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PubMed2000

Evidence for the coincident initiation of homolog pairing and synapsis during the telomere-clustering (bouquet) stage of meiotic prophase.

To improve knowledge of the prerequisites for meiotic chromosome segregation in higher eukaryotes, we analyzed the spatial distribution of a pair of homologs before and during early meiotic prophase. Three-dimensional images of fluorescence in situ hybridization (FISH) were used to localize a single pair of homologs in diploid nuclei of a chromosome-addition line of oat, oat-maize9b. The system provided a robust assay for pairing based on cytological colocalization of FISH signals. Using a triple labeling scheme for simultaneous imaging of chromatin, telomeres and the homolog pair, we determined the timing of pairing in relation to the onset of three sequential hallmarks of early meiotic prophase: chromatin condensation (the leptotene stage), meiotic telomere clustering (the bouquet stage) and the initiation of synapsis (the zygotene stage). We found that the two homologs were mostly unpaired up through middle leptotene, at which point their spherical cloud-like domains began to transform into elongated and stretched-out domains. At late leptotene, the homologs had completely reorganized into long extended fibers, and the beginning of the bouquet stage was conspicuously marked by the de novo clustering of telomeres at the nuclear periphery. The homologs paired and synapsed during the bouquet stage, consistent with the timing of pairing observed for several oat 5S rDNA loci. In summary, results from analysis of more than 100 intact nuclei lead us to conclude that pairing and synapsis of homologous chromosomes are largely coincident processes, ruling out a role for premeiotic pairing in this system. These findings suggest that the genome-wide remodeling of chromatin and telomere-mediated nuclear reorganization are prerequisite steps to the DNA sequence-based homology-search process in higher eukaryotes.

Avena↗
PubMed1978

A carmine-Giemsa staining technic for meiotic prophase chromosomes of the genus Beta L.

For a detailed study of chromosome morphology in meiotic prophase stages of Beta species, a special double staining technic has been developed. It consists of combined maceration-staining in an ethanol-hydrochloric acid-carmine mixture followed by poststaining of the squashed material in a diluted Giemsa solution. The technic yields well-spread prophase meiotic nuclei showing detailed structures both in weaker stained chromosome segments and in threadlike chromatin structures. This technic proved to be especially favorable for stages which are difficult to interpret, such as pachytene, schizotene and diffuse stages.

Azure Stains↗
PubMed1976

[Duration of meiotic prophase stages during oogenesis in the newt Triturus cristatus cristatus].

The duration of the early stages of meiotic prophase was determined in the oogenesis of T. cristatus cristatus by means of autoradiography. The oocytes were being investigated during 39 days from the moment of 3H-thymidine injection. It was shown that preleptotene lasts 1--2, leptotene ca. 4, zygotene 5 and pachytene 26 days. When studying the preparations obtained 1 day after the injection of 3H-thymidine, the silver grains were found to be localized over the nuclei at all stages of meiotic prophase; this suggests the amplification of rDNA which begins in leptotene-zygotene and ends in early diplotene.

Animals↗
PubMed2000

[Oogenesis of Tilapia mossambica. I. Oogonia and meiotic prophase oocytes].

Using light and electron microscopy and autoradiography, the morphology and synthesis of DNA, RNA and proteins in oogonia and early meiotic prophase oocytes in Tilaria mossabique were studied. According to dimensions and morphological features observed it is possible to distinguish between two groups of oogonia: large oogonia corresponding to type A spermatogonia of mammals, and small actively dividing oogonia, located in groups and identical to type B spermatogonia. The morphology of oogonia and of the early meiotic prophase oocytes well compares with the pattern described for other species of bony fishes. In the cytoplasm of these cells dense bodies, nuage-material, free ribosomes, large mitochondria with lamellar cristae and Golgi cisterns are available. In the oocyte nuclei at zygotene and pahytene stages 3H-thymidine incorporation was seen mainly into the nucleolus-associated chromatin. Besides, the formation of a heterochromatin cape and the synaptonemal complex was observed. Incorporation of 3H-uridine and 3H-leucine in the nuclei of these cells was very poor.

