scholarly journals Effect of enucleation procedures and maturation conditions on the development of nuclear-transferred rabbit oocytes receiving male fibroblast cells

Reproduction ◽  
2002 ◽  
pp. 41-47 ◽  
Author(s):  
XJ Yin ◽  
Y Kato ◽  
Y Tsunoda
Keyword(s):  
Cell Cycle ◽  
Nuclear Transfer ◽  
Chromosomal Abnormalities ◽  
Mature Male ◽  
Fibroblast Cells ◽  
Spindle Fibre ◽  
Implantation Sites ◽  
Low Efficiency ◽  
Successful Production

Enucleated oocytes matured in vitro, from which chromosomes were removed by treatment with ionomycin and demecolcine, were used as recipient oocytes for nuclear transfer of fibroblast cells from a mature male rabbit. The enucleated oocytes with donor nuclei were electrically activated 2 h after fusion. The potential of nuclear-transferred oocytes matured in vitro and ovulated oocytes to develop into blastocysts was high (33-55%), except for oocytes cultured for 8.0 (19%) and 8.5 h (25%) in vitro. After transfer of nuclear-transferred oocytes to recipients, ten of 62 (16%) and one of eight (13%) recipients that received in vitro-matured and ovulated oocytes, respectively, had 19 (1%) and one (0.6%) implantation sites at the time of laparotomy on days 8-17 after transfer. Four fetuses, including two with beating hearts, were obtained on day 15 of gestation after transfer of nuclear-transferred oocytes matured in vitro. The reason for the low efficiency of fetus production was not clear. One possibility is chromosomal abnormalities of nuclear-transferred oocytes, as most (21 of 22) of the oocytes had chromosomes dispersed along the spindle fibre at the first cell cycle. This is the first report of successful production of fetuses after nuclear transfer of rabbit somatic cells.

30 NUCLEAR AND MICROTUBULE DYNAMICS IN SOMATIC CELL NUCLEAR TRANSFER SHEEP EMBRYOS ACTIVATED WITH T-DIMETHYLAMINOPURINE AND CYCLOHEXIMIDE

2006 ◽  
Vol 18 (2) ◽  
pp. 123
Author(s):  
G. Coppola ◽  
B.-G. Jeon ◽  
B. Alexander ◽  
E. St. John ◽  
D. H. Betts ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Laser Scanning ◽  
Cell Cycle Progression ◽  
Treated Group ◽  
Blastocyst Development ◽  
Fetal Fibroblasts

The early reprogramming events following somatic cell nuclear transfer (SCNT) determine the fate of the cloned embryo and its development to a healthy viable offspring. In the present study, we undertook a detailed immunocytochemical study of the patterns of both microtubules and chromatin during the first cell cycle of sheep nuclear transfer embryos after fusion and artificial activation using either 6-dimethylaminopurine (6-DMAP) or cycloheximede (CHX). Sheep oocytes were collected from abattoir ovaries and matured in vitro for 18-20 h and enucleated; fetal fibroblasts were transplanted using standard SCNT techniques. Reconstructed cell-cytoplast couplets were fused and activated with ionomycin, followed by culture in two separate groups containing 6-DMAP (2 mM) or CHX (10 �g/mL) for 3 h. Following activation, embryos were cultured in in vitro culture (IVC) medium for blastocyst development. Embryos (n = 15, 3 replicates) were randomly removed from culture at various time points and stained using standard immunocytochemical methods to observe microtubule and nuclear configurations. Images were captured using laser scanning confocal microscopy. Results reveled that at 1 h post-fusion, 63.3% of reconstructed embryos underwent nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC) was apparent as chromosomes were situated on a non-polar spindle. The remaining embryos showed abnormal spindle and DNA configurations including chromosome outliers, congression failure, and non-NEBD. At 1 h post-activation (hpa), the embryos treated with 6-DMAP had already formed a clearly visible pronucleus (diameter 6-8 �m), whereas in the CHX-treated group, none of the embryos were at pronuclear stage; instead most of the latter embryos showed two masses of chromatin. At 1 hpa, 6-DMAP- and CHX-treated embryos showed one swelled pronucleus with a mean diameter of 8.4 � 1.3 �m and 25.8 � 0.8 �m, respectively (P < 0.05). At 16 hpa, embryos from both treatment groups still showed one swelled pronucleus. In the 6-DMAP-treated embryos, most of the embryos showed a metaphase spindle with aligned chromosomes of the first mitotic division as early as 18-10 hpa, whereas in the CHX-treated group embryos were still at the pronuclear stage. Typical 2-cell division was seen in most of the 6-DMAP-treated embryos between 24 and 30 hpa, but it was slightly delayed in CHX-treated embryos (32-35 hpa). Blastocyst development rates in the 6-DMAP- and CHX-treated groups were 21.4 � 5.6% and 14.0 � 6.3%, respectively (P < 0.05). In summary, artificial activating agents 6-DMAP and CHX exhibited different effects on chromatin remodeling, cell cycle progression, and the degree of pronuclear swelling which may explain the poor developmental rates and abnormal chromosome complements observed for cloned embryos. This work was funded by NSERC, OMAF, and International Council for Canadian Studies.

