Microtubule arrays in fucoid zygotes are sensitive to cytoplasmic pH

Regulation of microtubule (MT) arrays and embryo‐genesis by cytoplasmic pH (pH_c) was investigated in zygotes of the brown alga Pelvetia compressa (J. Agardh) De Toni. pH_c was clamped to (set to) acidic values using a weak acid, propionic acid (PA), and to alkaline values using a weak base, methylamine (MA). Acidification of pH_c from the normal value of 7.4–7.5 to about 7.0 caused disruption of microtubule arrays. The nucleating activity was delocalized from the centrosomes and dispersed over the nuclear envelope, the number of MTs decreased, and MTs failed to extend into the cell cortex. Alkalinization to about pH 8.0 also caused dispersal of nucleating activity, but distinct centrosomes remained. MTs coursed in various directions following MA treatment, giving the array a disorganized appearance. Two MT‐dependent processes, rhizoid morphogenesis and cell division, were found to be perturbed by small changes in pH_c.     

Ultrastructural and immunocytological studies on the rhizoplast in the chrysophycean alga Ochromonas danica

The rhizoplast, a striated band elongating from the flagellar basal body to the nucleus, is conspicuous in cells of Ochromonas danica Prings. In interphase cells, it runs from the basal body of the anterior flagellum to the space between the nucleus and the Golgi body. In O. danica, the rhizoplast duplicates during mitosis and the two rhizoplasts serve as mitotic poles. In the present study, we reinvestigated mitosis of O. danica using transmission electron microscopy and immunofluorescence microscopy, especially focusing on the rhizoplast. The nuclear envelope became dispersed during metaphase, and the rhizoplasts from two sets of the flagellar basal bodies functioned as the mitotic poles. Immunofluorescence microscopy using anti‐α‐tubulin, anti‐centrin and anti‐γ‐tubulin antibodies showed that centrin molecules were localized at the flagellar basal bodies, whereas γ‐tubulin molecules were detected at the rhizoplast during the whole cell cycle.     

Derivation of the membrane comprising the male pronuclear envelope in inseminated sea urchin eggs

Investigations were conducted in an effort to determine the origin of the membrane comprising the male pronuclear envelope of inseminated sea urchin eggs. The events of fertilization in zygotes treated with 200 μg/ml of puromycin are not impaired even though incorporation of [³H]leucine is inhibited up to 80% when compared to control specimens. Developing male pronuclei in zygotes treated with puromycin form nuclear envelopes structurally similar to and within the same period as controls. In puromycin-treated and untreated zygotes morphologically recognizable portions of the sperm nuclear envelope are incorporated into the structure of the male pronuclear envelope. Pronuclear development was also examined in inseminated ova where most of the endoplasmic reticulum (ER) was confined to a specific area of the zygote. Eggs were centrifuged in order to stratify their organelles into specific layers (stratified eggs); with further centrifugation stratified eggs are bisected to form nucleate (rich in ER) and nonnucleate halves (containing little ER). Observations of inseminated stratified eggs and nucleate and nonnucleate halves demonstrate an inverse relation between the amount of ER present in the vicinity of a reorganizing sperm nucleus and the time it takes to form the male pronuclear envelope. Computation of the maximum quantity of membrane in the male pronucleus that may be derived from the sperm nuclear envelope is approximately 15%. These investigations suggest that a major portion of the male pronuclear envelope is derived from endoplasmic reticulum within the egg and only a small portion (up to 15%) originates from the sperm nuclear envelope.     

Phosphorylation of centrin during the cell cycle and its role in centriole separation preceding centrosome duplication

Once during each cell cycle, mitotic spindle poles arise by separation of newly duplicated centrosomes. We report here the involvement of phosphorylation of the centrosomal protein centrin in this process. We show that centrin is phosphorylated at serine residue 170 during the G(2)/M phase of the cell cycle. Indirect immunofluorescence staining of HeLa cells using a phosphocentrin-specific antibody reveals intense labeling of mitotic spindle poles during prophase and metaphase of the cell division cycle, with diminished staining of anaphase and no staining of telophase and interphase centrosomes. Cultured cells undergo a dramatic increase in centrin phosphorylation following the experimental elevation of PKA activity, suggesting that this kinase can phosphorylate centrin in vivo. Surprisingly, elevated PKA activity also resulted intense phosphocentrin antibody labeling of interphase centrosomes and in the concurrent movement of individual centrioles apart from one another. Taken together, these results suggest that centrin phosphorylation signals the separation of centrosomes at prophase and implicates centrin phosphorylation in centriole separation that normally precedes centrosome duplication.     

