/. Embryo/, exp. Morph. Vol. 26, 3, pp. 611-622, 1971
Printed in Great Britain
611
Synthesis of nuclear and cytoplasmic proteins in the
early development offishesand echinoderms
By MAYA R. KRIGSGABER, 1 ALLA A. KOSTOMAROVA, 2
TAMARA A.TEREKHOVA 3 AND TATIANA A. BURAKOVA 4
From the Institute of Developmental Biology, Academy of Sciences of the
U.S.S.R., Moscow
SUMMARY
Synthesis of nuclear and cytoplasmic proteins was studied biochemically and autoradiographically in early loach (Misgurnus fossilis) and sea-urchin (Strongylocentrotus nudus)
embryos.
After incubation with [14C]amino acids for 5-120 min the ratio of the specific activities of
nuclear, mitochondrial and 12000g supernatant proteins was shown to be equal approximately to 6:1:2 in loach embryos and to 8:4:3 in sea-urchin embryos independently of the
duration of labelling.
After incubation with [3H]amino acids the number of silver grains per unit section was on
the average 2-4 times higher for nuclei than it was for cytoplasm at mid-blastula and midgastrula stages. At the mid-gastrula the vegeto-animal gradient of protein synthesis was found.
A higher level of the synthesis of nuclear proteins as compared with that of cytoplasmic
proteins appears to be related to an increase in the nuclear volume and the nucleocytoplasmic ratio during the early development of the loach and sea-urchin embryos.
INTRODUCTION
Study of protein synthesis in the development of animal embryos is important
for elucidation of the mechanisms of differentiation, since protein synthesis is
considered as an indispensable step in the expression of genetic information. A
great many investigations are devoted to the analysis of the intensity of total
protein synthesis in sea-urchin (Gross, Malkin & Mayer, 1964; Berg, 1965), frog
(Smith, Ecker & Subtelny, 1966), starfish, mollusc (Monroy &Tolis, 1964) and
fish(Krigsgaber &Neyfakh, 1968) embryos at different stages of development. At
the same time, the study of protein synthesis in individual subcellular fractions
allows us to characterize this process more precisely. However, data concerning
the comparative intensity of nuclear and cytoplasmic protein synthesis have
been obtained mostly in sea-urchin embryos (Kedes, Gross, Gognetti & Hunter,
3>a 4
' Authors' address: Institute of Developmental Biology, Academy Sciences U.S.S.R.,
Vavilov Street 26, Moscow, 117-133, U.S.S.R.
3
Author's address: Institute of Cytology, Academy Sciences U.S.S.R., Maklin Avenue, 32,
Leningrad, 190-121, U.S.S.R.
39
E M B 26
612
M. R. KRIGSGABER AND OTHERS
1969). Little is known so far about nuclear and cytoplasmic protein synthesis in
the other classes of animals.
The present article describes the intensity of the synthesis of nuclear and
cytoplasmic proteins in loach (Misgurnus fossihs) embryos at blastula and gastrula stages and in sea-urchin {Strongylocentrotus nudus) embryos at cleavage
and blastula stages.
The preliminary data were presented as demonstrations at the IX International
Embryological Conference (Krigsgaber & Terekhova, 1969).
MATERIALS AND METHODS
The eggs were obtained from the loach (Misgurnus fossilis) females 40 h after
injection of gonadotropic hormone choriogonin (G. Richter, Budapest,
Hungary) according to Neyfakh (1959). Developmental stages of the loach
were expressed in hours of development after fertilization at the temperature
21-5 °C (Neyfakh, 1959).
Biochemistry. To obtain a large amount of material at the same stage blastoderms were isolated from the yolk by means of centrifugation of shell-free eggs
in double-layered sucrose density gradient (Kostomarova, 1969). Several
thousand isolated blastoderms were incubated in the mixture of [14C]amino
acids [14C]lysine, specific activity 39 mCi/g (5/*Ci/ml); [14C]valine, sp.act. 44
mCi/g (3 /*Ci/ml); [14C]leucine, sp.act. 57 mCi/g (2 /^Ci/ml) to give a final
concentration of 10/tCi/ml per sample.
