Early changes in embryonic nuclei fused to chemically enucleated mouse oocytes.
Mouse oocytes were chemically enucleated by subjecting them to etoposide and cycloheximide treatment during the first meiotic division (Fulka, Jr. and Moor, Mol. Reprod. Dev. 34:427-430, 1993) and thereafter electrofused to karyoplasts prepared from: (i) two-cell stage embryos at the G2-phase; (ii) four-cell stage blastomeres (S- or G2-phase); or (iii) embryonic stem (ES) cells. In the first series of experiments we used fusion conditions which do not induce egg activation to define the series of nuclear changes that are initiated immediately following fusion. Although fusion is evident within 5-10 min of induction, nuclei remain visible for up to 20 min prior to chromatin condensation and the formation of metaphase plates (60-90 min post fusion). After activation, the anaphase-telophase transition is completed within 1-2 h, followed thereafter by cleavage of 75% of reconstituted eggs into two equal nucleated blastomeres, irrespective of the origin of the nuclei used for fusion. We conclude from the first study that a protocol involving fusion without activation, followed 90 min later by activation, is likely to be optimal for nuclear transplantation using MII-phase cytoplasts. In the second series of experiments the above optimized protocol was used to study the effects of different cell cycle combinations on chromosome organization in eggs reconstituted by nuclear transplantation. Both G1- and S-phase karyoplasts fused to MII-phase cytoplasts exhibited spindle abnormalities in all eggs studied. Characteristic abnormalities in these cell cycle combinations included chromatin fragmentation and joining or aggregations of chromatin.(ABSTRACT TRUNCATED AT 250 WORDS)
Fulka
J
J
Development and Differentiation Laboratory, AFRC Babraham Institute, Cambridge, United Kingdom.
Notarianni
E
E
Passoni
L
L
Moor
R M
RM
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000502:physiology*; D002875:Chromosomes; D004622:Embryo, Mammalian / Q000648:ultrastructure*; D007399:Interphase / Q000502:physiology; D008561:Membrane Fusion / Q000502:physiology; D051379:Mice; D008938:Mitosis / Q000502:physiology; D009865:Oocytes / Q000502:physiology*