Cytological research on the argentophilic nucleolus-organizer regions of the metaphase chromosomes in parthenogenetic mouse embryos]

Silver staining technique visualizing argentophilic nucleolus organizer regions (Ag-NORs) was used for studying parthenogenetic mouse embryos produced by artificial activation of oocytes in Ca(2+)-Mg(2+)-free medium. Ag-NOR-containing chromosomes were detected in metaphases of parthenogenetic embryos during six successive cleavage divisions starting with the two-cell stage. The frequency of metaphases with varying AG-NOR number in diploid parthenogenones was similar to that in the control (fertilized) embryos. Average number of metaphase Ag-NOR chromosomes (calculated per diploid chromosome set) in haploid parthenogenones exceeded that in the control; in some cases all NORs were stained by silver. This is evidence that latent ribosomal cistrons in some chromosomes can be activated.     

Behavior of sperm nuclei incorporated into parthenogenetic mouse eggs prior to the first cleavage division.

When artificially activated mouse eggs are inseminated in the middle of the first cell cycle, sperm nuclei remain condensed until the first mitosis. During mitosis of the first cleavage division sperm nuclei decondense, subsequently recondense and are passively displaced to the daughter blastomeres. In the 2-cell embryos sperm nuclei form interphase nuclei which are able to replicate DNA and to condense into discrete chromosomes during the following mitotic division. These observations suggest that the mitotic cytoplasm of 1-cell embryos creates similar conditions for the transformation of sperm nuclei into male pronuclei as the cytoplasm of metaphase II oocytes.     

Cell-cycle-dependent subcellular localization of cyclin B1, phosphorylated cyclin B1 and p34cdc2 during oocyte meiotic maturation and fertilization in mouse

M phase or maturation promoting factor (MPF), a kinase complex composed of the regulatory cyclin B and the catalytic p34cdc2 kinase, plays important roles in meiosis and mitosis. This study was designed to detect and compare the subcellular localization of cyclin B1, phosphorylated cyclin B1 and p34cdc2 during oocyte meiotic maturation and fertilization in mouse. We found that all these proteins were concentrated in the germinal vesicle of oocytes. Shortly after germinal vesicle breakdown, all these proteins were accumulated around the condensed chromosomes. With spindle formation at metaphase I, cyclin B1 and phosphorylated cyclin B1 were localized around the condensed chromosomes and concentrated at the spindle poles, while p34cdc2 was localized in the spindle region. At the anaphase/telophase transition, phosphorylated cyclin B1 was accumulated in the midbody between the separating chromosomes/chromatids, while p34cdc2 was accumulated in the entire spindle except for the midbody region. At metaphase II, both cyclin B1 and p34cdc2 were horizontally localized in the region with the aligned chromosomes and the two poles of the spindle, while phosphorylated cyclin B1 was localized in the two poles of spindle and the chromosomes. We could not detect a particular distribution of cyclin B1 in fertilized eggs when the pronuclei were initially formed, but in late pronuclei cyclin B1 was accumulated in the pronuclei. p34cdc2 and phosphorylated cyclin B1 were always concentrated in one pronucleus after parthenogenetic activation or in two pronuclei after fertilization. At metaphase of 1-cell embryos, cyclin B1 was accumulated around the condensed chromosomes. Cyclin B1 was accumulated in the nucleus of late 2-cell embryos but not in early 2-cell embryos. Furthermore, we also detected the accumulation of p34cdc2 in the nucleus of 2- and 4-cell embryos. All these results show that cyclin B1, phosphorylated cyclin B1 and p34cdc2 have similar distributions at some stages but different localizations at other stages during oocyte meiotic maturation and fertilization, suggesting that they may play a common role in some events but different roles in other events during oocyte maturation and fertilization.     

Onset of nucleolus organizer activity in early mouse embryogenesis and evidence for its regulation

Ovulated mouse oocytes and preimplantation embryos were examined for NOR activity by means of selective silver staining. Evidence of the first staining activity appeared in two cell embryos, which was later followed by an increase in nucleolar activity, whereas the ovulated oocytes and pronuclei showed no such activity whatsoever. The staining of chromosomes was restricted to the nucleolus organizing region. Our results agree with earlier observations that genes for ribosomal RNA (rRNA) are transcribed as early as in the 2-cell stage in mouse embryogenesis. In addition to the nuclear staining we also observed some silver staining within the cytoplasm, at least from 4-cell stages onwards. Cytoplasmic staining was resistant to incubation with cycloheximide and actinomycin D. Nuclear staining was depressed, or even totally blocked, after actinomycin D incubation but was not blocked by cycloheximide. The onset of silver staining depends not on a specific embryonic stage but on the time interval following ovulation. This appears to indicate that the initiation of ribosomal cistrons is regulated by molecules which are activated or synthesized within the oocyte soon after ovulation.     

