J. Cell Sci. s, 333-349 (1969)
Printed in Great Britain
333
MOVEMENT OF CYTOPLASMIC PROTEINS
INTO NUCLEI INDUCED TO ENLARGE AND
INITIATE DNA OR RNA SYNTHESIS
R. W. MERRIAM
Department of Biological Sciences, State University of Netv York,
Stony Brook, New York, U.S.A.
SUMMARY
In other studies it has been shown that somatic cell nuclei, which normally do not divide,
are induced to enlarge and synthesize DNA when introduced into the cytoplasm of egg cells of
Xenopus laevis. Introduction of such nuclei into the cytoplasm of large oocytes, however,
causes nuclei to enlarge in a different way and to synthesize RNA but not DNA.
In this study the proteins of eggs and oocytes were labelled with radioactive amino acids.
Brain or blastula nuclei were then injected into cells containing labelled proteins under conditions in which protein synthesis was inhibited. Movement of cytoplasmic proteins was studied
by observing the increase in acid-insoluble label over the injected nuclei by quantitative autoradiography.
To prevent nuclear protein synthesis during the experiments, puromycin was injected with
the nuclei. An estimation of the size of the labelled amino acid pool, a demonstration of the
inhibitory effects of puromycin, and comparison of the amount of labelled material with and
without puromycin all showed that protein synthesis in the nuclei played an insignificant role
during the course of the experiments.
A movement of acid-insoluble label from cytoplasm of egg cells into injected brain nuclei
was noted even before they had begun to swell or synthesize DNA. In the initial period of
nuclear enlargement there was a disappearance of the heterochromatic clumps characteristic
of brain nuclei. This period coincided with a very rapid uptake of label to concentrations about
twice that of the surrounding cytoplasm. A subsequent phase of nuclear swelling was characterized by a dilution of stainable nuclear material, loss of basophilia, and establishment of
acidophilia of the nuclear contents. Cytoplasmic proteins continued to enter nuclei during this
phase, but at a slower rate. Extraction of the soluble materials of labelled eggs with 001 M
NaCl at pH y 8 and a subsequent fractionation of the extract showed that there were many
radioactive compounds present with molecular weights greater than 5000.
The synthesis of DNA was initiated even before nuclear swelling could be detected, proceeding at least through the early stages of swelling.
The induction of enlargement in blastula nuclei by oocyte cytoplasm containing labelled
proteins was also accompanied by an uptake of label from the cytoplasm. In this case, although
the uptake of unlabelled acidophilic material was much greater, the uptake of labelled proteins
was much slower than that observed in the egg.
The results are discussed in terms of chromosomal changes which occur during nuclear
enlargement and during concomitant changes in nucleic acid metabolism.
INTRODUCTION
It has now been demonstrated with several cell types that a somatic cell nucleus
which normally does not synthesize DNA can be experimentally induced to do so.
For example, human lymphocytes are activated by phytohaemagglutinin to enlarge,
334
R- W. Merriam
synthesize DNA and undergo cell divisions (for a review see Robbins, 1964); avian
erythrocyte nuclei become metabolically activated upon introduction into HeLa cell
cytoplasm, initiating both RNA and DNA syntheses (Harris, 1965); spermatozoa and
somatic cell nuclei from adult Xenopus laevis tissues are induced to synthesize DNA
when introduced into the cytoplasm of eggs of the same species (Graham, Arms &
Gurdon, 1966); there is evidence that mouse liver and hen erythrocyte nuclei, in vitro,
can be stimulated to increase their incorporation of thymidine when treated with cytoplasmic extracts of dividing cells (Thompson & McCarthy, 1968). In all of these cases
except that of human lymphocytes the induction was shown to result from contact
of the nucleus with the inducing cytoplasm.
In the case of the Xenopus egg cytoplasm, the factors inducing DNA synthesis have
been shown to operate on nuclei from a variety of cell types as well as on nuclei from
mouse liver (Graham et al. 1966). Gurdon (1967) has been able to show in addition
that these cytoplasmic factors become capable of acting on injected nuclei only after
maturation of the oocyte when the contents of the germinal vesicle become mixed
with the cytoplasm; they remain active for at least 1 h after egg activation.
It must be emphasized that this effect of the Xenopus egg cytoplasm on injected
nuclei is similar to its effect on the sperm and egg nuclei during the course of normal
fertilization. Shortly after sperm entry both egg and sperm nuclei swell in the egg
cytoplasm and commence replication of their DNA (Graham, 1966). Injected nuclei
also swell and synthesize DNA at the same time. The ability of injected nuclei to
mimic these normal events thus provides a convenient experimental system for investigating the nature and mode of action of the cytoplasmic factors which cause them.
