/. Embryol. exp. Morph. Vol. 28, 3, pp. 491-509, 1972
Printed in Great Britain
491
Investigation of the mode of
nuclear control over protein synthesis in early
development of loach and sea urchin
By MAYA R. KRIGSGABER 1 AND A. A. NEYFAKH 1
From the Institute of Developmental Biology,
Academy of Sciences of USSR, Moscow
SUMMARY
It is shown that in loach embryos the incorporation of precursors into protein takes place
in the blastoderm cells only. The change of the rate of incorporation of labelled amino acids
into protein of the blastoderm separated from the yolk at successive developmental stages
reflects the changes in the level of protein synthesis in intact embryos of the same developmental stages. Typical periodic changes of the intensity of protein synthesis in early embryogenesis of the loach are detected: low incorporation of amino acids at blastula stages is
followed by an increase of synthesis during gastrulation and by a decrease with the onset of
organogenesis.
To study the genetic control over protein synthesis at various developmental stages of
loach and sea-urchin embryos the effects of ionizing radiation and long-term treatment with
actinomycin D have been examined. X-Irradiation doses produce an insignificant direct effect
on protein synthesis, while radiation damage of the nuclear apparatus results in a gradual
but ever increasing inhibition of protein synthesis. The inhibition of RNA synthesis with
actinomycin or ionizing radiation damage of the nuclei produce essentially the same effect on
the intensity of protein synthesis. Protein synthesis in androgenetic haploid hybrid embryos
(loach ?x goldfish cJ) and in loach androgenetic haploid embryos after producing a partial
elimination of chromosomes does not differ from 'enucleated' loach embryos completely
deprived of chromosomes. These data suggest that high-polymeric RNA formed after the
elimination of some chromsomes is unable to provide a normal level of protein synthesis.
Protein synthesis is not controlled by the nuclei up to the stages of early blastula (sea urchin)
and of late blastula (loach), being evidently programmed in oogenesis. To provide a rapid
activation of protein synthesis in the course of gastrulation in the loach the function of the
nuclei has to be realized during mid-blastula stages. An increase of the rate of the incorporation of amino acids at the stages of mesenchyme blastula in the sea urchin depends on the
synthesis of RNA at the early blastula. At the same time protein synthesis during gastrulation
in the loach and at mesenchyme blastula in the sea urchin is much less dependent on the
simultaneous RNA synthesis. Protein synthesis at these stages seems to be provided by the
long-living templates and controlled by non-gene mechanisms of the regulation of translation.
Thus early embryonic differentiation in the loach and sea-urchin development is related to
the activation of protein synthesis. The latter is provided by the preceding morphogenetic
nuclear function, which makes protein synthesis relatively independent of simultaneous
synthesis of templates that ensures subsequent development stages.
1
Authors' address: Institute of Developmental Biology, Ac. Sci. USSR, Vavilov Street 26,
Moscow 117334, USSR.
492
M. R. KRIGSGABER AND A. A. NEYFAKH
INTRODUCTION
A complicated control of protein synthesis is known to occur in early development. It proceeds on RNA templates stored in oogenesis, and on new long- and
short-living ones synthesized in the nuclei of the embryo. The intensity of protein
synthesis and possibly the selection of templates are controlled by translation
mechanisms whose nature is mostly unknown. Recently a particular emphasis
has been placed upon the special phenomenon of the sudden intensification of
protein synthesis after fertilization of sea-urchin eggs. Little is known so far
about protein synthesis in other species of animals or about protein synthesis at
advanced stages of sea-urchin development.
Arrest of RNA synthesis after actinomycin D treatment (Gross & Cousineau,
1963) provides a method which allows the assessment of the contribution of
direct gene control over protein synthesis. The present work was aimed at studying the contribution of the genetic apparatus by its inactivation at successive
developmental stages by means of both actinomycin treatment and large doses of
ionizing radiation. The latter has already been used to evaluate the participation
of the nuclei in early embryogenesis of various species (Neyfakh, 1959, 1964).
Besides sea-urchin eggs loach embryos were used - a convenient material which
has long been used in this laboratory. Some additional data on the relationship
between RNA and protein synthesis were obtained on lethal fish hybrids.