Developmental regulation of an snRNP core protein epitope during pig embryogenesis and after nuclear transfer for cloning.

The appearance and stabilization of a core protein epitope of the snRNP is developmentally regulated during pig embryogenesis. The epitope recognized by the monoclonal antibody Y12 is present in the germinal vesicle of mature oocytes and interphase nuclei of late 4-cell stage (24 to 30 hours post cleavage to the 4-cell stage) to blastocyst stage embryos. There was no antibody localization within pronuclei, or nuclei of 2-cell or early 4-cell stage embryos. Zygotes or 2-cell stage embryos cultured in the presence of alpha-amanitin to the late 4-cell stage showed no immunoreactivity, whereas control embryos had immunoreactivity. Thus antibody localization was correlated with RNA synthesis and RNA processing that begins by 24 hours post cleavage to the 4-cell stage. A final experiment showed no detectable immunoreactivity in 16-cell stage nuclei that had been transferred to enucleated activated meiotic metaphase II oocytes. Since immunoreactivity is associated with active RNA synthesis and RNA processing, it suggests that the 16-cell stage nucleus, which is RNA synthetically active, does not process RNA after nuclear transfer to an enucleated activated meiotic metaphase II oocyte.     

Cytogenetic study of silver-staining NOR in 8-cell-stage mouse blastomeres fused to 1-cell-stage embryos

Isolated blastomeres from 8- to 16-cell-stage embryos were fused by standard micromanipulatory means with either unfertilized eggs or fertilized or haploid parthenogenetically activated pronuclear-stage embryos. The hybrid eggs/embryos were incubated overnight in the presence of Colcemid until they had entered the first cleavage division. Air-dried chromosome preparations were then stained with silver nitrate in order to detect active nucleolar organizing regions (NOR). While control unfertilized eggs and 1-cell-stage fertilized and parthenogenetically activated embryos showed no evidence of silver-staining NOR-positive regions, the metaphase plates from 8- to 16-cell embryos showed characteristic NOR-positive regions, while their interphase nuclei also showed a characteristic reticular staining appearance. When hybrids between blastomere nuclei and unfertilized eggs were examined, none of the blastomere nuclei entered mitosis. However, when hybrids between blastomere nuclei and fertilized embryos were examined, in two thirds of the embryos, a single blastomere-derived diploid metaphase plate was present in association with two pronuclear-derived haploid metaphase plates. In most instances, the blastomere-derived chromosomes did not display silver-nitrate-staining NOR. Similar findings were observed when the blastomere-derived chromosomes in hybrids between blastomere nuclei and haploid parthenogenetic embryos were analysed. In the majority of cases, when blastomere nuclei remained in interphase, the characteristic silver-nitrate-staining fine reticular material either was not seen, or the nuclear contents were dispersed into clumps of chromatin-like material. Occasionally, the diploid chromosomes in the hybrids displayed morphological abnormalities. Our findings suggest that the cytoplasm of activated (but not nonactivated) 1-cell embryos is capable of influencing the nucleolar activity of the introduced 8- to 16-cell nuclei, effectively erasing from their chromosomes the memory of at least three previous rounds of rRNA synthesis.     

Kinetochore appearance during meiosis,fertilization and mitosis in mouse oocytes and zygotes