A provocative experiment by Arms (1968) has provided evidence that proteins
synthesized during early embryogenesis can enter nuclei injected into the early
cleavage-stage embryos. Some of the nuclei which protein entered had also enlarged
and synthesized DNA. In this communication we report on studies to find out if
proteins, accumulated in the egg cytoplasm during gonadotropic hormone-stimulated
maturation, enter injected nuclei. We have examined in detail the relationship between
the entry of cytoplasmic protein into nuclei and the experimentally induced nuclear
swelling and DNA or RNA synthesis.
MATERIALS AND METHODS
Preparation of 'pre-labellea" eggs and oocytes
Proteins of eggs and oocytes of Xenopiu laevit were labelled with radioactive amino acids
during the maturational changes induced by gonadotropic hormones. A mixture of tritiumlabelled amino acids was injected into the peritoneal cavity in a saline solution. At the same
time the initial dose of gonadotropin ('Pregnyl' of the Organon Laboratories Ltd.) was given
to induce maturation and ovulation. Thus, whatever label was incorporated into protein of the
eggs was synthesized during the 12—16 h between the time of hormone administration and the
time of egg shedding. Freshly extruded eggs were either extracted, after being de-jellied with
cysteine-activated papain (Spiegel, 1951), or were injected with nuclei.
The amino acids used consisted of a mixture, labelled with tritium, from the Radiochemical
Centre, Amersham, England. A total of 480/iCi of the following composition was injected:
80 fid of L-leucine (1 Ci/mM); 100 /tCi DL-lysine (8-2 Ci/mM); 100 fid of L-alanine (169 mCi/
HIM); 100 /tCi of L-histidine (500 mCi/mM); ioo /iCi of L-aspartic acid (217 mCi/mM).
Movement of cytoplasmic proteins into nuclei
335
Labelled oocytes were obtained by removal of the ovary from the same female which had
produced labelled eggs, 4 days after ovulation. Groups of large oocytes were dissected free of
the ovary for injection of nuclei. Such eggs and oocytes, labelled before the experimental
injection of nuclei, are termed 'pre-labelled'.
Additional experiments were done to determine the pattern of protein synthesis in nuclei
during their induced enlargement and DNA synthesis. In this case labelling was accomplished
by mixing the nuclear suspension with an equal volume of a solution containing DL-[4,5- 3 H]leucine (35-i Ci/mM). About 0-025 /*Ci were injected into each unlabelled egg, giving an internal
concentration of about 44 /tCi per ml of total cell water. This compares with about 0-021 /tCi
per egg (37 /iCi per ml of total cell water) taken up by ovarian oocytes to produce the prelabelled eggs. The latter estimate comes from a calculation which assumes that half the total
injected isotope went uniformly into all cells of the ovary and is undoubtedly an upper limit.
Thus, there was more labelled amino acid in cells which became labelled during changes in
injected nuclei than there was originally introduced into pre-labelled eggs.
Preparation of nuclei
Nuclei from two types of cells were injected into eggs or oocytes: nuclei from adult Xenopus
laevis brain and nuclei from X. laevis blastulae. Brain nuclei were isolated in 0-25 M sucrose
plus 3 mM MgClj after gentle homogenization in the cold and centrifugation at low speeds.
This was the 'crude pellet' of Graham et al. (1966). Blastula nuclei were prepared by sucking
cells from the animal hemisphere of a blastula into the injection pipette. When extruded into
the cytoplasm of oocytes, cell membranes rupture so that nuclei, surrounded with cytoplasm
but no cell membrane, were placed into the oocyte cytoplasm.
Injection of nuclei and puromycin
Injections of nuclei were performed as previously described (Graham et al. 1966), about
50 nl containing roughly 50—200 nuclei being introduced into each egg or oocyte. Nuclei in
10-15 cells were examined after each period of incubation.
In experiments where protein synthesis during the enlargement of injected nuclei was not
wanted, puromycin (Nutritional Biochemicals Corp., Cleveland, Ohio) was injected with the
nuclei. Nuclei to be injected were pre-incubated in the antibiotic for 20-50 min and at the time
of injection more puromycin was injected in solution. The total intracellular concentration
introduced by this method was about 70 ng per egg or 012 mg per ml of total cell water.
Microscopic and autoradiographic analysis
Eggs or oocytes containing injected nuclei were incubated at room temperature in saline
solution, fixed in Perenyi's fluid, embedded in paraffin, serially sectioned at 7 /tm, and stained
with haemalum and light green. Autoradiographs were prepared by dipping the slides in diluted
Ilford K 2 emulsion and exposing for 10-56 days, depending on the experiment.
