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PubMed1991

Chromosome condensation from prophase to late metaphase: relationship to chromosome bands and their replication time.

As chromosomes condense during early mitosis, their subbands fuse in a highly coordinated fashion. Subband fusion occurs when two large subbands flanking one minor subband come together to form one band, which takes on the cytological characteristics of the original flanking subbands. Using four different banding techniques--GTG (G-bands obtained with trypsin and Giemsa), GBG (G-bands obtained with BrdU and Giemsa), RHG (R-bands obtained by heating and Giemsa), and RBG (R-bands obtained with BrdU and Giemsa)--we studied subband fusion from prophase (1,250 bands per haploid set) to late metaphase (300 bands). To quantify the condensation process, a fusion index was established. We found that chromosomes contain preferential zones of condensation. From prophase to late metaphase, the early replicating subbands (R-subbands) fuse more readily with each other than do the late-replicating subbands (G-subbands). R-bands usually replicate early and condense late independently of the adjacent G-bands, which replicate late but condense early. Therefore, chromosome bands can undergo DNA replication and chromatin condensation relatively autonomously. Our data suggest that (1) chromosome replication and condensation are closely connected in time, (2) the metaphase bands represent independent units of chromatin condensation, and (3) the condensation process is an important feature of chromosome organization.

Cells, Cultured↗
PubMed1995

Precise ordering of 26 cosmid markers on chromosome region 3p23-->p21.3 by two-color FISH on human prophase chromosomes and stretched DNAs.

To construct a detailed cytogenetic map of human chromosome region 3p23-->p21.3, we determined the order of 26 cosmid markers (cCI 3 series) previously localized within this region by fluorescence in situ hybridization (FISH). Two-color pairwise FISH analysis of prophase chromosomes provided the order of these markers as follows: pter - 245 (D3S647) - 872 (D3S1018) - 818 (D3S996) - 905 (D3S1022) - 515 (D3S685) - 1195 (D3S1125) - 718 (D3S935) - 911 (D3S1025) - 878 (D3S1020) - 717 (D3S934) - 401 (D3S664) - [708 (D3S926)/524 (D3S686)] - 848 (D3S1011) - 771 (D3S966) - 917 (D3S1029) - 533 (D3S688) - 470 (D3S676) - 940 (D3S1037) - 785 (D3S974) - 810 (D3S988) - 9 (D3S643) - 382 (D3S660) - 769 (D3S965) - 792 (D3S978) - 604 (D3S705) - cen. The two-color signals of 524 (D3S686) and 708 (D3S926) were visualized as an overlapping pattern on prophase chromosomes, and, further, the string signals also overlapped on stretched DNAs, allowing us to determine their precise order as pter - D3S926 - D3S686 - cen. The precise order of 26 DNA markers on 3p23-->p21.3 can provide useful information for the positional cloning of tumor suppressor gene(s) and cancer breakpoint(s) encompassed in this region.

Chromosome Mapping↗
PubMed2003

The p27cip/kip ortholog dacapo maintains the Drosophila oocyte in prophase of meiosis I.

Animal oocytes undergo a highly conserved developmental arrest in prophase of meiosis I. Often this marks a period of rapid growth for the oocyte and is necessary to coordinate meiotic progression with the developmental events of oogenesis. In Drosophila, the oocyte develops within a 16-cell germline cyst. Throughout much of oogenesis, the oocyte remains in prophase of meiosis I. By contrast, its 15 mitotic sisters enter the endocycle and become polyploid in preparation for their role as nurse cells. How germline cysts establish and maintain these two independent cell cycles is unknown. We demonstrate a role for the p21(CIP)/p27(Kip1)/p57(Kip2)-like cyclin-dependent kinase inhibitor (cki) dacapo in the maintenance of the meiotic cycle in Drosophila oocytes. Our data indicate that it is through the differential regulation of the cki Dacapo that two modes of cell-cycle regulation are independently maintained within the common cytoplasm of ovarian cysts.

Animals↗
PubMed1998

Oscillatory nuclear movement in fission yeast meiotic prophase is driven by astral microtubules, as revealed by continuous observation of chromosomes and microtubules in living cells.

