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Joseph G Gall - One of the best experts on this subject based on the ideXlab platform.

  • An inducible nuclear body in the Drosophila Germinal Vesicle.
    Nucleus (Austin Tex.), 2011
    Co-Authors: Alison B. Singer, Joseph G Gall
    Abstract:

    When living egg chambers of Drosophila are isolated in a saline solution and gently squashed between a microscope slide and coverslip, prominent nuclear bodies (1 - 20 mm diameter) can be seen inside the oocyte nucleus or Germinal Vesicle (GV).  These bodies do not pre-exist within the GV and are not seen in material that is fixed in paraformaldehyde before squashing.  Instead, they form spontaneously within minutes after an egg chamber is damaged and the cytoplasm is exposed to the isolation medium.  Electron microscopy shows that the bodies lack an investing membrane and consist of closely packed, irregular particles 30-50 nm in diameter.  We used GFP-tagged proteins from the Carnegie Protein Trap Library to identify 22 proteins that are either enriched in the bodies or excluded from them.  We were unable to discern common features of proteins that are concentrated in the bodies, such as isoelectric point, molecular weight, or biological process.  Induced bodies are formed in GVs of flies that are null ...

  • Nuclear bodies in the Drosophila Germinal Vesicle
    Chromosome Research, 2006
    Co-Authors: Michael Buszczak, Joseph G Gall
    Abstract:

    The Germinal Vesicle of the Drosophila oocyte is transcriptionally quiescent during the latter part of the first meiotic prophase. Concomitant with silencing of the genome, the nucleolus disappears at an early stage and the chromatin condenses into a compact mass called the karyosome. A prominent Cajal body (endobody) is present during most of prophase, attached to the karyosome. Components of the U7 small nuclear (sn) RNP reside in a separate body, the histone locus body, which is also attached to the karyosome. The histone locus body is no longer detectable with probes for the U7 snRNP after about stage 5 of oogenesis. Several other nuclear bodies of unknown nature can be detected by phase contrast, differential interference contrast, and electron microscopy.

  • Structure in the amphibian Germinal Vesicle.
    Experimental cell research, 2004
    Co-Authors: Joseph G Gall, Christine Murphy, Hongjuan Gao
    Abstract:

    Abstract The Germinal Vesicle (GV) of Xenopus laevis is an enormous nucleus that contains 18 giant lampbrush chromosomes and thousands of inclusions. The inclusions are primarily of three types: ∼1500 extrachromosomal nucleoli, 50–100 Cajal bodies, and several thousand B-snurposomes, which correspond to speckles or interchromatin granule clusters in other nuclei. The large size and abundance of the GV organelles, as well as the ease with which they can be studied both in vivo and in vitro, make the GV an ideal object for analysis of nuclear structure and function.

  • dynamics of coilin in cajal bodies of the xenopus Germinal Vesicle
    Proceedings of the National Academy of Sciences of the United States of America, 2004
    Co-Authors: Svetlana Deryusheva, Joseph G Gall
    Abstract:

    Cajal bodies (CBs) are complex organelles found in the nuclei of a wide variety of organisms, including vertebrates, invertebrates, plants, and yeast. In most cell types CBs are <1 μm in diameter, severely limiting the range of experimental observations that can be made on them. By contrast, CBs in the amphibian oocyte nucleus (also called the Germinal Vesicle) are 2–10 μm in diameter. We have taken advantage of this large size to carry out kinetic studies on coilin, a protein that is specifically enriched in CBs. We labeled coilin with photoactivatable green fluorescent protein and analyzed the movement of the protein by confocal microscopy in unfixed Germinal Vesicles isolated in oil. We showed that coilin leaves the CB relatively slowly (minutes rather than seconds) with kinetics similar to earlier measurements on its entrance. We also showed that coilin diffuses very slowly within the CB, consistent with its being in a large macromolecular complex. Finally, we found that the movement of coilin is not directly affected by the transcriptional state of the nucleus or ongoing nucleocytoplasmic exchange. These data on the kinetics of coilin reinforce the conclusion that CB components are in a constant state of flux, consistent with models that postulate an active role for CBs in nuclear physiology.

