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PubMed1992

[A method for demonstrating chromosomes from human sperm and its application].

A cytogenetic technique for analysis of human sperm chromosomes is described in this article. It is based on penetration of hamster oocytes after enzymatic treatment and inhibition of cell division during pronuclei stage. Two different methods of capacitation of sperm, that is necessary for them to penetrate the eggs have been considered. Attention is paid to manipulations and technical difficulties on consecutive steps of realization of this method. This method is applied mainly in analysis of chromosome segregation in males, that are carriers of chromosome aberrations. The possibility of using this method in other clinical cases and basic research has been presented.

Animals↗
PubMed2006

Evidence of allopolyploidy in Brachiaria brizantha (Poaceae: Paniceae) through chromosome arrangement at metaphase plate during microsporogenesis.

In the hexaploid (2n = 6x = 54) accession B176 of Brachiaria brizantha, one cytological characteristic differentiated it from the other accessions previously analyzed with the same ploidy level. Nearly 40% of meiocytes displayed the chromosome set arranged at two metaphase plates at the poles of the cell, close to the membrane. In these cells, both metaphase plates were arranged in an angle to form a typical tripolar spindle. Therefore, cells did not show normal chromosome segregation at anaphase I. Only nine univalent chromosomes migrated from each plate to the opposite pole with the remainder staying immobile on the plate. As a result of such spindle orientation and chromosome behavior, trinucleate telophases I were recorded. After telophase, cytokinesis eliminated the small nuclei into a microcyte. The second division proceeded normally, with the presence of microcytes in all phases. The origin of such an abnormality was explained on the hexaploid level of the accession which could have resulted by chromosome doubling of a triploid derived from species that did not display the same behavior for spindle organization. The high percentage of meiotic abnormalities recorded in this accession compromises fertility and renders it inadequate for the breeding program.

Brachiaria↗
PubMed1986

Recessive lethal t haplotypes increase the frequency of the partial trisomy of chromosome 17 (including the T-t complex) among offspring of T(16;17)43H female mice.

The male-sterile reciprocal autosomal translocation T(16;17)43H displays a high frequency of adjacent-2 disjunction in meiosis of translocation heterozygotes. One haploid product of this abnormal chromosome segregation leads to viable partial trisomy of chromosome 17 after fertilization. The frequency of trisomics increases in the progeny of T43H/+ females when the wild-type allelic form of T-t complex is substituted for t12, tw32 or t6 recessive lethal haplotypes. Indirect evidence supports the idea that the suppression of crossing-over by t haplotypes is linked with the increase in adjacent-2 disjunction and subsequent Ts43H trisomy. The possible use of Ts43H trisomy for genetic dissection of the T-t complex is briefly discussed.

Animals↗
PubMed1994

[A method for visualization of chromosomes from human sperm and its application].

A cytogenetic technique for analysis of human sperm chromosomes is described in this article. It is based on penetration of hamster oocytes after enzymatic treatment and inhibition of cell division during pronuclei stage. Two different methods of capacitation of sperm, that is necessary for them to penetrate the eggs have been considered. Attention is paid to manipulations and technical difficulties on consecutive steps of realisation of this method. This method is applied mainly in analysis of chromosome segregation in males, that are carriers of chromosome aberrations. The possibility of using this method in other clinical cases and basic research has been presented.

Animals↗
PubMed2006

Mitotic phosphatases: no longer silent partners.

Recent work has highlighted the important role played by protein phosphatase complexes in the regulation of mitosis from yeast to mammals. There have been important advances in defining the roles of the protein serine/threonine phosphatases PP1 and PP2A and the dual specificity protein tyrosine phosphatases CDC25 and Cdc14. Three independent studies defined a regulatory role for PP2A in the control of sister chromatid cohesion, involving a direct interaction with shugoshin. A chromatin targeting subunit has been identified for PP1 and the complex shown to play an essential role in chromosome segregation. Key regulatory residues within CDC25 have been mapped and its activity tied both to the initial activation of cyclin-dependent kinases at the centrosome and to DNA damage checkpoints. Novel roles have been defined for Cdc14, including regulation of rDNA and telomere segregation and participation in spindle assembly. These exciting advances show that protein phosphatases are not merely silent partners to kinases in regulating the control of cell division.

