Abnormal behavior of the yolk centrosomes during early embryogenesis of Drosophila melanogaster

After the 10th nuclear cycle the yolk centrosomes follow an irregular pathway. Unlike the somatic centrosomes, which move to the opposite poles of the nuclei to form the bipolar spindles, the yolk centrosomes remain as pairs at one pole of the yolk nuclei or shift feebly and nucleate irregular spindles, most of which have only one main pole. The yolk centrosomes are no longer observed near the yolk nuclei, but progressively move away into the surrounding cytoplasm. Despite the irregular behavior of the centrosomes and although the yolk nuclei cease to divide, the yolk centrosome duplication cycle continues. The early development of Drosophila thus provides an excellent natural system for the study of the uncoupling of the nuclear and centrosomal cycles.     

Assembly of yolk spindles in the early Drosophila embryo

The development of the early Drosophila embryo is marked by the separation of two nuclear lineages, yolk and somatic nuclei, each having its own division program despite residing in a common cytoplasm. We show that the failure of nuclear division of the yolk nuclei is a consequence of dysfunction in bipolar spindle organization during mitosis 10 and 11. Yolk spindle organization defects are directly correlated to centrosome behaviour, which is abnormal in at least three sequential aspects. First, the yolk centrosomes do not migrate properly along the nuclear envelope during nuclear cycles 10 and 11 and give rise to non-functional monopolar spindles. Second, the centrosomes detached from the poles spindle at the end of nuclear cycle 11, leaving the spindles anastral. Third, the free centrosomes duplicate in the absence of nuclear division during last mitoses and early gastrulation, but do not separate properly. In spite of their reduced nucleating properties, beyond the nuclear cycle 12, the yolk centrosomes contain typical centrosomal antigens, suggesting that their structural organization has not been changed after they disperse in the cytoplasm. Our findings also demonstrate that the centrosome dynamics are spatially and temporally regulated in the yolk region. This observation is consistent with the presence of rate-limiting levels of maternally provided key molecular components, needed for centrosome duplication and positioning. The presence of normal and abnormal centrosomes in the same cytoplasm provides an useful model for investigating the common regulators of the nucleus and centrosome cycle which ensure precise spindle pole duplication.     

Drop out: a third chromosome maternal-effect locus required for formation of the Drosophila cellular blastoderm.

drop out (dop) is a recessive maternal-effect locus identified in a screen for female-sterile mutations in Drosophila polytene region 71C-F. Phenotypic analyses of the dop mutation indicate that the gene is required for proper formation of the cellular blastoderm. In embryos derived from either homozygous or hemizygous dop mothers, cytoplasmic clearing, nuclear migration and division, and pole cell formation appear normal. However, developmental defects are observed prior to and during cellularization of the blastoderm. At the beginning of nuclear cycle 14, the distinct separation of the internal yolk mass and the cortical cytoplasm breaks down. Subsequently, a population of somatic nuclei located at the periphery of the syncytial blastoderm becomes irregularly spaced and nonuniform in their distribution. Despite a somewhat regular formation of the cortical actin network, cellularization in mutant embryos is extremely variable. Such embryos fail to gastrulate normally and produce variable amounts of defective cuticle. Overall, our analyses suggest that the dop gene functions in maintaining the separation of yolk and cortical cytoplasm and in stabilizing the distribution of somatic nuclei in the Drosophila syncytial blastoderm.     

Nuclear division and migration during early embryogenesis of Bradysia tritici coquillet (syn. Sciara ocellaris) (diptera : Sciaridae)

Nuclear division and migration of cleavage nuclei in the embryos of Bradysia tritici (Diptera : Sciaridae) have been studied by light microscopy and nuclear staining. There are 8 cleavage cycles up to the syncytial blastoderm stage (4.5 hr), and during the 11th cycle cellularization begins (6.5 hr). The first 3 divisions take about 30 min each. During the 5th and 6th cycles, the maximum rate of division is reached (12 min/cycle at 22°C). After pole cell formation, the duration of the following mitotic cycles increases progressively. During nuclear migration, the presumptive germ line nuclei reach the egg cortex first, followed by anterior somatic nuclei and finally, posterior somatic nuclei reach the egg cortex. Possibly as a result of this region-specific nuclear migration, nuclear divisions become parasynchronous after 3 hr of embryogenesis (4th cycle). Several mitotic cycles later, between the 8th and 10th cycle in different embryos, X-chromosome elimination in somatic nuclei begins at the anterior egg pole and progresses in anteroposterior direction. Our observations suggest that the observed region-specific differences may be due to the activity of localized factors in the egg that control migration and nuclear cycle of the somatic nuclei.     