Using a computerized fluorescence microscope system to observe fluorescently stained cellular structures in vivo, we have examined the dynamics of chromosomes and microtubules during the process of meiosis in the fission yeast Schizosaccharomyces pombe. Fission yeast meiotic prophase is characterized by a distinctive type of nuclear movement that is led by telomeres clustered at the spindle-pole body (the centrosome-equivalent structure in fungi): the nucleus oscillates back and forth along the cell axis, moving continuously between the two ends of the cell for some hours prior to the meiotic divisions. To obtain a dynamic view of this oscillatory nuclear movement in meiotic prophase, we visualized microtubules and chromosomes in living cells using jellyfish green fluorescent protein fused with alpha-tubulin and a DNA-specific fluorescent dye, Hoechst 33342, respectively. Continuous observation of chromosomes and microtubules in these cells demonstrated that the oscillatory nuclear movement is mediated by dynamic reorganization of astral microtubules originating from the spindle-pole body. During each half-oscillatory period, the microtubules extending rearward from the leading edge of the nucleus elongate to drive the nucleus to one end of the cell. When the nucleus reversed direction, its motion during the second half of the oscillation was not driven by the same microtubules that drove its motion during the first half, but rather by newly assembled microtubules. Reversible inhibition of nuclear movement by an inhibitor of microtubule polymerization, thiabendazole, confirmed the involvement of astral microtubules in oscillatory nuclear movement. The speed of the movement fluctuated within a range 0 to 15 micron/minute, with an average of about 5 microm/minute. We propose a model in which the oscillatory nuclear movement is mediated by dynamic instability and selective stabilization of astral microtubules.

Cell Division↗
PubMed2002

Homolog pairing and two kinds of bouquets in the meiotic prophase of rye, Secale cereale.

Chromosome configurations and structures during meiotic prophase were investigated by staining large repeated DNA sequences localized in the subtelomeric regions of all the chromosomes in rye, Secale cereale, in order to clarify when and how homolog pairing and bouquet formation occur. The changes of the spatial locations of chromosomes in the nucleus were investigated by the use of laser confocal microscopy, together with the surface-spreading method of silver nitrate staining to detect the formation of the synaptonemal complex. Homolog pairing in which homologs of four chromatids of a pair of homologs were coaligned in parallel but remained distinctly separate was microscopically detected for the first time in the present study. Homolog pairing showed the following characteristics: (1) it occurred at the leptotene-zygotene transition stage, prior to the formation of nodules and the synaptonemal complex; (2) the chromatin structure of chromosomes was in a state of decondensation; (3) it required no telomere clustering. These data suggest that homolog pairing represents a structure that indicates incipient recombination. After the homolog pairing stage, two kinds of bouquet configuration were found in zygotene. The commonly observed type was a loose bouquet, in which the subtelomeric regions were loosely aggregated. The other type was a definite bouquet, in which almost all the subtelomeric regions were conjugated, but this type was observed only in a limited number of the meiotic prophase cells of some individuals. It was concluded that the former represents the configuration of homologous recombination and the latter that of ectopic recombination.

Cell Nucleus↗
PubMed1992

[Changes in the relative arrangement of the chromosomes and nucleolus in developing mammalian oocytes during meiotic prophase I in relation to functional changes in the oocytes].

Data on chromosome transformation in meiotic prophase I during mammalian oogenesis are summarized. The main peculiarity of the female meiosis in mammals is an unusually long diplotene stage which may be subdivided into four periods: 1) the early diplotene (up to the beginning of follicle formation); 2) the dictyotene or "diffuse diplotene", implying primordial follicle oocytes; 3) the most pronounced lampbrush chromosome stage coinciding with the large growth period; 4) the stage of chromosome inactivation and karyosphere formation corresponding to the terminal stage of oocyte development before ovulation. These stages are associated with changes in the transcriptional chromosome activity. A correlation is revealed between the spatial chromosome arrangement in the oocyte nucleus and the transcriptional activity. Some regularities are followed in the transformation of the main nucleolar component arrangement during meiotic prophase I in mammalian oocytes. At the late pachytene and at the early diplotene, a segregation of the main nucleolar components has been observed. These components are disposed in the direction: chromatin--fibrillar center--dense fibrillar component--granulo-fibrillar component. At the dictyotene, signs of nucleolar segregation are still observed. At the lampbrush chromosome stage, when the nucleus is most highly transcriptionally active, an integration of nucleolar components occurs. At the late diplotene--prediakinesis stage, i.e. in the course of transcriptional activity lowering and karyosphere formation, the secondary segregation of the main nucleolar components occurs. These move to the nucleolar periphery to be disposed around a large fibrillar mass which is gradually displacing the rest of the nucleolar components. The fibrillar mass formation in the preovulatory oocyte nucleoli is one of the peculiarities of the diplotene and prediakinetic mammalian oocytes.

Animals↗
PubMed1990

The mechanism of kinetochore-spindle attachment and polewards movement analyzed in PtK2 cells at the prophase-prometaphase transition.

