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Abstract
In general oocytes arrested at metaphase of the second
meiotic division (MII) are used as recipient cytoplasts
for nuclear transfer (NT) procedures. MII oocytes contain
high levels of maturation promoting factor (MPF) and
mitogen activated protein kinase (MAPK) which cause
nuclear envelope breakdown (NEBD) and premature chromosome
condensation (PCC) in the transferred nucleus and have
been implicated in 'nuclear reprogramming'. However, the
occurrence of NEBD and the extent of PCC are variable
between individual oocytes and species and are dependent
on donor cell type and cell cycle stage. Enucleation,
which removes oocyte cytoplasm may reduce MPF and MAPK
activities and reduce 'reprogramming', conversely
increasing kinase activities may increase 'reprogramming'.
We compared the effects of enucleation of ovine oocytes at
anaphase/telophase of the first meiotic division (AI-TI)
and at MII. MPF and MAPK activities were maximal at MII;
blind enucleation at AI-TI was more efficient than at MII
and removed a smaller volume of cytoplasm. Neither
protocol significantly affected the activity of either
kinase and the fate of the donor nucleus, however
enucleation per se significantly reduced the occurrence of
NEBD in NT embryos. Treatment with 10 mM caffeine
significantly increased the activities of both kinases and
the occurrence of NEBD but did not affect the frequency of
development to the blastocyst stage, however, a
significant increase in total cell numbers was observed.
The results show that caffeine can increase MPF and MAPK
activities in ovine oocytes and that this may contribute
to an increased 'reprogramming' in NT embryos.
Key words:
Embryo •
Kinases •
Meiosis •
Oocyte development •
Phosphatases
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