The Experts below are selected from a list of 939 Experts worldwide ranked by ideXlab platform
Hiroko Yamagishi - One of the best experts on this subject based on the ideXlab platform.
-
establishment of a tetraploid cell line from mouse h 1 es cells highly polyploidized with Demecolcine
Cell Proliferation, 2007Co-Authors: Kohzaburo Fujikawayamamoto, Minoru Miyagoshi, Hiroko YamagishiAbstract:. Objective: Establishment of tetraploid ES cells. Materials and methods: Mouse H-1 (ES) cells were polyploidized by Demecolcine and released from the drug. Results: A tetraploid cell line (4nH1 cells) was established from mouse H-1 (ES) cells (2nH1 cells) highly polyploidized by treatment with Demecolcine. Cell cycle parameters of 4nH1 cells were almost the same as those of 2nH1 cells, suggesting that the rate of DNA synthesis was about twice that of the diploid cells. Mode of chromosome number of 4nH1 cells was 76, about twice that of 2nH1 cells. Cell volume of 4nH1 cells was about twice of that of diploid cells, indicating that 4nH1 cells contained about twice as much total intracellular material as 2nH1 cells. Morphology of the 4nH1 cells was flagstone-like, thus differing from that of the spindle-shaped 2nH1 cells, suggesting that the transformation had occurred during the diploid–tetraploid transition. 4nH1 cells exhibited alkaline phosphatase activity and formed teratocarcinomas, implying that they would be pluripotent. Conclusion: A pluripotent tetraploid cell line (4nH1 cells) was established.
-
Establishment of a tetraploid cell line from mouse H‐1 (ES) cells highly polyploidized with Demecolcine
Cell proliferation, 2007Co-Authors: Kohzaburo Fujikawa-yamamoto, Minoru Miyagoshi, Hiroko YamagishiAbstract:. Objective: Establishment of tetraploid ES cells. Materials and methods: Mouse H-1 (ES) cells were polyploidized by Demecolcine and released from the drug. Results: A tetraploid cell line (4nH1 cells) was established from mouse H-1 (ES) cells (2nH1 cells) highly polyploidized by treatment with Demecolcine. Cell cycle parameters of 4nH1 cells were almost the same as those of 2nH1 cells, suggesting that the rate of DNA synthesis was about twice that of the diploid cells. Mode of chromosome number of 4nH1 cells was 76, about twice that of 2nH1 cells. Cell volume of 4nH1 cells was about twice of that of diploid cells, indicating that 4nH1 cells contained about twice as much total intracellular material as 2nH1 cells. Morphology of the 4nH1 cells was flagstone-like, thus differing from that of the spindle-shaped 2nH1 cells, suggesting that the transformation had occurred during the diploid–tetraploid transition. 4nH1 cells exhibited alkaline phosphatase activity and formed teratocarcinomas, implying that they would be pluripotent. Conclusion: A pluripotent tetraploid cell line (4nH1 cells) was established.
-
polyploidization of mouse h 1 es cells by Demecolcine and k 252a
Cytologia, 2006Co-Authors: Kohzaburo Fujikawayamamoto, Minoru Miyagoshi, Hiroko YamagishiAbstract:Mouse H-1 (ES) cells (H-1 cells) were examined for polyploidization by Demecolcine and K-252a. H-1 cells were highly polyploidized by both Demecolcine and K-252a, and died 3 d after their addition, suggesting that polyploidized H-1 cells are sensitive to these drugs. The polyploidized H-1 cells showed alkaline phosphatase activity, suggesting that the pluripotent potential of H-1 cells was maintained through out polyploidization. Although the polyploidized H-1 cells became tentatively diploid or tetraploid after the drug removal, the tetraploid H-1 cells reverted to diploid cells within a month.