Animals↗
PubMed1990

Subcellular structure of prenatal human ovary: mitochondrial distribution during meiotic prophase.

The dynamics of mitochondrial population during the first meiotic prophase in the fetal human ovary was studied in sequential stages of development, from week 13 to week 23 of intrauterine life; the study was performed by means of electron microscopy. Two mitochondrial translocations were observed: first, from a random cytoplasmic distribution during leptotene, the mitochondria translocate toward a perinuclear location during zygotene. Second, from this perinuclear position they migrate toward a pole of the cell where they contribute during diplotene to form the Balbiani's vitelline body. Coincident with these mitochondrial redistributions, changes in mitochondrial cristae morphology were observed. From a tubular profile during early leptotene, they turn parallel with a more electron dense matrix during zygotene and pachytene, and finally arciform in diplotene. Since meiosis is a process that probably requires high levels of energy, these dynamic changes in mitochondrial population and the close association between mitochondria and nuclear membrane may be related with energy producing reaction that may be necessary for insuring the completion of the first meiotic prophase.

Cell Cycle↗
PubMed1999

Chromosome movement during meiotic prophase in crane-fly spermatocytes: IV. Actin and the effects of cytochalasin D.

Cytochalasin D (CD) was applied to crane-fly spermatocytes at late diakinesis with the aim of perturbing actin structure and actin function, thereby testing the hypothesis that intranuclear chromosome movement during late diakinesis is actin-based. Isolated tests were incubated in a range of CD concentrations (2-100 microM) for 1 or 2 h. None of those treatments resulted in cessation of prophase movements in living cells. An immediate effect of 10-100 microM CD at late diakinesis was the formation of highly refractile, actin-containing cables within the nonchromosomal nucleoplasm. No such cables were observed in vehicle-treated control cells. CD treatments caused autosomal bivalents in unusually large numbers of spermatocytes to become aggregated into densely-packed clusters; for example, with 40 microM CD about 80% of late diakinesis spermatocytes had clustered autosomes, vs. about 25% clustering in untreated cells. We conclude from these data that the mechanism of chromosome positioning at the nuclear envelope is CD-sensitive. Rhodamine-conjugates of phalloidin and DNase I were used to assess the status of actin in untreated cells as well as the effect of CD on actin distribution. Differences in nucleoplasmic staining with phalloidin and DNase I conjugates suggest that nucleoplasm at late diakinesis contains actin in a nonfilamentous form.

Actins↗
PubMed1985

Chromosome movement during meiotic prophase in crane-fly spermatocytes: III. Microtubules and the effects of Colcemid, nocodazole, and vinblastine.

The effects of Colcemid, nocodazole, and vinblastine on microtubules and on the movement of chromosomes during late diakinesis were investigated in spermatocytes of the crane fly Nephrotoma suturalis. The kinds of movements observed in untreated cells--sex bivalent rotations, sex bivalent excursions, and rotations and positional changes of autosomal bivalents--also were observed in drug-treated cells. These results were obtained in living cells in which the disruption and inhibition of microtubule assembly had been confirmed with polarized light microscopy. Effects of Colcemid and nocodazole also were assessed in fixed cells using electron microscopy. The results are in agreement with a hypothesis that microtubules are not a force-generating component of the molecular machinery that brings about prophase movements.

Animals↗
PubMed1992

Arrest in late G2 or prophase of cell cycle induced by 4,4-(1,2-ethanediyl) bis (1-isobutoxycarbonyloxymethyl 2, 6-piperazinedione) (MST-16) in cultured L1210 cells.