Centrosome attachment to the C. elegans male pronucleus is dependent on the surface area of the nuclear envelope

A close association must be maintained between the male pronucleus and the centrosomes during pronuclear migration. In C. elegans, simultaneous depletion of inner nuclear membrane LEM proteins EMR-1 and LEM-2, depletion of the nuclear lamina proteins LMN-1 or BAF-1, or the depletion of nuclear import components leads to embryonic lethality with small pronuclei. Here, a novel centrosome detachment phenotype in C. elegans zygotes is described. Zygotes with defects in the nuclear envelope had small pronuclei with a single centrosome detached from the male pronucleus. ZYG-12, SUN-1, and LIS-1, which function at the nuclear envelope with dynein to attach centrosomes, were observed at normal concentrations on the nuclear envelope of pronuclei with detached centrosomes. Analysis of time-lapse images showed that as mutant pronuclei grew in surface area, they captured detached centrosomes. Larger tetraploid or smaller histone::mCherry pronuclei suppressed or enhanced the centrosome detachment phenotype respectively. In embryos fertilized with anucleated sperm, only one centrosome was captured by small female pronuclei, suggesting the mechanism of capture is dependent on the surface area of the outer nuclear membrane available to interact with aster microtubules. We propose that the limiting factor for centrosome attachment to the surface of abnormally small pronuclei is dynein.     

Cytoplasmic and sperm nuclear transformations in fertilized ammonia-activated sea urchin (Arbacia punctulata) eggs

Studies examining cytoplasmic and sperm nuclear transformations in sea urchin (Arbacia punctulata) eggs inseminated at different periods after ammonia activation have been caried out at the light- and electron-microscopic levels of observation. Arbaca eggs treated with ammonia-seawater demonstrated chromosome condensation after DNA synthesis and underwent a chromosome cycle similar to that described for Lytechinus [Mazia, 1947]. Cortical granule reaction, fertilization cone formation, and sperm aster development in eggs fertilized at 20 (interphase), 50 (prometaphase), and 180 (interphase) min after ammonia activation were structurally simialr to processes in untreated zygotes. Cyclical changes in the formation of fertilization cones and sperm asters, as reported for eggs fertilized after activation by agents that induce a cortical granule reaction, were not observed. Although sperm nuclear transformations were prolonged (14 vs 18 min), male pronuclei that developed in eggs fertilized 20 min after ammonia activation were morphologically similar to those observed in fertilized, untreated ova and incorporated ³H-thymidine. Sperm incorporated into eggs at 50 min after ammonia activation underwent nuclear envelope breakdown and chromatin despersion; however, ³H-thymidine incorporation was not observed, and male pronuclei rarely developed (less than 5% of all specimens examined). Subsequent to dispersion, the paternal chromatin condensed into chromosomes which were associated with an aster. These results demonstrate that although ammonia-activated eggs inseminated at interphase or prometaphase undergo similar cytoplasmic alterations, sperm nuclear transformations vary with the chromosome cycle of the egg.     

CDC25B Overexpression Stabilises Centrin 2 and Promotes the Formation of Excess Centriolar Foci

CDK-cyclin complexes regulate centriole duplication and microtubule nucleation at specific cell cycle stages, although their exact roles in these processes remain unclear. As the activities of CDK-cyclins are themselves positively regulated by CDC25 phosphatases, we investigated the role of centrosomal CDC25B during interphase. We report that overexpression of CDC25B, as is commonly found in human cancer, results in a significant increase in centrin 2 at the centrosomes of interphase cells. Conversely, CDC25B depletion causes a loss of centrin 2 from the centrosome, which can be rescued by treatment with the proteasome inhibitor MG132. CDC25B overexpression also promotes the formation of excess centrin 2 “foci”. These foci can accumulate other centrosome proteins, including γ-tubulin and PCM-1, and can function as microtubule organising centres, indicating that these represent functional centrosomes. Formation of centrin 2 foci can be blocked by specific inhibition of CDK2 but not CDK1. CDK2-mediated phosphorylation of Monopolar spindle 1 (Mps1) at the G1/S transition is essential for the initiation of centrosome duplication, and Mps1 is reported to phosphorylate centrin 2. Overexpression of wild-type or non-degradable Mps1 exacerbated the formation of excess centrin 2 foci induced by CDC25B overexpression, while kinase-dead Mps1 has a protective effect. Together, our data suggest that CDC25B, through activation of a centrosomal pool of CDK2, stabilises the local pool of Mps1 which in turn regulates the level of centrin 2 at the centrosome. Overexpression of CDC25B may therefore contribute to tumourigenesis by perturbing the natural turnover of centrosome proteins such as Mps1 and centrin 2, thus resulting in the de novo assembly of extra-numerary centrosomes and potentiating chromosome instability.     