-
establishment of a tetraploid meth a cell line through polyploidization by Demecolcine but not by staurosporine k 252a and paclitaxel
Cell Proliferation, 2001Co-Authors: Kohzaburo Fujikawayamamoto, Hiroko Yamagishi, Chie Ohdoi, H Murano, S. Wang, Teruaki IkedaAbstract:: Polyploid cells are made by DNA reduplication without cell division, however, it is not easy to establish polyploid mammalian cell lines. It is worth studying the difference in cell character between hyperploid and parent cell lines. Meth-A cells were polyploidized by Demecolcine, K-252a, staurosporine and paclitaxel. The cell-cycle responses of highly polyploid Meth-A cells after the removal of the drugs were examined by flow cytometry (FCM). Meth-A cells were highly polyploidized by these drugs. The polyploid Meth-A cells gradually decreased in ploidy after the drug release. A tetraploid Meth-A cell line was established only from the Demecolcine-induced polyploid Meth-A cells. The duration of G1, S and G2/M phases of the tetraploid cell line were mostly the same as those of the parent diploid cells, except that the G2/M phase was 1.5 h longer. The chromosome number of tetraploid Meth-A cell line was about twice of the diploidy. A tetraploid Meth-A cell line was established.
-
Polyploidization of K562 Cells by Demecolcine, K-252a and Staurosporine.
Cytologia, 2000Co-Authors: Kohzaburo Fujikawa-yamamoto, Hiroko Yamagishi, Zhi-ping Zong, Chie Ohdoi, Shiyong WangAbstract:The polyploidization of K562 cells was examined by flowcytometry (FCM). M562 cells were polyploidized by Demecolcine (colcemid) at a concentration of 81 nM. At this concentration, apoptosis was induced, as identified by a sub G1 peak in DNA histograms and by a DNA ladder in gel-electrophoresis. Staurosporine and K-252a polyploidized K562 cells without causing apoptosis, and altered the morphology from spherical to fibroblast-like. The adherency of K562 cells to dishes increased on exposure to staurosporine and K-252a, but not Demecolcine. The rate of polyploidization by these drugs was almost the same. It was suggested that the mechanism of polyploidization of K562 cells was independent of the drug-induced change in adherency.
Kohzaburo Fujikawa-yamamoto - One of the best experts on this subject based on the ideXlab platform.
-
Establishment of a tetraploid cell line from mouse H‐1 (ES) cells highly polyploidized with Demecolcine
Cell proliferation, 2007Co-Authors: Kohzaburo Fujikawa-yamamoto, Minoru Miyagoshi, Hiroko YamagishiAbstract:. Objective: Establishment of tetraploid ES cells. Materials and methods: Mouse H-1 (ES) cells were polyploidized by Demecolcine and released from the drug. Results: A tetraploid cell line (4nH1 cells) was established from mouse H-1 (ES) cells (2nH1 cells) highly polyploidized by treatment with Demecolcine. Cell cycle parameters of 4nH1 cells were almost the same as those of 2nH1 cells, suggesting that the rate of DNA synthesis was about twice that of the diploid cells. Mode of chromosome number of 4nH1 cells was 76, about twice that of 2nH1 cells. Cell volume of 4nH1 cells was about twice of that of diploid cells, indicating that 4nH1 cells contained about twice as much total intracellular material as 2nH1 cells. Morphology of the 4nH1 cells was flagstone-like, thus differing from that of the spindle-shaped 2nH1 cells, suggesting that the transformation had occurred during the diploid–tetraploid transition. 4nH1 cells exhibited alkaline phosphatase activity and formed teratocarcinomas, implying that they would be pluripotent. Conclusion: A pluripotent tetraploid cell line (4nH1 cells) was established.
-
Polyploidization of K562 Cells by Demecolcine, K-252a and Staurosporine.
