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At least 19 recordsLinked to original sources
PubMed1985

Inhibition by dibutyryl cyclic AMP of the transition to metaphase of mouse oocyte nuclei and its reversal by cell fusion to metaphase oocytes.

Mouse oocytes at metaphase I of meiotic maturation were treated with puromycin, which caused the condensed chromosomes to become decondensed to form an interphase nucleus. The chromosomes returned to a metaphase state 6.3 hr after the oocytes were transferred to puromycin-free medium [H. J. Clarke and Y. Masui (1983) Dev. Biol. 97, 291-301]. In contrast, the chromosomes of the puromycin-treated oocytes remained decondensed within the nucleus if dibutyryl cyclic AMP (dbcAMP) was included in the puromycin-free medium. This implies that dbcAMP inhibited the development of conditions in the oocytes that were required for the transition to metaphase. The chromosomes of puromycin-treated oocytes that were incubated for 7.5 hr in dbcAMP-containing medium returned to metaphase just 1.9 hr after transfer to dbcAMP-free medium. Therefore, the protein synthesis-dependent process that is required for the transition to metaphase could occur in the presence of dbcAMP. Fusion to metaphase II oocytes, or to puromycin-treated oocytes that had returned to metaphase, rapidly induced transition of the nuclei of dbcAMP-inhibited oocytes to metaphase, despite the presence of the inhibitor. These results suggest that the transition of nuclei to metaphase can be induced by a cytoplasmic factor that is present in metaphase oocytes, and that dbcAMP inhibits the development of this factor.

Animals↗
PubMed1991

Progression of oocyte maturation from metaphase I to metaphase II is disturbed by previous immunological interference with cumulus cell function.

The effects of an antibody preparation reacting with preovulatory mouse cumuli oophori (anticumulus Ig) on oocyte maturation in vivo and in vitro were studied. Continuous presence of anticumulus Ig in culture medium did not impair oocyte maturation in vitro. Similarly, no effect on oocyte maturation in vivo was observed when anticumulus Ig was given to females superovulated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) at the time of hCG treatment. However, when administered earlier, anticumulus Ig brought about serious disturbances of oocyte meiotic competence, since only immature oocytes were ovulated after anticumulus Ig injection at the time of PMSG treatment and as much as 70% of the ovulated oocytes were immature when the antibody was applied 24 hr later. Previous absorption of anticumulus Ig with isolated cumulus cells removed the inhibitory effect of this preparation on oocyte meiotic competence to the same extent as absorption with whole cumuli oophori, despite the persistence of a strong reactivity of the cumulus cell-absorbed antibody preparations with the cumulus intercellular matrix. The ability of oocytes obtained from antibody-injected animals to mature in vitro was also considerably impaired when the injection was made at the time of PMSG treatment. In all cases the maturation defect concerned the progression of meiosis from metaphase I to metaphase II, while the ability of oocytes to undergo germinal vesicle breakdown (GVBD) was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Absorption↗
PubMed1981

[Identification of isolated metaphase chromosomes. II. A comparison with the recognizability of chromosomes in metaphase plates].

The metaphase chromosomes (MC) isolated from the Chinese hamster cells were identified with the aid of differential staining (G-bands). It was shown that differences in the relative recognizability of MC in metaphase plates and after their isolation are determined by changes in composition of isolated MC, rather than by those in staining capacity of MC after their isolation. The frequencies of identified MC are constant and independent upon the type of MC preparations and relation between identified and unidentified MC in certain preparations. At allows to apply the described method for the analysis of chromosome fractionation, using changes in frequencies of identified MC as a criterion of efficiency of the fractionation method. Possible ways of increasing the recognizability level of isolated MC are discussed.

Animals↗
PubMed1975

[Ultrastructure of sheep metaphase chromosomes identified by study of total preparations of metaphase plates].

The data obtained suggest wide possibilities of using the elaborated method of the whole metaphase plates investigation under the electron microscope to disclose the specific characters of the structure of individual chromosomes. Each chromosome has a definite number of bands of condensed DNP material. The number and disposition of bands are essentially the same in homologous chromosomes. The Giemsa-positive disks which could be seen after differential chromosome staining correspond to the bands of condensed material.

