri's [5] observations and interpretations of the centrosome. Centrosomes have shapes. The shapes may be imparted to the microtubular structures that they generate. The formation of two separate centrosomes from one, in the formation of mitotic poles, is describable as a sequence of changes in shape.     

Centrosome reduction during gametogenesis and its significance

Animal spermatids and primary oocytes initially have typical centrosomes comprising pairs of centrioles and pericentriolar fibrous centrosomal proteins. These somatic cell-like centrosomes are partially or completely degenerated during gametogenesis. Centrosome reduction during spermiogenesis comprises attenuation of microtubule nucleation function, loss of pericentriolar material, and centriole degeneration. Centrosome reduction during oogenesis is due to complete degeneration of centrioles, which leads to dispersal of the pericentriolar centrosomal proteins, loss of replicating capacity of the spindle poles, and switching to acentrosomal mode of spindle organization. Oocyte centrosome reduction plays an important role in preventing parthenogenetic embryogenesis and balancing centrosome number in the embryonic cells.     

The Plk1 target Kizuna stabilizes mitotic centrosomes to ensure spindle bipolarity

Formation of a bipolar spindle is essential for faithful chromosome segregation at mitosis. Because centrosomes define spindle poles, defects in centrosome number and structural organization can lead to a loss of bipolarity. In addition, microtubule-mediated pulling and pushing forces acting on centrosomes and chromosomes are also important for bipolar spindle formation. Polo-like kinase 1 (Plk1) is a highly conserved Ser/Thr kinase that has essential roles in the formation of a bipolar spindle with focused poles. However, the mechanism by which Plk1 regulates spindle-pole formation is poorly understood. Here, we identify a novel centrosomal substrate of Plk1, Kizuna (Kiz), depletion of which causes fragmentation and dissociation of the pericentriolar material from centrioles at prometaphase, resulting in multipolar spindles. We demonstrate that Kiz is critical for establishing a robust mitotic centrosome architecture that can endure the forces that converge on the centrosomes during spindle formation, and suggest that Plk1 maintains the integrity of the spindle poles by phosphorylating Kiz.     

Regulation of the paternal inheritance of centrosomes in starfish zygotes

In most animals, fertilized eggs inherit one centrosome from a meiosis-II spindle of oocytes and another centrosome from the sperm. However, since first proposed by Boveri [Sitzungsber. Ges. Morph. Phys. Münch. 3 (1887) 151-164] at the turn of the last century, it has been believed that only the paternal (sperm) centrosome provides the division poles for mitosis in animal zygotes. This uniparental (paternal) inheritance of centrosomes is logically based on the premise that the maternal (egg) centrosome is lost before the onset of the first mitosis. For the processes of the selective loss of the maternal centrosome, three models have been proposed: One stresses the intrinsic factors within the centrosome itself; the other two emphasize external factors such as cytoplasmic conditions or the sperm centrosome. In the present study, we have examined the validity of one of the models in which the sperm centrosome overwhelms the maternal centrosomes. Because centrosomes cast off into both the first and the second polar bodies (PB) are known to retain the capacity for reproduction and cell-division pole formation, we observed the behavior of those PB centrosomes with reproductive capacity and the sperm centrosome in the same zygotic cytoplasm. We prepared two kinds of fertilized eggs that contain reproductive maternal centrosomes, (1) by micromanipulative transplantation of the PB centrosomes into fertilized eggs, and (2) by suppression of the PB extrusions of fertilized eggs with cytochalasin B. In both types of eggs, the PB centrosomes could double and form cell-division poles, indicating that they are not suppressed by the sperm centrosome, which in turn indicates that selective loss of the maternal centrosome is due to intrinsic factors within the centrosomes themselves.     

