/. Embryol. exp. Morph. Vol. 48, pp. 101-108, 1978
Printed in Great Britain © Company of Biologists Limited 1978
A method for isolating uncontaminated
nuclei from all stages of developing Xenopus
laevis embryos*
By F. FARZANEH 1 AND C. K. PEARSON 2
From the Department of Biochemistry, University of Aberdeen
SUMMARY
A method for isolating nuclei from Xenopus laevis embryos has been developed. This
procedure enables the isolation of nuclei, free from contamination with yolk and pigment
granules, at all stages of embryonic development. Using this method the nuclear yield is
60-70% of the estimated number of cells in the embryo. The DNA, RNA, histone and
non-histone protein content of these nuclei during embryogenesis (from early cleavage to
the swimming tadpole stage) has been measured.
INTRODUCTION
Previously published methods for the isolation of nuclei from amphibian
embryos (Mariano, 1964; Arms, 1971; Claycomb & Villee, 1971; Destree,
d'Adelhart & Charles, 1973; Kohl, Norman & Brooks, 1973; Ramage & Barry,
1975; Theriault & Landesman, 1974) are unsatisfactory in one or more of the
following ways: They usually produce nuclei which are heavily contaminated
with yolk and/or pigment granules; they are only successful in obtaining
nuclei from embryos of stage 10 (Nieuwkoop & Faber, 1956) onwards, and the
yields are generally poor. The only published method capable of isolating
nuclei from early embryos is that of Destree et ah (1973). Nuclei isolated by
this method are, however, highly contaminated with pigment granules and the
yield is poor. In this paper we describe a simple method whereby uncontaminated nuclei can be isolated from Xenopus laevis embryos at all stages of
development, and in good yield.
*A preliminary account of this work was presented at the 36th meeting of the British
Society for Developmental Biology (1977).
1
Authors' address: Biochemistry Laboratory, School of Biological Sciences, University of
Sussex, Falmer, Brighton BN1 9QG, U.K.
2
Authors' address: Department of Biochemistry, University of Aberdeen, Marischal
College, Aberdeen AB9 IAS, Scotland, U.K.
102
F. FARZANEH AND C. K. PEARSON
MATERIALS AND METHODS
Production of embryos
Pairs of sexually mature toads (purchased from Harris's Biological Supplies
Limited) were injected into the dorsal lymph sac with 1000 units of chorionic
gonadotropin hormone per toad, and then placed in 81. of 1/10 modified
Barth-X solution (Gurdon, 1974) at 22 °C. Spawning commences 8—12 h later
and continues for 2-4 h. The advantage of one high dose of hormone instead
of the more commonly employed procedure of injecting a number of smaller
doses over a period of time, is that on subsequent incubation of the fertilized
eggs a large number of embryos which are all at approximately the same stage
of development could be obtained. This is due to the short duration of spawning,
without a marked decrease in the number of eggs produced. This procedure also
minimizes the number of over-ripe eggs which are shed. About 85 % of eggs
produced by this procedure are fertilized, and usually 95 % of these develop
to the tadpole stage. Alternatively, very well synchronized embryos could be
obtained by artificial fertilization of the eggs (Wolf & Hedrick, 1971). The
method of artificial fertilization is particularly useful for isolating nuclei from
pre-blastula embryos. This is because unless these embryos are at the interphase of a cell cycle, all or a large number of the nuclei could be in metaphase
or anaphase of mitosis and hence the yield will be poor.
The fertilized eggs were incubated in the 1/10 modified Barth-X medium,
and were allowed to develop to the required developmental stage under continuous aeration of the medium at 22 °C.
