/. Embryo!. exp. Morph. Vol. 20, 3, pp. 367-74, November 1968
With 2 plates
Printed in Great Britain
367
Cytonucleoproteins in cleaving eggs of
Xenopus Icevis
By K. ARMS 1
Department of Zoology, University of Oxford, England
The cytoplasm of unfertilized eggs of Xenopus laevis induces DNA synthesis
in a high proportion of adult and embryonic nuclei introduced into it (Graham,
Arms & Gurdon, 1966). This induction of DNA synthesis is not due merely to
the fact that the egg cytoplasm supplies precursors of DNA synthesis which
might be absent from the nuclei, since adult liver nuclei, at least, of those nuclei
which are induced to synthesize DNA after their injection into eggs, do not
do so if incubated in vitro with DNA precursors (Arms, 1968). One of the ways
in which the cytoplasm of unfertilized eggs might induce DNA synthesis is by
supplying other molecules, such as various enzymes, to the introduced nuclei.
There are an increasing number of reports describing the transfer of proteins
from cytoplasm to nucleus (Zetterberg, 1966) or from one nucleus to another
in a binucleate cell (Byers, Platt & Goldstein, 1963; Goldstein, 1964). Byers and
his collaborators have named these proteins 'cytonucleoproteins'. These facts
suggested that cytonucleoproteins might be detectable during early embryonic
development and might be implicated in the induction of DNA synthesis.
The experiments described here show that protein passes from the cytoplasm
of cleaving eggs into adult nuclei which are induced to synthesize DNA after
their injection into embryos. Proteins of cleaving eggs were labelled with tritiated leucine, and it was shown that unlabelled nuclei introduced into such
embryos became labelled with the amino acid and can be induced to synthesize
DNA under conditions in which no further protein synthesis occurred in the
system. It was concluded that such labelling of introduced nuclei was due to
transfer of labelled proteins from the embryonic cytoplasm.
MATERIALS AND METHODS
Ovulation and mating of Xenopus laevis (Daudin), rearing of embryos,
isolation of nuclei, micro-injection and autoradiographic techniques have been
described in a preceding paper (Graham et al. 1966).
1
Author's address: Department of Biological Sciences, Stanford University, Stanford,
California 94305, U.S.A.
24-2
368
K. ARMS
Embryonic stages are described by the numerical system of Nieuwkoop &
Faber (1956).
DL-Leucine-4,5-T (35-1 c/mM; 1 mc/l-2ml) from the Radiochemical Centre,
Amersham, Bucks., was used to label proteins. Puromycin was obtained from
the Nutritional Biochemical Corporation, Cleveland, Ohio and was injected
into embryos in a solution of 1 or 2 mg/ml in 50 mM Tris-HC 1 (pH 7-8).
RESULTS
Induction of DNA synthesis in nuclei injected into cleaving eggs
To investigate the possibility that cytonucleoproteins are involved in the
induction of DNA synthesis, cytoplasmic proteins were labelled before DNA
synthesis was induced. Unfertilized eggs, which have been used previously for
the induction of DNA synthesis in adult nuclei, are not suitable for these
experiments, since their condition deteriorates visibly over the 4 h period after
activation that would be necessary both to label cytoplasmic proteins and to
observe DNA synthesis induced in injected nuclei. If, however, DNA synthesis
could be induced in nuclei injected into cleaving eggs, this difficulty would be
overcome, since a high proportion of embryos develop apparently normally
after injection of various substances. The possibility that DNA synthesis would
be induced in nuclei injected into cleaving eggs was therefore investigated.
Table 1. Incorporation of3H-TdR by nuclei isolated from the liver of
young frogs after their injection into 2- to 12-cell cleaving eggs
Time after
injection
Number of
nuclei
counted
60min
90min
6h
140
71
16
Degree of labelling
wL*
L*
Total
nuclei
labelled
(%)
13
12
1
16
21
5
20-71
46-48
37-5
c
* Nuclei in each category (number counted) were classified as unlabelled, weakly labelled
(wL= labelled up to twice background level), or labelled (L = labelled more than twice
background level).
About 30 n\[i\ of a suspension of isolated young frog liver nuclei in Tris-HCl
(pH 7-8) containing tritiated thymidine (3H-TdR) at a concentration of about
1 me/ml were injected into cleaving eggs between the 2- and 12-cell stages.
