scholarly journals Effects of Synchronization of Donor Cell Cycle on Embryonic Development and DNA Synthesis in Porcine Nuclear Transfer Embryos

2007 ◽  
Vol 53 (2) ◽  
pp. 237-246 ◽  
Author(s):  
Kei MIYAMOTO ◽  
Yoichiro HOSHINO ◽  
Naojiro MINAMI ◽  
Masayasu YAMADA ◽  
Hiroshi IMAI
Keyword(s):  
Cell Cycle ◽  
Embryonic Development ◽  
Dna Synthesis ◽  
Nuclear Transfer ◽  
Donor Cell

scholarly journals CLONAGEM POR TRANSFERÊNCIA NUCLEAR EM BUBALINOS

2017 ◽  
Vol 13 (Especial 2) ◽  
pp. 110-117
Author(s):  
Aline Sousa Camargos ◽  
Ariane Dantas
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Somatic Nucleus ◽  
Number Of Species ◽  
Stage Of Development ◽  
Cellular Development

The success of core transfer (CT) depends on the origin of the donor cell, on the stage of development of the recipient cytoplast and on the synchronization between the cell cycle of the donor and recipient cells. The somatic nucleus must be reprogrammed after CT, thus restoring the totipotent state, and then resuming cellular development. However, it is noted that the efficiency of CT is still low, especially with a deficiency of the overall gene expression of the cloned embryo. However, the number of species of cloned mammals has been increasing in the last years, being this technique an important tool that does not aid in the effectiveness of buffalo reproduction. Thus, this review focuses on the description of the main processes pertinent to this process, as well as to analyze as future implications, as well as some factors that affect the success of nuclear transfer.

Control of cell cycle length in amphibian eggs: evidence for a temporal relationship between the nucleus and egg cytoplasm

Development ◽  
1988 ◽  
Vol 104 (3) ◽  
pp. 415-422
Author(s):  
C. Aimar
Keyword(s):  
Cell Cycle ◽  
Embryonic Development ◽  
Early Phase ◽  
Nuclear Transfer ◽  
Cycle Length ◽  
Temporal Relationship ◽  
Activation Time ◽  
Pleurodeles Waltl ◽  
Cell Cycle Length

The role of the cytoplasm and nucleus in the control of the length of the division cycle was investigated in Pleurodeles waltl eggs. Injection of spermine into enucleated eggs showed that the ability to cleave was not restricted just to the period of normal cytokinesis (T=1.0) but was possible throughout most of the first egg cycle. The cytoplasmic components required for cytokinesis seem to increase progressively during the first division cycle. Nuclear transfer experiments indicated that the timing of cleavage was normal only when the nucleus and egg cytoplasm were reassociated between T=0.0 (activation time) and T=0.50. Delayed associations, after T=0.50, provoked an alteration in the chronology of first cleavage and led to abnormal embryonic development. In the absence of a nucleus, the egg cycle seemed to stop at T=0.50. These different observations suggest that the normal timing of cleavage not only depends on a ‘cytoplasmic clock’ but is also determined by an isochronous nucleocytoplasmic relationship during the early phase of egg development.

scholarly journals 43 DNA SYNTHESIS, PREIMPLANTATION DEVELOPMENT AND Oct-4 EXPRESSION OF BOVINE CLONES RECONSTRUCTED WITH OOCYTES PREACTIVATED OR ENUCLEATED AFTER SPINDLE DISASSEMBLY

2005 ◽  
Vol 17 (2) ◽  
pp. 171
Author(s):  
S. Kurosaka ◽  
K.J. McLaughlin
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Dna Synthesis ◽  
Nuclear Transfer ◽  
Blastocyst Stage ◽  
Immunofluorescence Assay ◽  
Donor Cells ◽  
Spindle Disassembly ◽  
Student’S T

