ABSTRACT
We studied the capacity of rabbit oocytes for electrofusion with morula blastomeres and fetal fibroblasts. The morula blastomeres fused with aging ooplasts more readily than the fetal fibroblasts: 92.9 versus 63.0%, p < 0.001. The fetal fibroblasts fused with young enucleated oocytes more efficiently than with the aging ones: 98.4 versus 63.0%, p < 0.001. Serum starvation of the fetal fibroblasts in DMEM medium for 7-14 days reduced their capacity for fusion with young ooplasts, as compared to that after starvation for 0-4 days: 67.2 versus 98.9%, p < 0.01). The increased time of "starvation" in an "impoverished" medium reduced the capacity of fetal fibroblasts with aging ooplasts as compared to the fibroblasts cultivated in the full medium and in the "impoverished" medium for one or two days: 64.5 versus 37.4%, p < 0.01. Hence, the efficiency of the fusion of the oocytes with nuclear donor cells depends on the age of the recipient oocyte, the origin of nuclear donor cells, and the conditions of cultivation.
Subject(s)
Blastomeres , Cell Fusion/methods , Cell Nucleus , Oocytes/physiology , Animals , Cells, Cultured , Electrophysiology/methods , Female , Fibroblasts , RabbitsABSTRACT
We studied the capacity of nuclei of rabbit fibroblasts taken from various developmental stages for reprogramming in the cytoplasm of mature aging enucleated oocytes and development of the cloned embryos to the preimplantation stages. A negative correlation was found between the age of an animal--donor of fibroblasts and efficiency of the development of cloned embryos (rmorula-blastocyst = -0.826, rblastocyst = -0.7139). A reliably decreased capacity for reprogramming of the nuclei of donor fibroblasts was shown upon transition from prenatal development to the postnatal one, as well as a trend to a decreased capacity of nuclei for reprogramming during aging. Aging of cells in the culture, at least until the 10th passage, did not affect the capacity of the nuclei of fetal fibroblasts for reprogramming and development of cloned embryos.
Subject(s)
Age Factors , Cloning, Organism , Embryonic and Fetal Development , Nuclear Transfer Techniques , Animals , Cells, Cultured , Cellular Senescence , Female , RabbitsABSTRACT
A review of the development of the method of nuclear transplantation and cloning of animals with the use of nuclei of embryonic and adult cells is presented. We also present the results of studies of nuclear remodeling and reprogramming in the reconstructed oocyte and of cytoplasmic factors that control these processes.
Subject(s)
Cell Nucleus , Cloning, Organism/methods , Oocytes/cytology , Oocytes/physiology , Animals , Cattle , Embryo, Mammalian/physiology , MiceABSTRACT
Successful transplantation of mammalian nuclei from differentiated cells has become possible after the application of original methods directed at the synchronization of cell cycles of the donor cell and recipient cytoplasm. We obtained a line of rabbit fetal fibroblasts which was used to study factors affecting the success of reprogramming. The nuclei of fetal fibroblasts (up to the 10th passage inclusive) proved to be capable of reprogramming and ensuring development of the cloned embryos until the preimplantation stages. The influence of synchronization of the cell cycles of the nucleus donor and recipient on the efficiency of reprogramming was studied. The rate of development of the cloned rabbit embryos to the morula-blastocyst stage reached 67% when the nuclei used were from stationary culture cells (G0-phase).
Subject(s)
Cell Nucleus/drug effects , Culture Media, Serum-Free/pharmacology , Fibroblasts/drug effects , Animals , Cell Culture Techniques/methods , Cell Cycle , Cell Differentiation , Cell Nucleus/ultrastructure , Cell Separation/methods , Cells, Cultured , Embryo, Mammalian , Female , Fibroblasts/ultrastructure , Nuclear Transfer Techniques , Pregnancy , RabbitsABSTRACT
Bovine oocytes were aspirated from ovaries within 1.6 to 2 hours after slaughter. They were then matured in TCM-199 medium drops under oil in CO(2)/air incubator at 39 degrees C. Spermatozoa were capacitated in SP-TALP medium with heparin. The percentage of embryos that developed in vitro to the 4- and 6- cell stages 48 hours post insemination and then reached the morula or blastocyst stage was 64.3% and 59.2%, respectively, while only 3.6% of the embryos that reached the 2-cell stage became morula or blastocysts. An average of 6.3+/-3.2 total in vitro fertilized embryos per cow were obtained (range 2 to 11). Maturation of bovine oocytes in vitro for 18 or 24 hours did not influence the percentage of cleaved embryos (71.0 and 75.9%, respectively) or that developed to the blastocyst stage (25.6 and 24.2%, respectively). The use of reindeer blood serum for in vitro culture of immature bovine oocytes and of dividing of embryos gave the following results: 57.4% of the oocytes cleaved after fertilization and 16.2% developed further to the blastocyst stage. In contrast in the control group, where cow serum was used, the values were 73.4% and 24.8%, respectively. Rabbit oviduct epithelium cell monolayers were able to support the development of 16.3% of the cleaved bovine embryos to the blastocyst stage as compared with 24.0% of the embryos on cow oviduct epithelium cell monolayers. After nonsurgical transplantation, 12 calves were produced from 91 in vitro fertilized embryos.