Sixty minutes, 90 min and 6 h later, embryos were fixed, sectioned at 6 (i and
examined autoradiographically. Many of the injected nuclei are found to be
labelled after this treatment (Plate Ib; Table 1). Ninety minutes after injection
these embryos are still undergoing cleavage and contain about 48 blastomeres.
After 4 h embryos have reached stage 8 (blastula). Plate 1 a shows nuclei fixed
J. Embryo I. exp. Morph., Vol. 20, Part 3
PLATE 1
Incorporation of 3 H-TdR by nuclei isolated from young frog liver and injected into cleaving
eggs.
(a) Liver nuclei, 90 min after their injection into a two-cell embryo. The nuclei lie within an
embryonic cell packed with yolk platelets. The nuclei are considerably swollen; their diameter,
before isolation from the liver, would have been 6-10/t.
(b) Autoradiograph of young frog liver nuclei injected with 3 H-TdR into a cleaving embryo
and fixed 60 min later. The nuclei in both these photographs have come to lie within embryonic
cells. This is not always the case; in Plate 2b injected nuclei have come to lie in an intercellular
space in the embryo. The position of the nuclei within the embryos was found to have no
effect on whether they became labelled or not, in any of the experiments described.
K. ARMS
facing p. 368
J. Embryol. exp. Morph., Vol. 20, Part 3
PLATE 2
(a) Autoradiograph of part of a stage 5 embryo, 60 min after it had been injected with
3
H-leucine (see text). Labelling appears to be heaviest around the periphery of the cells and
in the nucleus (indicated by the arrow).
r
(b) Autoradiograph of young frog liver nuclei fixed 60 min after their injection into an
eight-cell embryo. The nuclei lie in an intercellular space between the embryonic cells. Two
labelled nuclei are indicated by open arrows and an unlabelled nucleus by a solid arrow. The
approximate positions of the cell membranes of two of the embryonic cells are indicated by
dotted lines.
K. ARMS
Cytonucleoproteins in Xenopus
369
90 min after their injection into cleaving embryos. Injected nuclei are, almost
invariably, distinguishable from embryonic nuclei by the fact that they are
smaller and stain more intensely with Mayer's haemalum. Furthermore, only
one nucleus per cell can be embryonic. However, where there could be any
doubt as to the identity of a nucleus it was scored as embryonic. When 3H-TdR
is injected into young frogs and their livers removed and examined by autoradiography 2 h later, about 10 % of the liver nuclei are found to be labelled.
A similar proportion of nuclei is found to be labelled if isolated young frog liver
nuclei are incubated in vitro with 3H-TdR and precursors of DNA synthesis for
90 min (Arms, 1968). Since 46 % of such nuclei are labelled 90 min after their
injection into cleaving eggs, it is clear that exposure to the material of the
embryo has induced DNA synthesis in a high percentage of these nuclei. The
proportion of injected frog liver nuclei found to be labelled 90 min after their
injection into cleaving eggs (Table 1) is, however, lower than the proportion
labelled 90 min after their injection into unfertilized eggs (46 % as opposed to
about 86 % (Graham et al. 1966)). The degree of labelling of individual nuclei
in the two cases appears comparable as far as can be ascertained from counting
the number of grains over a nucleus, and the reason for the difference in the
proportion of nuclei labelled in the two cases is unknown.
The above experiment shows that nuclei are induced to synthesize DNA after
their injection into cleaving eggs and the possibility that transfer of proteins
from the embryonic cytoplasm into such nuclei might occur in the course of
such induction was tested.
Protein synthesis in cleaving eggs
The pattern of incorporation of 3H-leucine into cleaving eggs was studied by
injecting about 100 m/tl of 3H-leucine into 4-64 cell embryos and fixing them 30,
60 and 90 min later. Autoradiography revealed that embryonic cells were
heavily labelled 60 min after the injection of 3H-leucine and that the label was
concentrated in the nucleus and round the periphery of cells (Plate Id). Labelling was not reduced by the injection of a concentration of unlabelled leucine
equal to about 100 times that of the labelled amino acid, 60 min after injection
of the label. The labelled amino acid, therefore, is not removed by a chase
administered 60 min after the label, as has also been found by Ecker & Smith
(1966) for cleaving embryos oiRana. This suggests that the label is incorporated
into protein which shows little or no turnover in a 60 min period.