Enucleation procedures applied in mammalian cloning remove not only the oocyte's chromosomes but presumably also the spindle-associated factors. If these factors are beneficial for reprogramming, alternative protocols that limit enucleation of factors in addition to the chromosomes may improve cloning efficiency. In this study, we evaluated the enucleation in combination with various activation protocols on clone development and gene expression. Clones produced by nuclear transfer into pre-activated bovine oocytes rather than non-activated oocytes can develop in vitro (Kurosaka et al. 2002 Biol. Reprod. 67, 643–647; Tani et al. 2003 Biol. Reprod. 69, 1890–1894). We produced bovine clones using four different nuclear transfer protocols and, in clones of all groups, examined timing of DNA synthesis in the first cell cycle, pre-implantation development, and gene expression at the blastocyst stage. Protocols applied were: (A) donor cells were fused with non-activated oocytes; (B) donor cells were fused with oocytes at 2 hours after activation with ethanol (7%, 7 min); (C) oocytes were enucleated after spindle disassembly with nocodazole treatment (0.3 μg/mL, 30 min) and donor cells were fused with non-activated oocytes; and (D) oocytes were enucleated after spindle disassembly and donor cells were fused with oocytes at 2 h after activation. Fused couplets in all treatment groups were treated with 10 μg/mL cycloheximide for 6 h, and cultured in vitro in SOF supplemented with fetal bovine serum at 39°C in an atmosphere of 5% CO2, 5% O2, and 90% N2. The onset of DNA synthesis was determined by an immunofluorescence assay of 5-bromo-deoxyuridine incorporation at 6 and 9 h post-fusion (hpf). Oct-4 mRNA distribution in clone blastocysts was examined by whole mount in situ hybridization using a bovine Oct-4-specific antisense riboprobe. Data were statistically analyzed with Student's t-test. In the majority of clones DNA synthesis had not commenced 6 hpf but had initiated 9 hpf. Although the cell cycle of activated oocytes (protocols B and D) was 2 hours advanced compared to non-activated oocytes (protocols A and C), clones produced by all protocols had a similar onset of DNA synthesis at 6 to 9 h post-fusion. Developmental rates to the blastocyst stage of clones were not significantly different between the four protocols (48.5%–57.7%, P < 0.05). Oct-4 distribution in clones produced by all four protocols was not different from that of IVF embryos used as a control in that Oct-4 mRNA signal was typically restricted to the ICM (87.0%–100.0%, P < 0.05). We conclude that in bovine clones produced in this study, nocodazole-treated enucleation and activation status of recipient oocyte did not influence the pre-implantation development and spatial pattern of Oct-4 expression. This work was supported by the Lalor Foundation.

scholarly journals Effect of the number of passages of fetal and adult fibroblasts on nuclear remodelling and first embryonic division in reconstructed horse oocytes after nuclear transfer

Reproduction ◽  
2003 ◽  
pp. 535-542 ◽  
Author(s):  
X Li ◽  
JL Tremoleda ◽  
WR Allen
Keyword(s):  
Embryonic Development ◽  
Nuclear Transfer ◽  
Metaphase Plate ◽  
Donor Cell ◽  
Fibroblast Cells ◽  
Cell Nuclei ◽  
Cell Stage ◽  
Metaphase Ii Oocytes ◽  
Adult Fibroblasts

The effects of repeated passage in vitro of fetal fibroblast cells (FFC) and adult fibroblast cells (AFC) on nuclear remodelling and first embryonic division when used to reconstruct horse oocytes, and the reasons for the developmental block in progression to the two-cell stage were investigated. A total of 463 metaphase II oocytes produced 427 fibroblast-cytoplasm couplets after nuclear transfer, which finally resulted in 319 reconstructed oocytes. With increasing numbers of passages, the rates of nuclear remodelling decreased in both types of donor cell; about half of the fused donor cell nuclei showed the S-G2-prometaphase stages of the first embryonic division 18-20 h after cell-fusion treatment, irrespective of the number of donor cell passages (FFC: 49%; AFC: 53%). The rates of first embryonic division in the reconstructed oocytes fell with increasing age of the donor cells (FFC: 32%-26%-23%; AFC: 27%-23%-24%) and these rates were significantly lower than those obtained from metaphase II oocytes activated parthenogenetically (79%, P < 0.05). Microscopic analysis of the organization of the first embryonic division in the developmentally blocked oocytes reconstructed with either FFC or AFC showed that most of these (FFC: 78%; AFC: 92%) could not form the mitotic spindle and the metaphase plate of chromosomes. These findings indicate that either fetal or adult fibroblasts that have undergone relatively few passages in vitro are most suitable as donors. However, both types of cell have lower potential to restart first embryonic development after nuclear transfer than do the equivalent cells in other species. Improvement in the rate of donor cell nuclear progression from S-G2-prometaphase to beyond the metaphase stage, and the normal organization of first embryonic development in reconstructed horse oocytes, would seem to be the key to the production of cloned embryos in this species.