By means of a camera lucida, the outlines of nuclei were drawn on paper of constant weight
per unit area. The number of grains over each nucleus and the number of grains in an equal
area of adjacent cytoplasm, as seen in the camera lucida projection, were counted. Finally, the
same area of background was counted. Most nuclei were found within a single section, while
much enlarged nuclei could be sliced in sectioning, appearing in adjacent sections. The largest
area of a sectioned nucleus was selected for measurement, slices in adjacent sections being
ignored.
The area of each measured nucleus was estimated by cutting out the outline drawn on paper
and weighing the cut-out. The resulting weights were converted to /tm1 by a conversion factor
obtained by the use of a stage micrometer.
The amount of labelled protein was expressed as grains per unit area, a measure of labelled
protein concentration. Cytoplasmic counts were subtracted from the adjacent nuclear counts
to reduce the variability of over-all labelling from cell to cell. Thus, labelled protein concentration means labelled protein more ( + ) or less ( —) than that of adjacent cytoplasm.
336
R. W. Merriam
RESULTS
Estimation of protein synthesis by injected nuclei during swelling in the cytoplasm of eggs
The primary objective of the present study was to determine whether egg proteins
enter nuclei injected into Xenopus egg cytoplasm. The method chosen was to label
cytoplasmic proteins of the egg before injection of nuclei and then to record whether
acid-insoluble label appeared in the nuclei during the course of their induced transformations. Since movement of acid-insoluble label was to be observed, incorporation
during the course of the transformation was inhibited with puromycin.
ifx
•«
200
600
1000
1400
1800
2200
Nuclear area (//mJ)
Fig. 1. The relationship between the size (area) of brain nuclei in egg cytoplasm and
the difference in concentration of labelled protein in them and in the cytoplasm. Each
point is the mean of 10 nuclei. Label injected ivith nuclei.
In this section preliminary model experiments are described in which protein
synthesis was not inhibited during the induced nuclear changes and in which a large
pool of labelled amino acids was available. Under such conditions the amount of label
appearing in the nuclei constitutes an upper limit for synthesis by the nuclei themselves—if one assumes that no transfer from the cytoplasm occurred. The effect of
puromycin on amino-acid incorporation was then measured and the relative size of
the radioactive free amino-acid pool determined. From these data it is possible to
assign an upper limit to the labelling of injected nuclei by nuclear incorporation under
the conditions of the principal experiment where the radioactive pool was smaller
and puromycin was used. Such labelling is found to be negligible.
Brain nuclei and 0-025 /*Ci of DL-[4,5-3H]leucine were injected into unlabelled eggs
at the same time. After either 10 or 90 min of incubation, groups of eggs were fixed
and sectioned for autoradiography. The number of grains per nucleus and the nuclear
area were determined for 100 injected nuclei in various stages of enlargement.
The concentration of grains over injected nuclei, minus that over the same area of
cytoplasm, is plotted as a function of nuclear area in Fig. i. It is seen that the nuclear
concentration of labelled protein rises rapidly in the early stages of swelling, reaching
a maximum at a nuclear area of about 175-300 /j.m2. The highest concentration is of
the order of 200-300 grains per unit area greater than the cytoplasm.
In the principal experiments using pre-labelled eggs, protein synthesis during
nuclear enlargement was undesirable. Since puromycin was found to have little or no
effect on nuclear enlargement or initiation of DNA synthesis (Arms, 1968), an experiment was done to determine how much effect puromycin had on protein synthesis in the
Movement of cytoplasmic proteins into nuclei
337
injected egg cell. Isolated brain nuclei were pre-incubated for 40 min in cold homogenizing medium containing puromycin. They were injected into eggs along with
L-^CJleucine and enough additional puromycin to bring the total to 12 ng per egg.
Controls received saline instead of puromycin. After o or 55 min of incubation, the
eggs were homogenized and extracted in one of two ways. Either they were extracted
and washed with 5 % trichloroacetic acid or else processed by the method of Mayne,
Barry & Riviera (1966). In both cases the acid-insoluble residue was collected on
Millipore filters for scintillation counting. The net incorporation was taken as the
counts after 55 min minus the counts at zero min. Results, expressed as disintegrations per min (dpm) per egg, are given in Table 1.
Table 1. The effect of puromycin* on h-[uC]leucine incorporation in Xenopus
eggs containing brain nuclei
dpm/egg
Extraction procedure
Trichloroacetic
acid
Method of Mayne
et al. (1966)
Puromycin
Saline
control
Inhibition
by puromycin
(%)
65
1894
97
12
950
98
• 12 ng per egg.