PtK2 cells at the prophase-prometaphase transition were analyzed to study the origin of kinetochore microtubules, the mode of kinetochore fiber construction and the mechanism of polewards movement. Attention was focused on chromosomes which, as deduced from video time-lapse tapes, had just started their initial characteristic movement towards one of the poles. In the same cell, the arrangement of microtubules in the vicinity of the kinetochore region was visualized either with indirect immunofluorescence and confocal fluorescence microscopy, or with electron microscopy in semithin sections of cells, immunostained for microtubules and embedded in epon. The results strengthen the evidence that kinetochore microtubules are nucleated in the centrosomal region. Bundles of microtubules, some ending at the kinetochore and others passing beyond it, are formed rapidly, seemingly without influencing the rapid rate of movement. They also show that microtubules often establish contact with kinetochores by lateral interaction, prior to kinetochore-pole orientation, and that kinetochores can move polewards along the microtubule wall of attached microtubules, independently of the latter's dynamics. These findings confirm and extend to the earliest chromosome movements at the prophase-prometaphase transition, the results of Rieder and Alexander (J. Cell Biol. 110, 81-95, (1990)), who studied the attachment and polewards movement of chromosomes strongly delayed in forming an attachment to the spindle. They are discussed in the light of recent evidence for the localization of dynein to kinetochores and contemporary models for kinetochore structure and function.

Animals↗
PubMed1986

[Electron microscopic study of preleptotene-stage oocyte nuclei in the prophase of meiosis in the hen].

The morphology of oocyte nuclei at preleptotene and early leptotene stages of meiotic prophase I in the chick embryo was examined by electron microscopy and light cytochemistry. The intrenuclear fibrillar body (IFB) of proteinaceous nature is described. The IFB has a spherical or irregular form and consists of the disoriented fibrils ranging from 3 to 18 nm in diameter and of globules lying along the course of fibrils. The condensed chromatin in the oocyte nuclei at the early--late preleptotene stages and the thread-like chromosomes in the oocyte nuclei at the middle preleptotene--early leptotene stages either closely adjoin to IFB, localized in the centre of the nucleus, or are situated at some distance from it, and then the fibrils or fibrillar filaments are seen between chromosome material and IFB. The chromosomes are attached to IFV by the telomer or interstitial segment. The chromosomes lose the connection with IFB after the attachment of both the telomeres to the nuclear envelope (middle--late leptotene), and IFB removes from the centre of the nucleus to its periphery. It is supposed that IFB represents a nuclear skeleton element and takes part in the spatial organization of the chromosomes in the oocyte nuclei at the early stages of meiotic prophase I.

Animals↗
PubMed1979

A method for the in vivo experimental study of meiotic prophase in the golden hamster (Mesocricetus auratus).

Gonads of 15 day-old hamster fetuses were grafted under the kidney capsule of adult ovariectomized females. In 81.8% of the grafts, the germ cells developed and completed meiotic prophase; they reached the diplotene stage and gave rise to primordial follicles. These grafts could survive well in the host for at least 20 days. Meiotic prophase was not initiated within 24 h of grafting in contrast to the in vivo condition where it is observed 24 h after birth. In grafted ovaries, 69% of the germ cells were at the leptotene stage on day 3. By day 5, most of them were either at the zygotene (15%) or pachytene stages (60%). Oocytes at the diplotene stage were found from day 5 onwards and on day 7, 30% of the germ cells had already reached this stage. The highest numbers of atretic germ cells could be found on days 1, 9 and 10 post graft. It is noteworthy that the number of germinal cells remaining in the ovary 10 and 20 days after grafting were 11.4% and 12.9% of the total number of germinal cells present in normal animals of the same ages post-partum. This point is discussed in detail.

Animals↗
PubMed1984

[Female germ cell development during the meiotic prophase in the rat: criteria for cytoplasmic and nuclear identification of the different stages on thin histological slides].

The different stages of meiotic prophase in germ cells of the rat ovary were studied cytologically at definite times when there was a dominant nuclear stage. Each stage was identified in 1 mu sections. The usual references for the definition of meiotic nuclear stages according to chromosomal structures wee used and have been described in more detail. It has also been shown that cytological observations such as the organization and distribution of cytoplasmic organelles (mitochondria, Golgi apparatus) equally contributes to the identification of the stages of meiotic prophase.

Animals↗
PubMed1984

[Ultrastructural changes in female germ cells during the meiotic prophase in the rat: a special study of membranes after cryofracture].