The effects of MST-16, a new antitumor agent derived from bis (2, 6-dioxopiperazine), on cell growth, cell-cycle progression and DNA synthesis, alone and in combination with other antitumor agents, were investigated in murine leukemia L1210 cells in vitro. The drug showed dose-dependent inhibition of cell growth, and this effect was cell-cycle phase-specific. Flow cytometric analysis indicated that the drug could retard-arrest the cells in late G2 phase or prophase and that it did not affect the progression from G1 to G2 phase. In the presence of MST-16, the change in 3H-thymidine incorporation was proportional to the retardation-arrest of the cells, suggesting that MST-16 has no direct action on DNA synthesis itself. MST-16 could continuously retard the cells which were arrested by etoposide (VP-16); and vincristine (VCR) could block the progression of the cells arrested by MST-16, but not vice versa. The addition of MST-16 followed by VCR was more effective than simultaneous addition of the 2 drugs on inhibition of cell growth. These results will be useful in designing a reasonable regimen of MST-16 chemotherapy for malignancies.

Animals↗
PubMed1996

Plant mitosis promoting factor disassembles the microtubule preprophase band and accelerates prophase progression in Tradescantia.

The regulation of mitosis in higher plant cells has been investigated by microinjecting protein kinase from the metaphase-arresting (met1) mutant of Chlamydomonas. Biochemical characterization of this enzyme complex confirms the presence of a p34cdc2/cyclin B-like kinase. The enzyme was injected into living stamen hair cells of Tradescantia virginiana in which microtubules (MTs) were visualized using fluorescent analogue cytochemistry and confocal laser scanning microscopy. Microinjection of this p34cdc2/cyclin B-like kinase caused rapid disassembly of the preprophase band of MTs but not of interphase-cortical, spindle or phragmoplast MTs. Effects of the enzyme on the cytomorphology of live prophase cells were also monitored using video microscopy. We found that injection of this enzyme accelerated chromatin condensation and nuclear envelope breakdown. This indicates the presence and function in plants of an enzyme that can initiate nuclear division similar to the maturation or mitosis promoting factor (MPF) of animal cells. These studies provide the first direct evidence that the mitotically-active form of plant MPF can drive disassembly of preprophase band MTs, chromosome condensation and initiation of mitosis in plant cells.

Animals↗
PubMed1997

Change in intracellular Ca2+ is not involved in serotonin-induced meiosis reinitiation from the first prophase in oocytes of the marine bivalve Crassostrea gigas.

In response to the neurohormone serotonin (5-hydroxytryptamine, 5-HT), prophase-arrested oocytes of the marine bivalve Crassostrea gigas (oyster) reinitiate meiosis, undergo germinal vesicle breakdown (GVBD), and are arrested again at metaphase I. We examined the pharmacological characteristics of 5-HT receptors and the signal transduction pathway following 5-HT stimulation in oyster oocytes. Among 5-HT agonists tested, only alpha-methyl 5-HT, a 5-HT2 agonist, induced GVBD although it was 1000 times less sensitive than 5-HT. The rank order of the potency of 5-HT antagonists to inhibit GVBD was propranolol, cyproheptadine > metoclopramide > mianserin. These results are quite different from those reported for other mollusks, suggesting the presence of unique 5-HT receptors on oyster oocytes. Using the fluorescent Ca2+ dyes fura 2 and calcium green and the pH indicator 1-hydroxypyrene-3,6,8-trisulfonic acid, we examined changes in intracellular Ca2+ ([Ca2+]i) and intracellular pH (pHi) during 5-HT-induced meiosis reinitiation. 5-HT did not trigger any changes in [Ca2+]i. However, an increase in pHi was observed during the 5-HT-induced meiosis reinitiation. The increased pHi level was rather small before GVBD and not necessary for GVBD, because lowering pHi by sodium acetate seawater (pH 7.0) did not prevent 5-HT-induced GVBD. Measurement of the kinase activity toward a peptide substrate specific to cdc2 demonstrated that maturation-promoting factor (MPF) was activated in accordance with the occurrence of GVBD in response to 5-HT. Therefore, it is likely that in oyster oocytes the signal transduction pathways and intracellular effectors participating in 5-HT-induced meiosis reinitiation via the activation of MPF are insensitive to [Ca2+]i and pHi.