  • the stem loop binding protein slbp1 is present in coiled bodies of the xenopus Germinal Vesicle
    Molecular Biology of the Cell, 1999
    Co-Authors: Jennifer Abbott, William F Marzluff, Joseph G Gall
    Abstract:

    The stem-loop binding protein (SLBP1) binds the 39 stem-loop of histone pre-mRNA and is required for efficient processing of histone transcripts in the nucleus. We examined the localization of SLBP1 in the Germinal Vesicle of Xenopus laevis oocytes. In spread preparations of Germinal Vesicle contents, an anti-SLBP1 antibody stained coiled bodies and specific chromosomal loci, including terminal granules, axial granules, and some loops. After injection of myc-tagged SLBP1 transcripts into the oocyte cytoplasm, newly translated myc-SLBP1 protein was detectable in coiled bodies within 4 h and in terminal and axial granules by 8 h. To identify the region(s) of SLBP1 necessary for subnuclear localization, we subcloned various parts of the SLBP1 cDNA and injected transcripts of these into the cytoplasm of oocytes. We determined that 113 amino acids at the carboxy terminus of SLBP1 are sufficient for coiled body localization and that disruption of a previously defined RNA-binding domain did not alter this localization. Coiled bodies also contain the U7 small nuclear ribonucleoprotein particle (snRNP), which participates in cleavage of the 39 end of histone pre-mRNA. The colocalization of SLBP1 and the U7 snRNP in the coiled body suggests coordinated control of their functions, perhaps through a larger histone-processing particle. Some coiled bodies are attached to the lampbrush chromosomes at the histone gene loci, consistent with the view that coiled bodies in the oocyte recruit histone-processing factors to the sites of histone pre-mRNA transcription. The non-histone chromosomal sites at which SLBP1 is found include the genes coding for 5 S rRNA, U1 snRNA, and U2 snRNA, suggesting a wider role for SLBP1 in the biosynthesis of small non-spliced RNAs.

Qing-yuan Sun - One of the best experts on this subject based on the ideXlab platform.

  • Cytoplasmic Determination of Meiotic Spindle Size Revealed by a Unique Inter-Species Germinal Vesicle Transfer Model.
    Scientific reports, 2016
    Co-Authors: Zhong-wei Wang, Guang-li Zhang, Heide Schatten, John L. Carroll, Qing-yuan Sun
    Abstract:

    Spindle sizes are different in diverse species and cell types. In frogs, the meiotic spindle size is positively correlated with the egg cell volume. Across species, relatively small mouse oocytes (70-80 μm) have a relatively large spindle while larger pig oocytes (about 120 μm) have a considerably smaller spindle. In this study we investigated whether species-specific oocyte spindle size was determined by cytoplasmic or nuclear factors. By exchanging the Germinal Vesicle between mouse and pig oocytes, we obtained two kinds of reconstructed oocytes: one with mouse ooplasm and pig GV (mCy-pGV oocyte), and the other with pig ooplasm and mouse GV (pCy-mGV oocyte). We show that the MII spindle size of the mCy-pGV oocyte is similar to that of the mouse meiotic spindle and significantly larger than that of the pig meiotic spindle. The timing of oocyte maturation also followed that of the species from which the oocyte cytoplasm arose, although some impact of the origin of the GV was observed. These data suggest that spindle size and the timing of meiotic progression are governed by cytoplasmic components rather than cytoplasmic volume and GV materials.

  • cyclin o regulates Germinal Vesicle breakdown in mouse oocytes
    Biology of Reproduction, 2013
    Co-Authors: Yingchun Ouyang, Heide Schatten, Yibo Luo, Zhenbo Wang, Yi Hou, Zhiming Han, Zhonghua Liu, Qing-yuan Sun
    Abstract:

    It is well accepted that oocyte meiotic resumption is mainly regulated by the maturation-promoting factor (MPF), which is composed of cyclin B1 (CCNB1) and cyclin-dependent kinase 1 (CDC2). Maturation-promoting factor activity is regulated by the expression level of CCNB1, phosphorylation of CDC2, and their Germinal Vesicle (GV) localization. In addition to CCNB1, cyclin O (CCNO) is highly expressed in oocytes, but its biological functions are still not clear. By employing short interfering RNA microinjection of GV-stage oocytes, we found that Ccno knockdown inhibited CDC2 (Tyr15) dephosphorylation and arrested oocytes at the GV stage. To rescue meiotic resumption, cell division cycle 25 B kinase (Cdc25b) and Ccnb1 were overexpressed in the Ccno knockdown oocytes. Unexpectedly, we found that Ccno knockdown did not affect CDC25B entry into the GV, and overexpression of CDC25B was not able to rescue resumption of oocyte meiosis. However, GV breakdown (GVBD) was significantly increased after overexpression of Ccnb1 in Ccno knockdown oocytes, indicating that GVBD block caused by cyclin O knockdown can be rescued by cyclin B1 overexpression. We thus conclude that cyclin O, as an upstream regulator of MPF, plays an important role in oocyte meiotic resumption in mouse oocytes.

