ABSTRACT
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 tri-tiated 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).
Embryonic stages are described by the numerical system of Nieuwkoop & Faber (1956).
DL-Leucine-4,5-T (351 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.
About 30 mμl of a suspension of isolated young frog liver nuclei in Tris-HCI (pH 7·8) containing tritiated thymidine (3H-TdR) at a concentration of about 1 mc/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 μ and examined autoradiographically. Many of the injected nuclei are found to be labelled after this treatment (Plate 1b;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 la shows nuclei fixed 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 auto- radiography 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.
Incorporation of3H-TdR by nuclei isolated from the liver of young frogs after their injection into 2-to 12-cell cleaving eggs

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 mμ1 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 2 a). 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 of Rana. 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 mμ1 of puromycin (1 mg/ml) were injected into embryos 30, 90 and 120 min before the label (about 50 μ1) was injected at the same time as a further 50 mμ1. of puromycin. This would give a concentration inside the embryo of about 50 mμM/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 Plewodeles embryos, where it was found that protein synthesis was not substantially reduced until 3 h after puro-mycin injection. However, the concentration of puromycin used by these authors was very low (about 40 μμM/ml inside the embryo) compared with that used here. The fact that puromycin suppresses 3H-Ieucine 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/d 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 autoradi-ography. 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 follow-ing 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 pro-tein synthesis in the system was prevented by puromycin.
About 100 mμ1 of a suspension of nuclei in Tris buffer, containing puromycin at a concentration of 2 mg/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 mμM/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 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 em-bryonic protein synthesis occurs, however, no labelling is detected in the system. This is demonstrated by the following experiment.
Fifty mμ1 of puromycin solution (1 mg/ml) were injected into embryos at the 2-to 4-cell stage. Thirty minutes later 50 mμ1 of the 3H-Ieucine solution were injected and 30, 60 and 90 min later about 50 mμ1 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 in-corporation 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 μc/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 puro-mycin 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.
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 syn-thesis 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 Arhacia 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
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.
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.
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.
RÉSUMÉ
Cytonucléoprotéines dans des œufs de Xenopus laevis en segmentation
L’examen autoradiographique d’œufs de Xenopus en segmentation injectés de leucine tritiée 60 min avant la fixation, montre une radioactivité élevée. Ce marquage n’est pas éliminé par l’injection du même précurseur froid, 60 min après administration du radioisotope. Ce fait, ainsi que la suppression du marquage par la puromycine, fait supposer que la radioactivité détectée correspond à l’incorporation de l’acide aminé tritié dans des protéines. Après injection d’une suspension de noyaux de foie de grenouille, dans des œufs ainsi marqués, on observe, 60 min plus tard, le marquage des noyaux introduits. Ce marquage de noyaux ‘etrangers’ dans le cytoplasme d’œufs radioactifs, s’effectue même lorsque la synthèse protéique est inhibée par la puromycine à un point tel qu’elle n’est pas décelable par autoradiographie. Ce transfert n’est donc pas le résultat d’une synthèse protéique dans les noyaux introduits. On peut dès lors conclure que les noyaux sont rendus radioactifs par le transfert de ‘cytonucléoprotéines’ ayant pour origine le cytoplasme embryonnaire.
On observe une induction de la synthèse du DNA dans des noyaux injectés dans des œufs en segmentation comparable à celle observée chez des noyaux transplantés dans des œufs non fécondés. L’influence éventuelle d’un transfert de cytonucléoprotéines sur l’induction de la synthèse du DNA est discutée.
Acknowledgements
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
PLATE I
Incorporation of 3H-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 6-10μ.
(b) Autoradiograph of young frog liver nuclei injected with 3H-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 Plate26 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.
(a) Autoradiograph of part of a stage 5 embryo, 60 min after it had been injected with 3H-lcucine (see text). Labelling appears to be heaviest around the periphery of the cells and in the nucleus (indicated by the arrow).
(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.