Amino Acid Sequence↗
PubMed1990

Prophase of meiosis in human spermatocytes analysed by EM microspreading in infertile men and their controls and comparisons with human oocytes.

Observations at the electron microscope (EM) level have been made on 1883 primary spermatocytes from 40 chromosomally normal subfertile men and 566 spermatocytes from 10 fertile controls, using the technique of microspreading. Spermatocytes of infertile men in general showed greater indications of degeneration including higher levels of background silver deposition, nucleolar organising region - XY associations, fragmentation of synaptonemal complexes and overproduction of XY excrescences. A few oligospermic men also showed an immature morphology of the XY pair and/or a reduced extent of XY synapsis. Dissociation of the sex chromosome axes at prophase was found to occur with a much lower frequency than that recorded for separated X and Y chromosomes at metaphase I. In a single spermatocyte, synaptonemal complex formation was observed between Xqter and Yqter, a situation that could enable rare XqYq interchange. A proteinaceous stalked body exists on the Y axis towards its non-pairing end; this structure might have a functional relationship with the gene for spermatogenesis, (AZF), located at the euchromatin/heterochromatin interface. Compared with human oocytes, spermatocytes show fewer anomalies of synapsis, i.e. asynapsed segments or whole axes, non-homologous associations, interchanges, interlocks. These latter data agree well with findings from the mouse.

Adult↗
PubMed1988

Presence of microtubules in isolated cortices of prophase I and metaphase II oocytes in Xenopus laevis.

An extensive array of microtubules has been shown to exist in the cortex of Xenopus laevis oocytes both at the prophase I and metaphase II stages. The cortical microtubules were visualized after the oocyte cortex was squashed and immunostained using anti-tubulin antibody. They are cold- and nocodazole-sensitive; their stability to both treatments decreases after meiotic maturation. Biochemical extraction of manually isolated oocyte cortices, in a microtubule-stabilizing buffer, confirms these cytological observations.

Animals↗
PubMed1987

The human inactivated X chromosome folds in early metaphase, prometaphase, and prophase.

The inactivated X chromosome has a site of unusually frequent folding in region Xq1, whereas a fold in Xq1 is uncommon on the active X. We investigated the pattern of X chromosome folding in high-resolution GTG- and RBG-stained preparations from four women. In early metaphase cells, slightly more than 50% of late-replicating Xs folded at Xq1----Xq21, compared with about 6% of early replicating Xs. The late-replicating X folded in about 80% of prometaphase cells; the early, in only about 14% of these cells. and the late-replicating X folded in 19 of 20 prophase cells. Occasionally, one X had an omega-shaped loop or apparent physical connection between Xq13 and Xq21.1. It is possible that a segment of Xq1 never completely uncoils and may help to provide continuity for the Barr body from one interphase to the next.

Chromosome Banding↗
PubMed1983

A light- and electron microscopic analysis of meiotic prophase in female mice.

In the paper we describe meiotic prophase of female mice on successive days of embryonic and early postnatal development. For this purpose we used three different techniques on ovarian material, i.e., Giemsa staining for the light microscopic study of chromatin, silver staining for the light microscopic study of the synaptonemal complex (SC), and agar filtration followed by uranyl acetate staining for the electron microscopic study of the SC. In all types of preparation it was impossible to distinguish leptotene stages, and we conclude that if leptotene really exists, it is of very short duration.--Two types of zygotene stages were found: the "normal" one, resembling zygotene stages in male mice, and a second type that has never been described in males and is characterized by, probably stable, unpaired regions together with totally unpaired axial elements of the SC.--The duration of pachytene was found to be 3-4 days, which is considerably shorter than in males. During early diplotene despiralization of the chromatin and disintegration of the axes of the SC were usually found together with desynapsis.--A considerable variation in distribution of meiotic stages was found between different litters in the same day of gestation. Fetuses in the same litter showed no significant variation. However, the oocytes in an ovary did not pass through meiosis synchronously, with differences up several days. The appearance of chromosomes in a highly contracted state could not be interpreted as a preleptotene condensation stage but probably is a mitotic phenomenon.