Cytologia, 2000Co-Authors: Kohzaburo Fujikawa-yamamoto, Hiroko Yamagishi, Zhi-ping Zong, Chie Ohdoi, Shiyong WangAbstract:The polyploidization of K562 cells was examined by flowcytometry (FCM). M562 cells were polyploidized by Demecolcine (colcemid) at a concentration of 81 nM. At this concentration, apoptosis was induced, as identified by a sub G1 peak in DNA histograms and by a DNA ladder in gel-electrophoresis. Staurosporine and K-252a polyploidized K562 cells without causing apoptosis, and altered the morphology from spherical to fibroblast-like. The adherency of K562 cells to dishes increased on exposure to staurosporine and K-252a, but not Demecolcine. The rate of polyploidization by these drugs was almost the same. It was suggested that the mechanism of polyploidization of K562 cells was independent of the drug-induced change in adherency.
-
Lack of synchrony among multiple nuclei induces partial DNA fragmentation in V79 cells polyploidized by Demecolcine.
Cell Proliferation, 1999Co-Authors: Kohzaburo Fujikawa-yamamoto, Hiroko Yamagishi, Zhi-ping Zong, Chie Ohdoi, M. Murakami, N. YamaguchiAbstract:. The nuclear morphology of polyploidized cells was examined in V79 Chinese hamster cells polyploidized by Demecolcine or K-252a, inhibitors of spindle fibre formation and protein kinases, respectively. A variety of nuclear morphologies, including multinuclei, were observed in V79 cells polyploidized by Demecolcine but not by K-252a, which produced mononuclear cells. A lack of synchrony in the nuclear cycle was observed among nuclei in multinuclear polyploidized cells. Partial DNA fragmentation, defined as DNA fragmentation of a nucleus in a multinuclear cell, was detected using the TUNEL method in V79 cells polyploidized by Demecolcine but not by K-252a. Apoptosis occurred earlier in cell populations treated with Demecolcine than in these treated with K-252a once the drugs were removed from the medium, suggesting that polyploidized cells with separate nuclei tend to apoptose earlier than those with mononuclei.
-
Different manner of DNA synthesis in polyploidizations of meth-A and B16F10 cell lines.
Cell structure and function, 1997Co-Authors: Kohzaburo Fujikawa-yamamoto, Zhi-ping Zong, Manabu Murakami, Shizuo OdashimaAbstract:Polyploidization of Meth-A and B16-F10 cells by Demecolcine was examined using flow cytometry (FCM). In the presence of Demecolcine, both cell lines were polyploidized to more than 16c DNA content. A marked difference was observed in the durations of S phase of polyploidy. The S-phase duration of Meth-A cells was doubly increased with ploidy, but that of B16F10 cells remained constant. When the rate of DNA synthesis in the polyploidizing cells was examined through the BrdU-uptake experiments, it was confirmed that the level of DNA-synthesis rate was constant in Meth-A cells but increased in B16F10 cells. The cellular content of c-Myc protein in polyploidized cells was also examined using anti-c-Myc monoclonal antibody. The c-Myc level of Meth-A cells was constant regardless of the ploidy but that of B16F10 cells increased with ploidy. Thus, the c-Myc content seems to be related to the duration of S phase in polyploidy.
-
Apoptosis by Demecolcine in V79 Cells.
Cell structure and function, 1994Co-Authors: Kohzaburo Fujikawa-yamamoto, Hiroko Yamagishi, Zhi-ping Zong, Kouhei Teraoka, Shizuo OdashimaAbstract:Demecolcine (Colcemid), an inhibitor of spindle fiber formation in M phase, induced apoptosis in V79 cells. At a concentration of 0.01 microgram/ml Demecolcine, V79 cells proliferated exponentially as well as controls, although temporal M phase accumulation occurred 6 h after the addition of Demecolcine. At 0.1 microgram/ml, the cells became hyperploid after remaining in the M phase for some time. Apoptosis occurred in V79 cells exposed to Demecolcine at a concentration of 0.03 microgram/ml. Apoptosis was defined as the appearance of a sub-G1 peak in DNA histograms and a ladder pattern of fragmented DNA in gelelectrophoresis.
Kohzaburo Fujikawayamamoto - One of the best experts on this subject based on the ideXlab platform.