Isolation of nuclei
Embryos of the desired developmental stages were dejellied by incubating
them for approximately 5 min in a solution of 0-14M-NaCl containing 1%
(w/v) cystein HC1 brought to pH 8-0 with NaOH. They were then washed,
at least 20 times, with 0-14M-NaCl to ensure the complete removal of the
jelly coat and its contaminating bacteria, etc. A suspension of 500-1000 dejellied embryos or 1 g of adult Xenopus liver in 1 -0 ml of the incubation medium
was mixed with 6-0 ml of a 2-5 M sucrose solution. All sucrose solutions contained 2-0 mM-MgCl2, 5-0 mM-Tris-HCl pH 8-0 at 2 °C, 2-0 mM 2-mercaptoethanol, 0-5 mM spermine and 0-4% (w/v) Triton-NlOl, unless otherwise
specified. All the nuclear isolation procedures were carried out at 0-4 °C. The
suspension of embryos or liver in the 2-5 M sucrose was homogenized in a
glass homogenizer, with an undersized teflon pestle to give a clearance of 1 mm,
using a Tri-R-Stir-R homogenizer operating at 1000 rev./min. Conditions for
homogenizing embryos at different stages of development varied slightly
(see Table 1). The homogenate, 7 ml, was filtered through a nylon mesh of
pore size 100 /mi. In order to reduce the losses through filtering, the mesh
was washed with 1 ml of 2-3 M sucrose which was added to the homogenate.
Isolation of uncontaminated nuclei from. Xenopus embryos 103
Table 1. Conditions for homogenizing embryos and adult Xenopus
liver at different stages of development
Stage of
development
No. of strokes
Rev./min.
No. of cells/embryo*
1-5
5-10
10-20
20-40
Adult Xenopus
2
4
6
10
20
1000
1000
2000
5000
5000
1-16
16-30000
30000-80000
80000-400000
* From Woodland & Gurdon, 1968.
A step gradient was set up with 8 ml of 2-5 M sucrose, overlaid with 22 ml of
2-3 M sucrose, in a cellulose nitrate or polyallomer centrifuge tube. The filtered
homogenate was layered onto the gradient and the interphase between the
2-3 M sucrose and homogenate was stirred with a glass rod. The tubes were
then centrifuged at 27000 rev./min (100000 # av.) at 2 °C for 2 h using a
Beckman SW-27 rotor. Under these conditions the pigment granules pelleted
at the bottom of the tube, and the yolk and cell debris formed a cuticle at the
top cf the gradient which was removed with a spatula. The nuclei, which form
an opaque band at the interphase between the 2-3 M and 2-5 M sucrose solutions,
were removed with a wide bore pipette after 18 ml of the 2-3 M sucrose was
first removed by careful aspiration. The nuclear suspension was gradually
diluted with two volumes of 0-3 M sucrose solution to avoid bursting them by
osmotic shock. The 0-3 M sucrose contained no Triton-NlOl. The nuclei were
then centrifuged for 10 min at 1000 # av. in an MSE 2L centrifuge, using siliconized glass tubes. The supernatant was discarded and the nuclear pellet
was washed in 20 ml of the 0-3 M sucrose solution twice. The washed nuclei
were suspended in a small volume of the 0-3 M sucrose by gentle mixing, and
displayed in a haemocytometer chamber for morphological studies and determination of the yield.
Determination of DNA, RNA, histone and non-histone protein content of the
nuclei.
The histones present in these nuclei were extracted with 0-25 M-HC1, according to the method of Lake, Goidl & Salzman (1972). The extracted histcnes
were precipitated with an equal volume of 40% ice-cold trichloroacetic acid.
This procedure was shown by Easton (1977) to extract and precipitate 86%
of the histones. The content of histones, non-histones, DNA and RNA of the
isolated nuclei was then determined. Proteins were assayed by the method of
Lowry, Rosebrough, Farr & Randall (1951), using calf thymus histones and
bovine serum albumin as standards for histones and non-histones, respectively.
DNA was measured by the method of Burton (1956), and RNA by the method
104
F. FARZANEH AND C. K. PEARSON
Fig. 1. Phase-contrast photograph of nuclei (in 03 M sucrose solution) isolated
from Xenopus laevis embryos, at stage 2-4. Arrows indicate some of the kidneyshaped nuclei.
of Munro & Fleck (1966), using calf thymus DNA and RNA as standards
respectively.