MATERIAL AND METHODS
The experiments were carried out on loach, Misgurnusfossilis, and sea-urchin,
Strongylocentrotus nudus, embryos. The eggs were obtained from loach females
40 h after the injection of gonadotrophic hormone, choriogonin. Developmental
stages of the loach were expressed in hours of development after fertilization at
21 °C according to Neyfakh (1959). The hybrid embryos were obtained by fertilizing loach eggs with goldfish sperm. Radiation inactivation of loach gametes and
embryos at different developmental stages was performed on X-ray machine
RUP-1 (180 kV, 15 mA, without filter).
To study the changes of protein synthesis in intact loach embryos and hybrid
embryos, loach? x goldfish^, portions of eggs were incubated in closed vessels
with [14C]carbonate to give a final concentration of 50/^Ci/ml in Flickinger
solution (Flickinger, 1954). The isolation of blastoderms of the loach and hybrid
of loach? x goldfish $ embryos according to Kostomarova (Kostomarova, 1969)
was performed immediately before the incubation with labelled amino acids,
which was carried out in double-strength Holtfreter's solution. The latter was
prepared with 0-05 M Tris-HCl buffer, pH 7-8, containing 100 i.u./ml of penicillin and 50 i.u./ml of streptomycin. The mixture of [14C]amino acids in a final
concentration of 2 /^Ci/ml per sample was used as precursor.
Artificially fertilized sea-urchin eggs were incubated at a concentration of
Nuclear control of protein synthesis
493
Table 1. Incorporation of [1<lC]carbonate into loach eggs protein
(cpmjmg protein)
Development time
(h)
Blastoderm
Intact eggs
Yolk
8
11
14
17
20
1720
2250
2950
6250
5250
131
178
270
504
483
20
22
15
30
46
5000-10000 eggs per ml under gentle manual swirling at 21-23 °G. The samples
were X-irradiated on machine RUM-3 (15 mA, 180 kV). Actinomycin D was
used at a concentration of 25 /*g/ml, puromycin at 30 /*g/ml. To determine the
intensity of protein synthesis the samples of 10000 eggs were taken and incubated with [14C]amino acids at a concentration of 0-5 /tCi/ml. Incubation was
carried out in sea water filtered through RUF-2 niters containing penicillin
(100 i.u./ml) and streptomycin (50 i.u./ml).
After incubation the eggs were fixed with 5 % TCA (trichloroacetic acid)
containing an excess of cold amino acids, washed from acid-soluble fraction,
nucleic acids and lipids by routine procedures. The residue was dissolved in
formic acid, protein concentration being determined according to Lowry
(Lowry, Rosenbrough, Farr & Randall, 1951), its radioactivity being counted in
a Geiger counter. The specific radioactivity was expressed as impulses per minute
per mg of protein.
The data on the incorporation of labelled amino acids into protein were
calculated from protein activity in incubated samples minus the absorption
value. To determine the absorption embryos were incubated with labelled amino
acids in ice for the appropriate time and treated as all the other samples.
RESULTS
Study of protein synthesis in the loach embryos
Impermeability of loach eggs to water-dissolved substances has complicated
the task. First, at all developmental stages except the earliest ones the nuclear
function was inactivated with high doses of ionizing radiation rather than with
specific inhibitors. This required a number of procedures to show that the effect
of ionizing radiation on protein synthesis was like that of actinomycin. Secondly,
to study protein synthesis in intact eggs, instead of amino acids, an unspecific
labelled precursor [14C]carbonate was used.
Loach eggs at blastula and gastrula stages were incubated in Flickinger acid
medium with a solution of [14C]carbonate. Then a portion of intact eggs was
fixed, another portion of shell-free eggs was separated in sucrose gradient into
494
M. R. KRIGSGABER AND A. A. NEYFAKH
I
II I I I
540
6000 - - 2560
405
4500 -- 1920
-= 270
3000 -• 12S0
135
1500 -r 640
11
14
17
~
20
Hours
14
14
Fig. 1. [ C]carbonate and [ C]amino acids as precursors. At given developmental
stages the eggs were incubated for 2 h with [14C]carbonate solution (50 /tCi/ml). One
portion wasfixedimmediately after the incubation and the specific activity of protein
was determined (curve 1, scale I). Blastoderms were isolated from the second portion
of eggs after their incubation with [14C]carbonate, and the specific activity of protein
was determined (curve 2, scale II). At the same developmental stages blastoderms were
isolated and incubated with mixture of [14C]amino acids, after that the specific
activity of protein in them was determined (curve 3, scale III).
blastoderm and yolk. Table 1 shows the results of such an experiment. After
fixation with TCA, protein was isolated and its specific activity determined. The
specific activity of protein of the blastoderm was found to be 10 times higher
than that of intact egg, and the incorporation into the protein of the yolk found
not to exceed the level in an unfertilized egg. Evidently protein synthesis proceeds only in the blastoderm and provides for its growth in the course of development. Low specific activity of the total protein of the whole egg seems to result
from at least a 10-fold dilution of blastoderm protein with unlabelled yolk
protein (for the yolk mass is about 10 times that of the blastoderm mass).