-
establishment of a tetraploid cell line from mouse h 1 es cells highly polyploidized with Demecolcine
Cell Proliferation, 2007Co-Authors: Kohzaburo Fujikawayamamoto, Minoru Miyagoshi, Hiroko YamagishiAbstract:. Objective: Establishment of tetraploid ES cells. Materials and methods: Mouse H-1 (ES) cells were polyploidized by Demecolcine and released from the drug. Results: A tetraploid cell line (4nH1 cells) was established from mouse H-1 (ES) cells (2nH1 cells) highly polyploidized by treatment with Demecolcine. Cell cycle parameters of 4nH1 cells were almost the same as those of 2nH1 cells, suggesting that the rate of DNA synthesis was about twice that of the diploid cells. Mode of chromosome number of 4nH1 cells was 76, about twice that of 2nH1 cells. Cell volume of 4nH1 cells was about twice of that of diploid cells, indicating that 4nH1 cells contained about twice as much total intracellular material as 2nH1 cells. Morphology of the 4nH1 cells was flagstone-like, thus differing from that of the spindle-shaped 2nH1 cells, suggesting that the transformation had occurred during the diploid–tetraploid transition. 4nH1 cells exhibited alkaline phosphatase activity and formed teratocarcinomas, implying that they would be pluripotent. Conclusion: A pluripotent tetraploid cell line (4nH1 cells) was established.
-
polyploidization of mouse h 1 es cells by Demecolcine and k 252a
Cytologia, 2006Co-Authors: Kohzaburo Fujikawayamamoto, Minoru Miyagoshi, Hiroko YamagishiAbstract:Mouse H-1 (ES) cells (H-1 cells) were examined for polyploidization by Demecolcine and K-252a. H-1 cells were highly polyploidized by both Demecolcine and K-252a, and died 3 d after their addition, suggesting that polyploidized H-1 cells are sensitive to these drugs. The polyploidized H-1 cells showed alkaline phosphatase activity, suggesting that the pluripotent potential of H-1 cells was maintained through out polyploidization. Although the polyploidized H-1 cells became tentatively diploid or tetraploid after the drug removal, the tetraploid H-1 cells reverted to diploid cells within a month.
-
establishment of a tetraploid meth a cell line through polyploidization by Demecolcine but not by staurosporine k 252a and paclitaxel
Cell Proliferation, 2001Co-Authors: Kohzaburo Fujikawayamamoto, Hiroko Yamagishi, Chie Ohdoi, H Murano, S. Wang, Teruaki IkedaAbstract:: Polyploid cells are made by DNA reduplication without cell division, however, it is not easy to establish polyploid mammalian cell lines. It is worth studying the difference in cell character between hyperploid and parent cell lines. Meth-A cells were polyploidized by Demecolcine, K-252a, staurosporine and paclitaxel. The cell-cycle responses of highly polyploid Meth-A cells after the removal of the drugs were examined by flow cytometry (FCM). Meth-A cells were highly polyploidized by these drugs. The polyploid Meth-A cells gradually decreased in ploidy after the drug release. A tetraploid Meth-A cell line was established only from the Demecolcine-induced polyploid Meth-A cells. The duration of G1, S and G2/M phases of the tetraploid cell line were mostly the same as those of the parent diploid cells, except that the G2/M phase was 1.5 h longer. The chromosome number of tetraploid Meth-A cell line was about twice of the diploidy. A tetraploid Meth-A cell line was established.
-
different responses of polyploidized v79 cells after removal of two drugs Demecolcine and k 252a
Cell Structure and Function, 2000Co-Authors: Kohzaburo Fujikawayamamoto, Hiroko Yamagishi, Zhi-ping Zong, Chie Ohdoi, Shiyong WangAbstract:To examine whether or not cells polyploidized by different mechanisms behave in a different manner after drug removal, V79 Chinese hamster cells were assessed by flow cytometry (FCM) after their polyploidization by Demecolcine and K-252a, inhibitors of spindle-fiber formation and protein kinase, respectively. Cell cycle analysis of DNA histograms of V79 cells before and after the drug release was performed. With both drugs, the ploidy of V79 cells increased just after the drug removal and was maintained for a week. A difference was evident 10 days after the release. Tetraploid cells were the main population from 10 to 18 days after the release of K-252a, but not Demecolcine. Cell cycle parameters were almost the same in pseudo diploid and tetraploid V79 cells, except for the tetraploid S phase which was 2h longer.