  • Inducible nitric oxide synthase-derived nitric oxide regulates Germinal Vesicle breakdown and first polar body emission in the mouse oocyte
    Reproduction (Cambridge England), 2005
    Co-Authors: Li-jun Huo, Cheng-guang Liang, Zhisheng Zhong, Zeng-ming Yang, Heng-yu Fan, Da-yuan Chen, Qing-yuan Sun
    Abstract:

    The present study investigated the subcellular localization of inducible nitric oxide synthase (iNOS) during mouse oocyte meiotic maturation and fertilization using confocal microscopy, and further studied the roles of iNOS-derived NO in oocyte maturation by using an iNOS-specific inhibitor aminoguanidine (AG) and iNOS antibody microinjection. In Germinal Vesicle-stage oocytes, iNOS immunoreactivity was mainly localized in the Germinal Vesicle. Shortly after Germinal Vesicle breakdown, the iNOS immunoreactivity accumulated around the condensed chromosomes. At metaphase I and metaphase II, with the organization of chromosomes to the equatorial plate, iNOS immunoreactivity was concentrated around the aligned chromosomes, putatively the position of the metaphase spindle. The accumulation of iNOS immunoreactivity could not be detected at anaphase I and anaphase II. However, at telophase I and telophase II, the staining of iNOS was concentrated in the region between the separating chromosomes/chromatids. Furthermore, the staining of iNOS also accumulated in the male and female pronuclei in fertilized eggs. Germinal Vesicle breakdown and the first polar body emission of the oocytes were significantly blocked by the iNOS-specific inhibitor AG in a dose-dependent manner. The Germinal Vesicle breakdown in oocytes injected with iNOS antibody was also inhibited. We found that the phosphorylation of mitogen-activated protein kinase in oocytes after Germinal Vesicle breakdown was inhibited by AG treatment. The control oocytes extruded a normal first polar body, while the AG-treated oocytes exhibited an elongated protrusion or no elongated protrusion. The results of confocal microscopy showed that the AG-treated oocytes were arrested at anaphase I-telophase I. Our results suggest that the iNOS-derived NO pathway plays important roles in mouse oocyte meiotic maturation, especially in Germinal Vesicle breakdown and the anaphase-telophase transition.

  • viable rabbits derived from reconstructed oocytes by Germinal Vesicle transfer after intracytoplasmic sperm injection icsi
    Molecular Reproduction and Development, 2001
    Co-Authors: Da-yuan Chen, Qing-yuan Sun, Li Lian, Minkang Wang, Jilong Liu, Zhiming Han
    Abstract:

    Abnormal oocyte spindle due to the improper function of ooplasm is associated with female infertility of advanced maternal age. A possible way to overcome this problem is to transfer an oocyte Germinal Vesicle (GV) which contains genetic materials of a patient with a history of poor embryo development to the cytoplast from a donor oocyte. Here we demonstrate that GV transfer is feasible using a rabbit model. When the GVs were transferred to auto- or hetero-cytoplasts of GV stage oocytes, around 80% of the reconstructed oocytes could mature in vitro and 7.1-9.4% of the oocytes developed to blastocyst stage after intracytoplasmic sperm injection (ICSI). Transfer of 93 fertilized eggs reconstructed via GV transfer into six recipients resulted in two live offspring. Results of this experiment indicate that GV transfer can potentially become a new approach in treatment of infertility because of advanced maternal age.

J Van Der Elst - One of the best experts on this subject based on the ideXlab platform.

  • embryo development after successful somatic cell nuclear transfer to in vitro matured human Germinal Vesicle oocytes
    Human Reproduction, 2007
    Co-Authors: Bjorn Heindryckx, P De Sutter, Jan Gerris, Marc Dhont, J Van Der Elst
    Abstract:

    BACKGROUND: Somatic cell nuclear transfer (SCNT) involves the transfer of somatic cell nuclei into enucleated oocytes. Because human in vivo matured oocytes are scarcely available, we investigated whether in vitro matured (IVM) Germinal Vesicle (GV) oocytes could also support preimplantation development of human cloned embryos. METHODS: Three groups were used for SCNT: in vitro matured GV oocytes (IVM oocytes), ‘in vivo’ matured oocytes (in vivo oocytes) and ‘failed fertilized’ oocytes after routine-ICSI (FF oocytes). After removal of the chromosome–spindle complex, cumulus cell nuclei were injected, and oocytes were artificially activated and cultured. RESULTS: In total 61, 54 and 45 metaphase II oocytes were used for SCNT in the three groups, respectively. Survival and pronuclear rates were 59, 78 and 58% and 61, 64 and 50%, respectively. Of the 22 activated IVM oocytes, 13 cleaved to the 2-cell stage, whereby 2 morulae were formed. For the in vivo oocytes, 17 of 27 activated oocytes cleaved to the 2-cell stage and 1 morula was observed. Cleavage to the 2-cell stage in the group of FF oocytes was compromised. CONCLUSIONS: To our knowledge, this is the first report describing development of cloned human embryos using IVM oocytes and non-autologous transfer using a conventional method of SCNT.

John Z H Zhang - One of the best experts on this subject based on the ideXlab platform.

  • metaphase ii nuclei generated by Germinal Vesicle transfer in mouse oocytes support embryonic development to term
    Human Reproduction, 2003
    Co-Authors: H Liu, John Z H Zhang, Jamie Grifo, H C Chang, L C Krey
    Abstract:

    BACKGROUND: Cytoplasmic defects are thought to cause aneuploidies in oocytes and embryos and oocyte ‘reconstruction’ by Germinal Vesicle (GV) transfer may circumvent such defects. In mice ‘reconstructed’ oocytes undergo meiosis and fertilize normally, but early embryonic development is compromised if their ooplasm matured in vitro. This study employs sequential MII spindle and/or pronucleus (PN) transfer to assess the embryonic potential of MII nuclei that form following GV transfer. METHODS AND RESULTS: Mouse embryos generated by these procedures were transferred to the oviducts of pseudopregnant mice to monitor pregnancy outcome. Following GV transfer, the resultant metaphase II (MII) nuclei were activated either in situ or transferred and activated in ooplasts from in-vivo matured oocytes. When exchanged with the female PN of a fertilized zygote, only the PNs that developed in in-vivo matured ooplasts generated live offspring. Viable offspring also resulted when MII nuclei were transferred to in-vivo matured ooplasts and fertilized by insemination with sperm or by artificial activation and male PN transfer. Significantly, the offspring displayed normal fertility as adults. CONCLUSION: This report of live births following GV transfer in mice illustrates the importance of the maturational history of the ooplasm at PN formation for normal embryonic and fetal development.

  • reconstruction of mouse oocytes by Germinal Vesicle transfer maturity of host oocyte cytoplasm determines meiosis
    Human Reproduction, 1999
    Co-Authors: H Liu, L C Krey, Chiawoei Wang, J Grifo, John Z H Zhang
    Abstract:

    We evaluated the maturational competence of mouse oocytes reconstructed by the transfer and electrofusion of Germinal Vesicles (GV) into anuclear cytoplasts of GV stage oocytes (both auto- and hetero-transfers), metaphase II stage oocytes or zygotes. Following in-vitro culture, the maturation rates of the reconstructed oocytes to metaphase II did not significantly differ between auto- and hetero-transfers (40/70 versus 95/144 respectively); these rates also did not differ from those of control oocytes (57/97) which were matured in vitro without micromanipulation and electrofusion. In contrast, when a GV was transferred into an enucleated metaphase II oocyte or a zygote, only a few reconstructed oocytes underwent Germinal Vesicle breakdown (5/30 and 2/21 respectively); moreover, none reached metaphase II stage. Cytogenetic and immunofluorescence analyses were conducted on hetero-GV oocytes that extruded a first polar body. Each oocyte showed two groups of chromosomes, one in the cytoplast and one in the polar body, as well as a bipolar spindle with twenty univalent chromosomes. Our findings suggest that oocytes reconstructed by GV transfer into a cytoplast of the same developmental stage mature normally in vitro through metaphase II. Such oocytes may be a useful research model to elucidate the cytoplasmic and nuclear mechanisms regulating meiosis and the relationships between meiotic errors and age-related changes in the oocyte.