Dynamics of microtubules and positioning of female pronucleus during bovine parthenogenesis

The zygote centrosome, consisting of both paternal and maternal centrosomal components, is the microtubule-organizing center necessary for pronuclear migration and positioning in fertilization. Maternal centrosomal function in microtubule organization and pronuclear positioning, however, remains unclear. In the present study, we sought to elucidate the function of maternal centrosomes during bovine parthenotes in the microtubule organizational processes required to move the pronucleus to the cell center without sperm centrosomal components. Microtubule organization, pronuclear position, and distribution of gamma-tubulin, which is thought to be the major component of maternal centrosomal material, were imaged by immunocytochemistry and conventional epifluorescence microscopy. In bovine parthenotes treated with paclitaxel, a microtubule-stabilizing drug, the cytoplasmic microtubule asters became organized after chemical activation, and the microtubules radiated dynamically toward the female pronucleus. The microtubule patterns correlated well with pronuclear movement to the cell center. Microtubules aggregated at regions of gamma-tubulin concentration, but gamma-tubulin did not localize to a spot until the first interphase of bovine parthenogenesis. These findings indicate that gamma-tubulin is responsible for microtubule organization as the maternal centrosome. In bovine parthenogenesis, the maternal centrosome then organizes cytoplasmic microtubules to move the female pronucleus into the cell center. We propose that the maternal centrosome plays a role as a functional centrosome despite the lack of a sperm contribution, making this structure less competent for microtubule organization in comparison with centrosomes containing sperm centrosomal components.     

Phosphorylation of a 225-kDa centrosomal component in mitotic CHO cells and sea urchin eggs

Components of centrosomes are those among cellular proteins that are phosphorylated at the transition from interphase to mitosis. Using an anti-phosphoprotein antibody (CHO3) directed against isolated mitotic CHO spindles, we identified a 225-kDa centrosomal phosphocomponent in mitotic CHO cells and in cleaving sea urchin eggs. The 225-kDa protein is tightly attached to the centrosome, which allowed us to separate it from other spindle-associated factors by high salt extraction. Phosphorylation of the 225-kDa protein occurred during mitosis. This was shown by isotope labeling on gels as well as by visualization of thiophosphorylated centrosomes with an anti-thiophosphoprotein antibody (M. Cyert, T. Scherson, and M. W. Kirschner, 1988, Dev. Biol. 129, 209) after preincubation with ATP-gamma-S in vivo and in vitro. Mitotic spindles isolated from CHO cells retained their ability to phosphorylate the centrosomal component, whereas sea urchin spindles did not, possibly due to loss or inactivation of protein kinase(s) during spindle isolation. The enzyme associated with isolated CHO spindles was extractable by high salt treatment and was capable of phosphorylating many spindle components, including the 225-kDa centrosomal protein of CHO cells and sea urchin embryos. Such high salt extracts contain protein kinases, including cell cycle control protein kinase p34cdc2, suggesting that the enzyme responsible for centrosomal phosphorylation could be p34cdc2 or other downstream mitotic kinases activated by the action of p34cdc2.     

A kinesin-related protein, KRP(180), positions prometaphase spindle poles during early sea urchin embryonic cell division

We have investigated the intracellular roles of an Xklp2-related kinesin motor, KRP(180), in positioning spindle poles during early sea urchin embryonic cell division using quantitative, real-time analysis. Immunolocalization reveals that KRP(180) concentrates on microtubules in the central spindle, but is absent from centrosomes. Microinjection of inhibitory antibodies and dominant negative constructs suggest that KRP(180) is not required for the initial separation of spindle poles, but instead functions to transiently position spindle poles specifically during prometaphase.     

Influence of centriole behavior on the first spindle formation in zygotes of the brown alga Fucus distichus (Fucales, Phaeophyceae)

The influence of centrioles, derived from the sperm flagellar basal bodies, and the centrosomal material (MTOCs) on spindle formation in the brown alga Fucus distichus (oogamous) was studied by immunofluorescence microscopy using anti-centrin and anti-beta-tubulin antibodies. In contrast to a bipolar spindle, which is formed after normal fertilization, a multipolar spindle was formed in polyspermic zygote. The number of mitotic poles in polyspermic zygotes was double the number of sperm involved in fertilization. As an anti-centrin staining spot (centrioles) was located at these poles, the multipolar spindles in polyspermic zygotes were produced by the supplementary centrioles. When anucleate egg fragments were fertilized, chromosome condensation and mitosis did not occur in the sperm nucleus. Two anti-centrin staining spots could be detected, microtubules (MTs) radiated from nearby, but the mitotic spindle was never produced. When a single sperm fertilized multinucleate eggs (polygyny), abnormal spindles were also observed. In addition to two mitotic poles containing anti-centrin staining spots, extra mitotic poles without anti-centrin staining spots were also formed, and as a result multipolar spindles were formed. When karyogamy was blocked with colchicine, it became clear that the egg nucleus proceeded independently into mitosis accompanying chromosome condensation. A monoastral spindle could be frequently observed, and in rare cases a barrel-shaped spindle was formed. However, when a sperm nucleus was located near an egg nucleus, the two anti-centrin staining spots shifted to the egg nucleus from the sperm nucleus. In this case, a normal spindle was formed, the egg chromosomes arranged at the equator, and the associated MTs elongated from one pole of the egg spindle toward the sperm chromosomes which were scattered. From these results, it became clear that paternal centrioles derived from the sperm have a crucial role in spindle formation in the brown algae, such as they do during animal fertilization. However, paternal centrioles were not adequate for the functional centrosome during spindle formation. We speculated that centrosomal materials from the egg cytoplasm aggregate around the sperm centrioles and are needed for centrosomal activation.     