High GATA-2 expression inhibits human hematopoietic stem and progenitor cell function by effects on cell cycle

Blood ◽  
2009 ◽  
Vol 113 (12) ◽  
pp. 2661-2672 ◽  
Author(s):  
Alex J. Tipping ◽  
Cristina Pina ◽  
Anders Castor ◽  
Dengli Hong ◽  
Neil P. Rodrigues ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cord Blood ◽  
Cell Function ◽  
Ki 67 ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Cord Blood Cells ◽  
Low Efficiency

Abstract Evidence suggests the transcription factor GATA-2 is a critical regulator of murine hematopoietic stem cells. Here, we explore the relation between GATA-2 and cell proliferation and show that inducing GATA-2 increases quiescence (G0 residency) of murine and human hematopoietic cells. In human cord blood, quiescent fractions (CD34+CD38−HoechstloPyronin Ylo) express more GATA-2 than cycling counterparts. Enforcing GATA-2 expression increased quiescence of cord blood cells, reducing proliferation and performance in long-term culture-initiating cell and colony-forming cell (CFC) assays. Gene expression analysis places GATA-2 upstream of the quiescence regulator MEF, but enforcing MEF expression does not prevent GATA-2–conferred quiescence, suggesting additional regulators are involved. Although known quiescence regulators p21CIP1 and p27KIP1 do not appear to be responsible, enforcing GATA-2 reduced expression of regulators of cell cycle such as CCND3, CDK4, and CDK6. Enforcing GATA-2 inhibited human hematopoiesis in vivo: cells with highest exogenous expression (GATA-2hi) failed to contribute to hematopoiesis in nonobese diabetic–severe combined immunodeficient (NOD-SCID) mice, whereas GATA-2lo cells contributed with delayed kinetics and low efficiency, with reduced expression of Ki-67. Thus, GATA-2 activity inhibits cell cycle in vitro and in vivo, highlighting GATA-2 as a molecular entry point into the transcriptional program regulating quiescence in human hematopoietic stem and progenitor cells.

Effect of delayed enucleation on the developmental potential of nuclear-transferred oocytes receiving adult and fetal fibroblast cells

Zygote ◽  
2002 ◽  
Vol 10 (3) ◽  
pp. 217-222 ◽  
Author(s):  
Xi Jun Yin ◽  
Yoko Kato ◽  
Yukio Tsunoda
Keyword(s):  
Somatic Cell ◽  
Cell Number ◽  
Heart Beat ◽  
Fibroblast Cells ◽  
Cell Nuclei ◽  
Average Cell ◽  
Developmental Potential ◽  
Membrane Protrusions ◽  
Implantation Sites