Animals↗
PubMed2000

Sister chromatid separation: falling apart at the seams.

Cohesion between sister chromatids must be dissolved at the time of chromosome segregation. Recent studies reveal that the principles of cohesion dissolution in mitosis and meiosis are the same, but that there are important differences that stem from the distinct natures of these two processes.

Animals↗
PubMed2003

Reproductive semi-cloning respecting biparental origin. A biologically unsound principle.

The original debate article proposed the use of "semi-cloning" as a viable method for assisted reproduction. This debate counters the proposal as being biologically unsound. Given the fundamental limitations of chromosomal segregation and genomic imprinting, the notion of using the MII oocyte to drive haploidization of a somatic cell genome and thereby obtain a substitute for authentic gametes is ill-conceived and untenable.

Chromosome Segregation↗
PubMed2006

The centrosome cycle.

Centrosomes are dynamic organelles involved in many aspects of cell function and growth. Centrosomes act as microtubule organizing centers, and provide a site for concerted regulation of cell cycle progression. While there is diversity in microtubule organizing center structure among eukaryotes, many centrosome components, such as centrin, are conserved. Experimental analysis has provided an outline to describe centrosome duplication, and numerous centrosome components have been identified. Even so, more work is needed to provide a detailed understanding of the interactions between centrosome components and their roles in centrosome function and duplication. Precise duplication of centrosomes once during each cell cycle ensures proper mitotic spindle formation and chromosome segregation. Defects in centrosome duplication or function are linked to human diseases including cancer. Here we provide a multifaceted look at centrosomes with a detailed summary of the centrosome cycle.

Animals↗
PubMed2004

The conserved kinetochore protein shugoshin protects centromeric cohesion during meiosis.

Meiosis comprises a pair of specialized nuclear divisions that produce haploid germ cells. To accomplish this, sister chromatids must segregate together during the first meiotic division (meiosis I), which requires that sister chromatid cohesion persists at centromeres. The factors that protect centromeric cohesion during meiosis I have remained elusive. Here we identify Sgo1 (shugoshin), a protector of the centromeric cohesin Rec8 in fission yeast. We also identify a homologue of Sgo1 in budding yeast. We provide evidence that shugoshin is widely conserved among eukaryotes. Moreover, we identify Sgo2, a paralogue of shugoshin in fission yeast, which is required for faithful mitotic chromosome segregation. Localization of Sgo1 and Sgo2 at centromeres requires the kinase Bub1, identifying shugoshin as a crucial target for the kinetochore function of Bub1. These findings provide insights into the evolution of meiosis and kinetochore regulation during mitosis and meiosis.

Amino Acid Sequence↗
PubMed2005

A dual role for Bub1 in the spindle checkpoint and chromosome congression.

The spindle checkpoint ensures faithful chromosome segregation by linking the onset of anaphase to the establishment of bipolar kinetochore-microtubule attachment. The checkpoint is mediated by a signal transduction system comprised of conserved Mad, Bub and other proteins. In this study, we use live-cell imaging coupled with RNA interference to investigate the functions of human Bub1. We find that Bub1 is essential for checkpoint control and for correct chromosome congression. Bub1 depletion leads to the accumulation of misaligned chromatids in which both sister kinetochores are linked to microtubules in an abnormal fashion, a phenotype that is unique among Mad and Bub depletions. Bub1 is similar to the Aurora B/Ipl1p kinase in having roles in both the checkpoint and microtubule binding. However, human Bub1 and Aurora B are recruited to kinetochores independently of each other and have an additive effect when depleted simultaneously. Thus, Bub1 and Aurora B appear to function in parallel pathways that promote formation of stable bipolar kinetochore-microtubule attachments.

Animals↗
PubMed2004

A puromycin-sensitive aminopeptidase is essential for meiosis in Arabidopsis thaliana.

