Effects of demecolcine and sucrose on the incidence of cytoplasmic protrusions containing chromosomes in pig oocytes matured in vitro.

The present study was carried out to examine whether demecolcine and sucrose affect the formation of a cytoplasmic protrusion containing chromosomes in pig oocytes independently or in combination. In the presence of 20 mM sucrose, the rates of oocytes with a cytoplasmic protrusion after culture for 60 min with 0.2-1.0 microg/ml demecolcine were significantly higher than those with 0.01-0.05 microg/ml demecolcine. When oocytes were cultured for 15 min in the presence of 0.2 microg/ml demecolcine and 20 mM sucrose, 35.1% of them extruded a cytoplasmic protrusion; this rate was significantly lower than those of oocytes cultured for 30-90 min. In the presence of 0.2 microg/ml demecolcine, significantly fewer oocytes extruded a cytoplasmic protrusion after culture for 30 min with 160 mM sucrose than with 0-80 mM sucrose. Significantly more oocytes extruded a cytoplasmic protrusion after culture for 30 min with 0.2 microg/ml demecolcine than without it, regardless of the presence or absence of 20 mM sucrose. In 88.9-100% of the oocytes, the cytoplasmic protrusions contained chromosomes with no significant differences among the different concentrations of demecolcine and sucrose and among the different treatment times. The results of the present study show that the cytoplasmic protrusion containing chromosomes in the pig oocyte is attributable to demecolcine, but sucrose does not affect its formation.

[1]  K. Miyoshi,et al.  Birth of cloned miniature pigs derived from somatic cell nuclear transferred embryos activated by ultrasound treatment , 2007, Molecular reproduction and development.

[2]  H. Shimada,et al.  Demecolcine-assisted enucleation for bovine cloning. , 2006, Cloning and stem cells.

[3]  A. Yabuuchi,et al.  Effect of demecolcine and nocodazole on the efficiency of chemically assisted removal of chromosomes and the developmental potential of nuclear transferred porcine oocytes. , 2003, Cloning and stem cells.

[4]  Ji-Long Liu,et al.  Hypertonicity-Induced Projections Reflect Cell Polarity in Mouse Metaphase II Oocytes: Involvement of Microtubules, Microfilaments, and Chromosomes1 , 2002, Biology of reproduction.

[5]  Rie Hasegawa,et al.  Production of Cloned Pigs from Adult Somatic Cells by Chemically Assisted Removal of Maternal Chromosomes1 , 2002, Biology of reproduction.

[6]  Kwang-Wook Park,et al.  Transgenic pig expressing the enhanced green fluorescent protein produced by nuclear transfer using colchicine‐treated fibroblasts as donor cells , 2002, Molecular reproduction and development.

[7]  Ji-Long Liu,et al.  Hypertonic Medium Treatment for Localization of Nuclear Material in Bovine Metaphase II Oocytes1 , 2002, Biology of reproduction.

[8]  A. Boquest,et al.  Production of Cloned Pigs from Cultured Fetal Fibroblast Cells , 2002, Biology of reproduction.

[9]  B. N. Day,et al.  Mosaic Gene Expression in Nuclear Transfer-Derived Embryos and the Production of Cloned Transgenic Pigs from Ear-Derived Fibroblasts1 , 2002, Biology of reproduction.

[10]  A. Archibald,et al.  Somatic Cell Nuclear Transfer in the Pig: Control of Pronuclear Formation and Integration with Improved Methods for Activation and Maintenance of Pregnancy1 , 2002, Biology of reproduction.

[11]  Yifan Dai,et al.  Targeted disruption of the α1,3-galactosyltransferase gene in cloned pigs , 2002, Nature Biotechnology.

[12]  Kwang-Wook Park,et al.  Production of α-1,3-Galactosyltransferase Knockout Pigs by Nuclear Transfer Cloning , 2002, Science.

[13]  W. Fodor,et al.  Cloned pigs generated from cultured skin fibroblasts derived from a H‐transferase transgenic boar , 2001, Molecular reproduction and development.

[14]  Kwang-Wook Park,et al.  PRODUCTION OF NUCLEAR TRANSFER-DERIVED SWINE THAT EXPRESS THE ENHANCED GREEN FLUORESCENT PROTEIN , 2001, Animal biotechnology.

[15]  Ji-Long Liu,et al.  Sucrose pretreatment for enucleation: An efficient and non‐damage method for removing the spindle of the mouse MII oocyte , 2001, Molecular reproduction and development.

[16]  Q. Sun,et al.  Effect of telophase enucleation on bovine somatic nuclear transfer. , 2000, Theriogenology.

[17]  M. Pfister-Genskow,et al.  Production of cloned pigs from in vitro systems , 2000, Nature Biotechnology.

[18]  Yifan Dai,et al.  Cloned pigs produced by nuclear transfer from adult somatic cells , 2000, Nature.

[19]  A Onishi,et al.  Pig cloning by microinjection of fetal fibroblast nuclei. , 2000, Science.

[20]  B. N. Day,et al.  Optimisation of porcine oocyte activation following nuclear transfer , 2000, Zygote.

[21]  G. Schatten,et al.  Dynamic Imaging of the Metaphase II Spindle and Maternal Chromosomesin Bovine Oocytes: Implications for Enucleation Efficiency Verification, Avoidanceof Parthenogenesis, and Successful Embryogenesis1 , 2000, Biology of reproduction.

[22]  Y. Takahashi,et al.  Preparation of young preactivated oocytes with high enucleation efficiency for bovine nuclear transfer. , 1999, Theriogenology.

[23]  T. Otoi,et al.  Cryopreservation of mature bovine oocytes by vitrification in straws. , 1998, Cryobiology.

[24]  V. Bordignon,et al.  Telophase enucleation: An improved method to prepare recipient cytoplasts for use in bovine nuclear transfer , 1998, Molecular reproduction and development.

[25]  K. Imai,et al.  Effect of sugars-addition on the survival of vitrified bovine blastocysts produced in vitro. , 1994, Theriogenology.

[26]  R. Petters,et al.  Culture of pig embryos. , 2020, Journal of reproduction and fertility. Supplement.

[27]  L. Smith Membrane and intracellular effects of ultraviolet irradiation with Hoechst 33342 on bovine secondary oocytes matured in vitro. , 1993, Journal of reproduction and fertility.

[28]  S. Leibo,et al.  Osmotic shock of fertilized mouse ova. , 1992, Journal of reproduction and fertility.

[29]  A. Széll,et al.  Osmotic and cryoprotective effects of glycerol-sucrose solutions on day-3 mouse embryos. , 1987, Journal of reproduction and fertility.

[30]  R. Heap,et al.  Production, chemical composition and prostaglandin F-2alpha content of uterine fluid in pregnant sheep. , 1976, Journal of reproduction and fertility.