Reorganisation du Type Endomixique chez les Loxodidae et chez les Centrophorella

New investigations of the nuclear cycle in the gymnostome ciliate Loxodes rostrum verify the long-neglected hypothesis proposed by Bütschli that in this multinucleated ciliate the macronuclei never divide. The consequences of this "caryosterose" are compensated for by a process of endomixis, that is, by the transformation of a certain number of the micronuclei, during vegetative multiplication, into new macronuclei.
The nuclear cycle in the gymnostome Centrophorella fistulosa does not show any aspect of nuclear division. Interpretation of this supposes the existence of polyenergid nuclei which fragment, during the interdivisional period, into subnuclei; among these some evolve in the somatic and macronuclear direction, before disappearance by karyolysis; others, corresponding to the micronuclei, grow by endomitosis, and reconstitute the polyploid and polyenergid nuclei. Thus the very particular nuclear cycle of C. fistulosa is believed to exhibit a new aspect in the endomictic process of nuclear reorganization.     

Initiation of DNA replication in nuclei from quiescent cells requires permeabilization of the nuclear membrane

We have investigated the replication capacity of intact nuclei from quiescent cells using Xenopus egg extract. Nuclei, with intact nuclear membranes, were isolated from both exponentially growing and contact- inhibited BALB/c 3T3 fibroblasts by treatment of the cells with streptolysin-O. Flow cytometry showed that > 90% of all contact- inhibited cells and approximately 50% of the exponential cells were in G0/G1-phase at the time of nuclear isolation. Intact nuclei were assayed for replication in the extract by incorporation of [alpha- 32P]dATP or biotin-dUTP into nascent DNA. Most nuclei from exponential cells replicated in the egg extract, consistent with previous results showing that intact G1 nuclei from HeLa cells replicate in this system. In contrast, few nuclei from quiescent cells replicated in parallel incubations. However, when the nuclear membranes of these intact quiescent nuclei were permeabilized with lysophosphatidylcholine prior to addition to the extract, nearly all the nuclei replicated under complete cell cycle control in a subsequent incubation. The ability of LPC-treated quiescent nuclei to undergo DNA replication was reversed by resealing permeable nuclear membranes with Xenopus egg membranes prior to extract incubation demonstrating that the effect of LPC treatment is at the level of the nuclear membrane. These results indicate that nuclei from G1-phase cells lose their capacity to initiate DNA replication following density-dependent growth arrest and suggest that changes in nuclear membrane permeability may be required for the initiation of replication upon re-entry of the quiescent cell into the cell cycle.     

Ion channels in murine nuclei during early development and in fully differentiated adult cells

Summary The nuclear envelope functions as a selective barrier between nucleus and cytoplasm. During cycles of cell division the nuclear envelope repeatedly disassembles and re-associates. Presumably, each cycle re-establishes the functional and structural integrity of the nuclear envelope. After repeated rounds of cell division, as occurs during differentiation, the selectivity and configuration of the envelope may change. We compare the ionic conductance and the nuclear pore density in four types of murine nuclei: germinal vesicles in oocytes, pronuclei in zygotes, nuclei from two-cell blastomeres, and somatic cell nuclei from the liver. A large-conductance ion channel is present in all nuclear envelopes. Liver cell nuclei have a greater number of these channels than those from earlier developmental stages, and they also have a higher density of nuclear pores. In this article we hypothesize an association between the ion channels and the nuclear pores.     

Generation of a transgenic medaka (Oryzias latipes) strain for visualization of nuclear dynamics in early developmental stages

In this study, we verified nuclear transport activity of an artificial nuclear localization signal (aNLS) in medaka fish (Oryzias latipes). We generated a transgenic medaka strain expresses the aNLS tagged enhanced green fluorescent protein (EGFP) driven by a medaka beta‐actin promoter. The aNLS‐EGFP was accumulated in the nuclei of somatic tissues and yolk nuclei of oocytes, but undetectable in the spermatozoa. The fluorescent signal was observed from immediately after fertilization by a maternal contribution. Furthermore, male and female pronuclei were visualized in fertilized eggs, and nuclear dynamics of pronuclear fusion and subsequent cleavage were captured by time‐lapse imaging. In contrast, SV40NLS exhibited no activity of nuclear transport in early embryos. In conclusion, the aNLS possesses a strong nuclear localization activity and is a useful probe for fluorescent observation of the pronuclei and nuclei in early developmental stage of medaka.     

