EARLY CHANGES IN EMBRYONIC NUCLEI FUSED TO CHEMICALLY ENUCLEATED MOUSE OOCYTES

Mouse oocytes were chemically enucleated by subjecting them to etoposi de and cycloheximide treatment during the first meiotic division (Fulk a, Jr. and Moor, Mol. Reprod Dev. 34:427-430,1993) and thereafter elec trofused to karyoplasts prepared from: (i) two-cell stage embryos at t he G2-phase; (ii) four-cell stage blastomeres (S- or G2-phase); or (ii i) embryonic stem (ES) cells. In the first series of experiments we us ed fusion conditions which do not induce egg activation to define the series of nuclear changes that are initiated immediately following fus ion. Although fusion is evident within 5-10 min of induction, nuclei r emain visible for up to 20 min prior to chromatin condensation and the formation of metaphase plates (60-90 min post fusion). After activati on, the anaphase-telophase transition is completed within 1-2 h, follo wed 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 li kely to be optimal for nuclear transplantation using MII-phase cytopla sts. 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 transplantati on. Both G1- and S-phase karyoplasts fused to MII-phase cytoplasts exh ibited spindle abnormalities in all eggs studied. Characteristic abnor malities in these cell cycle combinations included chromatin fragmenta tion and joining or aggregations of chromatin. An entirely different s pindle morphology was associated with G2- to MII-phase fusion: morphol ogically normal spindles were found in 45% of eggs in this group while the remainder showed abnormalities characterized by chromosomes dispe rsed (36%) or detached (12%) from the spindle. Although elevated MPF l evels during metaphase II are considered necessary for nuclear remodel ling, our results demonstrate the highly restricted requirements neede d for normal spindle organization and subsequent cloning success after nuclear transplantation.