It is apparent that during the first 55 min after injection, puromycin at 12 ng per
egg inhibits the system by more than 95 %. The amount of puromycin injected into
the pre-labelled eggs of the principal experiments was about 70 ng per egg, an amount
more than 5 times greater. Clearly, not much synthesis could occur in eggs under
these conditions.
In the cells of the experiment shown in Fig. 1 labelling occurred in the presence of
about 44/tCi of radioactivity (specific activity 35-1 Ci/miw) per ml of total cell water.
Determinations of total radioactivity remaining in the free amino-acid pool of prelabelled eggs at the time of nuclear injection revealed that no more than 13 /<.Ci per ml
total cell water (sp.act. 2 Ci/mM) could have been present. Thus on the grounds of pool
activity alone, any nuclear incorporation within pre-labelled eggs should be less than
one-third of that of the cells represented in Fig. 1.
In Fig. 1 the maximum nuclear label concentration due to nuclear amino-acid
incorporation was of the order of 300 grains per unit area. In the pre-labelled eggs of
the principal experiments with a specific activity of the free amino-acid pool less than
one-third that in the eggs represented in Fig. 1 and in the presence of 95 % inhibition
by puromycin, the maximum was about 600 grains per unit area. Assuming the
maximum nuclear incorporation under these same conditions one could expect less
than 5 grains per unit area. This amount would be negligible in the plot. We conclude
therefore, that the incorporation of amino acids by injected nuclei in pre-labelled eggs
containing puromycin is not detectable with the methods used and that the increase
22
Cell Sci. 5
338
R. W. Merriam
in nuclear labelling must be due to transfer of cytoplasmic proteins. This conclusion
was borne out by a comparison of nuclear labelling with and without the presence of
puromycin. There was no difference, indicating also that injected nuclei do not
incorporate amino acids in detectable amounts during their transformation.
The contribution of non-incorporative adsorption of radioactive amino acids to the
labelling of injected nuclei
It was possible to make a rough estimate of the amount of labelled material in fixed
nuclei which was not actually incorporated into acid-insoluble form. The concentration of label was compared in injected nuclei of the same size after 10 and 90 min in
the egg cytoplasm labelled with pHJleucine. Assuming that true incorporation occurs
linearly with time between 10 and 90 min after injection of the label, one can extrapolate the resulting line to zero time. The concentration of grains at zero time was
considered a rough estimate of label which was made acid-insoluble by simple complexing with nuclear solids.
In two such determinations the zero time values were 64 and 78 grains per unit
area under the conditions of the experiment shown in Fig. 1. This concentration
amounts to less than one grain for a small nucleus of 100/tm2 and about 6 grains for
a large nucleus of 1000 /tm2. These values are only about 5 % of the values observed
in Fig. 1 and lead to the conclusion that non-incorporative complexing of label to acidinsoluble material plays only a negligible role in these experiments.
Movement of acid-insoluble label from pre-labelled egg cytoplasm into unlabelled brain
nuclei
Do cytoplasmic proteins of the egg cytoplasm go into injected nuclei which are
induced to swell and begin DNA synthesis? To approach this question unlabelled
brain nuclei were injected into the cytoplasm of pre-labelled eggs with or without
puromycin at about 65 ng per cell. Groups of the injected eggs were fixed immediately
or after 30, 50 or 80 min of incubation. The range of sizes of unswollen nuclei, fixed
immediately after injection and showing a mode of about 100 /im2, is presented as
a frequency distribution in Fig. 2. This size heterogeneity is a reflexion of the fact that
-whole brain contains many different cell types.
Autoradiograms showed that label did in fact appear over injected nuclei, so a
relationship was sought between the size of a swelling nucleus and the number of
grains over it. Areas and grains were measured for nuclei after all times of incubation.
The nuclei were then arranged in order of increasing area, the mean nuclear area and
•grains per unit area being determined for each successive group of ten. A plot of grains
per nuclear area and per identical cytoplasmic area is presented in Fig. 3 A as a function
•of nuclear size (area).
From these plots two things can be seen. There are always more grains over injected
nuclei than over an identical area of adjacent cytoplasm. Although cytoplasmic concentration remains about the same, grains over nuclei increase in concentration rapidly
from about 70 to 120 per unit area in the early stages of enlargement, while nuclei
swell from about 100 to 300 /im2 in cross-sectional area.