Selective disappearance of maternal centrioles after fertilization in the anisogamous brown alga Cutleria cylindrica (Cutleriales, Phaeophyceae): Paternal inheritance of centrioles is universal in the brown algae

The behavior of centrioles in zygotes and female gametes developing parthenogenetically in the anisogamous brown alga Cutieria cyiindrica Okamura was studied using electron and immunofluorescence microscopy. Two pairs of centrioles, detected using anti-centrin antibody, were observed in the vicinity of the male and female nuclei, respectively, just after plasmogamy. The fluorescence intensity of one of the two centrin foci became weak 6 h after plasmogamy and finally disappeared. It was impossible to determine whether the male- or female-derived centrioles disappeared in zygotes, because there was nothing to detect morphological differences between the two centrioles. However, a prominent anti-centrin staining focus was located at the condensed male nucleus in zygotes in which karyogamy had not occurred yet. As a result, it was considered that the maternally inherited centrioles had selectively disappeared during development in C. cylindrica. The paternal inheritance of centrioles in zygotes was also confirmed by electron microscopy. Considering previous observations from oogamous and isogamous species of brown algae, we concluded that the paternal inheriance of centrioles could be universal in the brown algae.     

Relationship between nuclear DNA synthesis and centrosome reproduction in sea urchin eggs

The importance of nuclear DNA synthesis for the doubling, or reproduction, of centrosomes in cells that are not growth-limited, such as sea urchin eggs, has not been clearly defined. Studies of enucleated, fertilized eggs show that nuclear activities are not required at each cell cycle for the normal reproduction of the complete centrosome. However, other studies report that the inhibition of nuclear DNA synthesis in intact eggs by the drug aphidicolin prevents centrosome reproduction and entry into mitosis as seen by nuclear envelope breakdown. To resolve this paradox, we systematically characterized the effect of aphidicolin on cell division in eggs from three species of sea urchins. Eggs were continuously treated with 5 or 10 micrograms/ml aphidicolin starting 5 min after fertilization. This blocked total incorporation of 3H-thymidine into DNA by at least 90%, as previously reported. We found that the sperm aster always doubles prior to first mitosis. Over a period of several hours, the centrosomes reproduce in the normal 2-4-8-16 fashion, with a period that is longer and more variable than normal. In every culture, a variable percentage of the eggs undergoes nuclear envelope breakdown. Once broken down, the nuclear envelope never visibly reforms even though centrosomes continue to double. Fluorescent labeling of DNA revealed that the chromatin does not condense into discrete chromosomes. Whether or not the nuclear envelope breaks down, the chromatin appears as an amorphous mass of fibers stretched between first two and then four asters. Later, the nuclear envelope/chromatin loses its association with some or all centrosomes. Our results were the same for all eggs at both drug concentrations. Thus, nuclear DNA synthesis is not required for centrosome reproduction in sea urchin eggs.     

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.     

Electron and immunofluorescence microscopy on the fertilization ofFucus distichus (Fucales,Phaeophyceae)

Summary Processes of fertilization and zygote development inFucus distichus were studied by indirect immunofluorescence microscopy using anti- tubulin antibody and electron microscopy. Just after plasmogamy, sperm aster formation occurs during migration of a sperm nucleus toward an egg nucleus at the center of cytoplasm. Only sparse microtubules (MTs) exist around the egg nucleus. The sperm aster can be observed till karyogamy, but afterwards vanishes. Accompanying sperm aster formation, cortical MTs which are reticulately arranged develop further in the zygotes. In 4 h-old zygotes, characteristic structures which are composed of fine granular masses and consist of intermixed dense and lighter staining areas appear around the nucleus. These structures cannot be detected with anti- tubulin immunofluorescence microscopy. The two centrioles derived from the sperm separate and migrate to both poles. In 4 h-and 8 h-old zygotes, there are no defined MT foci around the zygote nucleus and MTs radiate from the circumference of it. In 12 h-old zygotes, each centriole has migrated to the poles and derivative centrioles are generated. The fine granular masses also migrate to both poles and finally disappear accompanying the appearance of numerous MTs radiating from the poles. Therefore, two distinct MT foci appear from 12 h onwards. Progressive stages of nuclear division were also examined with electron and immunofluorescence microscopy in 16 h-old zygotes. The sperm chloroplast with an eyespot and the sperm mitochondria with an intercristal tubular structure, which are distinctive from those of egg, can be detected after plasmogamy and karyogamy. The sperm chloroplast is still present in 16 h-old zygotes.     

