-210 Nottingham - Nottingham eTheses - The University of Nottingham

Caffeine has been reported to enhance reprogramming in porcine SCNT embryos
using MII enucleated oocytes, however, the mechanism of action is unknown but may
involve MPF and MAPK as discussed in Chapter 1. Kawahara et al. (2005) added 2.5
mM caffeine to the handling medium when porcine oocytes were enucleated (at 44
hpm) for 3h until NT embryos were activated, resulting in a higher frequency of
development to the blastocyst stage of NT embryos as compared to the non-treated
control. Similarly Iwamoto et al. (2005) reported that addition of 5 mM caffeine in to
porcine oocyte maturation media from 36 hpm to 60 hpm (without removal of the
cumulus cells) prior to enucleation promoted nuclear remodelling in cloned embryos.
Kwon et al. (2008) suggested that a lower apoptotic cell index was found in NT
blastocysts made from enucleated oocytes treated with 5 mM caffeine from 42 hpm
for 2.5 h and released from caffeine for 0.5 h before cell transfer. Moreover, similar
results were obtained using TI enucleated ovine oocytes treated with 10 mM caffeine
(Lee and Campbell, 2006) and live lambs have been produced (Campbell, manuscript
in preparation).
MPF activity can be measured using biological or biochemical assays. Analysis of
MPF activity by phosphorylation of histone HI was first introduced in studies on
amphibian oocytes (Mailer et al., 1977; Doree et al., 1983). In mammalian studies,
this method was first applied in porcine oocytes (Naito and Toyoda, 1991), followed
by mouse (Choi et al., 1992), cattle (Collas et al., 1993), rabbit (Jelinkovä et al., 1994)
and ovine oocytes (Bogliolo et al., 2000). It involves three main steps: (1) transfer of
[7_32 PI ATP to the substrates by in vitro reaction of oocyte lysate with [_t_32 P] ATP
and the substrates, (2) separation of phosphorylated substrates from unincorporated
[y_32PI ATP by SDS-PAGE and (3) quantification of radioactively labeled substrates.
Modification of the method allows both MPF and MAPK activities to be measured
simultaneously in oocytes by a SDS-PAGE and autoradiography using
histone HI
(Anion et al., 1988) and bovine myelin basic protein (MBP) (Sanghera et al., 1990) as
86