Movement of cytoplasmic proteins into nuclei
339
These changes in the nuclear concentration when compared with the cytoplasmic
concentration are emphasized by the plot in Fig. 3B, where the difference between
nuclear and cytoplasmic concentration is plotted as a function of nuclear area. In this
plot there appears to be a break in the kinetics of uptake of label into nuclei, the fast
uptake of the early stages of enlargement being quite distinct from the subsequent
20
60
95
135 170 210
Nuclear area classes (/im2)
Fig. 2. Frequency distribution histogram of nuclear sizes (areas) of brain nuclei in egg
cytoplasm fixed before swelling or DNA synthesis could occur.
130 r-
90
° 50
x
re
I; 70
50
30
10
100
300
500
700
900
Nuclear area (//m2)
1100
1300
Fig. 3. A, The relationship between the size (area) of brain nuclei in pre-labelled egg
cytoplasm and the concentration (grains per unit area) of labelled protein in them
(•—#) and in adjacent egg cytoplasm ( x — x ). Puromycin was present in the injected
eggs. Each point is the mean of 10 nuclei, B, The same data as in A but plotted as the
difference in concentration of labelled protein between injected brain cell nuclei and
adjacent egg cytoplasm. The dotted line connects points in the size range of unswollen
nuclei.
R. W. Merriam
34°
slower uptake. The interpretive lines drawn through the points emphasize this change
in the kinetics. When these data are plotted as the ratio of nuclear/cytoplasmic concentration of grains, the rapid increase is again noted in the size range of nuclei from
about ioo to 300 /im2. Thereafter the ratio remains essentially constant until a marked
increase is associated with the largest nuclei of the last point.
This last point representing the very largest nuclei is significantly different from
the adjacent size class. Moreover, its remarkably high label concentration is an underestimate since the concentration and number of grains were too high to allow accurate
counting of all grains. Although these large nuclei were rare and represent only one
point on the plots of Fig. 3, it is possible that they do not actually fall into the graphed
relationship. Terminal stages of swelling could cause an increased accumulation of
labelled material, or these nuclei could represent the host egg pronuclei.
8 1-
•10
30
50
70
Min after injection
90
Fig. 4. Changes in the concentration of labelled proteins of cytoplasmic origin in unswollen brain nuclei (40—140 /an9 in area) as a function of time in pre-labelled egg
cytoplasm. The number of nuclei per point was 17-25, each point representing the
mean ± standard error of the mean.
One obvious question arising from these data is whether labelled protein enters
injected nuclei simply as a passive consequence of the enlargement or whether the
entering protein is involved in causing the observed changes. A partial answer can be
obtained from the fact that some injected nuclei do not enlarge and synthesize DNA
even after 80 min in the egg cytoplasm. If cytoplasmic proteins enter nuclei only as a
consequence of nuclear enlargement, then these unswollen nuclei should show little
or no uptake of label. If, on the other hand, cytoplasmic proteins are involved in producing the changes, labelled proteins might be expected to enter nuclei even before
they begin to swell or synthesize DNA.
Small unswollen nuclei of the size range 40-140 /im2 were selected from the measured
nuclei and the mean concentration of grains per unit area plotted as a function of time
spent in the pre-labelled egg cytoplasm. In Fig. 4 these data show that protein does
enter unswollen nuclei. This finding rules out the possibility that protein uptake is
simply a consequence of nuclear swelling and, although not proving the point, leaves
open the possibility that entering proteins are involved as inducing agents. The fact
that some nuclei do not enlarge even when cytoplasmic factors enter them could mean
Movement of cytoplasmic proteins into nuclei
341
that they have not yet responded but will do so, or that they are incapable of responding
for either biological or experimental reasons.
The swelling of injected brain nuclei, and their uptake of egg proteins, is accompanied by morphological changes in their chromatin. As seen in Figs. 8 and 9,
unswollen nuclei contain prominent heterochromatic clumps which are strongly
basophilic. During the early stages of swelling, when cytoplasmic protein is entering
rapidly, the heterochromatic clumps disappear (Figs. 10—12). In these early stages a
basophilic region in the nuclear periphery persists while the centre becomes progressively empty of stainable material. During further enlargement (Figs. 13, 14),
when cytoplasmic proteins are entering at a slower rate, the last dense basophilic
regions disappear and the diluted nuclear material becomes definitely acidophilic.
Sometimes these nuclear substances are concentrated in one area with wispy strands
radiating out. The autoradiographic grains are concentrated over the condensed area
and over the wispy material of the rest of the nucleus (Fig. 13). One gains the impression that the dispersed basophilic material acquires acidophilic properties by the
adsorption of labelled proteins.