The events of mammalian fertilization overlap with the completion of meiosis and first mitosis; the pronuclei never fuse, instead the parental genomes first intermix at the mitotic spindle equator at metaphase. Since kinetochores are essential for the attachment of chromosomes to spindle microtubules, this study explores their appearance and behavior in mouse oocytes, zygotes and embryos undergoing the completion of meiosis, fertilization and mitoses. Kinetochores are traced with immunofluorescence microscopy using autoimmune sera from patients with CREST (CREST = calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia) scleroderma. These sera cross-react with the 17 kDa centromere protein (CENP-A) and the 80 kDa centromere protein (CENP-B) found at the kinetochores in human cell cultures. The unfertilized oocyte is ovulated arrested at second meiotic metaphase and kinetochores are detectable as paired structures aligned at the spindle equator. At meiotic anaphase, the kinetochores separate and remain aligned at the distal sides of the chromosomes until telophase, when their alignment perpendicular to the spindle axis is lost. The female pronucleus and the second polar body nucleus each receive a detectable complement of kinetochores. Mature sperm have neither detectable centrosomes nor detectable kinetochores, and shortly after sperm incorporation kinetochores become detectable in the decondensing male pronucleus. In pronuclei, the kinetochores are initially distributed randomly and later found in apposition with nucleoli. At mitosis, the kinetochores behave in a pattern similar to that observed at meiosis or mitosis in somatic cells: irregular distribution at prophase, alignment at metaphase, separation at anaphase and redistribution at telophase. They are also detectable in later stage embryos. Colcemid treatment disrupts the meiotic spindle and results in the dispersion of the meiotic chromosomes along the oocyte cortex; the chromosomes remain condensed with detectable kinetochores. Fertilization of Colcemid-treated oocytes results in the incorporation of a sperm which is unable to decondense into a male pronucleus. Remarkably kinetochores become detectable at 5 h post-insemination, suggesting that the emergence of the paternal kinetochores is not strictly dependent on male pronuclear decondensation.     

A maternal store of macroH2A is removed from pronuclei prior to onset of somatic macroH2A expression in preimplantation embryos

MacroH2A histones are variants of canonical histone H2A that are conserved among vertebrates. Previous studies have implicated macroH2As in epigenetic gene-silencing events including X chromosome inactivation. Here we show that macroH2A is present in developing and mature mouse oocytes. MacroH2A is localized to chromatin of germinal vesicles (GV) in both late growth stage (lg-GV) and fully grown (fg-GV) stage oocytes. In addition, macroH2A is associated with the chromosomes of mature oocytes, and abundant macroH2A is present in the first polar body. However, maternal macroH2A is lost from zygotes generated by normal fertilization by the late 2 pronuclei (2PN) stage. Normal embryos at 2-, 4-, and 8-cell stages lack macroH2A except in residual polar bodies. MacroH2A protein expression reappears in embryos after the 8-cell stage and persists in morulae and blastocysts, where nuclear macroH2A is present in both the trophectodermal and inner cell mass cells. We followed the loss of macroH2A from pronuclei in parthenogenetic embryos generated by oocyte activation. Abundant macroH2A is present upon the metaphase II plate and persists through parthenogenetic anaphase, but macroH2A is progressively lost during pronuclear decondensation prior to synkaryogamy. Examination of embryos generated by intracytoplasmic sperm injection (ICSI) revealed that macroH2A is associated exclusively with female pronuclei prior to loss in late pronucleus stage embryos. These results outline a surprising finding that a maternal store of macroH2A is removed from the maternal genome prior to synkaryogamy, resulting in embryos that execute three to four mitotic divisions in the absence of macroH2A prior to the onset of embryonic macroH2A expression.     

The effects of proteasome inhibitor lactacystin on mouse oocyte meiosis and first cleavage

Many studies have shown that the ubiq-uitin-proteasome pathway (UPP) for the degradation of short-lived proteins plays a key role in regulating cell cycle progression[1—3]. At least two distinct prote-olytic pathways are required for cell cycle process. The first pathway promotes transition from G1 to S phase, and the second initiates the onset of anaphase and exit from mitosis. The inhibition of UPP will re-sult in the blockage of cell cycle process. The knowl-edge of the role of UPP in…     

The effects of proteasome inhibitor lactacystin on mouse oocyte meiosis and first cleavage

In order to study the effects of ubiquitin-proteasome pathway (UPP) on mouse oocyte meiosis and cleavage, oocytes undergoing maturation and parthenogenetic activation and 1-cell embryos were treated with lactacystin, a specific inhibitor of proteasome. The results indicated that the rate of GVBD was not influenced by the treatment, but polar body extrusion, parthenogenesis and first cleavage were inhibited. Immunofluorescent staining using anti β-tubulin antibody indicated that the continuous treatment of lactacystin from GV stage disorganized microtubules and spindle assembly. When metaphase stage oocytes were treated with the drug, the already formed spindle structure was not affected, but the oocytes were arrested at metaphases. The 1-cell embryos were arrested at interphase or metaphase of first mitosis when they were incubated in the drug. Proteasome regulatory subunit PA700 was located in the spindle region, as indicated by immunofluorescence. These results suggest that UPP has effects on the process of oocyte meiosis and early cleavage in many aspects, including normal organization of spindle at prophase and segregation of chromosomes at anaphase for normal meiosis.     