Animals↗
PubMed1992

Changes in the metaphase transit times and the pattern of sister chromatid separation in stamen hair cells of Tradescantia after treatment with protein phosphatase inhibitors.

Stamen hair cells from the spiderwort plant, Tradescantia virginiana, exhibit remarkably predictable metaphase transit times, making them uniquely suitable for temporal studies on mitotic regulation. In this study, we describe two kinds of experiments that test whether protein phosphatase activity is a necessary prerequisite for entry into anaphase in living, mitotic cells. We treated cells at specific points during prophase, prometaphase and metaphase with the broad-spectrum protein phosphatase inhibitor, alpha-naphthyl phosphate (administered by microinjection), or with the naturally occurring, potent phosphatase inhibitors okadaic acid, microcystin-LR or microcystin-RR (administered by perfusion), and we have observed changes in the metaphase transit time that are primarily dependent on the time of initial exposure to the inhibitor. Maximal extensions of the metaphase transit time result from alpha-naphthyl phosphate microinjections initiated in mid-metaphase, 10-20 min after nuclear envelope breakdown. Perfusions with okadaic acid started during a specific interval in mid-metaphase, 15-20 min after nuclear envelope breakdown, resulted in a statistically significant extension of the metaphase transit time. Perfusions with either microcystin-LR or microcystin-RR initiated 15-26 min after nuclear envelope breakdown extended the metaphase transit times significantly. Treatments of cells with okadaic acid or with either of the microcystins initiated outside this mid-metaphase interval either were without effect or, alternatively, resulted in a significant shortening of the metaphase transit time. In addition to their effects on the timing of anaphase onset, treatments with these protein phosphatase inhibitors also resulted in a remarkable change in the way in which these cells enter anaphase. Sister chromatid separation in stamen hair cells typically requires only 5 seconds, but after treatment with any of these inhibitors some, but not all, of the chromatids split apart at anaphase onset. Those that split begin to migrate toward the spindle pole regions, while those that fail to split remain at the metaphase plate. Later, more of the paired chromatids split apart and begin moving toward the spindle pole regions. Those that fail to separate remain at the metaphase plate. This process can be repeated several times before all of the chromatids have separated. Thus, entry into anaphase becomes extremely asynchronous, and as much as 30 min can transpire between the centromeric separation of the first and last chromosomes. Some of the chromosomes complete their anaphase movements before others have even split apart at the metaphase plate. Asynchronous separation did not result in a permanent segregation anomaly.(ABSTRACT TRUNCATED AT 400 WORDS)

Anaphase↗
PubMed1988

Scaffold attachment of DNA loops in metaphase chromosomes.

We have examined the higher-order loop organization of DNA in interphase nuclei and metaphase chromosomes from Drosophila Kc cells, and we detect no changes in the distribution of scaffold-attached regions (SARs) between these two phases of the cell cycle. The SARs, previously defined from experiments with interphase nuclei, not only are bound to the metaphase scaffold when endogenous DNA is probed but also rebind specifically to metaphase scaffolds when added exogenously as cloned, end-labeled fragments. Since metaphase scaffolds have a simpler protein pattern than interphase nuclear scaffolds, and both have a similar binding capacity, it appears that the population of proteins required for the specific scaffold-DNA interaction is limited to those found in metaphase scaffolds. Surprisingly, metaphase scaffolds isolated from Drosophila Kc cells contain both the lamin protein and a pore-complex protein, glycoprotein (gp) 188. To study whether lamin contributes to the SAR-scaffold interaction, we have carried out comparative binding studies with scaffolds from HeLa metaphase chromosomes, which are free of lamina, and from HeLa interphase nuclei. All Drosophila SAR fragments tested bind with excellent specificity to HeLa interphase scaffolds, whereas a subset of them bind to HeLa metaphase scaffolds. The maintenance of the scaffold-DNA interaction in metaphase indicates that lamin proteins are not involved in the attachment site for at least a subset of Drosophila SARs. This evolutionary and cell-cycle conservation of scaffold binding sites is consistent with a fundamental role for these fragments in the organization of the genome into looped domains.