哺乳动物核移植中供核与受体卵胞质细胞周期的相互关系

就供核与受体卵胞质细胞周期的相互关系问题进行了综述.核移植技术不管是在基础理论,还是在应用研究中都具有广泛的应用价值,但核移植的效率却很低,其根本原因是与核移植相关的许多基础理论问题尚不清楚,对这些问题的研究发现,维持重构卵核的正确倍性,并使其重新程序化是核移植成功的关键,不同的胞质受体及不同的供体细胞及其状态均对重构胚的发育有影响.     

DNA polymerase epsilon may be dispensable for SV40- but not cellular-DNA replication.

The contributions of DNA polymerases alpha, delta, and epsilon to SV40 and nuclear DNA syntheses were evaluated. Proteins were UV-crosslinked to nascent DNA within replicating chromosomes and the photolabelled polymerases were immunopurified. Only DNA polymerases alpha and delta were detectably photolabelled by nascent SV40 DNA, whether synthesized in soluble viral chromatin or within nuclei isolated from SV40-infected cells. In contrast, all three enzymes were photolabelled by the nascent cellular DNA. Mitogenic stimulation enhanced the photolabelling of the polymerases in the alpha>delta>epsilon order of preference. The data agree with the notion that DNA polymerases alpha and delta catalyse the principal DNA polymerisation reactions at the replication fork of SV40 and, perhaps, also of nuclear chromosomes. DNA polymerase epsilon, implicated by others as a cell-cycle checkpoint regulator sensing DNA replication lesions, may be dispensable for replication of the small, fast propagating virus that subverts cell cycle controls.     

The use ofXenopus oocytes and embryos as a route towards cell replacement

When nuclei of somatic cells are transplanted to enucleated eggs ofXenopus, a complete reprogramming of nuclear function can take place. To identify mechanisms of nuclear reprogramming, somatic nuclei can be transplanted to growing meiotic oocytes ofXenopus, and stem cell genes activated without DNA replication. The combination of somatic cell nuclear transfer with morphogen signalling and the community effect may lead towards the possibility of cell replacement therapy. When mechanisms of nuclear reprogramming are understood, it may eventually be possible to directly reprogramme human somatic cell nuclei without the use of eggs.     

The stability of the yolk granules of Artemia. An improved method for their isolation and study

The yolk granules of Artemia behave as unstable structures during isolation, especially after hatching. A stringent dechorionization of the cysts is required for an easy homogenization and good extraction of the yolk granules. The use of Ficoll and the avoidance of high dilutions in the homogenization process allows the isolation in an intact state of a pure yolk granules fraction without nuclear contamination. Previously described methods for the isolation of yolk granules lead to low recoveries of these structures in preparations with nuclear contamination. Lipovitellin, the major yolk protein, can be used as a biochemical marker to monitor the recovery of yolk granules and the possible yolk contamination of other subcellular fractions.The isolation of nuclei is also improved, their double membrane preserved in an enriched fraction with less yolk contamination than usually obtained with previously described methods.The other major storage structure, the lipoid bodies, can be isolated intact in these conditions, whereas they are also disrupted in common homogenization media.     

Telophase-stage host ooplasts support complete reprogramming of roscovitine-treated somatic cell nuclei in cattle

Nuclear-cytoplasmic incompatibilities are known to play a significant role in the developmental outcome of embryos produced by nuclear transfer, particularly when metaphase arrested oocytes are used as hosts for interphase donor nuclei. To further our understanding of how cell cycle coordination affects somatic cell cloning, somatic cells at different stages of the cell cycle were fused to host oocytes either before (metaphase II, M-II) or after (telophase II, T-II) activation. To obtain cells at different stages of the cell cycle, fetal fibroblast (FF) and granulosa cells (GC) were treated with roscovitine, an inhibitor of cyclin-dependent kinases (CDKs) resulting in a large percentage of cells in S/G(2)-phase. In contrast to the M-II group, which did better with confluent cells, embryos reconstructed with T-II cytoplasts resulted in higher rates of blastocyst formation when fused to cells recovered at 16-24 h after passage. Embryos reconstructed with FF treated with roscovitine and T-II cytoplasts (Rosc/T-II) resulted in similar blastocyst rate compared to those produced with confluent cells and M-II cytoplasts (Conf/M-II). Transfer of blastocysts to surrogate heifers resulted pregnancies and birth of healthy calves from Rosc/T-II and Conf/M-II reconstructed embryos. These results indicate that, when combined with nuclear donor cells at specific cell cycle stages, M-II and T-II bovine oocytes are similarly effective in supporting the reprogramming of somatic cell nuclei.     