To test this further, 100 m/tl of puromycin (1 mg/ml) were injected into
embryos 30, 90 and 120 min before the label (about 50 /ic) was injected at the
same time as a further 50 m/d. of puromycin. This would give a concentration
inside the embryo of about 50 m/tM/ml of puromycin. Under these conditions,
no labelling above background was detected in embryos fixed 60 or 90 min
after 3H-leucine was injected. This is a swifter inhibition of protein synthesis
than that found by Legros & Brachet (1965) for Pleurodeles embryos, where it was
370
K. ARMS
found that protein synthesis was not substantially reduced until 3 h after puromycin injection. However, the concentration of puromycin used by these authors
was very low (about 40 ii/iuijwl inside the embryo) compared with that used
here. The fact that puromycin suppresses 3H-leucine incorporation in cleaving
Xenopus embryos, as demonstrated above, strongly suggests that the label has
been incorporated into protein, since puromycin is known to inhibit protein
synthesis in a wide range of systems (e.g. Nemeth & de la Haba, 1962; Allen &
Zamecnik, 1962).
Evidence of protein transfer
Embryos were incubated for 60 min after they had been injected at the 4- to
12-cell stage with 3H-leucine. About 50 m/tl of a suspension of young frog liver
nuclei in 50 mM Tris-HCl (pH 7-8) were then injected into each embryo and the
embryos incubated for a further 60 min before they were fixed for autoradiography. Subsequent examination of injected nuclei showed that nearly all were
quite heavily labelled as is shown in Plate 2b. In a control experiment part of
the same nuclear suspension was injected with 3H-TdR, as described above, into
other embryos of the same stage and from the same mating and also fixed 60 min
later. Autoradiographic examination of these nuclei showed that 35 % of them
had synthesized DNA in this time, as would be predicted.
These nuclei have thus been induced to synthesize DNA by their injection into
embryos and have also become labelled with leucine which has been incorporated
into an acid-insoluble form. It is clear that the labelled leucine must have
entered the nuclei from the embryo which was labelled before the nuclei were
introduced. Nuclear labelling might be due to transfer of labelled protein from
the embryonic cytoplasm or to the fact that the nuclei synthesized protein after
their injection into embryos and incorporated the label in this way. The following experiments were performed to decide between these two possibilities.
Possibility that nuclei synthesize protein after their injection into embryos
This possibility was investigated by an experiment designed to show whether
or not transfer of 3H-leucine from the labelled embryonic cytoplasm into
unlabelled, introduced nuclei still occurred under conditions in which all protein synthesis in the system was prevented by puromycin.
About 100 m/A of a suspension of nuclei in Tris buffer, containing puromycin
at a concentration of 2mg/ml, were injected into 16-cell embryos which had
been labelled 30 and 60 min before by injections of 3H-leucine. This would give
a concentration of about 100 m/*M/ml of puromycin inside the embryo. It has
been shown above that a concentration of puromycin somewhat lower than this
is sufficient to depress protein synthesis within the embryo to a level at which it
is not detectable by autoradiography. Nuclei injected with puromycin in this
way still became labelled with leucine within 60 min of their injection even if they
had been allowed to soak in the puromycin solution for up to 30 min before
Cytonucleoproteins in Xenopus
371
their injection. Thus transfer of label from the embryonic cytoplasm occurs
even when protein synthesis in the system has been prevented by puromycin.
When protein synthesis in the system is suppressed by puromycin before embryonic protein synthesis occurs, however, no labelling is detected in the system.
This is demonstrated by the following experiment.
Fifty m/d of puromycin solution (1 mg/ml) were injected into embryos at the
2- to 4-cell stage. Thirty minutes later 50 m/d of the 3H-leucine solution were
injected and 30, 60 and 90 min later about 50 m/tl of a suspension of liver
nuclei were injected into the same embryos. Embryos were fixed 60 min after
the final injection. Under these conditions negligible label was detected in the
embryos or in the injected nuclei. This further confirms that 3H-leucine incorporation represents protein synthesis and not unspecific adsorption of the
labelled amino acid.
DNA synthesis in the presence of puromycin
It is possible that, in the above experiment, puromycin suppresses all activity
of the injected nuclei and not merely protein synthesis. An experiment was
therefore designed to test whether puromycin suppresses DNA as well as
protein synthesis in this system.