Animals↗
PubMed1992

Three-group metaphase as a morphologic criterion of progressive cervical intraepithelial neoplasia.

OBJECTIVE: The purpose of our study was to investigate the presence of three-group metaphase in progressive cervical intraepithelial neoplasia. STUDY DESIGN: This was a retrospective histologic study on the conization specimens of 41 women with microinvasive cervical carcinoma, 28 of whom were enrolled in the study. Three-group metaphase was scored in the invasive part of the lesion and in the adjacent cervical intraepithelial neoplasia. RESULTS: Three-group metaphase was found in 93% of cervical intraepithelial neoplasia adjacent to the invasive part of the lesion. However, three-group metaphase was found in 11% of the microinvasive cervical carcinoma cases with an infiltration depth of less than 2.5 mm and in 60% of the microinvasive cervical carcinoma cases with an infiltration depth of between 2.5 and 5.0 mm. CONCLUSION: The chance of finding three-group metaphase seems to be limited by the area of the lesion examined for three-group metaphase on the slide. Given the relation between three-group metaphase and aneuploid cervical intraepithelial neoplasia found in the literature and the occurrence of three-group metaphase in the cervical intraepithelial neoplasia adjacent to the microinvasive cervical carcinoma in this study, three-group metaphase can be considered a morphologic criterion for progressive cervical intraepithelial neoplasia and can be of value for practical use.

Cervix Uteri↗
PubMed1986

Transformation of sperm nuclei to metaphase chromosomes in the cytoplasm of maturing oocytes of the mouse.

Zona-free oocytes of the mouse were inseminated at prometaphase I or metaphase I of meiotic maturation in vitro, and the behavior of the sperm nuclei within the oocyte cytoplasm was examined. If the oocytes were penetrated by up to three sperm, maturation continued during subsequent incubation and became arrested at metaphase II. Meanwhile, each sperm nucleus underwent the following changes. First, the chromatin became slightly dispersed. By 6 h after insemination, this dispersed chromatin had become coalesced into a small mass, from which short chromosomal arms later became projected. Between 12 and 18 h after insemination, each mass of chromatin became resolved into 20 discrete metaphase chromosomes. In contrast, if oocytes were penetrated by four to six sperm, oocyte meiosis was arrested at metaphase I, and each sperm nucleus was transformed into a small mass of chromatin rather than into metaphase chromosomes. If oocytes were penetrated by more than six sperm, the maternal chromosomes became either decondensed or pycnotic, and the sperm nuclei were transformed into larger masses of chromatin. As control experiments, immature and fully mature metaphase II oocytes were inseminated. In the immature oocytes, which were kept immature by exposure to dibutyryl cyclic AMP, no morphological changes in the sperm nucleus were observed. On the other hand, in the fully mature oocytes, which were activated by sperm penetration, the sperm nucleus was transformed into the male pronucleus. Therefore, the cytoplasm of the maturing oocyte develops an activity that can transform the highly condensed chromatin of the sperm into metaphase chromosomes. However, the capacity of an oocyte is limited, such that it can transform a maximum of three sperm nuclei into metaphase chromosomes. Furthermore, the presence of more than six sperm causes a loss of the ability of the oocyte to maintain the maternal chromosomes in a metaphase state.

Animals↗
PubMed1979

[Formation of structural chromosome mutations in metaphase of mitosis].

The rate of structural chromosome mutations at metaphase of the first mitosis was determined in culture of embrionic mouse fibroblasts after UV-irradiation during the S-period (lambda = 265 nm at an incident dose of 40 erg/mm2). It is established that the mutation rate is higher at late metaphase than at early metaphase. After the cell treatment with intercalating compounds (actinomycin D, acridine orange or ethidium bromide) at metaphase, the rate of UV-induced chromosome aberrations was decreased (about 2-fold). It is concluded from the results obtained that the majority of aberrations arise during metaphase after UV-irradiation in the process of DNA synthesis. After the cell treatment with o-methylhydroxylamine (OMHA) during the S-period the rate of structural mutations was the same at late and early metaphases. This rate was not affected by the caffeine treatment at metaphase; during this stage the acentric chromosome fragments lie outside the equatorial plate, which is an indication that the OMHA-induced aberrations, in contrast to the UV-induced aberrations, are formed before the beginning of metaphase, possibly during the interphase. It is suggested that the chromosome condensation during metaphase is of importance in the formation of structural mutations.

