-210 Nottingham - Nottingham eTheses - The University of Nottingham

5.4 DISCUSSION
In the first experiment, I further developed an MPF/MAPK double kinase assay
protocol. MPF and MAPK are thought to be important to oocytes and nuclear
reprogramming of the NT embryos. Also, MPF and MAPK double kinase assay gives
an overview about MPF and MAPK activities in hourly intervals during in vitro
maturation.
Naito and Toyoda (1991) found repeated freezing and thawing reduced the kinase
activities and hence in this experiment all oocytes were subjected to
snap-freeze-thawing only once for 30 sec, as was different from Lee and Campbell
(2006). Oocytes were stored at -80°C to break the membrane until
snap-freeze-thawing. This was also based on Naito and Toyoda (1991), which was
also different from Ye et al. (2003) and Lee and Campbell (2006). In addition, the
recipe of the kinase assay buffer was slightly different from Ye et al. (2003) and Lee
and Campbell (2006).
Caffeine treatment of TI enucleated oocytes may contribute to the efficiency of
nuclear transfer. In experiments two and three, the effects of different concentrations
of caffeine was examined. Early MII oocytes were chosen for these two experiments
to help select the caffeine concentration because TI arrested oocytes progressed to
MII within 30 min after denuding the cumulus cells.
As seen in the second experiment, 5,10 or 20 mM caffeine did not increase either of
MPF and MAPK activities of porcine oocytes selected at TI and early MII stage
statistically after 6 h. However, Kikuchi et al. (2000) reported that treatment of 72
h-cultured aged oocytes with 5 mM caffeine (last 10 h of culture) elevated MPF
activity. Kawahara et al. (2005) found that the addition of 2.5 mM caffeine in the
maturation medium of 44 h-matured oocytes for 8h significantly increase the MPF
level. The reason for the results observed in these studies could be that those oocytes
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