Zhi-ping Zong - One of the best experts on this subject based on the ideXlab platform.
-
Polyploidization of K562 Cells by Demecolcine, K-252a and Staurosporine.
Cytologia, 2000Co-Authors: Kohzaburo Fujikawa-yamamoto, Hiroko Yamagishi, Zhi-ping Zong, Chie Ohdoi, Shiyong WangAbstract:The polyploidization of K562 cells was examined by flowcytometry (FCM). M562 cells were polyploidized by Demecolcine (colcemid) at a concentration of 81 nM. At this concentration, apoptosis was induced, as identified by a sub G1 peak in DNA histograms and by a DNA ladder in gel-electrophoresis. Staurosporine and K-252a polyploidized K562 cells without causing apoptosis, and altered the morphology from spherical to fibroblast-like. The adherency of K562 cells to dishes increased on exposure to staurosporine and K-252a, but not Demecolcine. The rate of polyploidization by these drugs was almost the same. It was suggested that the mechanism of polyploidization of K562 cells was independent of the drug-induced change in adherency.
-
different responses of polyploidized v79 cells after removal of two drugs Demecolcine and k 252a
Cell Structure and Function, 2000Co-Authors: Kohzaburo Fujikawayamamoto, Hiroko Yamagishi, Zhi-ping Zong, Chie Ohdoi, Shiyong WangAbstract:To examine whether or not cells polyploidized by different mechanisms behave in a different manner after drug removal, V79 Chinese hamster cells were assessed by flow cytometry (FCM) after their polyploidization by Demecolcine and K-252a, inhibitors of spindle-fiber formation and protein kinase, respectively. Cell cycle analysis of DNA histograms of V79 cells before and after the drug release was performed. With both drugs, the ploidy of V79 cells increased just after the drug removal and was maintained for a week. A difference was evident 10 days after the release. Tetraploid cells were the main population from 10 to 18 days after the release of K-252a, but not Demecolcine. Cell cycle parameters were almost the same in pseudo diploid and tetraploid V79 cells, except for the tetraploid S phase which was 2h longer.
-
Lack of synchrony among multiple nuclei induces partial DNA fragmentation in V79 cells polyploidized by Demecolcine.
Cell Proliferation, 1999Co-Authors: Kohzaburo Fujikawa-yamamoto, Hiroko Yamagishi, Zhi-ping Zong, Chie Ohdoi, M. Murakami, N. YamaguchiAbstract:. The nuclear morphology of polyploidized cells was examined in V79 Chinese hamster cells polyploidized by Demecolcine or K-252a, inhibitors of spindle fibre formation and protein kinases, respectively. A variety of nuclear morphologies, including multinuclei, were observed in V79 cells polyploidized by Demecolcine but not by K-252a, which produced mononuclear cells. A lack of synchrony in the nuclear cycle was observed among nuclei in multinuclear polyploidized cells. Partial DNA fragmentation, defined as DNA fragmentation of a nucleus in a multinuclear cell, was detected using the TUNEL method in V79 cells polyploidized by Demecolcine but not by K-252a. Apoptosis occurred earlier in cell populations treated with Demecolcine than in these treated with K-252a once the drugs were removed from the medium, suggesting that polyploidized cells with separate nuclei tend to apoptose earlier than those with mononuclei.
-
Different manner of DNA synthesis in polyploidizations of meth-A and B16F10 cell lines.