When isolating blastoderms from yolk the surface bordering the yolk becomes
naked. This surface being suitably permeable, the blastoderms were used for the
incubation with labelled amino acids. Fig. 1 compares the intensity of protein
Nuclear control of protein synthesis
14
495
synthesis in intact embryos incubated with [ C]carbonate, in blastoderms isolated from these embryos and in blastoderms incubated with [14C]amino acids.
The scales are so chosen that the values of the intensity of protein synthesis at
the developmental stage of 14 h coincide on Fig. 1 for all three experimental
series. The slopes of these curves also turned out to be close to each other.
The intensity of protein synthesis was also studied under the same incubation
conditions in the eggs irradiated at the stage of late blastula. The change of the
intensity of amino acid incorporation observed after irradiation is virtually the
same in intact eggs, in embryos separated from the yolk incubated with
[14C]carbonate and in those isolated after the incubation with [14C]amino acids.
Similar changes of the intensity of protein synthesis in intact eggs (precursor
14
CO2) and in isolated blastoderms (precursor [14C]amino acids) were also found
for diploid and haploid embryos of the loach.
It seems possible to interpret the change in the rate of protein synthesis in
terms of the incorporation of labelled amino acids into protein only if the
specific activity of amino acid pool at the developmental stages studied remains
constant. The specific activity of amino acids in the cell, when amino acids are
added to the medium, depends both on the rate of their penetration into the cell,
and on its pool in the cell. When [14C]carbonate is used the problem of the change
of permeability seems to be insignificant. The specific activity of intracellular
amino acid pool in this case will depend on the rate of carboxylation (with the
participation of biotin) of keto acids on the one hand and on the rate of amination on the other. In the course of development all these parameters may change.
However, the similarity of the curves obtained applying various precursors
suggests that, first, carboxylation of keto acids with formation of amino acids
does not limit the rate of protein synthesis in loach embryos, and secondly, that
the specific activity of intracellular amino acid pool is changed insignificantly in
the course of development. It was also concluded after chromatographic analysis
and direct determination of the specific activity of intracellular amino acid pool
(Kukhanova & Neyfakh, unpublished, Neyfakh, 1971) that the latter is invariable
in the embryos isolated at various stages of early loach development. Consequently the level of the incorporation of amino acids into protein during 1 h incubation of embryos isolated from the yolk at successive developmental stages
reflects the intensity of their protein synthesis in each given moment of development. These results allowed further studies on protein synthesis in early development of the loach by the incubation of blastoderms with labelled amino acids.
The change in the intensity of protein synthesis in the course of development
is presented in Fig. 2, showing the results of six experiments. The incorporation
of amino acids into embryos' protein insignificantly increases during mid-late
blastula, but with the onset of gastrulation the rate of protein synthesis rapidly
increases. During the developmental period from mid-blastula up to the late
gastrulathe rate of protein synthesis increases 5-6 times. At subsequent developmental stages the intensity of protein synthesis decreases and reaches the
496
M. R. KRIGSGABER AND A. A. NEYFAKH
4500
3000
E. 1500
6
Blastula
9
12
Gastrulation
15
18
21
24
27
30 h
Organogenesis
Fig. 2. Intensity of protein synthesis in the development of loach embryos (the data
of six experiments). At given developmental stages blastoderms were isolated and
incubated with mixture of [14C]amino acids, and the specific activity of protein was
determined.
minimum at the stages of 3-7 somites (22-24 h after fertilization). At this stage
the intensity of the incorporation of amino acids amounts to one half of the
maximum in early development at the stage of late gastrula and is equal to the
rate of protein synthesis at early gastrula stage, 24 h after which the intensity
increases again.
Effect of irradiation on protein synthesis in loach embryos at various
developmental stages
As demonstrated above, the intensity of protein synthesis in loach embryos
changes regularly, which allowed us to consider it as one of biochemical criteria
of differentiation. To study the dependence of the intensity of protein synthesis
on nuclear control, loach eggs were irradiated at various developmental stages
at a dose of 20 kr (516 C/kg air) that completely inactivated the morphogenetic
function of the nuclei (Neyfakh, 1959).