Ultrastructural changes in the nuclear and cytoplasmic elements in the germ cells of female rats were followed before meiotic prophase (15.50 days post-coïtum and 17.25 days post-coïtum) and during it (17.75 days post-coïtum to birth). We observed: modifications in the nuclear envelope which was thick during the oogonial stage, becoming thinner when the chromosomes entered preleptotene stage. The thinning of the envelope was due to the disappearance of the chromatin material lining it; variations in the number and distribution of germ cell nuclear pores according to stage; the pores were first scattered in small clusters of 6 to 8 over the entire nuclear membrane. From the preleptotene to zygotene stage, these clusters enriched in pores to form large areas. Finally, in the pachytene and diplotene stages, clusters of more than 100 pores were seen; nucleolar fragmentation from the preleptotene stage, followed by the formation of a new active nucleole in the diplotene; polarization of the mitochondria in the oldest oogonia just before the beginning of meiotic prophase. This polarization disappeared after the onset of the meiotic processes, then appeared again near the developing Golgi apparatus at the end of the pachytene stage; the formation of large gap junctions and numerous bands of tight junctions between the somatic cells; these formations contrasted with small gap junctions, and the tight junctions became scarce just before the meiotic process began. These observations, as well as those concerning nuclear pore distribution were made using the cryofracture technique.

Animals↗
PubMedDate not supplied

Relationship between G band number and chromosome length from prophase to metaphase.

The statistical analysis of the distribution of human chromosomes length served as a basis for their classification according to the suggestions of the Denver Conference (1960). Standards of metaphasal chromosome length were used for programmes of computer-assisted classification. The introduction of banded chromosomes in the study provided a new measurable feature which was used for classification standards accepted at the Paris Conference (1971). The measurements of these two features have been used also in the mathematical models of computer analysis and classification of chromosomes. In the present study the relationship between the number of bands and the length of chromosomes was studied in the period of cell division from prophase to metaphase in the cultures of leucocytes of healthy people. This relationship is strongly correlated and can be approximated in description to simple regression in the period from prophase to metaphase. The relationship will facilitate the construction of dynamic patterns for the needs of computer-assisted classification of chromosomes and detection of structural aberrations.

Cells, Cultured↗
PubMed1980

[Dynamics of chromosome rearrangements in the meiotic prophase in human oogenesis].

A quantitative analysis of germ cell population development in human female embryonic gonads has been carried out, with special attention being paid to chromosomal changes in the prophase of the first meiotic division. 97 embryos at the age of 6 to 40 weeks were investigated. The morphology of nuclei at different prophase stages is described, temporal dynamics of all the stages is presented, dynamics of germ cell degeneration is outlined.

Chromosomes, Human↗
PubMed1994

Cryopreservation of human prophase I oocytes collected from unstimulated follicles.

OBJECTIVE: To evaluate the cryopreservation of immature human oocytes obtained from unstimulated ovarian tissue. DESIGN: Immature prophase I oocytes were obtained from unstimulated follicles and were either cryopreserved or cultured as controls. Cryopreservation was performed in a programmable freezing machine using one of two protocols. Method I (n = 133) used a one-step addition of cryoprotectant followed by a slow freeze and thaw protocol. With method II (n = 95), the cryoprotectant was added in a stepwise manner with cryopreservation performed in the presence of 0.2 M sucrose followed by rapid freezing and thawing. SETTING: Basic research center at a medical school. PATIENTS: Patients undergoing oophorectomy for nonovarian pathology. MAIN OUTCOME MEASURES: Rates of survival and maturation to metaphase II were compared between control oocytes and oocytes cryopreserved with methods I and II. RESULTS: With method I, a survival rate of 15.6% was obtained with 58.3% of surviving oocytes reaching metaphase II after culture compared with 50.0% of nonfrozen control oocytes. Method II produced a survival rate of 43.3% with 27.3% maturing to metaphase II. Maturation of control oocytes for method II was 46.4%. Although the survival rate with method II was significantly higher than with method I, the rate of in vitro maturation to metaphase II showed no difference. CONCLUSIONS: These results demonstrate that human prophase I oocytes obtained from unstimulated antral follicles are capable of meiotic maturation after cryopreservation.

Cell Survival↗
PubMed1992

A coiled-coil related protein specific for synapsed regions of meiotic prophase chromosomes.