Part of a suspension of nuclei, soaked in puromycin for 30 min exactly as in
the experiment described above, was injected with 3H-Tdr (to a final concentration inside the embryo of about 30 /*c/ml) into embryos at the 16-cell stage and
the embryos fixed 60 min later. These nuclei incorporated thymidine as usual.
Puromycin per se, therefore, does not prevent DNA synthesis in this system. It
has, however, been shown by Black, Baptist & Piland (1967), that puromycin
may prevent DNA synthesis in embryos as a by-product of its inhibition of
protein synthesis.
DISCUSSION
Incorporation of injected labelled amino acid detected by autoradiography
may be considered to represent protein synthesis in these embryos since puromycin suppresses incorporation. Furthermore, it is not possible to reduce leucine
labelling of these embryos by an injection of unlabelled amino acid 60 min later.
Frog liver nuclei injected into embryos labelled in this way with amino acid
themselves become labelled. There is no evidence that young frog liver nuclei
ever synthesize protein either in vivo or when incubated in a protein synthesis
system in vitro. It therefore seems unlikely that the labelling of frog liver nuclei
observed after their introduction into labelled embryos is due to protein synthesis by the nuclei themselves. This is confirmed by the fact that these nuclei
become labelled in this system even under conditions in which all detectable
protein synthesis is suppressed by puromycin. It must be concluded, therefore,
that the nuclei are labelled by the transfer of labelled protein from the embryos
into which they have been injected.
372
K. ARMS
Another result of injecting nuclei into unfertilized eggs (Graham et al. 1966)
or into embryos is that nuclei treated in this way are induced to synthesize DNA.
It is of interest to inquire whether there is any causal connexion between the
cytonucleoprotein transfer demonstrated here and the induction of DNA synthesis in these systems. The experiments described here provide no evidence for
such a causal connexion nor do they rule out its possibility. Gurdon (1967) has
shown that, during its development, the egg of Xenopus first acquires the power
to induce DNA synthesis in nuclei injected into it after the release of pituitary
hormone in the gravid female, which leads to breakdown of the oocyte germinal
vesicle (Detlaff, Nikitina & Stroeva, 1964) and eventually to ovulation. The
mechanism and significance of germinal vesicle breakdown are little understood. It is known, however, that at this time a massive increase in protein
synthesis by the oocyte occurs (Smith, Ecker & Subtelny, 1966). It is possible
that this leads to the supply of various proteins that are important in activation and cleavage of the egg. This is supported by the fact that Black et al.
(1967) found that puromycin applied after fertilization, at a concentration
sufficient to suppress phenylalanine incorporation by 99 % in 6 min, caused
negligible reduction in thymidine incorporation during the first period of DNA
synthesis by the fertilized Arbacia egg. In view of these facts, it seems a reasonable hypothesis that some of the proteins synthesized at the time of germinal
vesicle breakdown are implicated in the induction of DNA synthesis by the
cytoplasm of unfertilized eggs and of embryos of Xenopus.
SUMMARY
1. When cleaving eggs of Xenopus, into which tritiated leucine has been
injected 60 min before, are examined autoradiographically, the embryonic cells
are found to be heavily labelled. It is not possible to reduce this labelling by a
chase injection of unlabelled leucine 60 min after the injection of label. This,
plus the fact that puromycin suppresses labelling in this system almost completely, indicates that the labelling which is detected represents incorporation
of the tritiated amino acid into protein.
2. If a suspension of isolated frog liver nuclei is injected into a cleaving egg
previously labelled in this way, the introduced nuclei are found to be labelled
60 min later. Such labelling of nuclei introduced into radioactive embryonic
cytoplasm occurs even under conditions in which protein synthesis in the system
is inhibited by puromycin to such an extent as to be undetectable by autoradiography. It therefore cannot be due to protein synthesis by the introduced
nuclei. It is concluded that introduced nuclei become labelled by the transfer
of' cytonucleoproteins' from the embryonic cytoplasm.
3. Nuclei injected into cleaving eggs are induced to synthesize DNA as are
nuclei injected into unfertilized eggs. The possible relationship of cytonucleoprotein transfer to the induction of DNA synthesis is discussed.