The inactivation of nuclei with heavy doses of ionizing radiation required a
study of possible damage to the cytoplasm which could directly inhibit protein
synthesis. To study a differential damage to the nucleus and cytoplasm of an
embryo and its effect on protein synthesis the experiments on haploid and
'enucleated' embryos were carried out. Diploid embryos were taken as a control.
After fertilizing the eggs irradiated at the dose of 20 kr with normal sperm
androgenetic haploid embryos were obtained in which the father's set of chromosomes was preserved, the mother's one was inactivated and the cytoplasm was
irradiated. While fertilizing normal eggs with the sperm irradiated at a dose of
40 kr gynogenetic haploid embryos were obtained, in which the father chromo-
Nuclear control of protein synthesis
497
2500
2000
£. 1500
'_
o
to
p
(='
8* IOOO
500
6
Blastula
12
Gastrulation
15 h
Fig. 3. Protein synthesis in loach embryos of various ploidy. At 6, 9, 12, 15 h after
fertilization blastoderms were isolated from diploid gyno- and androgenetic haploid
and 'enucleated' embryos of the loach, incubated with labelled amino acids for an
hour, after that the specific activity of protein was determined. 1, Control; 2,
gynogenetic haploids; 3, androgenetic haploids; 4, 'enucleated' embryos.
somes were inactivated, and the mother ones and the cytoplasm remained
undamaged. Andro- and gynogenetic haploids developed with increasing
abnormality up to hatching and then died at early larva stages (Neyfakh &
Radziyevskaya, 1967).
After fertilizing irradiated eggs with irradiated sperm, 'enucleated' embryos
virtually deprived of chromosomes were obtained. These eggs cleaved outwardly normally, reaching the blastula stages, although the rate of cleavage was
slower than that of controls. After 13-15 h of development they perished. At
the same period the control embryos reached the stage of mid-gastrula.
The dynamics of the incorporation of amino acids into protein in the above
four series is shown on Fig. 3. The intensity of total protein synthesis in both
haploid and diploid embryos differed little from one another. It increased
drastically during gastrulation, though in androgenetic haploids the intensity of
protein synthesis was usually somewhat lower than in gynogenetic ones. Protein
synthesis in 'enucleated' embryos was much lower than in the control and
shortly before the death (12 h after fertilization) reached approximately the level of
9 h control embryos at the late blastula stage. Consequently drastic inhibition
32
E M B 28
498
M. R. KRIGSGABER AND A. A. NEYFAKH
2400 -
2100 -
1800
1500
1200
900
600
9
11
13
15
Hours
Fig. 4. Protein synthesis in loach haploids, haploid hybrids, 'enucleated' embryos,
and haploid embryos with partial elimination of chromosomes. The experiment was
carried out on blastoderms isolated 7, 9, 11, 13 and 15 h after fertilization. The
experimental conditions are the same as in Fig. 3. 1, Loach haploids; 2, haploid
hybrids; 3, 'enucleated' embryos; 4, embryos with partial elimination of chromosomes.
of protein synthesis due to the damage of nuclear structures was combined
with the relatively insignificant direct effect of ionizing radiation on protein
synthesis resulting from irradiation of cytoplasm. Thus the irradiation dose of
20 kr could be used while analysing the role of nuclei in the control of protein
synthesis.
When fertilizing loach eggs with the sperm of goldfish diploid hybrids
developing up to the larval stages were obtained, with foreign chromosomes
functioning from early developmental stages. Androgenetic haploid hybrids,
being a combination of goldfish chromosomes and loach cytoplasm, were
incapable of differentiation, and their development was arrested at the stage of
late blastula like that of 'enucleated' embryos of the loach. Thus goldfish
chromosomes function in the cytoplasm in the presence of loach chromosomes, but they do not manifest themselves morphogenetically in the loach
cytoplasm when the loach chromosomes are absent. The count of chromosomes
showed that some of them were eliminated in diploid hybrids and in androgenetic haploid ones as well (Neyfakh & Radziyevskaya, 1967).
