Effects of Preactivation of Ooplasts or Synchronization of Blastomere Nuclei in G1 on Preimplantation Development of Rabbit Serial Nuclear Transfer Embryos1

Piotrowska, Karolina; Modliński, Jacek A.; Korwin-Kossakowski, Maciej; Karasiewicz, Jolanta

doi:10.1095/biolreprod63.3.677

Cited by 23 publications

(10 citation statements)

References 28 publications

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“…Our current NT procedures apparently do not reprogram the differentiated, somatic nuclei of fetal fibroblasts; therefore, NT with donor cells expressing, for example, green fluorescence protein driven by exogenous or endogenous preimplantation, embryo-specific promoters could be useful in allowing assessment of embryonic genome activation [36]. Parenthetically, preactivation of the cytoplast was beneficial for the unsynchronized embryonic cell NT embryos in this study (Table 4), which is in agreement with previous results in monkeys [20], cattle [4,26], and rabbits [37]. We show here a high blastocyst formation rate based on a significant number of NT embryos (n ϭ 240) derived from 8-to 16-cell stage blastomeres.…”

Section: Discussionsupporting

confidence: 91%

Rhesus Monkey Embryos Produced by Nuclear Transfer from Embryonic Blastomeres or Somatic Cells1

Mitalipov

Yeoman

Nusser

et al. 2002

Biology of Reproduction

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Production of genetically identical nonhuman primates would reduce the number of animals required for biomedical research and dramatically impact studies pertaining to immune system function, such as development of the human-immunodeficiency-virus vaccine. Our long-term goal is to develop robust somatic cell cloning and/or twinning protocols in the rhesus macaque. The objective of this study was to determine the developmental competence of nuclear transfer (NT) embryos derived from embryonic blastomeres (embryonic cell NT) or fetal fibroblasts (somatic cell NT) as a first step in the production of rhesus monkeys by somatic cell cloning. Development of cleaved embryos up to the 8-cell stage was similar among embryonic and somatic cell NT embryos and comparable to controls created by intracytoplasmic sperm injection (ICSI; mean +/- SEM, 81 +/- 5%, 88 +/- 7%, and 87 +/- 4%, respectively). However, significantly lower rates of development to the blastocyst stage were observed with somatic cell NT embryos (1%) in contrast to embryonic cell NT (34 +/- 15%) or ICSI control embryos (46 +/- 6%). Development of somatic cell NT embryos was not markedly affected by donor cell treatment, timing of activation, or chemical activation protocol. Transfer of embryonic, but not of somatic cell NT embryos, into recipients resulted in term pregnancy. Future efforts will focus on optimizing the production of somatic cell NT embryos that develop in high efficiency to the blastocyst stage in vitro.

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Section: Discussionsupporting

confidence: 91%

Rhesus Monkey Embryos Produced by Nuclear Transfer from Embryonic Blastomeres or Somatic Cells1

Mitalipov

Yeoman

Nusser

et al. 2002

Biology of Reproduction

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“…After blind enucleation (Yang et al, 1992;Piotrowska et al, 2000), the oocytes were transferred with a Pasteur pipette to the drop of medium containing nuclear donor cells. A sufficient number of donor cells was added to another drop of B-Ham's without CCB in the same micromanipulation plate.…”

Section: Nuclear Transfermentioning

confidence: 99%

Oocyte age and nuclear donor cell type affect the technical efficiency of somatic cloning in rabbits

Cervera

García-Ximénez

2003

Zygote

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The present study in rabbits compared, in the first experiment, the effect of two commonly used oocyte ages, 13 h and 17 h after ovulation induction treatment, on the technical efficiency of somatic nuclear transfer steps, using fresh cumulus cells as nuclear donors. Recently ovulated metaphase II oocytes (13 h) showed higher fusion (13 h: 83% vs 17 h: 67%, p < 0.05) and in vitro development rates than in vivo slightly aged metaphase II oocytes (morula, 13 h: 74% vs 17 h: 25%, p < 0.05; blastocyst, 13 h: 16% vs 17 h: 8%; p < 0.05). In contrast, activation rate was higher in the 17 h group (13 h: 45% vs 17 h: 67%; p < 0.05). In a second experiment, using recently ovulated oocytes (13 h) as recipients, two donor cell types (from primary cultures of either cumulus cells or fetal fibroblasts) were tested to evaluate their effects on the efficiencies of the different technical steps of somatic nuclear transfer procedure. A better fusion rate was obtained when fetal fibroblasts were used as nuclear donors (cumulus cells: 45% vs fetal fibroblasts: 67%, p < 0.05). No statistically significant differences were detected in cleavage rate regardless of the cell type used (cumulus cells: 44% vs fetal fibroblasts: 60%, p > 0.05). However, in vitro development to morula (cumulus cells: 41% vs fetal fibroblasts: 14%, p < 0.05) and to blastocyst stage (cumulus cells: 27% vs fetal fibroblasts: 3%, p < 0.05) were different between cell types.

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“…As mentioned, so synchronized cell populations become less synchronous after progression through G 1 . Furthermore, undesirable effects such as cytotoxicity and growth imbalance are seen during arrest in mitosis (26–28). …”

Section: Introductionmentioning

confidence: 99%

Cell Synchronization by Inhibitors of DNA Replication Induces Replication Stress and DNA Damage Response: Analysis by Flow Cytometry

Darzynkiewicz

Halicka

Zhao

et al. 2011

Methods in Molecular Biology

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Cell synchronization is often achieved by inhibition of DNA replication. The cells cultured in the presence of such inhibitors as hydroxyurea, aphidicolin or thymidine become arrested at the entrance to S-phase and upon release from the block they synchronously progress through S, G 2 and M. We recently reported that exposure of cells to these inhibitors at concentrations commonly used to synchronize cell populations led to phosphorylation of histone H2AX on Ser139 (induction of γH2AX) through activation of ataxia telangiectasia mutated and Rad3-related protein kinase (ATR). These findings imply that the induction of DNA replication stress by these inhibitors activates the DNA damage response cell signaling pathways and caution about interpreting data obtained with the use of cells synchronized such way as representing unperturbed cells. The protocol presented in this chapter describes the methodology of assessment of phosphorylation of histone H2AX-Ser139, ATM/ATR substrate on Ser/Thr at SQ/TQ cluster domains as well as ataxia telangiectasia mutated (ATM) protein kinase in cells treated with inhibitors of DNA replication. Phosphorylation of these proteins is detected in individual cell immunocytochemically with phospho-specific Ab and measured by flow cytometry. Concurrent measurement of cellular DNA content and phosphorylated proteins followed by multiparameter cytometric analysis allows one to correlate extent of their phosphorylation with cell cycle phase.

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Effects of Preactivation of Ooplasts or Synchronization of Blastomere Nuclei in G1 on Preimplantation Development of Rabbit Serial Nuclear Transfer Embryos1

Cited by 23 publications

References 28 publications

Rhesus Monkey Embryos Produced by Nuclear Transfer from Embryonic Blastomeres or Somatic Cells1

Rhesus Monkey Embryos Produced by Nuclear Transfer from Embryonic Blastomeres or Somatic Cells1

Oocyte age and nuclear donor cell type affect the technical efficiency of somatic cloning in rabbits

Cell Synchronization by Inhibitors of DNA Replication Induces Replication Stress and DNA Damage Response: Analysis by Flow Cytometry

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