scholarly journals Cell cycle analysis and interspecies nuclear transfer of cat cells treated with chemical inhibitors

2014 ◽  
Vol 62 (2) ◽  
pp. 233-242 ◽  
Author(s):  
Manita Wittayarat ◽  
Akira Fujiwara ◽  
Kaywalee Chatdarong ◽  
Mongkol Techakumphu ◽  
Yoko Sato ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Fusion Rate ◽  
Cell Cycle Analysis ◽  
Control Group ◽  
Fibroblast Cells ◽  
Cell Stage ◽  
Chemical Inhibitors ◽  
M Phase

This study investigated the effect of chemical inhibitors on the cell-cycle synchronisation in cat fibroblast cells and evaluated the development of interspecies embryos reconstructed from cat donor cells and enucleated bovine oocytes. Cat fibroblast cells were treated with 15 μg/mL roscovitine or 0.05 μg/mL deme-colcine prior to cell cycle analysis and nuclear transfer. The percentage of cat fibroblast cells arrested at the G0/G1 phase in the roscovitine group was similar to that in the control group without any treatment. The percentage of cells arrested at the G2/M phase was significantly higher in the demecolcine group than in the control group. The fusion rate of interspecies couplets was significantly greater in the roscovitine group than in the control group. Most embryos stopped the development at the 2- or 4-cell stage, and none developed into blastocysts. Chemical inhibitor-induced donor cell cycle synchronisation did not overcome developmental arrest in interspecies cloned embryos.

scholarly journals Effect of Fibroblast Donor Cell Age and Cell Cycle on Development of Bovine Nuclear Transfer Embryos In Vitro1

2001 ◽  
Vol 64 (5) ◽  
pp. 1487-1493 ◽  
Author(s):  
Poothapillai Kasinathan ◽  
Jason G. Knott ◽  
Pedro N. Moreira ◽  
Amy S. Burnside ◽  
D. Joseph Jerry ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Cell Age

scholarly journals Centrosome changes during meiosis in horse oocytes and first embryonic cell cycle organization following parthenogenesis, fertilization and nuclear transfer

Reproduction ◽  
2006 ◽  
Vol 131 (4) ◽  
pp. 661-667 ◽  
Author(s):  
Xihe Li ◽  
Y Qin ◽  
Sandra Wilsher ◽  
W R Allen
Keyword(s):  
Cell Cycle ◽  
Nuclear Transfer ◽  
Germinal Vesicle ◽  
Donor Cell ◽  
Embryonic Cell ◽  
Immunofluorescent Staining ◽  
Sperm Chromatin ◽  
Germinal Vesicle Breakdown ◽  
Embryonic Cell Cycle

Various types of cell cycle organization occur in mammals. In this study, centrosome changes during meiosis in horse oocytes, and first cell cycle organization following fertilization, parthenogenesis and nuclear transfer, were monitored. Cumulus oocyte complexes harvested from horse ovaries obtained from slaughtered mares were cultured in vitro. Meiotic oocytes of germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I and II (MI and MII) stages were selected at various set times during in vitro maturation. Embryos at the first cell cycle stage were generated by subjecting MII stage oocytes to fertilization by intracytoplasmic sperm injection (ICSI), parthenogenetic treatment or nuclear transfer. Centrosome changes during meiosis and the first cell cycle organization were detected by indirect immunofluorescent staining, using a mouse anti-α-tubulin antibody for microtubules and a rabbit anti-γ-tubulin antibody for centrosomes. These examinations showed that the centrosomes of the horse oocyte reorganize themselves from the beginning of GV stage to leave only PCM of γ-tubulin surrounding both poles of the MI and MII stage spindles. These MII oocytes can organize the separation of metaphase chromosomes during the first embryonic cell cycle by parthenogenetic treatment. When the MII oocytes were subjected to ICSI or nuclear transfer, one or two red-stained centrosomes of γ-tubulin were introduced by the fertilising spermatozoon or the donor cell which associated with the sperm chromatin in the fertilized embryos and with the donor cell chromatin and microtubules in the cloned embryos. This finding suggests that centrosomes are not an essential component in the formation of the metaphase spindle during meiotic maturation of horse oocytes, but they can be introduced from the spermatozoon or donor cell and are necessary for the organization of normal embryonic development.

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