CEP215 is involved in the dynein-dependent accumulation of pericentriolar matrix proteins for spindle pole formation

CEP215 is a human orthologue of Drosophila centrosomin which is a core centrosome component for the pericentriolar matrix protein recruitment. Recent investigations revealed that CEP215 is required for centrosome cohesion, centrosomal attachment of the g-TuRC, and microtubule dynamics. However, it remains to be obscure how CEP215 functions for recruitment of the centrosomal proteins during the centrosome cycle. Here, we investigated a role of CEP215 during mitosis. Knockdown of CEP215 resulted in characteristic mitotic phenotypes, including monopolar spindle formation, a decrease in distance between the spindle pole pair, and detachment of the centrosomes from the spindle poles. We noticed that CEP215 is critical for centrosomal localization of dynein throughout the cell cycle. As a consequence, the selective centrosomal proteins were not recruited to the centrosome properly. Finally, the centrosomal localization of CEP215 also depends on the dynein-dynactin complex. Based on the results, we propose that CEP215 regulates a dynein-dependent transport of the pericentriolar matrix proteins during the centrosome maturation.     

VDAC3 regulates centriole assembly by targeting Mps1 to centrosomes

Centrioles are duplicated during S-phase to generate the two centrosomes that serve as mitotic spindle poles during mitosis. The centrosomal pool of the Mps1 kinase is important for centriole assembly, but how Mps1 is delivered to centrosomes is unknown. Here we have identified a centrosome localization domain within Mps1 and identified the mitochondrial porin VDAC3 as a protein that binds to this region of Mps1. Moreover, we show that VDAC3 is present at the mother centriole and modulates centriole assembly by recruiting Mps1 to centrosomes.     

VDAC3 regulates centriole assembly by targeting Mps1 to centrosomes

Centrioles are duplicated during S-phase to generate the two centrosomes that serve as mitotic spindle poles during mitosis. The centrosomal pool of the Mps1 kinase is important for centriole assembly, but how Mps1 is delivered to centrosomes is unknown. Here we have identified a centrosome localization domain within Mps1 and identified the mitochondrial porin VDAC3 as a protein that binds to this region of Mps1. Moreover, we show that VDAC3 is present at the mother centriole and modulates centriole assembly by recruiting Mps1 to centrosomes.     

Microtubule and centrosome distribution during sheep fertilization

The distribution of microtubules and centrosomes was studied during sheep fertilization by electron and immunofluorescence microscopy. Tubulin and centrosomal material was identified with monoclonal anti-alpha-tubulin and MPM-2 antibodies, respectively. In ovulated eggs, microtubules were exclusively found in the meiotic spindle and centrosomal material at each of its poles. At fertilization, sperm centrosomes were incorporated into the egg and organized the sperm astral microtubules. During pronuclear development and migration, the sperm aster increased in size; microtubules of the sperm aster extended from the male pronucleus to the egg center and towards the female pronucleus. The position of the sperm aster during pronuclear migration suggests that it plays a role in this process. When the pronuclei were in apposition in the egg center, a dense array of microtubules and the centrosomal material were present between the two pronuclei. The proximal centriole of the sperm was identified by electron microscopy, between the apposed pronuclei. The centrosomal material extending around the centriole and the sperm neck and proximal mid-piece, apparently contained several foci from which microtubules radiated. These data suggest that in sheep unlike in mice, centrosomal material originating from the sperm is involved in the fertilization events.     