Changes in microtubule structures during the first cell cycle of physiologically polyspermic newt eggs

The unfertilized egg of the newt, Cynops pyrrhogaster, has a second meiotic spindle at the animal pole and numerous cortical cytasters. After physiologically polyspermic fertilization, all sperm nuclei incorporated into the egg develop sperm asters, and the cortical cytasters change into bundles of cortical microtubules. The size of the sperm asters in the animal hemisphere is ∼5.6-fold larger than that in the vegetal hemisphere. Only one sperm nucleus moves toward the center of the animal hemisphere to form a zygote nucleus with the egg nucleus. This movement is inhibited by nocodazole, but not by cytochalasin B. The centrosome in the zygote nucleus divides into two parts to form a bipolar spindle for the first cleavage synchronously with the nuclear cycle, but centrosomes of accessory sperm nuclei in the vegetal hemisphere remained to form monopolar interphase asters and subsequently degenerate around the first cleavage stage. The size of sperm asters in monospermically fertilized Xenopus eggs was ∼37-fold larger than those in Cynops eggs. Since sperm asters that formed in polyspermically fertilized Xenopus eggs exclude each other, the formation of a zygote nucleus is inhibited. Cynops sperm nuclei form larger asters in Xenopus eggs, whereas Xenopus sperm nuclei form smaller asters in Cynops eggs compared with those in homologous eggs. Since there was no significant difference in the concentration of monomeric tubulin between those eggs, the size of sperm asters is probably regulated by a component(s) in egg cytoplasm. Smaller asters in physiologically polyspermic newt eggs might be useful for selecting only one sperm nucleus to move toward the egg nucleus. Mol. Reprod. Dev. 47:210–221, 1997. © 1997 Wiley-Liss, Inc.     

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.     

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.     

MITOSIS IN DUNALIELLA BIOCULATA (CHLOROPHYTA): CENTRIN BUT NOT BASAL BODIES ARE AT THE SPINDLE POLES

Centrin, a 20 kDa calmodulin-like protein, is located in various basal body-associated fibers in protists. We used indirect immunofluorescence of isolated cytoskeletons or methanol-fixed cells to analyze the distribution of centrin during mitosis of the biflagellate green alga Dunaliella bioculata (Butcher). The distance between the nucleus and the basal apparatus decreased in late interphase, presumably caused by the contraction of the two centrin-containing nucleus–basal body connectors (NBBCs). During prophase, centrin accumulated on the new basal bodies as shown by postembedding immunogold labeling of serial thin sections. The new basal bodies were in close contact with plaque-like structures on the nuclear envelope. In mitotic cells, basal body pairs were separated and positioned at a considerable distance from the poles of the mitotic spindle. At this stage, we observed four separated centrin dots, two associated with the pairs of basal bodies and two located at the spindle poles as shown by double immunofluorescence, including anti-tubulin staining. The latter signals corresponded to an accumulation of centrin between the plasma membrane and the nuclei, indicating that centrin could be involved in mitotic movements of the nuclei. In telophase, centrin was observed along the nuclear surface and one new NBBC developed in each cell half. Our results demonstrate that centrin is present at the acentriolar spindle poles of Dunaliella independently from its localization in the basal apparatus.     

Taxol inhibits the nuclear movements during fertilization and induces asters in unfertilized sea urchin eggs

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.     

Sperm nuclear dispersion coordinate with meiotic maturation in fertilized Spisula solidissima eggs

Investigations were carried out with fertilized Spisula solidissima eggs, in which changes in incorporated sperm nuclei were determined by measurement of the diameter of dispersing paternal chromatin. Results of such an analysis demonstrated that sperm nuclear dispersion does not proceed at a constant rate and consists of four phases (1–4), coordinate with major changes in the status of the maternal chromatin. (1) The first phase was a short lag period prior to germinal vesicle breakdown in which the size of the sperm nucleus increased only slightly. (2) This was followed by a rapid dispersion of the sperm nucleus coordinate with germinal vesicle breakdown. With the development of the first meiotic spindle, sperm chromatin dispersion slowed dramatically; this phase (3) lasted until the completion of the meiotic divisions at which time the sperm chromatin underwent a second rapid increase in size (4) that was correlated with development of the female pronucleus. When zygotes were treated with agents that inhibited germinal vesicle breakdown (verapamil, sodium-free seawater, and chloroquine), sperm nuclear dispersion did not occur. Evidence is presented indicating that nucleocytoplasmic interactions coincident with germinal vesicle breakdown induce sperm nuclear dispersion in Spisula zygotes.     