To enhance the probability of reprogramming somatic cell nuclei, fibroblast cells from an adult male rabbit and a 12-day-old fetus were fused with oocytes at the second metaphase. The chromosomes of recipient oocytes were then removed by treatment with demecolcine for 1 or 2 h after fusion. Demecolcine treatment of fused oocytes induced membrane protrusions that contained all the maternal chromosomes, thus making it possible to remove the chromosomes. The potential of nuclear-transferred oocytes to develop into blastocysts was high (48% and 59%) and the average cell number of the blastocysts was large (149 and 159) 96 h after in vitro culture. The proportions of nuclear-transferred oocytes enucleated 1 h after fusion and implanted after transfer to pseudopregnant recipients were relatively high (2.8% and 4.9%) compared with our previous reports (1.7%: Yin et al., 2000; 0.6% and 1.0%: Yin et al., 2002a) where donor cells were fused with previously enucleated oocytes. Of 34 adult somatic cell implantation sites, 6 had fetuses on day 12 or 14 of pregnancy, but none of the fetuses had a heart beat or developed to term. None of the nuclear-transferred oocytes whose chromosomes were removed 2 h after demecolcine treatment implanted after transfer to recipients. The possible reasons why the high-quality nuclear-transferred oocytes did not develop to term are discussed.

TRIM28 down-regulation on methylation imprints in bovine preimplantation embryos

Zygote ◽  
2018 ◽  
Vol 26 (6) ◽  
pp. 449-456 ◽  
Author(s):  
Xin Ma ◽  
Sheng Zhang ◽  
Meiling Zhang ◽  
Yiran Zhu ◽  
Panpan Ma ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Preimplantation Embryos ◽  
Differentially Methylated Region ◽  
Fibroblast Cells ◽  
Mrna Sequence ◽  
Cell Stage ◽  
H19 Gene

SummaryTRIM28/KAP1/TIF1β was identified as a universal transcriptional co-repressor and is critical for regulating post-fertilization methylation reprogramming in preimplantation embryos. In this study, three siRNAs (si647, si742, and si1153) were designed to target the TRIM28 mRNA sequence. After transfection of the mixture of the three siRNA (siMix) into bovine fibroblast cells, the most effective one for TRIM28 knockdown was selected. By injecting RNAi directed against TRIM28 mRNA, we found that TRIM28 knockdown in oocytes had the most effect on the H19 gene, in which differentially methylated region (DMR) methylation was almost completely absent at the 2-cell stage (1.4%), while control embryos showed 74% methylation. In addition, global H3K9me3 levels at the 2-cell stage were significantly higher in the in vitro fertilization (IVF) group than in the TRIM28 knockdown group (P<0.05). We further show that TRIM28 is highly expressed during oocyte maturation and reaches peak levels at the 2-cell stage. In contrast, at this stage, TRIM28 expression in somatic cell nuclear transfer (SCNT) embryos decreased significantly (P<0.05), suggesting that Trim28 transcripts are lost during SCNT. TRIM28 is required for the maintenance of methylation imprints in bovine preimplantation embryos, and the loss of TRIM28 during SCNT may contribute to the unfaithful maintenance of imprints in cloned embryos.

Expression of IGF2 and IGF receptor mRNA in bovine nuclear transferred embryos

Zygote ◽  
2003 ◽  
Vol 11 (3) ◽  
pp. 245-252 ◽  
Author(s):  
Dong-Wook Han ◽  
Sang-Jin Song ◽  
Sang Jun Uhum ◽  
Jeong-Tae Do ◽  
Nam-Hyung Kim ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Donor Cell ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Pcr Method ◽  
Igf Receptor ◽  
Polymerase Chain ◽  
Low Efficiency ◽  
Insight Into

Incomplete reprogramming of the donor cell nucleus after nuclear transfer (NT) probably leads to the abnormal expression of developmentally important genes. This may be responsible for the low efficiency of cloned animal production. Insulin-like growth factor 2 (IGF2) and IGF2 receptor (IGF2R) are imprinted genes that play important roles in preimplantation development. To obtain an insight into abnormal gene expression after nuclear transfer, we assessed the transcription patterns of IGF2-IGF2R in single in vitro fertilised and cloned embryos by reverse-transcription polymerase chain reaction (RT-PCR). IGF2R expression did not differ significantly but IGF2 was more highly expressed in cloned embryos than in IVF embryos (p < 0.05). This was confirmed by a quantitative RT-PCR method. Thus, incomplete reprogramming may induce abnormal transcription of IGF2 in cloned embryos.