The initiation of DNA synthesis in relation to induced nuclear swelling
In order to determine the point in the events described above at which the synthesis
of DNA begins, brain nuclei were mixed with tritiated thymidine and injected into
unlabelled eggs. Autoradiographic emulsions were exposed for a relatively short time
and the number of grains and nuclear area were measured for all nuclei with few
enough grains to count.
I 90
70
50
£ 30
10
50
100
150
Nuclear area
200
250
Fig. 5. The relationship between nuclear size (area) and the number of associated
grains due to incorporated ['HJthymidine. Each point represents the mean and
standard error of the mean for 5 nuclei. All values corrected for background.
Graham et al. (1966) had previously shown a relationship between nuclear size and
DNA synthesis. In this experiment also a rough relationship was noted between
grains per nucleus and nuclear size as demonstrated in Fig. 5. Here we see that nuclei
showing only a few grains, and hence just starting DNA synthesis, have the typical
R. W. Merriam
342
appearance of unswollen nuclei (Figs. 15, 16). Using the nuclear area intercept as a
rough indication of the size at which synthesis begins, it is seen that the average
nucleus begins thymidine incorporation at an area of about 100 /im2. This value is
close to the mode of the size distribution of unswollen nuclei seen in Fig. 2. We conclude that DNA synthesis starts very early in the swelling process, even before enlargement can be detected. The synthesis continues at least during the early stages of
enlargement when labelled proteins from the cytoplasm are entering most rapidly.
Observation on the nature of the labelled substances in pre-labelled eggs
Acid-insoluble substances containing radioactivity, after administration of labelled
amino acids, have been referred to in this report as labelled protein. A partial justification for this comes from a preliminary fractionation of labelled material obtained from
pre-labelled eggs.
20
1-6
1-2
<u
u
c
n
o
< 08
0-4
250
290
330
Wavelength (nm)
Fig. 6. Absorbance spectrum of soluble compounds of molecular weight greater than
5000 from pre-labelled Xenopus eggs.
Eggs were homogenized in the cold in 10 mM tris-HCl, pH 7-8, containing 2 mM
ethylenediaminetetra-acetic acid (EDTA), 10 mM NaCl, and 2 mM sodium azide.
Coarse particulates were removed by centrifugation at 27000g for 30 min and the
supernatant centrifuged at 135000^ for 3 h at o °C. When this supernatant, containing substances likely to be in a soluble state in the living cell, was put through a
Sephadex G-25 column, over 95 % of the radioactivity was found in two distinct
peaks. One small peak was retarded, emerging at the location of free amino acids. The
major peak was of excluded substances with molecular weights greater than 5000.
This result indicated that there were no soluble oligopeptides or other small sub-
Movement of cytoplasmic proteins into nuclei
343
stances below a molecular weight of about 5000 which bore the label, other than free
amino acids. The ultraviolet absorption of the excluded peak is shown in Fig. 6.
The curve shows a predominantly protein profile, with relatively small amounts of
nucleic acid present.
A preliminary fractionation of the excluded material was initiated on DEAE
cellulose. The solutes were removed from a 0-9x21 cm column with a convex
gradient from o to 0-4 M NaCl at pH 7-8. Many peaks of radioactivity were observed,
indicating that there are probably many soluble substances bearing the label whose
molecular weight is greater than 5000. The run-off peak showed much radioactivity
and contained enough solute to allow a determination of its ultraviolet absorption
spectrum. The curve had a maximum at 278 nm with a typical protein profile.
Movement of acid-insoluble label from pre-labelled oocyte cytoplasm into unlabelled
blastula nuclei
Gurdon (1968) has found that isolated blastula nuclei behave differently when
injected into oocytes than when injected into eggs. After 80 min in the oocyte cytoplasm, the incubation period used with eggs, the injected nuclei have undergone no
0
3
-20
Q.
o
-60 _
Blastula / .
nuclei
•S
-100
Z
-140 _
/Il i
-
A
'
Germinal vesicle
-180 V
o
4
8
1 1 1 I
12 16 20 24
Hours
Fig. 7. The concentration of nuclear label in blastula cell nuclei and in the germinal
vesicle of the host oocyte, relative to that of the cytoplasm, as a function of time.