Cell structure and function, 1997Co-Authors: Kohzaburo Fujikawa-yamamoto, Zhi-ping Zong, Manabu Murakami, Shizuo OdashimaAbstract:Polyploidization of Meth-A and B16-F10 cells by Demecolcine was examined using flow cytometry (FCM). In the presence of Demecolcine, both cell lines were polyploidized to more than 16c DNA content. A marked difference was observed in the durations of S phase of polyploidy. The S-phase duration of Meth-A cells was doubly increased with ploidy, but that of B16F10 cells remained constant. When the rate of DNA synthesis in the polyploidizing cells was examined through the BrdU-uptake experiments, it was confirmed that the level of DNA-synthesis rate was constant in Meth-A cells but increased in B16F10 cells. The cellular content of c-Myc protein in polyploidized cells was also examined using anti-c-Myc monoclonal antibody. The c-Myc level of Meth-A cells was constant regardless of the ploidy but that of B16F10 cells increased with ploidy. Thus, the c-Myc content seems to be related to the duration of S phase in polyploidy.
-
Apoptosis by Demecolcine in V79 Cells.
Cell structure and function, 1994Co-Authors: Kohzaburo Fujikawa-yamamoto, Hiroko Yamagishi, Zhi-ping Zong, Kouhei Teraoka, Shizuo OdashimaAbstract:Demecolcine (Colcemid), an inhibitor of spindle fiber formation in M phase, induced apoptosis in V79 cells. At a concentration of 0.01 microgram/ml Demecolcine, V79 cells proliferated exponentially as well as controls, although temporal M phase accumulation occurred 6 h after the addition of Demecolcine. At 0.1 microgram/ml, the cells became hyperploid after remaining in the M phase for some time. Apoptosis occurred in V79 cells exposed to Demecolcine at a concentration of 0.03 microgram/ml. Apoptosis was defined as the appearance of a sub-G1 peak in DNA histograms and a ladder pattern of fragmented DNA in gelelectrophoresis.
E.w. Overstrom - One of the best experts on this subject based on the ideXlab platform.
-
Activated bovine cytoplasts prepared by Demecolcine-induced enucleation support development of nuclear transfer embryos in vitro.
Molecular reproduction and development, 2005Co-Authors: D. Fischer Russell, Elena Ibáñez, David F. Albertini, E.w. OverstromAbstract:Demecolcine-induced enucleation (IE) of mouse oocytes has been shown to improve development to term of cloned mice. In this study, we characterized the kinetics and morphological progression of bovine oocytes subjected to IE, and evaluated their ability to support embryo development to the blastocyst stage after nuclear transfer (NT). In vitro matured bovine oocytes were parthenogenetically activated and subsequently exposed to Demecolcine at various times post-activation. Onset and duration of Demecolcine treatment significantly altered activation and IE frequencies, which varied from 7.1% to 100% and 33.3% to 91.7%, respectively, at 5 hr post-activation. A significant decrease in IE frequencies was observed at 17 hr post-activation (3.4%-46.1%), possibly due to reincorporation of chromosomes into the oocyte after incomplete second polar body (PB) extrusion. Oocytes were reconstructed by NT before (treatment 1) or after (treatment 2) activation and Demecolcine treatment, and cultured in vitro. Cleavage (48.1%-54.2%) and blastocyst rates (15.7%-19%) were equivalent for the two treatments, as well as the total cell number in NT blastocysts. Furthermore, most of the blastocysts were completely diploid (treatment 2) or heteroploid but with a majority of diploid nuclei (treatment 1). Our results demonstrate that the IE method can be successfully used to produce enucleated bovine cytoplasts that are competent to support development to the blastocyst stage after NT. This technically simple approach may provide a more efficient method to enhance the success rate of NT procedures. Further studies are needed to improve the in vitro development efficiency and to expand our understanding of the mechanism(s) involved in Demecolcine-induced enucleation.