The effects of chemical enucleation combined with whole cell intracytoplasmic injection on panda–rabbit interspecies nuclear transfer

Zygote ◽  
2004 ◽  
Vol 12 (4) ◽  
pp. 315-320 ◽  
Author(s):  
Man-Xi Jiang ◽  
Cai-Xia Yang ◽  
Li-Sheng Zhang ◽  
Yue-Liang Zheng ◽  
Shu-Zhen Liu ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Giant Panda ◽  
Whole Cell ◽  
Intracytoplasmic Injection ◽  
Low Efficiency ◽  
Interspecies Cloning ◽  
Interspecies Nuclear Transfer

Conventional methods of somatic cell nuclear transfer either by electrofusion or direct nucleus injection have very low efficiency in animal cloning, especially interspecies cloning. To increase the efficiency of interspecies somatic cell nuclear transfer, in the present study we introduced a method of whole cell intracytoplasmic injection (WCICI) combined with chemical enucleation into panda–rabbit nuclear transfer and assessed the effects of this method on the enucleation rate of rabbit oocytes and the in vitro development and spindle structures of giant panda–rabbit reconstructed embryos. Our results demonstrated that chemical enucleation can be used in rabbit oocytes and the optimal enucleation result can be obtained. When we compared the rates of cleavage and blastocyst formation of subzonal injection (SUZI) and WCICI using chemically enucleated rabbit oocytes as cytoplasm recipients, the rates in the WCICI group were higher than those in the SUZI group, but there was no statistically siginificant difference (p>0.05) between the two methods. The microtubule structures of rabbit oocytes enucleated by chemicals and giant panda–rabbit embryos reconstructed by WCICI combined with chemical enucleation were normal. Therefore the present study suggests that WCICI combined with chemical enucleation can provide an efficient and less labor-intensive protocol of interspecies somatic cell nuclear transfer for producing giant panda cloned embryos.

scholarly journals Inactivation of mouse Hus1 results in genomic instability and impaired responses to genotoxic stress

2000 ◽  
Vol 14 (15) ◽  
pp. 1886-1898 ◽  
Author(s):  
Robert S. Weiss ◽  
Tamar Enoch ◽  
Philip Leder
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Replication ◽  
Chromosomal Abnormalities ◽  
Genotoxic Stress ◽  
Eukaryotic Cell ◽  
Cell Cycle Checkpoint ◽  
Cell Cycle Checkpoints ◽  
Genome Damage

The eukaryotic cell cycle is overseen by regulatory mechanisms, termed checkpoints, that respond to DNA damage, mitotic spindle defects, and errors in the ordering of cell cycle events. The DNA replication and DNA damage cell cycle checkpoints of the fission yeastSchizosaccharomyces pombe require the hus1+(hydroxyurea sensitive) gene. To determine the role of the mouse homolog of hus1+ in murine development and cell cycle checkpoint function, we produced a targeted disruption of mouse Hus1. Inactivation of Hus1results in mid-gestational embryonic lethality due to widespread apoptosis and defective development of essential extra-embryonic tissues. DNA damage-inducible genes are up-regulated inHus1-deficient embryos, and primary cells fromHus1-null embryos contain increased spontaneous chromosomal abnormalities, suggesting that loss of Hus1 leads to an accumulation of genome damage. Embryonic fibroblasts lackingHus1 fail to proliferate in vitro, but inactivation ofp21 allows for the continued growth of Hus1-deficient cells.Hus1−/−p21−/−cells display a unique profile of significantly heightened sensitivity to hydroxyurea, a DNA replication inhibitor, and ultraviolet light, but only slightly increased sensitivity to ionizing radiation. Taken together, these results indicate that mouse Hus1 functions in the maintenance of genomic stability and additionally identify an evolutionarily-conserved role for Hus1 in mediating cellular responses to genotoxins.

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