Centrosome inheritance in starfish zygotes: selective loss of the maternal centrosome after fertilization

The mature egg inherits a centrosome from the second meiotic spindle, and the sperm introduces a second centrosome at fertilization. Since only one of these centrosomes survives to be used in development, specific mechanisms must exist to control centrosome inheritance. To investigate how centrosome inheritance is controlled we used starfish eggs as a model system, because they undergo meiosis after fertilization. As a result, the fate of the maternal and paternal centrosomes can be followed by light microscopy and experimentally manipulated in vivo. We show initially that only the paternal centrosome is used in starfish zygote development; the maternal centrosome retained from meiosis II is functionally lost before first mitosis. We then tested a number of possible ways in which the zygote could exert this differential control over the stability of centrosomes initially residing in the same cytoplasm. The results of these experiments can be summarized as follows: (1) Although the microtubule organizing center activity of the maternal centrosome is not degraded after meiosis, the ability of this centrosome to double at successive mitoses is lost. (2) The sperm centrosome is not "masked" from cytoplasmic conditions which could destabilize all centrosomes during or after the meiotic sequence. (3) The functional loss of the maternal centrosome is not due to its cortical location. (4) The loss of this doubling capacity is determined by the egg, not by putative inhibitory factors from the fertilizing sperm. (5) The destabilization of the maternal centrosome is not due to the complete loss of its centrioles. Together, these results demonstrate that all maternal centrosomes are equivalent and that they are intrinsically different from the paternal centrosome. This intrinsic difference, in concert with a change in cytoplasmic conditions after meiosis, determines the selective loss of the maternal centrosome inherited from the meiosis II spindle.     

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.     

The centrosomin protein is required for centrosome assembly and function during cleavage in Drosophila.

Centrosomin is a 150 kDa centrosomal protein of Drosophila melanogaster. To study the function of Centrosomin in the centrosome, we have recovered mutations that are viable but male and female sterile (cnnmfs). We have shown that these alleles (1, 2, 3, 7, 8 and hk21) induce a maternal effect on early embryogenesis and result in the accumulation of low or undetectable levels of Centrosomin in the centrosomes of cleavage stage embryos. Hemizygous cnn females produce embryos that show dramatic defects in chromosome segregation and spindle organization during the syncytial cleavage divisions. In these embryos the syncytial divisions proceed as far as the twelfth cycle, and embryos fail to cellularize. Aberrant divisions and nuclear fusions occur in the early cycles of the nuclear divisions, and become more prominent at later stages. Giant nuclei are seen in late stage embryos. The spindles that form in mutant embryos exhibit multiple anomalies. There is a high occurrence of apparently linked spindles that share poles, indicating that Centrosomin is required for the proper spacing and separation of mitotic spindles within the syncytium. Spindle poles in the mutants contain little or no detectable amounts of the centrosomal proteins CP60, CP190 and (gamma)-tubulin and late stage embryos often do not have astral microtubules at their spindle poles. Spindle morphology and centrosomal composition suggest that the primary cause of these division defects in mutant embryos is centrosomal malfunction. These results suggest that Centrosomin is required for the assembly and function of centrosomes during the syncytial cleavage divisions.     

Functional role of centrosomes in spindle assembly and organization

The centrosome is the main MT organizing center in animal cells, and has traditionally been regarded as essential for organization of the bipolar spindle that facilitates chromosome segregation during mitosis. Centrosomes are associated with the poles of the mitotic spindle, and several cell types require these organelles for spindle formation. However, most plant cells and some female meiotic systems get along without this organelle, and centrosome‐independent spindle assembly has now been identified within some centrosome containing cells. How can such observations, which point to mutually incompatible conclusions regarding the requirement of centrosomes in spindle formation, be interpreted? With emphasis on the functional role of centrosomes, this article summarizes the current models of spindle formation, and outlines how observations obtained from spindle assembly assays in vitro may reconcile conflicting opinions about the mechanism of spindle assembly. It is further described how Drosophila mutants are used to address the functional interrelationships between individual centrosomal proteins and spindle formation in vivo. © 2004 Wiley‐Liss, Inc.