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spontaneous apoptosis of X. laevis egg extracts was fi rst described
[ 1 ]; however, it is not used as often today in favor of faster, more
quantitative assays.- Prepare egg extract from Subheading 3.1 with EM.
- Add 100× sperm chromatin at a 1:100 dilution, and incubate
at room temperature. Nuclei should begin to form after
20–30 min and be fully formed soon after. Well-formed nuclei
will appear round and homogenous. - After 2 h of incubation in egg extract, these synthetic nuclei
should be observed at 30 min intervals for a further 4–6 h. - To obtain images of nuclei at desired time points, take an
aliquot of extract and mix 1:1 with Hoechst stain on a micro-
scope slide. Add a cover slip and observe the nuclei on a fl uo-
rescent microscope ( see Note 19 ).
Stage VI X. laevis oocytes are arrested in prophase of meiosis 1,
undergoing a G2-like growth phase. Priming of the frogs with
PMSG ( see Subheading 3.1 ) induces production of more stage VI
oocytes. This protocol will describe how to harvest and isolate
stage VI oocytes from female X. laevis frogs ( see Note 20 ).
Induction of apoptosis can also be studied in these whole oocytes,
providing a primary, whole-cell model to validate results from
experiments conducted in the egg extract.- Euthanize one mature female frog in 1 L of aquarium water
containing 10 mL of 10 % benzocaine (0.1 % fi nal
concentration). - A secondary method of euthanasia should also be used, such
as cervical dislocation. - Place the frog with its abdomen facing up, and make a vertical
incision along the midline of the frog’s abdomen. Peel back the
skin and underlying tissue, and excise the ovaries and oocytes.
3.10 Isolation
of Stage VI Oocytes
from Mature, Female
X. laevis Frogs
Fig. 3 PARP cleavage in X. laevis egg extracts. Egg extract was incubated at
room temperature, and samples were taken at indicated time points and ana-
lyzed for PARP cleavage by immunoblotting as described in Subheading 3.8
(McCoy and Nutt, unpublished data)Francis McCoy et al.http://www.ebook3000.com