Incubation of isolates was carried out in double-strength Holtfreter solution
(isotonic for loach eggs) with Tris-buffer, pH7-8 containing lOOi.u./ml of
penicillin and 50i.u./ml of streptomycin (medium I) at 21-5C 0 . Time of
incubation is given in Table 1.
To obtain the isolated nuclei blastoderms were stirred at 0 °C in 0-25 M
sucrose, containing 0-05 M Tris-buffer, pH 7-4; 0-01 M-MgCl2 and 00025 M-KCI
(medium II) with addition of 0-1 % solution of Tween 80. Cytoplasm was
isolated by centrifugation at 3000g for lOmin. Nuclei distributed over the
surface of the precipitate as a thin hght layer were carefully collected. In order
to obtain mass nuclear material the precipitate was homogenized (Teflon-glass)
in a small volume of the medium II (without Tween) and centrifuged at lOOOg
for 10 min. This procedure was repeated several times.
To obtain mitochondria blastoderms were homogenized in the medium II
(Teflon-glass) and twice centrifuged at 3000g and 14000g for 10-15 min.
Isolated nuclei and mitochondria were twice washed with the medium II.
Sea-urchin (Strongylocentrotus nudus) embryos (1-5 millions eggs per sample)
were incubated in the mixture of [14C]amino acids (leucine, sp.act. 477 mCi/g
(2 /*Ci); lysine, sp.act. 38 mCi/g (1 -5/*Ci); alanine, sp.act. 31 mCi/g (0-5 /id)) to
give a final concentration of 4 /*Ci/ml. Incubation of eggs with precursors was
carried out in sea water, filtered through RUF-2 filters, containing penicillin
Nuclear and cytoplasmic proteins in early development
613
(100 i.u./ml) and streptomycin (50 i.u./ml) under gentle manual swirling at
21-23 °C. After incubation eggs were washed with filtered sea water (at 0 °C)
containing an excess of unlabelled amino acids. Homogenization was carried
out in the medium II in ice (Teflon-glass). Intact cells were isolated at 300 rpm;
nuclei were precipitated at 1000-3000 rpm and mitochondria were isolated from
the supernatant at 12 000 rpm.
Subcellular fractions were fixed with 5 % TCA and washed from acid-soluble
material, nucleic acids and lipids. The residue was dissolved in formic acid, its
radioactivity counted in the Geiger counter. Concentration of protein was
determined according to Lowry (Lowry, Rosebrough, Farr & Randall, 1951);
specific radioactivity was expressed as counts per minute per mg of protein.
Intensity of the amino acid incorporation into protein was calculated with
correction for absorption. To determine the absorption embryos were incubated
with labelled amino acids in ice. Time of incubation was equal to that at
optimal temperatures. After incubation cell constituents were isolated by means
of the technique described and the specific activity of protein was counted in
each fraction.
Autoradiography. Loach blastoderms were isolated surgically from the yolk
at mid-blastula (6 h of development) and mid-gastrula (14 h of development)
stages. Isolated blastoderms (about 30 isolates per sample) were incubated for
120 min in double-strength Holtfreter's solution with [3H]alanine (sp.act. 80
mCi/mM (11 /tCi/ml)) and [3H]leucine (sp.act. 70 mCi/mM (11 /*Ci/ml)) to give a
final concentration of 22 /tCi/ml. The total volume of each sample was 0-5 ml.
After incubation blastoderms were fixed with Carnoy fluid and embedded in
paraffin wax by the routine method. All blastoderms were stained totally with
carmine and the sections 5-7 /<m thick were stained with Carrachi haematoxylin
(after development). Serial longitudinal sections were covered with liquid
radiosensitive emulsion (type ' M ' , Nil ChimFoto, Moscow) and incubated for
21 days at 4 °C. Amidol was used as developer.