Animals↗
PubMed1988

Two major components of synaptonemal complexes are specific for meiotic prophase nuclei.

Monoclonal antibody II52F10 was elicited against purified synaptonemal complexes (SCs); it recognizes two major components of the lateral elements of SCs, namely an Mr = 30,000 and an Mr = 33,000 protein. We studied the distribution of the antigens of II52F10 within tissues and cells of the male rat by immunoblot analysis and immunocytochemical techniques. Nuclear proteins from various cell types, including spermatogonia and spermatids, did not react with antibody II52F10 on immunoblots; the same holds for proteins from isolated mitotic chromosomes. As expected, an Mr = 30,000 and an Mr = 33,000 protein from spermatocyte nuclei did react with the antibody. In cryostat sections of liver, brain, muscle and gut we could not detect any reaction with II52F10. In the testis the reaction was confined to SCs or SC fragments. Partly on the basis of indirect evidence we identified the antigen-containing cells as zygotene up to and including post-diffuse diplotene spermatocytes. The persistence of some antigen-containing fragments in the earliest stages of spermatids could not be excluded. We conclude that the lateral elements (LEs) of SCs are not assembled by rearrangement of pre-existing components of the nucleus: at least two of their major components are newly synthesized, presumably during zygotene. Furthermore we conclude partly from indirect evidence that the major components of the LEs of SCs are not involved in the chromosome condensation processes that take place during the earliest stages of meiotic prophase.

Animals↗
PubMed1979

Light microscope analysis of meiotic prophase chromosomes by silver staining.

A method is described for the silver staining of the synaptonemal complex in surface-spread mammalian spermatocytes for light microscope examination. The method is quick, reliable, of broad applicability, and provides a means of making karyotype analysis at meiotic prophase. Many hundreds of suitable cells can be examined in an average preparation in a relatively short space of time. It has so far been applied only to mammalian spermatocytes, but could be used for karyotype analysis in oocytes of mammals and also applied to gonocytes of non-mammalian species.

Animals↗
PubMed1985

The prophase stages in human foetal oocytes studied by light and electron microscopy.

The progression of the prophase stages of meiosis in human foetal ovaries is reported from a series of aborted foetuses spanning the first two trimesters of gestation. A surface-spreading technique allowed cells to be examined at both light and electron microscope levels. The pachytene stage was specifically examined for evidence of synaptic or other abnormalities. Two observations of interest are the relatively high incidence with which errors of pairing occur and second the state of thickening observed when such unpaired axes are stained with silver.

Cells, Cultured↗
PubMed1990

Telomere and centromere DNA are associated with the cores of meiotic prophase chromosomes.

Mouse (Mus musculus) whole-mount, surface-spread, meiotic prophase chromosomes have an axial core structure, the synaptonemal complex, SC, from which extend chromatin loops. This arrangement permits a novel approach to the analysis of chromosome structure. Using in situ hybridization, the types of DNA sequences preferentially associated with the SC and the types located primarily in the chromatin loops can be determined. With biotinylated probes, detected by avidin conjugated to FITC, we present evidence for differential chromatin-SC interaction. The telomere sequence (TTAGGG)n is associated exclusively with the two ends of each autosomal SC rather than with the chromatin loops. The minor satellite DNA sequences are predominantly localized to the centromeric region of the SC, as defined by CREST serum anti-centromere antibodies. In contrast, the major satellite DNA probe hybridizes to the chromatin loops of the centromeric heterochromatin, and a probe containing a LINE sequence hybridizes to chromatin loops in general with no obvious preference for the SC. These observations demonstrate that, depending on the type of DNA sequence, the chromatin has different properties in regard to its association with the SC.

Animals↗