Puromycin-sensitive aminopeptidases (PSAs) participate in a variety of proteolytic events essential for cell growth and viability, and in fertility in a broad range of organisms. We have identified and characterized an Arabidopsis thaliana mutant (mpa1) from a pool of T-DNA tagged lines that lacks PSA activity. This line exhibits reduced fertility, producing shorter siliques (fruits) bearing a lower number of seeds compared with wild-type plants. Cytogenetic characterization of meiosis in the mutant line reveals that both male and female meiosis are defective. In mpa1, early prophase I appears normal, but after pachytene most of the homologous chromosomes are desynaptic, thus, by metaphase I a high level of univalence is observed subsequently leading to abnormal chromosome segregation. Wild-type plants treated with specific inhibitors of PSA show a very similar desynaptic phenotype to that of the mutant line. A fluorescent PSA-specific bioprobe, DAMPAQ-22, reveals that the protein is maximally expressed in wild-type meiocytes during prophase I and is absent in mpa1. Immunolocalization of meiotic proteins showed that the meiotic recombination pathway is disrupted in mpa1. Chromosome pairing and early recombination appears normal, but progression to later stages of recombination and complete synapsis of homologous chromosomes are blocked.

Amino Acid Sequence↗
PubMed2004

Occurrence of maternal and paternal spindles in unfertilized human oocytes: possible relationship to nucleation defects after silent fertilization.

Experience with conventional clinical IVF indicates that a first cleavage can occur in the absence of detectable pronuclear formation (so-called silent fertilization). In these instances, the first division is often asymmetrical and delayed when compared with normally fertilized siblings. In this study, DNA configurations and spindle organization were examined by fluorescence microscopy in metaphase II human oocytes that remained unfertilized after conventional IVF and were considered likely candidates for silent fertilization. The results show comparatively high frequencies of penetration in the absence of detectable pronuclear evolution, and that both a maternal meiotic and a sperm-derived mitotic-like spindle can coexist in the same oocyte. Patterns of cell division and blastomere nucleation in silent fertilizations suggest the possibility that this division may involve uniparental chromosomal segregation in which maternal and paternal DNA is differentially partitioned into daughter blastomeres. This pattern of inheritance may generate certain types of ploidy and nucleation defects detected at the 2- to 4-cell stage.

Blastomeres↗
PubMed2005

Silencing of human Int-6 impairs mitosis progression and inhibits cyclin B-Cdk1 activation.

The Int-6 protein has been originally identified as the product of a mouse gene being a frequent integration site of the mouse mammary tumour virus. Here, we show that reducing Int-6 expression by RNA interference in HeLa cells markedly alters mitosis progression. Defects in spindle formation, chromosome segregation and cytokinesis were observed. These abnormalities of mitosis completion are correlated with an inhibition of cyclin B-Cdk1 kinase activity, due to a prolonged inhibitory phosphorylated state of Cdk1. In line with this observation, the Wee1 tyrosine kinase that negatively controls Cdk1 was less efficiently inactivated during G2 in Int-6-depleted cells. These findings support the notion that the oncogenic properties associated with alteration of Int-6 originate from chromosomal instability.

CDC2 Protein Kinase↗
PubMed1998

The POLO kinase is required at multiple stages during spermatogenesis in Drosophila melanogaster.

The polo gene of Drosophila melanogaster is the founding member of the polo-like kinase family which is conserved among eukaryotes. POLO has been implicated in the organisation and function of the mitotic apparatus. Furthermore, POLO has been shown to be required for normal spermatogenesis. To characterize further the role of POLO in spermatogenesis, polo mutants were analysed by immunostaining with specific antibodies and phase contrast microscopy. Immunofluorescence shows that POLO localises to the centrosomes, the centromere/kinetochore and the spindle midzone. The meiotic phenotype of various mutant allelic combinations was also studied in detail. Observation of mutant live testes indicates cytological abnormalities in all meiotic cell types, including variable DNA content and multipolar spindles. Primary spermatocytes in polo mutant testes contain an abnormal DNA content, suggesting failure of chromosome segregation during gonial division. Immunostaining of polo mutant cells with alpha-tubulin shows several abnormalities of the meiotic spindle, including a significantly reduced central spindle. Our results suggest that polo has multiple functions during spermatogenesis.

Animals↗
PubMed2004

Meiotic recombination: sealing the partnership at the junction.

Crossovers ensure proper chromosome segregation in meiosis. A heterodimer of MutS proteins, hMSH4-hMSH5, has recently been found to interact with recombination intermediates in a manner that suggests a mechanism for directing meiotic DNA double strand break repair towards a crossover pathway.