Acridine Orange↗
PubMed1990

The fate of DNA-protein crosslinks formed in gamma-irradiated metaphase cells.

The induction of DNA-protein crosslinks (DPC) was compared in gamma-irradiated metaphase and asynchronous Chinese hamster V79 cells. Unirradiated metaphase cells were found to have a higher level of background DPC than unirradiated asynchronous cells, and the metaphase cells were less susceptible to radiation-induced DPC production than were asynchronous cells. SDS-PAGE analysis of crosslinked proteins prepared from the two cell populations, both irradiated and unirradiated, showed very similar protein patterns. Crosslinked DNA was isolated and probed with radioactively labelled interphase poly(A+)RNA. The results indicated that the hypersensitivity of interphase actively transcribing DNA sequences to radiation-induced DPC formation was maintained at metaphase when the chromosomes are highly condensed. In contrast to asynchronous cells, radiation-induced DPC formed in metaphase cells were not removed during a 4 h post-irradiation period. However, metaphase cells appear to be able to remove the active DNA involved in DPC as indicated by a depletion of the probed sequences in the unrepaired DPC. Cell size analysis as well as cytological examination of the irradiated metaphase cells showed an absence of cell division during post-irradiation incubation. Furthermore, about 50% of the irradiated metaphase cells grew into giant cells which contain multiple nuclei and micronuclei, an indication of aberrant chromosome segregation.

Animals↗
PubMed1987

Dose-dependent relationship between oocyte cytoplasmic volume and transformation of sperm nuclei to metaphase chromosomes.

We have studied the chromosome condensation activity of mouse oocytes that have been inseminated during meiotic maturation. These oocytes remain unactivated, and in those penetrated by up to three or four sperm, each sperm nucleus is transformed, without prior development of a pronucleus, into metaphase chromosomes. However, those penetrated by more than four sperm never transform any of the nuclei into metaphase chromosomes (Clarke, H. J., and Y. Masui, 1986, J. Cell Biol. 102:1039-1046). We report here that, when the cytoplasmic volume of oocytes was doubled or tripled by cell fusion, up to five or eight sperm nuclei, respectively, could be transformed into metaphase chromosomes. Conversely, when the cytoplasmic volume was reduced by bisection of oocytes after the germinal vesicle (GV) had broken down, no more than two sperm could be transformed into metaphase chromosomes. Thus, the capacity of the oocyte cytoplasm to transform sperm nuclei to metaphase chromosomes was proportional to its volume. The contribution of the nucleoplasm of the GV and the cytoplasm outside the GV to the chromosome condensation activity was investigated by bisecting oocytes that contained a GV and then inseminating the nucleate and anucleate fragments. The anucleate fragments never induced sperm chromosome formation, indicating that GV nucleoplasm is required for this activity. In the nucleate fragments, the capacity to induce sperm chromosome formation was reduced as compared with whole oocytes, in spite of the fact that the fragments contained the entire GV nucleoplasm. This implies that non-GV cytoplasmic material also was required for chromosome condensation activity. When inseminated oocytes were incubated in the presence of puromycin, the sperm nuclei were transformed into interphase-like nuclei, but no metaphase chromosomes developed. However, when protein synthesis resumed, the interphase nuclei were transformed to metaphase chromosomes. These results suggest that the transformation of sperm nuclei to metaphase chromosomes in the cytoplasm of mouse oocytes requires both the nucleoplasm of the GV and non-GV cytoplasmic substances, including proteins synthesized during maturation.

Animals↗
PubMed1988

Regulation of meiotic metaphase by a cytoplasmic maturation-promoting factor during mouse oocyte maturation.

