J. Fulka et al., EARLY CHANGES IN EMBRYONIC NUCLEI FUSED TO CHEMICALLY ENUCLEATED MOUSE OOCYTES, The International journal of developmental biology, 37(3), 1993, pp. 433-439
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.