Transformations of sperm nuclei incorporated into sea urchin (Arbacia punctulata) embryos at different stages of the cell cycle

In order to test the hypothesis that regulators of male pronuclear development may have a more general role, sharing some relation to factors involved with the cell cycle, Arbacia zygotes and 2- to 8-cell stage embryos were inseminated during different phases of the cell cycle and examined by light and electron microscopy. Differences in the development and morphology of fertilization cones and sperm asters were observed in embryos inseminated during different stages of the cell cycle. Extremely large fertilization cones, approximately four times the length of those found in fertilized eggs, formed in embryos inseminated during metaphase to telophase. Sperm asters developed only in embryos inseminated during prophase to anaphase. These variations are believed to reflect changes in the status of the cortex and cytoskeletal system of the embryo. Although sperm nuclei underwent morphological changes subsequent to incorporation, in general, they failed to develop into male pronuclei. There was a consistent correlation in sperm nuclear transformations and the cell cycle which was expressed in two patterns of morphogenesis: (1) sperm nuclei incorporated into embryos just prior to prophase and at telophase failed, for the most part, to disperse and transformed into aggregations of chromatin granules approximately 40 nm in diameter; and (2) sperm nuclei incorporated into prometaphase-anaphase embryos dispersed and then condensed into chromatin masses, morphologically similar to chromosomes of the embryo. Evidence is discussed which indicates that following the normal period of fertilization changes occur in the zygote, rendering it unable to fully support the transformation of sperm nuclei into male pronuclei.     

REINSEMINATION OF FERTILIZED SEA URCHIN (ARBACIA PUNCTULATA) EGGS

Embryos of Arbacia punctulata , treated to remove the fertilization membrane and hyaline layer, were mixed with sperm (reinseminated) at 20–30 min (streak stage) and at 70 min (2-cell stage) postinsemination. Sperm, incorporated into embryos reinseminated at the streak stage, metamorphosed into male pronuclei which subsequently migrated to and fused with the zygote nucleus. Although blastomeres were capable of incorporating sperm and forming fertilization cones, less than 10% of the 2-cell stage embryos reinseminated. Sperm, were found entrapped within an amorphous material along the surface of 2-cell stage embryos; many had failed to undergo an acrosome reaction. These results indicate that conditions necessary for incorporation and metamorphosis of sperm nuclei into male pronuclei are present in the embryo after the normal period of fertilization.     

Unique Pattern of ORC2 and MCM7 Localization During DNA Replication Licensing in the Mouse Zygote

In eukaryotes, DNA synthesis is preceded by licensing of replication origins. We examined the subcellular localization of two licensing proteins, ORC2 and MCM7, in the mouse zygotes and two-cell embryos. In somatic cells ORC2 remains bound to DNA replication origins throughout the cell cycle, while MCM7 is one of the last proteins to bind to the licensing complex. We found that MCM7 but not ORC2 was bound to DNA in metaphase II oocytes and remained associated with the DNA until S-phase. Shortly after fertilization, ORC2 was detectable at the metaphase II spindle poles and then between the separating chromosomes. Neither protein was present in the sperm cell at fertilization. As the sperm head decondensed, MCM7 was bound to DNA, but no ORC2 was seen. By 4 h after fertilization, both pronuclei contained DNA bound ORC2 and MCM7. As expected, during S-phase of the first zygotic cell cycle, MCM7 was released from the DNA, but ORC2 remained bound. During zygotic mitosis, ORC2 again localized first to the spindle poles, then to the area between the separating chromosomes. ORC2 then formed a ring around the developing two-cell nuclei before entering the nucleus. Only soluble MCM7 was present in the G2 pronuclei, but by zygotic metaphase it was bound to DNA, again apparently before ORC2. In G1 of the two-cell stage, both nuclei had salt-resistant ORC2 and MCM7. These data suggest that licensing follows a unique pattern in the early zygote that differs from what has been described for other mammalian cells that have been studied.