The number of silver grains was counted over the nuclear and cytoplasmic
area equal to 25 jtim2, in basal, median and outer parts of the embryo. The
number of silver grains was counted in 30 areas in four embryos, i.e. in 120
areas for each observation point in each part of the embryo.
Mean value and standard error were calculated for each observation point.
RESULTS
(1) Biochemical analysis. The data obtained on loach embryos are given in
Table 1. Table 1 shows that the specific activity of the total nuclear proteins is
severalfold that of the cytoplasmic proteins. At the same time, the specific
activity of the mitochondrial protein is less than that of the 12000 g supernatant
protein. In Exp. 6 (Table 1, Fig. 1) incubation of blastoderms with labelled
amino acids lasted 5, 15 and 120 min. It can be seen that the ratio of the
39-2
614
M. R. KRIGSGABER AND OTHERS
p
M
Nuclei
12000g supernatant
Mitochondria
o
x
&0
1" 2
u
15
120
Time (min)
Fig. 1. Amino acid incorporation into nuclear, mitochondrial and 12000g
supernatant proteins in loach embryos. Blastoderms isolated at the mid-gastrula
stage (16 h of development) were incubated with [14C]amino acids for 5,15,120 min.
After labelling subcellular fractions were isolated and the specific radioactivity of
proteins was determined in each fraction.
Table 1. Distribution of protein radioactivity between subcellular fractions
in loach blastoderm (cis/min/nig protein)
Fractions
Time of
Stage of
No. of ex- development labelling
periment
(min)
GO
1
2
3
4
5
6
6
6
9
11
12-13
14
15-16
16
16
16
60
60
120
120
120
5
15
120
A
t
Nuclei
16000
2170
10000
2680
18000
1400
6000
50000
12000 £
Cytoplasm Mitochondria supernatant
3300
—
3000
—
8000
540
2000
15000
620
—
630
—
200
1300
—
1230
—
730
—
530
2500
12000
Nuclear and cytoplasmic proteins in early development
615
incorporation of the amino acids into the nuclear, mitochondrial and 12000 g
supernatant protein is equal, approximately, to 6:1:2 respectively and remains
constant independently of the duration of labelling.
The results of the same experiments carried out on sea-urchin embryos are
summarized in Table 2. In this case the specific activity of the total nuclear
proteins is also severalfold that of the total protein of the 12000g supernatant.
However, in contrast to the above model (loach embryos), the incorporation of
amino acids into mitochondrial proteins is greater as compared with that of the
12000g supernatant proteins. The ratio of the specific activities of the nuclear,
mitochondrial and 12 000 g supernatant protein is equal approximately to
8:4:3, respectively. As can be seen from Table 2, the specific activity of the
12000g supernatant proteins is close to that of the total proteins of the intact
embryo, probably due to the fact that the total amount of the 12000g supernatant proteins accounts for 90 % of the total proteins of the embryo.
Table 2. Distribution of protein radioactivity between subce/lular fractions
in sea-urchin embryos (ctslmin/mg protein)
Fractions
i
Stage of
No. of ex- developperiment ment (h)
1
2
2
Time of
labelling
(min)
Time of
chasing
(min)
15
15
15
5
15
75
5
—
—
150
—
—
—
—
—
—
—
—
3
4
5
2-5
3-5
3-5
3-5
3-5
6
7
5
7
15
5
5
6
7
7
8
30
60
30
3
Intact
embryos
400
600
770
—
820
—
—
500
—
—
—
Nuclei
1100
1680
1960
725
2800
5200
515
1200
2120
4200
10500
Mitochondria
12000g
supernatant
—
750
960
500
2100
2900
240
360
470
640
270
600
650
140
720
425
1480
2240
3920
790
1890
2900
In Exp. 3 (Table 2) eggs were incubated with labelled amino acids for 5 and
15 min and in Exp. 5 for 5, 15, 30 and 60 min (Table 2; Fig. Id). In both
experiments the specific activities of the mitochondrial and 12000 g supernatant
proteins were about 50 and 25 % respectively of that of the nuclear proteins,
independently of the duration of labelling.