RESULTS
Nuclei obtained by this procedure are seen by phase-contrast microscopy
to be intact and free from contamination by other cellular organelles (see Fig. 1).
A haemocytometer was used to display the nuclei shown because they are very
fragile, particularly from embryos earlier than stage 10, and are easily damaged
when put under an ordinary coverslip.
For isolation of nuclei from early cleavage embryos it is extremely important
to homogenize the embryos immediately after one of the synchronous cell
divisions. In our experience when the embryos were not homogenized after a
division few nuclei were recovered. However, when the early cleavage embryos
were homogenized soon after a cleavage, in four consecutive preparations the
nuclear yield was 55 %, 74 %, 67 % and 63 % from embryos of stages 2-4, 2-3,
3-4 and 2-4 respectively. In each case at least 75 % of the embryos used were of
only one developmental stage. These embryos were the products of four
separate matings, and were sorted into their corresponding stage of development
whilst being kept on ice.
The size of nuclei isolated from early cleavage embryos was about 15-20
/mi x 8-12 pirn, and some appeared to be kidney shaped (see Fig. 1). These
301
29-6
34-8
33-1
301
361
31-6
32-4
28-3
Early-cleavage stage 2-4
Late blastula stage 7-9
Late gastrula stage 12-13
Early neurula stage 15-16
Mid-neurula stage 18-19
Late neurula stage 22-23
Tail-bud stage 27-28
Swimming tadpole stage 33-34
Adult Xenopus liver
60
5-4
5-1
6-9
61
5-8
5-8
6-6
5-2
Histories
25-1
25-9
26-6
27-4
26-4
26-4
25-4
260
25-9
Non-histone:s
10
1-2
1-9
1-8
1-6
1-7
2-1
2-2
1-6
RNA
Protein/DNA
51
4-6
5-7
60
50
7-1
5-8
5-3
4-8
DNA
60
6-5
61
5-5
60
51
5-5
61
5-9
0-2
0-2
0-3
0-3
0-3
0-3
0-4
0-4
0-3
RNA/DNA
The figures represent values obtained from nuclei (approximately 106 in each case) isolated during one experiment from a batch of developing
embryos. Protein and DNA determinations were carried out routinely, however, and absolute values varied only slightly, for example, protein
content per nucleus (mixed stages of development) was 32 ± 2-2 pg (S.D. from 18 experiments) and DNA content was 5-8 ± 0-4 pg (S.D. from 18 experiments).
The total DNA recovered per embryo: early cleavage 15 pg; late blastula 34 ng; late gastrula 182-3 ng; early neurula 185-8 ng; mid neurula 3140
ng; late neurula 2991 ng; tail-bud 481-6 ng and swimming tadpole 811-6 ng.
Total
proteins
Embryonic stage of development
Table 2. DNA, UNA, histone and non-histone protein content of the nuclei (pg/nucleus)
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106
F. FARZANEH AND C. K. PEARSON
nuclei did not contain nucleoli, due to the absence of ribosomal RNA synthesis
during cleavage and blastula stages (Brown & Littna, \964a). Nuclei isolated
from embryos of stage 12 and beyond did not have the kidney shaped appearance
of some of the nuclei isolated from pre-gastrula embryos, and contained
zero to two nucleoli. These nuclei were smaller, and at stage 25 (tail-bud)
they were only half the size of the nuclei isolated from stage-2 to stage-4 embryos
(i.e. 8-10 /im x 4-6 /im). The isolated liver nuclei had a diameter of 4-5 /.im.
No significant change was observed in the amount of either histones, nonhistones, or DNA per nucleus during the embryonic growth (Table 2). However, the RNA content of the isolated nuclei increased during embryogenesis
from 1-0 pg/nucleus at early cleavage to 2-2 pg/nucleus in the swimming
tadpole (Table 2). The RNA content of adult Xenopus liver nuclei was 1-6
pg/nucleus.