During mouse oocyte maturation the regulation of the activity of a cytoplasmic maturation-promoting factor (MPF) was examined. The mouse MPF activity was determined based on its ability to induce maturation in immature starfish oocytes after microinjection with the cytoplasm from mouse oocytes. MPF appeared initially at germinal vesicle breakdown (GVBD), and its activity fluctuated in exact correspondence with meiotic cycles, reaching a peak at each metaphase and almost disappearing at the time of emission of the first polar body. Cycloheximide affected neither the initial MPF appearance nor GVBD. Thereafter, however, in the presence of cycloheximide the meiotic spindle was not formed and MPF disappeared, although the chromosomes remained condensed. After removing cycloheximide, MPF reappeared and was followed by the first metaphase and subsequently by polar body emission. Finally the meiotic cycle progressed to the second metaphase. Thus, for the appearance of MPF, there is a critical period shortly before the first metaphase, after which protein synthesis is required. In the presence of either cytochalasin D or colcemid, MPF activity remained at elevated levels. Addition of cycloheximide to such cytochalasin-treated oocytes, in which the meiotic cycle was arrested at the first metaphase, caused the MPF levels to decrease and was followed by movement of chromosomes to both poles where they decondensed and two nucleus-like structures were formed. Thus, the disappearance of MPF may initiate the metaphase-anaphase transition. Furthermore, detailed cytological examination revealed that chromosomes in cytochalasin-treated oocytes were monovalent while those treated only with cycloheximide were divalent, suggesting that dissociation of the synapsis is a prerequisite for chromosome decondensation after the disappearance of MPF. In all these respects, MPF seems to be a metaphase-promoting factor rather than just a maturation-promoting factor.

Animals↗
PubMed1992

Time-saving in biological dosimetry by using the automatic metaphase finder Metafer2.

The amount of time-saving by using the Metafer2 metaphase finder for routine analysis of radiation-induced chromosome aberrations (biological dosimetry) was determined. Metaphases were prepared by standard methods from cultures of human peripheral blood lymphocytes and stained either with Giemsa or with the FPG method. The metaphase finder was used for detecting metaphases on the microscope slides and for automatically processing the evaluation data. In our laboratory, standardized analysis of 1000 metaphases requires at least 3 working days for cell culturing and slide preparation and 51.5 working hours for cytogenetic analysis. When using the metaphase finder the time required for cytogenetic analysis is reduced to 17.3 working hours (time-saving factor: 51.5/17.3 h = 3.0). In our prolonged method, including more than one scoring of each slide and karyotyping of metaphases with chromosome aberrations, the analysis times for 1000 cells are 132 and 70 working hours, respectively (time saving factor: 132/70 h = 1.9).

Automation↗
PubMed1989

Murine oocyte cryopreservation: comparison between fertilization success rates of fresh and frozen metaphase I and II oocytes.

The surplus of oocytes obtained during in vitro and gamete intrafallopian transfer creates the need to either (1) cryopreserve (196 degrees C) or (2) fertilize these surplus oocytes and then freeze the resulting embryos. During this pilot study, C57BL6 X CBA female mice (F1 generation) were superovulated by means of hormone stimulation, resulting in adequate numbers of murine oocytes. Oocytes thus obtained were classified according to their nuclear status. The maturation stage of oocytes was evaluated and classified as metaphase I and metaphase II. The success of oocyte cryopreservation in each group was evaluated according to fertilization rate after the insemination procedure. Metaphase II oocytes had a fertilization rate of 33% (frozen) and 84% (fresh), whereas metaphase I oocytes had a 24% (frozen) and a 79% (fresh) fertilization rate. Metaphase I oocytes can mature to the metaphase II stage after having been frozen, metaphase II oocytes may be more vulnerable to chromosome damage, and the nuclear stage prior to freezing has no significant effect on the fertilizability of the frozen-thawed oocytes.

Animals↗
PubMed1991

Stage-specific damage to synaptonemal complexes and metaphase chromosomes induced by X rays in male mouse germ cells.