IMMUNOFLUORESCENCE MICROSCOPY OF FERTILIZATION AND PARTHENOGENESIS IN LAMINARIA ANGUSTATA (PHAEOPHYTA)1

Normal fertilization and parthenogenesis of unfertilized eggs were observed in Laminaria angustata Kjellman by indirect immunofluorescence microscopy using a tubulin antibody. Sperm aster formation did not occur at plasmogamy. The centrosome of the egg gradually disappeared. Shortly after karyogamy, one centrosome reappeared near the zygote nucleus. During mitosis, the centrosome replicated and the daughter centrosomes migrated to opposite poles. The mitotic spindle was formed by microtubules that elongated from both poles. After the first cell division, each of the daughter cells received one centrosome that persisted throughout the development of the sporophyte. During parthenogenetic development, abnormal mono-, tri-, and multi-polar spindles were formed. These abnormal spindles caused abnormal nuclear and cytoplasmic division. Thus, cells were produced with 1) no nuclei, 2) multiple nuclei, 3) irregular numbers of chromosomes, and/or 4) no centrosomes. This is one of the reasons for the abortion and abnormal morphogenesis during parthenogenesis. Ultrastructural observations showed that, although cells of some parthogenetic sporophytes have centrioles, cells of almost all abnormally shaped parthenogenetic sporophytes lack centrioles. These results suggest that centrioles are required for normal centrosomal functions in Laminaria. Although centrioles are inherited paternally, some centrosomal material appears to be present or produced de novo in unfertilized eggs.     

Centrin 2 localizes to the vertebrate nuclear pore and plays a role in mRNA and protein export

Centrins in vertebrates have traditionally been associated with microtubule-nucleating centers such as the centrosome. Unexpectedly, we found centrin 2 to associate biochemically with nucleoporins, including the Xenopus laevis Nup107-160 complex, a critical subunit of the vertebrate nuclear pore in interphase and of the kinetochores and spindle poles in mitosis. Immunofluorescence of Xenopus cells and in vitro reconstituted nuclei indeed revealed centrin 2 localized at the nuclear pores. Use of the mild detergent digitonin in immunofluorescence also allowed centrin 2 to be clearly visualized at the nuclear pores of human cells. Disruption of nuclear pores using RNA interference of the pore assembly protein ELYS/MEL-28 resulted in a specific decrease of centrin 2 at the nuclear rim of HeLa cells. Functionally, excess expression of either the N- or C-terminal calcium-binding domains of human centrin 2 caused a dominant-negative effect on both mRNA and protein export, leaving protein import intact. The mRNA effect mirrors that found for the Saccharomyes cerevisiae centrin Cdc31p at the yeast nuclear pore, a role until now thought to be unique to yeast. We conclude that in vertebrates, centrin 2 interacts with major subunits of the nuclear pore, exhibits nuclear pore localization, and plays a functional role in multiple nuclear export pathways.     

Reconstitution of Sperm Nuclei of Zebrafish (Danio rerio) in Xenopus Egg Extracts

Cell-free extracts of Xenopus eggs cause cyclic change in permeabilized sperm nucleus, nuclear envelope breakdown, chromosome condensation, and reformation of nuclei. In this study, the ability of cell-free extracts to cause similar changes in zebrafish sperm was examined. When lysolecithin-treated sperm from zebrafish were incubated in Xenopus egg extracts, a series of changes in sperm nuclear morphology were observed periodically. These changes correlated with maturation-promoting factor (MPF) activity. Furthermore, sperm nuclei of zebrafish replicated DNA during reconstitution in Xenopus egg extracts. These results showed that cell-free extracts of Xenopus egg possess the ability to cause cell-cycle-dependent changes in zebrafish sperm, implying the possibility of generating transgenic zebrafish in a similar way to transgenic Xenopus. Received October 21, 1999; accepted July 18, 2000.