Influence of cell cycle stage of the donor nucleus on development of nuclear transplant rabbit embryos.
We evaluated the influence of the stage of the cell cycle of the donor nucleus on development in vitro of nuclear transplant rabbit embryos. The developmental potential of nuclei in early, mid-, and late stages of the cell cycle was determined. Duration of the G1 phase in early embryos was determined, and a procedure for reversibly synchronizing donor embryos in the G1 phase was developed. In addition, the extent of development in vitro of nuclear transplant embryos with donor nuclei synchronized in the G1 phase was evaluated. Development to blastocysts was greatly affected by the stage of the cycle of the donor nucleus. Use of early-stage nuclei led to 59% nuclear transplant blastocysts, whereas 32% and 3% were obtained with mid- and late-stage nuclear donors, respectively (p less than 0.001). The short duration of the G1 phase in 16- and 32-cell-stage embryos (approximately 30 min) necessitated a procedure for synchronizing blastomeres in the G1 phase. This entailed, first, a 10-h incubation in 0.5 micrograms/ml colcemid to arrest embryos in metaphase. After release from colcemid, embryos were allowed to cleave in 0.1 microgram/ml of the DNA synthesis inhibitor, aphidicolin, and remained blocked at the G1/S transition. This treatment was reversible, as assessed by the resumption of DNA synthesis, cleavage rate, and development to blastocysts of treated embryos. The beneficial effect of using early-stage donor blastomeres was confirmed by the enhanced rate of development of manipulated embryos to blastocysts with donor nuclei in the G1 phase (71%), as opposed to the late S phase (15%, p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)
Collas
P
P
Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst 01003.
Balise
J J
JJ
Robl
J M
JM
D000818:Animals; D001755:Blastocyst / Q000378:metabolism / Q000648:ultrastructure; D001757:Blastomeres / Q000378:metabolism / Q000648:ultrastructure; D002453:Cell Cycle / Q000502:physiology*; D002970:Cleavage Stage, Ovum / Q000378:metabolism / Q000648:ultrastructure; D004247:DNA / Q000096:biosynthesis; D004622:Embryo, Mammalian / Q000378:metabolism / Q000648:ultrastructure*; D005260:Female; D016193:G1 Phase / Q000502:physiology; D008938:Mitosis / Q000502:physiology; D009028:Morula / Q000378:metabolism / Q000648:ultrastructure; D053652:Nuclear Transfer Techniques; D011817:Rabbits; D016196:S Phase / Q000502:physiology
D004247:DNA