Synaptonemal complexes reveal mutagen-induced effects in germ cell meiotic chromosomes. This study was aimed at characterizing relationships between damage to synaptonemal complexes and metaphase I chromosomes following radiation exposure at various stages of spermatogenesis. Male mice were irradiated with doses of 0, 2, or 4 Gy, and spermatocytes were harvested at times consistent with earlier exposures as spermatogonial stem cells, preleptotene cells (premeiotic DNA synthesis), or meiotic prophase cells. After stem-cell exposure, twice as many rearrangements were observed in synaptonemal complexes as in metaphase I chromosomes. Irradiation during premeiotic DNA synthesis resulted in dose-related increases in synaptonemal complex breakage and rearrangements (including novel forms) and in metaphase chromosomal aberrations. Following prophase exposure, various types and levels of damage to synaptonemal complexes and metaphase chromosomes were observed. Irradiation of zygotene cells led to high frequencies of chromosome multivalents in metaphase I without a correspondingly high level of damage in preceding prophase synaptonemal complexes. Thus irradiation of premeiotic and meiotic cells results in variable relationships between damage to synaptonemal complexes and metaphase chromosomes. Interpretations of these relationships are based upon what is known about both radiation clastogenesis and the structural/temporal relationships between synaptonemal complexes at prophase and chromosomes at metaphase I of meiosis.

Animals↗
PubMed1982

Spreading and staining of human metaphase chromosomes on aminoalkylsilane-treated glass slides.

The properties of aminoalkylsilane-treated glass slides for the preparation of metaphase spreads and their staining quality have been studied and compared with those of slides which had only been cleaned in ethanol/ether. The parameters investigated were: (1) the average area of metaphases from cultures of blood from both healthy donors and haematology patients; (2) the influence of the positively charged 'coating' on the quality of quinacrine- and Giemsa-banding patterns; (3) non-specific background staining for these banding methods; (4) the number of metaphases as compared to the number of interphase cell nuclei per area of preparation; and (5) the Feulgen-staining intensities of chromosomes and chicken erythrocyte nuclei. The quality of metaphase preparations and the differential staining of chromosomes is better on aminoalkysilane-treated glass slides than that of preparations on routinely cleaned normal microscope slides. In the preparations on aminoalkylsilane-treated slides, the distribution of the cells over the glass surface is more homogeneous; and no influence could be detected on the relative frequency of metaphases as compared to the number of non-divided cell nuclei; the average area per metaphase is increased by about 10% and consequently the number of overlapping chromosomes is decreased. Preparations on aminoalkylsilane-treated glass, after Q-, G- and DAPI-banding procedures, always showed less binding of the staining compounds to the glass slide (a cleaner background) than those on routinely cleaned microscope glass slides. The Feulgen-pararosaniline staining intensities of human metaphase chromosomes and chicken erythrocyte nuclei are the same on aminoalkylsilane-treated slides and on routinely cleaned glass slides. Furthermore, the reproducibility and constancy of quinacrine banding was improved by development of an equilibrium staining method which does not require a washing procedure. The medium, containing 0.002% quinacrine, allows optimal staining results to be obtained for microphotography purposes within 30 min of staining (for visual inspection at least 90 min is required) and is used as the embedding medium. In combination with aminoalkylsilane-treated glass slides, this procedure leads to a clean background and reproducible banding patterns of excellent quality, the results being better and more constant than those of methods described before.

Azure Stains↗
PubMed1987

Metaphase chromosome folds and X-inactivation.

To evaluate the suggested nonrandom folding of Xq13-q21 (center of Barr body condensation) of the inactivated X chromosomes, metaphases from nine subjects with or without X chromosome abnormalities (eight females and one male) were investigated with RBG-staining. A significant increase (p less than .05) in the number of arm folds (Xq13-q21) of the late-replicating X chromosome, particularly in early to mid-metaphase, was observed in four of eight females. Therefore, the stage of chromosome contraction was an important factor with more folds observed at the centromeres and in longer chromosomes in early metaphase compared with mid- to late metaphase. X chromosome folds were present in cells of subjects treated with or without bromodeoxyuridine. While our study agrees with the relationship of Xq13-q21 fold with the X-inactivation center, the correlation of extended chromosomes and folding limits this method as a sole indicator of X-inactivation in routine mid-metaphase, but is useful in the analysis of early metaphase chromosomes.

Dosage Compensation, Genetic↗