In order to study possible redistribution of newly synthesized proteins
between cell constituents, labelled amino acids were washed out after 15 min
incubation and replaced by an excess of unlabelled amino acids (Table 2,
Exp. 2; Fig. 2b). The assay of the radioactivity after 2-5 h chase showed that the
specific activity of each fraction increased approximately by 20 %, probably at
the extent of the label which had not been completely washed from the cells.
616
M. R. KRIGSGABER AND OTHERS
o
X
4-*
U
30
Time (min)
[JJ Nuclei
\^/\ Mitochondria
^
12000g supernatant
o
m
4
u
if1
I
15
Chase 150
Time (min)
Nuclear and cytoplasmic proteins in early development
617
However, the ratio of the specific activities of proteins of the subcellular fractions did not change.
The data obtained allow the conclusion that in loach and sea-urchin embryos
amino acid incorporation into nuclear proteins is more intensive than into
cytoplasmic proteins. Furthermore, in sea-urchin embryos amino acid incorporation into mitochondrial proteins is greater than into other cytoplasmic
proteins. On the contrary, in loach embryos the amino acid incorporation into
mitochondrial proteins is less than into the other cytoplasmic proteins.
(2) Autoradiography. Grain counts in the nuclei and cytoplasm of the loach
embryos at mid-blastula and mid-gastrula stages are given in Table 3. At the
mid-blastula stage the number of silver grains per unit section is 2-3-2-7 times
higher for nuclei than it is for cytoplasm (see also Fig. 3B). It can be seen that
the grain counts both in nuclei and cytoplasm are virtually the same in all parts
of the embryo. Table 3 and Fig. 3 A show that there are no significant differences
between the amount of the label in the basal, median and outer parts of the
embryo.
As can be seen from Table 3, at the mid-gastrula stage the grain counts per
unit section increased by 1 -8 times in nuclei and by 2-5 times in cytoplasm of the
cells of the basal part of the embryo as compared with those at the mid-blastula
stage. In the median part the grain number increased by 1 -6 times in nuclei and
by 1 -7 times in cytoplasm, respectively. In the outer part no reliable increase of
the grain number is found. At the mid-gastrula stage the label is distributed
unevenly throughout the embryo: the greatest amount of the label is localized
in the basal part. In the median part the grain counts are less than in the basal
part and in the outer part the amount of label is the least. Criterion of significance^) of different mean values of grain counts is equal to 7-1 and6-5(P < 0-01)
for nuclei of the basal-median and median-outer parts, respectively. For the
mean values of the grain counts in cytoplasm the criterion of significance (t) is
equal to 8-5 and 4-3, respectively. The number of observations for each mean is
120 (see also Table 3).
Uneven distribution of the label throughout the embryo at the mid-gastrula
stage is demonstrated in Fig. 3C. At the mid-gastrula stage the grain number
per unit section is 1-4-2-4 times higher for nuclei of basal, median and outer
parts than it is for cytoplasm of these parts of the embryo (Table 3).
Fig. 2. Amino acid incorporation into proteins of subcellular fractions in sea-urchin
embryos, (a) Embryos were incubated with [14C]amino acids for 5, 15, 30, 60 min at
the mid-blastula stage (7 h of development). After labelling the specific radioactivity
of proteins of subcellular fractions was determined, (b) Embryos were incubated
with [14C]amino acids for 15 min at the morula stage (3-5 h of development). After
labelling the specific radioactivity of proteins of subcellular fractions was determined
in one portion of eggs (1). In another portion of eggs (II) the label was washed and
the eggs were incubated with an excess of labelled amino acids for 2-5 h. After 2-5 h
chasing the specific radioactivity of proteins of subcellular fractions was determined.
