119Peter V. Bozhkov and Guy Salvesen (eds.), Caspases, Paracaspases, and Metacaspases: Methods and Protocols,
Methods in Molecular Biology, vol. 1133, DOI 10.1007/978-1-4939-0357-3_8, © Springer Science+Business Media New York 2014
Chapter 8
Methods for the Study of Caspase Activation
in the Xenopus laevis Oocyte and Egg Extract
Francis McCoy , Rashid Darbandi , and Leta K. Nutt
Abstract
The study of apoptosis and caspases has advanced greatly over recent decades. Studies conducted in the
Xenopus laevis egg extract and oocyte model system have signifi cantly contributed to these advances. Twenty
years ago, Newmeyer and colleagues fi rst showed that the X. laevis egg extract, when incubated at room
temperature, reconstituted the key molecular events of cellular apoptosis including cytochrome c release,
nuclear condensation, internucleosomal fragmentation, and caspase activation. The biochemical tractability
of the egg extract system allows for robust study of apoptotic events and caspase activation. Its nature as a
cell-free extract system allows substrates to be very simply added by pipette, and their effects on apoptosis
and caspase activation and their placement in the apoptotic signaling pathway (e.g., pre- or post- mitochondrial)
are subsequently very simply studied using the techniques described in this chapter. Also described in this
chapter are assays that allow the study of caspase activation in intact oocytes, another valuable tool available
when using the X. laevis model organism. Overall, the X. laevis egg extract/oocyte model is a robust,
effi cient, and biochemically tractable system that is ideal for the study of apoptosis and caspase activation.
Key words Xenopus laevis , Egg extract , Oocytes , Caspases , Apoptosis1 Introduction
The Xenopus laevis egg extract is an ideal model system for the study
of apoptosis. This cell-free in vitro system was fi rst shown to reca-
pitulate key apoptotic events nearly 20 years ago. After incubation at
room temperature, these egg extracts underwent apoptosis, display-
ing nuclear condensation and internucleosomal fragmentation of
synthetically formed nuclei. Apoptosis in these extracts was blocked
by addition of BCL-2 and also required the presence of mitochon-
dria [ 1 ]. Since this initial discovery, studies in the X. laevis egg
extract have helped uncover some of the key mechanisms underlying
the regulation of apoptosis: showing that BCL-2 inhibits apoptosis
by blocking release of cytochrome c from mitochondria [ 2 ], identi-
fying cytochrome c -induced activation of caspase-3 and the key
structural features of cytochrome c involved in this activation [ 3 ],