  • in vitro maturation of human preovulatory oocytes reconstructed by Germinal Vesicle transfer
    Fertility and Sterility, 1999
    Co-Authors: John Z H Zhang, Chiawoei Wang, L C Krey, H Liu, Li Meng, A Blaszczyk, Alexus Adler, Jamie Grifo
    Abstract:

    Abstract Objective: To describe a micromanipulation-electrofusion procedure for transferring Germinal Vesicles (GVs) between immature human oocytes. Design: Pilot study to assess oocyte maturation after an invasive micromanipulation procedure. Setting: Research laboratory at a university medical center. Patient(s): Immature oocytes were discarded from intracytoplasmic sperm injection (ICSI)-IVF cycles of patients 23–48 years of age. Intervention(s): Initially, GV removal and transfer were performed on the same oocyte; these "self-reconstructed" oocytes were then cultured in vitro for up to 50 hours and examined periodically for maturation as judged by the extrusion of the first polar body. In a second study, GVs from oocytes of "old" patients (>38 years old) were successfully transferred into enucleated immature oocytes of "young" patients ( Main Outcome Measure(s): Extrusion of the first polar body was monitored in "reconstructed" and control oocytes; karyotypes also were analyzed at meiosis II. Result(s): From 48 oocytes from old patients, 12 GVs were successfully removed, transferred, and fused into previously enucleated oocytes from young patients. After in vitro culture, 7 of these "reconstructed" oocytes matured to meiosis II, a maturation rate not significantly different from that observed in nonmanipulated controls. A normal, second meiotic metaphase chromosome complement was observed in 4 of 5 reconstructed oocytes. Conclusion(s): Normal meiosis can occur after the transfer of a GV into an enucleated host oocyte. Germinal Vesicle transfer may be a valuable research procedure that generates cell models to characterize the cytoplasmic-nuclear interplay for cell cycle regulation, maturation, and fertilization in the human oocyte; it also may be a potentially attractive alternative to oocyte donation.

Shane C Burgess - One of the best experts on this subject based on the ideXlab platform.

  • proteomics based systems biology modeling of bovine Germinal Vesicle stage oocyte and cumulus cell interaction
    PLOS ONE, 2010
    Co-Authors: Divyaswetha Peddinti, Erdogan Memili, Shane C Burgess
    Abstract:

    Background: Oocytes are the female gametes which establish the program of life after fertilization. Interactions between oocyte and the surrounding cumulus cells at Germinal Vesicle (GV) stage are considered essential for proper maturation or ‘programming’ of oocytes, which is crucial for normal fertilization and embryonic development. However, despite its importance, little is known about the molecular events and pathways involved in this bidirectional communication. Methodology/Principal Findings: We used differential detergent fractionation multidimensional protein identification technology (DDF-Mud PIT) on bovine GV oocyte and cumulus cells and identified 811 and 1247 proteins in GV oocyte and cumulus cells, respectively; 371 proteins were significantly differentially expressed between each cell type. Systems biology modeling, which included Gene Ontology (GO) and canonical genetic pathway analysis, showed that cumulus cells have higher expression of proteins involved in cell communication, generation of precursor metabolites and energy, as well as transport than GV oocytes. Our data also suggests a hypothesis that oocytes may depend on the presence of cumulus cells to generate specific cellular signals to coordinate their growth and maturation. Conclusions/Significance: Systems biology modeling of bovine oocytes and cumulus cells in the context of GO and protein interaction networks identified the signaling pathways associated with the proteins involved in cell-to-cell signaling biological process that may have implications in oocyte competence and maturation. This first comprehensive systems biology modeling of bovine oocytes and cumulus cell proteomes not only provides a foundation for signaling and cell physiology at the GV stage of oocyte development, but are also valuable for comparative studies of other stages of oocyte development at the molecular level.

  • bovine Germinal Vesicle oocyte and cumulus cell proteomics
    Reproduction, 2007
    Co-Authors: Erdogan Memili, Divyaswetha Peddinti, Leslie A Shack, Bindu Nanduri, Fiona M Mccarthy, H Sagirkaya, Shane C Burgess
    Abstract:

    Germinal Vesicle (GV) breakdown is fundamental for maturation of fully grown, developmentally competent, mammalian oocytes. Bidirectional communication between oocytes and surrounding cumulus cells (CC) is essential for maturation of a competent oocyte. However, neither the factors involved in this communication nor the mechanisms of their actions are well defined. Here, we define the proteomes of GVoocytes and their surrounding CC, including membrane proteins, using proteomics in a bovine model. We found that 4395 proteins were expressed in the CC and 1092 proteins were expressed in oocytes. Further, 858 proteins were common to both the CC and the oocytes. This first comprehensive proteome analysis of bovine oocytes and CC not only provides a foundation for signaling and cell physiology at the GV stage of oocyte development, but are also valuable for comparative studies of other stages of oocyte development at the molecular level. Furthermore, some of these proteins may represent molecular biomarkers for developmental potential of oocytes.