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and discovering that the Drosophila melanogaster protein, reaper,
could function in a vertebrate system to induce apoptosis upstream
of mitochondria [ 4 ]. These studies and others have utilized the egg
extract to study apoptosis in great detail [ 5 – 11 ].
Of all the caspases, caspase-2 is one of the least studied and
least well understood. In the past this has likely been due to an
inability to fi nd defi ned contexts under which it is important for
induction of apoptosis. However, caspase-2 knockout mice were
shown to have an excess number of oocytes that were chemo-
resistive [ 12 ], implicating caspase-2 as being important for oocyte
death. The use of various techniques to study caspase activation in
the X. laevis oocyte and egg extract has since uncovered that cas-
pase- 2 appears to function under defi ned contexts and stimuli; in
particular caspase-2 appears to be important for inducing apoptosis
in response to metabolic cues [ 13 – 17 ].
These studies showed that oocyte cell death is metabolically
regulated through CaMKII (Ca 2+ /calmodulin-dependent protein
kinase II)-mediated phosphorylation of caspase-2, resulting in
inhibition of caspase-2 activation upstream of Bax/Bak activation
and cytochrome c release. Specifi cally, the generation of NADPH
through the pentose-phosphate pathway activates CaMKII that in
turn phosphorylates and inactivates caspase-2 [ 13 , 14 ]. Conversely,
under metabolic deprivation, CaMKII is inactive, allowing for the
dephosphorylation of caspase-2 by protein-phosphatase-1 (PP1).
The model of metabolically regulated caspase-2-mediated oocyte
apoptosis has been further expanded, with recent studies showing
that metabolism acts to inhibit PP1, preventing dephosphorylation
of CaMKII, and allowing for sustained phosphorylation of cas-
pase- 2 by CaMKII [ 17 ].
These and future fi ndings rest on the techniques described
below to properly measure caspase activity in the highly tractable
X. laevis oocyte and egg extract. The process of collecting X. laevis
eggs and the preparation of interphase and cytosolic egg extract are
fi rst covered (Subheading 3.1 ). As key markers of apoptosis, the
assessment of caspase-3 and caspase-7 in extract (Subheadings 3.3
and 3.4 ) and in intact oocytes (Subheadings 3.10 and 3.11 ) is
described. As discussed previously, caspase-2 is a critical point of
control in the metabolic regulation of oocyte cell death, and the
assessment of its activity in extract using a^35 S-labeled in vitro-
translated protein is explained (Subheading 3.5 ).
The tractability of X. laevis egg extract has allowed for the
development of techniques that enable complete dissection of each
of the key steps in the apoptotic cascade. As a cell-free extract sys-
tem, substrates can simply be added by pipette to the extract, and
using the techniques described in this chapter, their effects on
apoptosis and their placement as pre- or post-mitochondrial in the
apoptotic cascade can be identifi ed (Subheadings 3.6 and 3.7 ).Francis McCoy et al.http://www.ebook3000.com