Dissimilar Staining Properties of Purified and Certified Toluidine Blue

Certified toluidine blue (National Aniline Co.). applied to sections of frog blastulae, stained the nuclei light blue and left the yolk platelets either unstained or light blue. Purified toluidine blue (also National Aniline Co.) stained the nuclei a deep blue and the yolk platelets a brilliant pink with deep blue borders. Some of the observations suggest that this difference in staining behavior is due to the presence of an inhibitor in the certified dye, which suppresses the metachromatic staining of the platelets and reduces the intensity of the nuclear staining. Unsuccessful attempts were made to remove the inhibitor by salting out the certified dye and washing it with alcohol or by extracting it with chloroform. Details of these attempts, and of other experiments designed to identify the stainable substrates in the yolk platelets are given in the text.     

The disappearance of the nuclear lamina during spermatogenesis: An electron microscopic and immunofluorescence study

The nuclear lamina is a proteinaceous layer lying directly beneath the inner nuclear membrane in somatic cells. Here we demonstrate by indirect immunofluorescence and electron microscopy that the lamina is completely absent from the nuclei of spermatocytes and spermatids of the chicken. The absence of a lamina in these cells can also be demonstrated in isolated nuclei lacking the two nuclear membranes. Implications of this finding for possible functions of the nuclear lamina are discussed.     

Identification of porcine oocyte proteins that are associated with somatic cell nuclei after co-incubation

Relatively little is known with respect to the oocyte proteins that are involved in nuclear reprogramming of somatic cells in mammals. The aim of the present study was to use a cell-free incubation system between porcine oocyte proteins and somatic cell nuclei and to identify oocyte proteins that remain associated with these somatic cell nuclei. In two separate experiments, porcine oocytes were either labeled with biotin to label total proteins at the germinal vesicle stage or metaphase II stage or they were labeled with 0.1 mM (35)S-methionine either during the first 6 h or 22-28 h of in vitro maturation to characterize protein synthesis during two distinct phases. To determine which oocyte proteins associate with somatic nuclei, labeled proteins were incubated in a collecting buffer and energy-regenerating system with isolated ovarian epithelial-like cell nuclei. After incubation, the nuclei were subjected to a novel affinity-binding system to recover biotin-labeled oocyte proteins or two-dimensional SDS-PAGE for separation and visualization of radiolabeled proteins. Proteins of interest were sent for identification using either matrix-assisted laser desorption/ionization time of flight or liquid chromatography-tandem mass spectrometry. Of the proteins that remain associated with isolated nuclei after incubation, 4 were identified using the affinity-binding system and 24 were identified using mass spectrometry and the two-dimensional gel interface. This study has identified porcine oocyte proteins that associate with somatic cell nuclei in a cell-free system using proteomics techniques, providing a novel way to identify oocyte proteins potentially functionally involved in nuclear reprogramming.     

The nuclear division and parasexual cycle inPenicillium

The vegetative nuclear division inPenicillium differs from classical mitosis, and a model for the division process is presented. In early divisional stages the interconnected chromosomes are arranged in a ring which breaks, giving rise to a linear configuration which divides by longitudinal splitting. The break may occur with equal probability between each of the chromosomes. At the end of the division process the daughter nuclei regain ring structure. One of the chromosomes is believed to represent a separation center for all the chromosomes. In diploid strains the two haploid genomes show a close somatic association and the nuclear configurations occurring during the divisional cycle are identical to those in the haploids. The double break of the ring structures in diploids will give recombinant nuclei in certain cases. The model explains the available data of the parasexual cycle, and both diploidization and haploidization are believed to represent singlestep processes.     

Cytological investigations of the parasexual cycle in fungi I. Nuclear fusion

Summary Investigations have been conducted to obtain specific, cytological evidence for nuclear fusions in heterokaryotic mycelium and in other vegetative (somatic) fungal cells. Results to-date have been inconclusive suggesting that such fusions possibly occur at a rate lower than 1 in 10⁷. It is also proposed that, in addition to the rare occurrence of somatic nuclear fusions, other processes may initiate the parasexual or some similar cycle. These processes are based upon the possible transfer of genetic material during pairing of unlike, vegetative nuclei or during close associations noted between mitochondria and nuclei.