Cytonucleoproteins in Xenopus
373
RESUME
Cytonucleoproteines dans des oeufs de Xenopus laevis en segmentation
1. L'examen autoradiographique d'oeufs de Xenopus en segmentation injectes
de leucine tritiee 60 min avant la fixation, montre une radioactivite elevee. Ce
marquage n'est pas elimine par l'injection du meme precurseur froid, 60 min
apres administration du radioisotope. Ce fait, ainsi que la suppression du
marquage par la puromycine, fait supposer que la radioactivite detectee
correspond a l'incorporation de l'acide amine tritie dans des proteines. Apres
injection d'une suspension de noyaux de foie de grenouille, dans des oeufs
ainsi marques, on observe, 60 min plus tard, le marquage des noyaux introduits.
Ce marquage de noyaux 'etrangers' dans le cytoplasme d'oeufs radioactifs,
s'effectue meme lorsque la synthese proteique est inhibee par la puromycine a
un point tel qu'elle n'est pas decelable par autoradiographie. Ce transfert n'est
done pas le resultat d'une synthese proteique dans les noyaux introduits. On
peut des lors conclure que les noyaux sont rendus radioactifs par le transfert de
'cytonucleoproteines' ayant pour origine le cytoplasme embryonnaire.
3. On observe une induction de la synthese du DNA dans des noyaux
injectes dans des oeufs en segmentation comparable a celle observee chez des
noyaux transplanted dans des oeufs non fecondes. L'influence eventuelle d'un
transfert de cytonucleoproteines sur l'induction de la synthese du DNA est
discutee.
This work was performed during the tenure of a Research Scholarship from the Science
Research Council.
I am most grateful to Dr J. B. Gurdon for his help and encouragement throughout the
course of this work.
REFERENCES
D. W. & ZAMECNIK, P. C. (1962). The effect of puromycin on rabbit reticulocyte
ribosomes. Biochim. Biophys. Acta 55, 865-74.
ARMS, K. (1968). DNA synthesis by isolated embryonic nuclei of Xenopus. Devel. Biol.
(submitted).
BLACK, R. E., BAPTIST, E. & PJLAND, J. (1967). Puromycin and cycloheximide inhibition of
thymidine incorporation into DNA of cleaving sea urchin eggs. Expl Cell Res. 48, 431-9.
BYERS, T. J., PLATT, D. B. & Goldstein, L. (1963). The cytonucleoproteins of amebae.
I. Some chemical properties and intracellular distribution. /. Cell Biol. 19, 453-66.
DETLAFF, T. A., NIKITINA, L. A. & STROEVA, O. G. (1964). The role of the germinal vesicle
in oocyte maturation in anurans as revealed by the removal and transplantation of nuclei.
/. Embryol. exp Morph. 12, 851-73.
ECKER, R. E. & SMITH, L. D. (1966). The kinetics of protein synthesis in early amphibian
development. Biochim. Biophys. Acta 129, 186-92.
GOLDSTEIN, L. (1964). RNA and protein in nucleocytoplasmic interactions. In Cell Growth
and Cell Division (R. J. C. Harris, ed.), Vol. 2, pp. 129-149. New York and London:
Academic Press.
GRAHAM, C. F., ARMS, K. & GURDON, J. B. (1966). The induction of DNA synthesis by frog
egg cytoplasm. Develop. Biol. 14, 439-60.
GURDON, J. B. (1967). On the origin and persistence of a cytoplasmic state inducing nuclear
DNA synthesis in frogs' eggs. Proc. natn. Acad. Sci. U.S.A. 58, 545-52.
ALLEN,
374
K. ARMS
LEGROS, F.
& BRACHET, J. (1964). Effets de la puromycine sur la mitose, la synthese des proteines et celle du DNA au cours de la segmentation de l'oeuf de Batracien. /. Embryol.
exp. Morph. 13, 195-206.
NEMETH, A. M. & DE LA HABA, G. (1962). The effect of puromycin on the development and
adaptive formation of tryptophan pyrrolase. /. biol. Chem. 237, 1190-3.
NIEUWKOOP, P. D. & FABER, J. (1956). Normal Table o/Xenopus laevis (Daudin). Amsterdam: North Holland Publishing Company.
SMITH, L. D., ECKER, R. E. & SUBTELNY, S. (1966). The initiation of protein synthesis in eggs
of Rana pipiens. Proc. natln. Acad. Sci. U.S.A. 56, 1724-8.
ZETTERBERG, A. (1966). Protein migration between cytoplasm and cell nucleus during interphase in mouse fibroblasts in vitro. Expl Cell Res. 43, 526-36.
(Manuscript received 16 April 1968)