618
M. R. KRIGSGABER AND OTHERS
>3
0-2 mm
0-2 mm
Fig. 3. [3H]amino acid incorporation into the nuclei and cytoplasm of the loach
blastoderms isolated from the yolk at early blastula (A, B) and mid-gastrula (C)
stages. Blastoderms were incubated with [3H]alanine and [3H]leucine for 120 min.
The label is higher in nuclei than it is in cytoplasm. The label is evenly distributed
throughout the blastoderm (A). Regional differences between grain counts in the
basal, median and outer parts of the embryo are present (C). 1, Basal; 2, median;
3, outer parts of the embryo.
Nuclear and cytoplasmic proteins in early development
619
Thus, it follows from the above data that, at mid-blastula and mid-gastrula
stages, the label is incorporated more intensively into proteins of the nuclei
than of the cytoplasm. Moreover, at the mid-gastrula stage the label is found to
be incorporated mainly into the nuclear and cytoplasmic proteins of the basal
part of the embryo. In the median part the intensity of labelling is less than in
the basal part. The cells of the outer part reveal the lowest intensity of labelling.
Table 3. Intensity of[sH]leucine and [3H]alanine incorporation into the
nuclear and cytoplasmic proteins in loach embryos
No. of silver grains per unit section (25 /tm2)
Stage of development
Early blastula
(6 h of development)
Mid-gastrula
(14 h of development)
Part of the embryo
Nuclei (n = 120)
Cytoplasm (//= 120)
Basal
Median
Outer
Basal
Median
Outer
41 ±1
39 ± 3
43 ± 1
73 ±1
63 ±1
43 ± 2
15 ±1
15 ± 1
19 ±1
38 ±1
26 ±1
20 ± 1
DISCUSSION
The data obtained here have shown that the early developmental stages of
loach and sea-urchin embryos are characterized by greater amino acid incorporation into nuclear proteins than into cytoplasmic proteins. What possible
explanation can be given for this phenomenon ? It is well known that during
early development the total volume and mass of the embryo remain virtually
constant. As can be seen from Table 4, the volume and mass of the loach
blastoderm increases by 1-3 times during the period from mid-blastula to midgastrula. During the same period the number of cells and nuclei increases,
approximately by 14 times (N. N. Rott & G. N. Sheveleva, unpublished). At the
same time, the volume of a single nucleus decreases more slowly than cell volume
and this results in an increase of the nucleo-cytoplasmic ratio by 4 times while the
total nuclear volume increases by 3 times (Pankova, 1963). Thus, the rate of
increase of the total nuclear mass is twice that of the cytoplasmic mass. Accordingly, one can expect the rate of synthesis of nuclear proteins to be higher than
that of cytoplasmic proteins. Hence, if the turnover rate of nuclear and cytoplasmic proteins is identical, the ratio of their specific activities must reflect
the rates of their synthesis.
In fact, our experiments have demonstrated that in loach embryos the incorporation of amino acids into nuclear proteins is 2-4 times more intensive than
into cytoplasmic proteins. These values are in good agreement with the expected
ones.
Autoradiography has revealed regional differences in protein synthesizing
activity in loach embryos at the mid-gastrula stage and a gradient pattern of
620
M. R. KRIGSGABER AND OTHERS
protein synthesis. Occurrence of the gradient of protein synthesizing activity
appears to be related to the primary differentiation of the embryo in the course
of gastrulation.
During the period from fertilization to hatching the number of cells in seaurchin embryos reaches 1000, while the amount of protein and volume of the
embryo remain constant (Harvey, 1956). These data suggest that while the
number of nuclei increases during the period of early sea-urchin development,
the amount of cytoplasmic proteins remains constant. Thus, the rate of nuclear
protein synthesis must exceed the rate of cytoplasmic protein synthesis.
High rates of nuclear protein synthesis were demonstrated in Lytechinus
pictus (Tyler, 1967) and Arbacia punctulata (Kedes et al. 1969) embryos. The
rate of synthesis of nuclear proteins was found to be sixfold that of cytoplasmic
proteins, in particular, histones were shown to be synthesized with the highest
rate (Kedes et al. 1969). It follows from Tyler's data (1967) that in Lytechinus
pictus zygotes the rate of nuclear protein synthesis is approximately 12 times as
great as that of cytoplasmic protein synthesis. (At the zygote stage the nuclear
volume accounts for 1/250 of the total cell volume (Harvey, 1956).)