Protein synthesis was inhibited with puromycin. Each point is the mean ± standard
error of the mean for 14-25 nuclei. The mean nuclear areas were 330, 860, and
2600 /tms for the 5-, 18-, and 24-h points, respectively.
morphological change (Fig. 17). In the course of 24 h and longer, however, they enlarge
many-fold and increase their rate of RNA synthesis. Morphologically the enlargement
produces a nucleus more like that of the germinal vesicle of the host cell (Fig. 18)
than that of enlarged somatic cell nuclei in egg cytoplasm. Thus, while the egg cytoplasm causes brain nuclei to cease RNA synthesis and commence DNA synthesis, the
oocyte cytoplasm induces rapidly dividing blastula cells to stop DNA and begin RNA
synthesis. An experiment was done to see if labelled proteins of oocyte cytoplasm enter
injected blastula nuclei undergoing this rather different type of transformation.
Oocytes were taken from the ovary of a female 4 days after she had ovulated pre-
344
R- W. Merriam
labelled eggs. Counts of autoradiographic grains in the cytoplasm of such oocytes
showed that they contained about 46% as much acid-insoluble labelled material as
the same area of pre-labelled eggs from the same female.
Unlabelled nuclei from the animal hemispheres of Xenopus blastulae were injected,
along with enough puromycin to bring the intracellular level up to the concentration
of the previous experiment. Groups of such oocytes were fixed after 5,18 and 24 h and
autoradiograms prepared as before. Nuclear areas were measured and the grains over
them counted. Grain counts were also made over equal areas of adjacent cytoplasm,
the germinal vesicle of the host cell, and background. The data were expressed as
grains per unit area in both injected and host nuclei as a function of time.
The results, in Fig. 7, show a slow entry of label into enlarging blastula nuclei,
compared with label in the cytoplasm. There is a roughly comparable entry into the
germinal vesicle of the host cell. This rate of entry is very much slower than that
observed for brain nuclei in eggs. Brain nuclei have a concentration of label slightly
exceeding that of the egg cytoplasm after only 5 min. In the oocyte even much enlarged blastula nuclei do not have a label concentration equal to that of the surrounding cytoplasm after 24 h.
DISCUSSION
The movement of cytoplasmic proteins into interphase nuclei has been reported for
several other cell types. For example diffusible, acid-insoluble substances which are
labelled with radioactive amino acids have been detected (first by Goldstein, 1958)
moving between nuclei and cytoplasm in non-dividing amoebae. Robbins & Borun
(1967) have done pulse-chase labelling experiments with HeLa cells which indicate
that histones are synthesized in the cytoplasm, subsequently moving into the nucleus
during interphase. Similarly in mouse fibroblast cells, a transfer of dry mass from
cytoplasm to nuclei during interphase could be inferred from combined microinterferometric and autoradiographic studies (Zetterberg, 1966). Thus, although the
functional significance of these protein movements is not yet known, it seems likely
that protein movements through the nuclear envelope, as described in this communication, may represent a rather general phenomenon in eukaryotic cells.
Labelled protein can be detected in brain nuclei at about the same concentration
as in the cytoplasm 10 min after injection. This occurs in nuclei which will ultimately
swell and synthesize DNA but does so well before these events begin. Thus, protein
entry is not simply a consequence of nuclear swelling. This does not prove that cytoplasmic proteins are the causative agents for nuclear transformation but certainly
leaves open the possibility.
Graham et al. (1966) have previously demonstrated a relationship between the
degree of nuclear swelling and the degree of thymidine incorporation. This has been
confirmed in the present study. It is also true that incorporation always occurs in
nuclei which are swelling. From this it seems that swelling is a necessary condition
for DNA synthesis. Yet it is doubtful whether swelling by itself is the immediate
cause of DNA synthesis because, in other systems in which it is induced, swelling
occurs well before the initiation of synthesis. Such a temporal dissociation of nuclear
Movement of cytoplasmic proteins into nuclei
345
swelling and DNA synthesis has been noted in heterokaryons (Bolund, Ringertz &
Harris, 1969), in plant cells undergoing tumorous transformation (Rasch, Swift &
Klein, 1959), and in phytohaemagglutinin-stimulated lymphocytes (Robbins, 1964).
The swelling observed in Xenopus egg cytoplasm probably corresponds to the nuclear
swelling which in other systems precedes the initiation of induced DNA replication.
Not sufficient in itself, it would result in replication when the necessary intermediates
are present and when DNA polymerase is made available. In Xenopus eggs all factors
seem to be present simultaneously.
The movement of cytoplasmic proteins into nuclei to reach higher concentrations
than that of the cytoplasm probably reflects some sort of binding to non-diffusible
nuclear structures such as chromosomes. The distribution of label most likely reflects
an equilibrium of labelled material between a bound, non-diffusible form and a freely
diffusible form in the nuclear and cytoplasmic compartments. Thus, the nuclearcytoplasmic distribution of label at the moment of fixation probably reflects the ratio
of bound label in each compartment plus a common distribution of diffusible label in
both compartments.