Adenosine Triphosphatases↗
PubMed2005

Distinct nuclear and cytoplasmic functions of the S. pombe Cdc14-like phosphatase Clp1p/Flp1p and a role for nuclear shuttling in its regulation.

Cdc14-like phosphatases regulate a variety of cell cycle events by dephosphorylating CDK sites. Their cell cycle-dependent changes in localization may be important to carry out distinct functions. Work in budding and fission yeast suggested that Cdc14-like phosphatases are inhibited by nucleolar sequestration. In S. cerevisiae, Cdc14p is released from the nucleolus by the FEAR network and Cdk1, whereas the S. pombe CDC14-like phosphatase Clp1p (also known as Flp1p) is released at mitotic entry by an unknown mechanism. The mitotic exit network (MEN) in S. cerevisiae and its homologous network, the septation initiation network (SIN), in S. pombe act through an unknown mechanism to keep the phosphatase out of the nucleolus in late mitosis. SIN-dependent cytoplasmic maintenance of Clp1p is thought to be essential for the cytokinesis checkpoint, which blocks further rounds of nuclear division until cytokinesis is completed. By targeting Clp1p to the nucleus or the cytoplasm, we demonstrate distinct functions for these pools of Clp1p in chromosome segregation and cytokinesis, respectively. Our results further suggest that the SIN does not keep Clp1p out of the nucleolus by regulating nucleolar affinity, as proposed for S. cerevisiae Cdc14p, but instead, Clp1p may be regulated by nuclear import/export.

Active Transport, Cell Nucleus↗
PubMed2000

Functional redundancy in the maize meiotic kinetochore.

Kinetochores can be thought of as having three major functions in chromosome segregation: (a) moving plateward at prometaphase; (b) participating in spindle checkpoint control; and (c) moving poleward at anaphase. Normally, kinetochores cooperate with opposed sister kinetochores (mitosis, meiosis II) or paired homologous kinetochores (meiosis I) to carry out these functions. Here we exploit three- and four-dimensional light microscopy and the maize meiotic mutant absence of first division 1 (afd1) to investigate the properties of single kinetochores. As an outcome of premature sister kinetochore separation in afd1 meiocytes, all of the chromosomes at meiosis II carry single kinetochores. Approximately 60% of the single kinetochore chromosomes align at the spindle equator during prometaphase/metaphase II, whereas acentric fragments, also generated by afd1, fail to align at the equator. Immunocytochemistry suggests that the plateward movement occurs in part because the single kinetochores separate into half kinetochore units. Single kinetochores stain positive for spindle checkpoint proteins during prometaphase, but lose their staining as tension is applied to the half kinetochores. At anaphase, approximately 6% of the kinetochores develop stable interactions with microtubules (kinetochore fibers) from both spindle poles. Our data indicate that maize meiotic kinetochores are plastic, redundant structures that can carry out each of their major functions in duplicate.

Biomechanical Phenomena↗
PubMed2002

Drosophila EB1 is important for proper assembly, dynamics, and positioning of the mitotic spindle.

EB1 is an evolutionarily conserved protein that localizes to the plus ends of growing microtubules. In yeast, the EB1 homologue (BIM1) has been shown to modulate microtubule dynamics and link microtubules to the cortex, but the functions of metazoan EB1 proteins remain unknown. Using a novel preparation of the Drosophila S2 cell line that promotes cell attachment and spreading, we visualized dynamics of single microtubules in real time and found that depletion of EB1 by RNA-mediated inhibition (RNAi) in interphase cells causes a dramatic increase in nondynamic microtubules (neither growing nor shrinking), but does not alter overall microtubule organization. In contrast, several defects in microtubule organization are observed in RNAi-treated mitotic cells, including a drastic reduction in astral microtubules, malformed mitotic spindles, defocused spindle poles, and mispositioning of spindles away from the cell center. Similar phenotypes were observed in mitotic spindles of Drosophila embryos that were microinjected with anti-EB1 antibodies. In addition, live cell imaging of mitosis in Drosophila embryos reveals defective spindle elongation and chromosomal segregation during anaphase after antibody injection. Our results reveal crucial roles for EB1 in mitosis, which we postulate involves its ability to promote the growth and interactions of microtubules within the central spindle and at the cell cortex.

Anaphase↗