Synaptonemal complexes (SCs) are structures that are formed between homologous chromosomes during meiotic prophase. They are probably involved in chromosome pairing and recombination. Using a monoclonal anti-SC antibody we isolated cDNAs encoding a major component of SCs which is localized specifically in synapsed segments of meiotic prophase chromosomes. The protein predicted from the nucleotide sequence of a full-length cDNA, named SCP1, consists of 946 amino acid residues and has a molecular weight of 111 kDa. It shares several features with nuclear lamins and some recently identified nuclear matrix proteins. The major part of SCP1 consists of long stretches capable of forming amphipathic alpha-helices. This region shows amino acid sequence similarity to the coiled-coil region of myosin heavy chain. A leucine zipper is included in this region. The carboxy-terminus has two small basic domains and several S/T-P-X-X motifs, which are characteristic of DNA-binding proteins. One of these motifs is a potential target site for p34cdc2 protein kinase. The amino-terminus is acidic and relatively proline-rich, but does not contain the S/T-P-X-X motif. The transcription of the gene encoding SCP1 is restricted to zygotene-diplotene spermatocytes. A polyclonal antiserum raised against the fusion protein of one of the cDNA clones recognizes a single protein on Western blots of isolated SCs, with an electrophoretic mobility identical to that of the antigen recognized by the original monoclonal antibody (mAb), IX5B2. From a detailed comparison of the immunogold labelling of rat SCs by mAb IX5B2 and the polyclonal anti-fusion protein antiserum respectively, we tentatively infer that the carboxy-terminus of SCP1 is orientated towards the lateral elements and that the other domains of the protein extend towards the central region between the lateral elements. We conclude that SCP1 is the major component of the transverse filaments of SCs, and speculate that it has evolved by specialization of a nuclear matrix protein.

Amino Acid Sequence↗
PubMed1979

Nucleolar organizers in human oocytes at meiotic prophase I, studied by the silver-NOR method and electron microscopy.

Use of the silver-NOR method to study the nucleolar organizers in human oocytes demonstrates that topographic and quantitative variations occur during meiotic prophase. In the oogonia nucleolus the nucleolar organizers are dispersed, whereas beginning at leptotene and throughout the remaining stages of meiotic prophase they occupy a marginal position in the nucleolus. At leptotene, a modal number of seven nucleolar organizers can be observed, whereas this number falls to 2.5 at pachytene and rises to ten at diplotene, thus showing that there is intense rRNA synthesis during the latter stage of meiosis. During pachytene, one end of the bivalents containing the ribosomal cistrons is always associated with the Ag-positive zone of the nucleolus. Observation of pachytene in the electron microscope shows that the secondary constriction region of D and G bivalents is constantly associated with the fibrillar center of the nucleolus. Comparison of these two methods of investigation reveals that the silver-stained regions of the nucleolus correspond to the fibrillar centers. The latter are surrounded by a layer of electron-dense fibrils corresponding to the zone of rDNA transcription. This electron-dense layer is absent during pachytene when the nucleolus displays spontaneous segregation of its components; this absence is related to temporary arrest of rDNA transcription. The affinity of the fibrillar centers for silver-NOR staining confirms that these structures contain ribosomal cistrons. During the diplotene stage, numerous micronucleoli are formed outside the nucleolar organizers of D and G chromosomes. Most of these micronucleoli present an Ag-positive granule on one of their margins, thus indicating that they contain an actively transcribed sequence of rDNA. This observation confirms the existence of amplification of ribosomal genes in the human oocyte.

Cell Nucleolus↗
PubMed1989

Nucleolar structures in chromosome and SC preparations from human oocytes at first meiotic prophase.

We describe a comparative study of the behavior of nucleolar structures and their relationship with nucleolar chromosomes and synaptonemal complexes at first meiotic prophase of human oocytes in an attempt to elucidate the nature of this cellular organization and to learn more about maternal nondisjunction. The number of main nucleoli varies along the different stages of prophase I and is usually low. It shows an increase from leptotene to pachytene and a decrease from pachytene to diplotene related to a decrease and an increase of main nucleoli volume, respectively. The methodology employed has enabled us to analyze in detail dark bodies, round bodies, dense bodies, and main nucleoli in chromosome or synaptonemal complex spreads. The relationship between nucleolar chromosomes or synaptonemal complexes and the nucleoli implies the existence, in a very reduced space, of chromosomal regions that contain homologous sequences and that are often unpaired. This situation may facilitate the production of heterologous pairing and chromosomal exchanges between nonhomologous chromosomes and finally result in aneuploidy. Thus, the situation explained above together with the differences between the oocyte and spermatocyte NOR cycles could be one of the reasons for the higher incidence of aneuploidies of maternal origin at meiosis I.

Cell Nucleolus↗