Table 4. Amount of nuclei and nucleo-cytoplasmic ratio in early
loach embryos
Amount of protein
in the blastoderm (y)
Amount of nuclei
in the embryo
Volume of a single
nucleus (/*m3)
Total nuclear volume
of the embryo (/tm3)
Nucleo-cytoplasmic
ratio
Mid-blastula
Mid-gastrula
15
20
Pattern of
change
Increases by
1 -3 times
l-7xlO 3
24 x 103
Increases by
14 times
1 x 103
0-2 xlO 3
l-7x!0 6
4-8 xlO 6
Decreases by \
5 times
Increases by
2-8 times
Increases by
4 times
'
2-7 xlO- 2
13 xlO" 2
Reference
Abramova &
Vasilieva
(unpubl.)
Rott &
Sheveleva
(unpubl.)
Pankova
(1963)
More intensive amino acid incorporation into mitochondrial proteins than
into other proteins of the cytoplasm had earlier been observed in sea-urchin
embryos by several authors (Giudice & Monroy, 1958; Nakano & Monroy,
1958; Tyler, 1967). In contrast to the above model, in loach embryos amino acid
incorporation into mitochondrial proteins is less than into both nuclear and
12000g supernatant proteins; this may be due to the fact that the number of
mitochondria does not change during the early development of the loach
(Abramova, Likhtman & Neyfakh, 1966). At the same time, the total volume and
mass of the cytoplasm increase.
Nuclear and cytoplasmic proteins in early development
621
The second problem to be discussed concerns the question of the localization
of the synthesis of nuclear and mitochondrial proteins in early sea-urchin and
loach embryos.
It is shown in the present paper that the specific activity of proteins increases
independently in each subcellular fraction and is proportional to the time of
labelling. After minimal exposure to labelled amino acids (5 min) the ratio
of radioactive proteins of nuclei, mitochondria and 12000g supernatant is the
same as after more prolonged pulses. The same observation was made by Tyler
(1967) on Lytechinus pictus embryos after brief pulse exposure (2-5-15 min) to
different labelled amino acids and amino acid mixtures.
In vitro experiments carried out on trout embryos revealed that in this species
the synthesis of nuclear proteins occurred in an intracellular compartment either
closely associated with or inside the nucleus (Trevithick, 1969). In vitro experiments on [32S]methionine incorporation showed that a certain proportion of
mitochondrial proteins of the Paracentrotus lividus embryos was synthesized in
mitochondria (Giudice, 1960). Thus the above data, as well as our experiments
on substitution of labelled amino acids by unlabelled ones, permit the suggestion
that protein synthesis might occur in each subcellular fraction autonomously.
However, our results can also be interpreted as an expression of redistribution
of labelled proteins between subcellular fractions since the time of labelling was
several minutes. Probably nuclear and mitochondrial proteins synthesized in
the cytoplasm have enough time to migrate from the cytoplasm to nuclei and
mitochondria during the period of experimentation. In fact, after a short pulse
new proteins of Arbacia punctulata embryos were confined to the cytoplasm
and were found neither in the nuclei nor in mitochondria. It was shown that
considerable fractions of nuclear proteins - e.g. histones - were synthesized on
polysomes of the cytoplasm while, after longer pulses or chasing, radioactive
proteins accumulated in nuclei (Kedes et al. 1969). Probably the migration of
nuclear proteins from the cytoplasm to the nuclei contributes to the high rate
of their synthesis.
The authors wish to thank Professor A. A. Neyfakh for his attention and interest in the
course of this work. The authors wish to express their gratitude to Professor G. V. Lopashov
for his helpful suggestions and criticism in the course of preparation of the manuscript for
publication.
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