The first frog egg extract was reported in 1983 by Lohka and Masui.[1] This pioneering work used eggs of the
Northern leopard frogRana pipiens to prepare an extract. Later, the same procedure was applied to eggs of Xenopus laevis, becoming popular for studying cell cycle progression and cell cycle-dependent cellular events.[2] Extracts derived from eggs of the
Japanese common toadBufo japonicus[3] or of the
Western clawed frogXenopus tropicalis[4] have also been reported.
Basics of extract preparation
The
cell cycle of unfertilized eggs of X. laevis is arrested highly synchronously at metaphase of meiosis II. Upon
fertilization, the metaphase arrest is released by the action of Ca2+ ions released from the
endoplasmic reticulum, thereby initiating early embryonic cell cycles that alternates
S phase (
DNA replication) and M phase (
mitosis).[5]
M phase extract
Figure 1. An egg extract is prepared by crushing X. laevis eggs by centrifugation
Unfertilized eggs in a buffer containing the Ca2+
chelatorEGTA (ethylene glycol tetraacetic acid) are packed into a centrifuge tube. After removing excess buffer, the eggs are crushed by
centrifugation (~10,000 g). A soluble fraction that appears between the lipid cap and the yolk is called an M phase extract. This extract contains a high level of
cyclin B-
Cdk1. When demembranated sperm nuclei are incubated with this extract, it undergoes a series of structural changes and is eventually converted into a set of M phase chromosomes with bipolar
spindles.
Interphase (S phase) extract
Different types of egg extracts
Cycling extract
Figure 2. An interphase nucleus (left) and a cluster of mitotic chromosomes (right) produced in a cycling extract. Bar, 10 μm.
More recently, the egg extracts have been used to study reprogramming of differentiated nuclei,[17] physical properties of spindles[18] and nuclei,[19] and theoretical understanding of cell cycle control.[20]
^Ohsumi K, Katagiri C (1991). "Characterization of the ooplasmic factor inducing decondensation of and protamine removal from toad sperm nuclei: involvement of nucleoplasmin". Dev. Biol. 148 (1): 295–305.
doi:
10.1016/0012-1606(91)90338-4.
PMID1936566.
^Görlich D, Prehn S, Laskey RA, Hartmann E (1994). "Isolation of a protein that is essential for the first step of nuclear protein import". Cell. 79 (5): 767–778.
doi:
10.1016/0092-8674(94)90067-1.
PMID8001116.
S2CID7539929.{{
cite journal}}: CS1 maint: multiple names: authors list (
link)