-
Demecolcine induced oocyte enucleation for somatic cell cloning coordination between cell cycle egress kinetics of cortical cytoskeletal interactions and second polar body extrusion
Biology of Reproduction, 2003Co-Authors: Elena Ibáñez, David F. Albertini, E.w. OverstromAbstract:Abstract Studies were designed to further explore the use of pharmacological agents to produce developmentally competent enucleated mouse oocytes for animal cloning by somatic cell nuclear transfer. Metaphase II oocytes from CF-1 and B6D2F1 strains were activated with ethanol and subsequently exposed to Demecolcine at various times postactivation. Chromosome segregation, spindle dynamics, and polar body (PB) extrusion were monitored by fluorescence microscopy using DNA-, microtubule-, and microfilament-selective probes. Exposure to Demecolcine did not affect rates of oocyte activation induced by ethanol but did disrupt the coordination of cytokinesis and karyokinesis, suppressing the extent and completion of spindle rotation and second PB extrusion in a strain-dependent manner. Moreover, strain- and treatment-specific variations in the rate of oocyte enucleation were also detected. In particular, CF1 oocytes were more efficiently enucleated relative to B6D2F1 oocytes, and Demecolcine treatments initiated ...
-
Demecolcine induced oocyte enucleation for somatic cell cloning coordination between cell cycle egress kinetics of cortical cytoskeletal interactions and second polar body extrusion
Biology of Reproduction, 2003Co-Authors: Elena Ibáñez, David F. Albertini, E.w. OverstromAbstract:Studies were designed to further explore the use of pharmacological agents to produce developmentally competent enucleated mouse oocytes for animal cloning by somatic cell nuclear transfer. Metaphase II oocytes from CF-1 and B6D2F1 strains were activated with ethanol and subsequently exposed to Demecolcine at various times postactivation. Chromosome segregation, spindle dynamics, and polar body (PB) extrusion were monitored by fluorescence microscopy using DNA-, microtubule-, and microfilament-selective probes. Exposure to Demecolcine did not affect rates of oocyte activation induced by ethanol but did disrupt the coordination of cytokinesis and karyokinesis, suppressing the extent and completion of spindle rotation and second PB extrusion in a strain-dependent manner. Moreover, strain- and treatment-specific variations in the rate of oocyte enucleation were also detected. In particular, CF1 oocytes were more efficiently enucleated relative to B6D2F1 oocytes, and Demecolcine treatments initiated early after activation resulted in higher enucleation rates than when treatment was delayed. The observed strain differences are possibly caused by a combination of factors, such as the time course of meiotic cell-cycle progression after ethanol activation, the degree of spindle rotation, and the extent of second PB extrusion. These results suggest that developmentally competent cytoplasts can be produced by timely exposure of activated oocytes to agents that disrupt spindle microtubules. However, the utility of the Demecolcine-induced enucleation protocol will require further investigation into factors linking karyokinesis to cytokinesis at the levels of cell-cycle control and oocyte cytoskeletal remodeling following artificial or natural means of egg activation.
-
Cloned Mice Derived from Embryonic Stem Cell Karyoplasts and Activated Cytoplasts Prepared by Induced Enucleation
Biology of reproduction, 2002Co-Authors: Bianca Gasparrini, E.w. Overstrom, Shaorong Gao, A. Ainslie, Judy Fletcher, Michelle Mcgarry, W. A. Ritchie, Anthea Springbett, Ian Wilmut, P.a. De SousaAbstract:Abstract Our objective was to induce enucleation (IE) of activated mouse oocytes to yield cytoplasts capable of supporting development following nuclear transfer. Fluorescence microscopy for microtubules, microfilaments, and DNA was used to evaluate meiotic resumption after ethanol activation and the effect of subsequent transient treatments with 0.4 μg/ml of Demecolcine. Using oocytes from B6D2F1 (C57BL/6 × DBA/2) donors, the success of IE of chromatin into polar bodies (PBs) was dependent on the duration of Demecolcine treatment and the time that such treatment was initiated after activation. Similarly, variations in Demecolcine treatment altered the proportions of oocytes exhibiting a reversible compartmentalization of chromatin into PBs. Treatment for 15 min begun immediately after activation yielded an optimized IE rate of 21% (n = 80) when oocytes were evaluated after overnight recovery in culture. With this protocol, 30–50% of oocytes were routinely scored as compartmentalized when assessed 90 min ...