In a model based on this concept, the relationship between the concentration of
nuclear label and nuclear size would assume structural meaning. The increase in
nuclear label, relative to cytoplasmic label, becomes a measure of the increase in
label-binding sites in the nucleus. The earliest stages of nuclear swelling, when heterochromatic clumps disappear, would consist of a loss of the coarsest level of chromosome
structure, the conversion of heterochromatin to a euchromatin-like state. This process
would be associated with such a rapid appearance of binding sites that label is taken
up faster than nuclear volume increases. A second level of chromosomal 'unwinding'
would proceed simultaneously with the loss of the heterochromatic state, or even
precede it in euchromatic regions. This second process would produce binding sites
for labelled protein at about the same rate as the increase in nuclear volume. When
the conversion of heterochromatin was complete, this process would still be continuing and could well correspond to the actual conversion of chromosomal DNA to
a replicable condition.
On this model, the two-phase kinetics of protein uptake into swelling nuclei would
reflect changes in chromosomal organization. If correct, the initial phase of rapid
protein uptake could be associated with the removal of lysine-rich histones (see, for
example, Littau, Burdick, Allfrey & Mirsky, 1965; Whitfield & Perris, 1968), perhaps
by their being complexed to anionic molecules (Whitfield & Perris, 1968) diffusing
in from the cytoplasm.
Labelled cytoplasmic proteins enter blastula nuclei injected into oocytes only very
slowly, but do get in. Two facts seem pertinent in this case. Nuclear swelling of this
type produces a much greater increase in acidophilic nuclear material than is seen in
the swelling of brain nuclei in eggs. This is just the opposite of what would be
expected if the comparatively elaborate isolation procedure used in preparing brain
nuclei were responsible for the differences in uptake patterns. From the scarcity of
grains over the enlarged blastula nuclei and from the fact that puromycin was present,
nuclear protein synthesis could not have accounted for this increase in acidophilic
346
R. W. Merriam
substances. Therefore, in this type of nuclear transformation there is a massive movement of acidophilic material from the oocyte cytoplasm into the enlarging blastula
nuclei. Being largely unlabelled, it is likely that this material was present in oocytes
before the labelling process and presumably would be present in eggs as well. Nuclear
transformation in oocytes thus seems to involve the transfer of much acidophilic but
unlabelled cytoplasmic material. Conversely, in eggs the enlarged nuclei contained
much less acidophilic but more labelled material which had been synthesized during
gonadotropic stimulation. It would appear that different types of cytoplasmic substances are associated with the different types of nuclear transformations.
This work was generously supported by a Fellowship from the Public Health Service,
National Institutes of Health, U.S.A., and by the State University of New York at Stony
Brook, New York, U.S.A.
The author gratefully acknowledges the many kindnesses and critical discussions afforded
him by Dr John B. Gurdon and his students during the course of this study. The data on the
effects of puromycin were obtained by Dr Gurdon.
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(Received 30 September 1968—Revised 21 February 1969)
Movement of cytoplasmic proteins into nuclei
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14
For legend see next page.
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R. W. Merriam
Figs. 8, 9. Brain nuclei in egg cytoplasm. Fixed 5 min after injection before swelling
or DNA synthesis had begun. Note prominent heterochromatic clumps. Nuclei are
basophilic. x 2200.
Figs. 10—12. Brain nuclei in egg cytoplasm fixed during early stages of enlargement.
Heterochromatic clumps have largely gone. Basophilic material is mainly at the nuclear
periphery, with central regions showing loss of basophilia. x 2200.
Fig. 13. Brain nucleus after much enlargement. Basophilia has been replaced by wispy
acidophilic material showing grains in the overlying autoradiogram. x 1300.
Fig. 14. Brain nucleus very much enlarged. It is acidophilic and shows autoradiographic grains in and out of focus, x 2200.
Fig. 15. Brain nuclei in egg cytoplasm showing small size and heterochromatic
clumps, x 2200.
Fig. 16. Same nuclei as in Fig. 15 but at a higher focal level to show autoradiographic
grains due to incorporated ['HJthymidine. x 2200.
Fig. 17. Blastula nuclei in oocyte cytoplasm surrounded by blastula cell cytoplasm.
Fixed before enlargement had begun. Basophilic. x 1600.
Fig. 18. A single blastula nucleus in oocyte cytoplasm after much enlargement. The
granular material in the nucleus is acidophilic. x 1600.
Movement of cytoplasmic proteins into nuclei
.• a
f L *
349