DISCUSSION
The nuclear isolation method described here offers a means of obtaining
early Xenopus embryonic nuclei in high yield (60-70%) and free from cytoplasmic contamination. This has been achieved by making the following
additions to the nuclear isolation method of Destree et al. (1973): inclusion of
spermine in the isolation media for stabilization of the nuclear membranes,
the use of a wider clearance homogenizer to reduce the nuclear damage during
homogenization, centrifugation of the nuclei through a one-step sucrose
gradient to remove the contaminating pigment granules and, finally, the use of
synchronously developing embryos for isolation of nuclei from early cleavage
stages.
The DNA, histone, and non-histone protein content of the isolated nuclei
did not change during the embryonic growth (Table 2). Since the nuclei in early
cleavage embryos are rapidly replicating, they would be expected to contain
more than the diploid amount of DNA. However, it can be seen (Table 2)
that the DNA content of the nuclei during early cleavage stages is the expected
diploid amount, i.e. 6 pg/nucleus. This could be due to the fact that early
cleavage embryos were homogenized immediately after one of the synchronous
cell divisions, i.e. during the early part of the cell cycle, when the DNA content
is close to the diploid amount. In late cleavage and blastula embryos the
cell divisions are asynchronous and cell numbers increase logarithmically.
Therefore, at any one time, there would be more cells in the early part of
the cell cycle than there are at the end of the cycle (Mitchison, 1973). Consequently, nuclei isolated from early embryos by this procedure have a DNA
content close to the expected 2C value. The protein/DNA ratio in these nuclei
was 5-6 ±0-7, which is similar to other published values for the later stages of
embryogenesis (Widnell & Tata, 1966; Yasuda, Shiokawa & Yamana, 1977).
The RNA content of the isolated nuclei, in contrast to protein and DNA,
increased during development from 1-0 pg/nucleus at early cleavage to 2-2
Isolation of uncontaminated nuclei from Xenopus embryos 107
pg/nucleus in the swimming tadpole. The RNA synthesized by early cleavage
embryos has been shown to be 'DNA-like' RNA (Brown & Littna, 19646;
Gurdon, 1968). It is presumably this 'DNA-like' RNA which contributes to
the RNA levels of early and mid-cleavage nuclei. The increase in the RNA
content during the later stages of development could be a reflexion of the
synthesis of large quantities of heterogeneous RNA and transfer RNA during
gastrulation (Woodland & Gurdon, 1968), and ribosomal RNA at postgastrulation stages (Brown & Littna, 1964a).
The poor yield, and the contamination of isolated nuclei by yolk and
pigment granules has hitherto been a major obstacle in studying some of the
major processes involved in the early development of Xenopus laevis embryos.
As a result, although there is a large accumulation of data on the post-gastrulation stages, up to date, there are no reported studies on the in vitro properties
of the nuclei in early cleavage and mid-blastula embryos of amphibia. The
high yield and absence of cytoplasmic contamination in nuclei isolated by
the procedure described should make the present method a useful tool in
studying such processes as RNA and DNA synthesis; analysis of the nuclear
RNA and protein species and their modifications; as well as determining the
sub-cellular location of such storage compounds as histones during early
embryogenesis. For instance, the early and mid-cleavage nuclei could be
used to study messenger RNA synthesis in vitro in the absence of ribosomal
RNA synthesis.
We are grateful to the Medical Research Council for their support, and would also like to
thank Dr J. McKenzie (Department of Developmental Biology, University of Aberdeen),
and our colleagues, Miss G. MacDermott and Mr H. M. Furneaux of this laboratory for
their helpful assistance during the course of this work. We would particularly like to acknowledge the helpful comments and advice of Dr C. C. Ford (School of Biological Sciences,
University of Sussex) during the preparation of the manuscript.
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