Nuclear control of protein synthesis
499
A biochemical study of some aspects of nuclear-cytoplasmic relations in the
hybrid loach $ x goldfish $ embryos was carried out. The data on the dynamics
of protein synthesis obtained on intact embryos and isolated blastoderms
showed that the intensity of protein synthesis was at the same level in diploid
loach embryos and diploid hybrid loach? x goldfish $ embryos at the blastula
and gastrula stages.
The dynamics of protein synthesis in haploid hybrids bears much resemblance
to that of 'enucleated' embryos, i.e. no intensification of protein synthesis was
observed, and 15-16 h after fertilization the level of the synthesis was nearly the
same as in the control at the stage of late blastula (Fig. 4). On the contrary,
synthesis of high-polymeric non-ribosomal RNA was much like that in the
loach haploids and strongly differed from that in' enucleated' embryos (Neyfakh,
Timofeeva, Krigsgaber & Svetaylo, 1968). The rate of RNA synthesis in
haploid hybrids was much higher than in the' enucleated' embryos; although they
did not significantly differ as to their early development, both groups of embryos
were incapable of gastrulation and their development was arrested at the late
blastula stage.
It follows from our data that the activation of the morphogenetic function of
nuclei in diploid hybrids at blastula stages is followed by the activation of
protein synthesis during gastrulation. However, the difference in protein
synthesis between androgenetic haploid hybrid loach ? x goldfish $ and androgenetic loach embryos, as well as the similar level of protein synthesis and the
arrest of development in loach haploid hybrids and 'enucleated' embryos,
suggest that RNA molecules synthesized on goldfish chromosomes were not
involved in protein synthesis. The level of the latter was apparently provided by
the templates stored in the loach cytoplasm during oogenesis.
Another means of chromosome 'elimination' was used to find out to what
extent the arrest of development at the late blastula stage in androgenetic
haploid hybrids was due to the elimination of goldfish chromosomes and to
what degree it resulted from them being strange to the loach cytoplasm. For this
purpose the chromosomes of loach male gametes in androgenetic haploids were
partly eliminated by irradiation. Therefore loach eggs after their nuclei had been
inactivated were inseminated with loach sperm previously irradiated in the dose
of 1-75 kr. This dose was chosen because the death of the 50 % of such embryos
took place 14-14-5 h after fertilization as in haploid hybrids, i.e. it was 2 h later
than the death of the 50 % of 'enucleated' embryos. The dynamics of protein
synthesis in such embryos did not virtually differ from androgenetic haploid
hybrids loach? x goldfish $ (Fig. 4). RNA synthesis also proceeded similarly in
both these types of embryos (Neyfakh, Timofeeva, Krigsgaber & Svetaylo 1968).
These phenomena show that loach androgenetic haploid embryos with partly
eliminated chromosomes can serve not only as a morphogenetic but also as a
biochemical model of haploid hybrid embryos in which an intensive synthesis
of high-polymeric RNA proceeds. Apparently a disagreement between the
32-2
500
M. R. KRIGSGABER AND A. A. NEYFAKH
3000
2500
2000
1500
1000
500
Blastula
12
Gastrukition
15h
Fig. 5. Effect of actinomycin treatment and ionizing irradiation on protein synthesis
in loach embryos. After fertilization a portion of loach eggs served as a control (1),
the second portion was placed into actinomycin D solution (100 /«g/ml) (2), and the
third one was irradiated at a dose of 20 kr (3). Blastoderms were isolated at blastula
and gastrula stages, incubated with labelled amino acids, after which the specific
activity of protein was determined.
goldfish chromosomes and loach cytoplasm first of all resulted in elimination
of some chromosomes and in a 'distortion' of RNA synthesis without a reliable
change in its quantitative characteristics. It again brings about inhibition of
protein synthesis, arrest of development and death of embryos.
The data on the dynamics of protein synthesis in loach embryos irradiated or
treated with actinomycin D 5-15 min after fertilization show that irradiation as
well as chemical inactivation of the nuclei results in similar inhibition of protein
synthesis (Fig. 5). Ionizing radiation applied in the developmental period of
0-5-5 h (early blastula) produced the same effect as irradiation or actinomycin D
treatment of the eggs immediately after fertilization (Fig. 6 A). In all the experimental series with the irradiated eggs the development was arrested at the late
blastula stage, which was specific of the 'enucleated' embryos (Neyfakh, 1959).
Protein synthesis was equally intense in the embryos in which the nuclei were
inactivated at different moments of cleavage and early blastula. At 12-15 h
after fertilization the intensity of protein synthesis corresponded to that of the
control embryos at the late blastula stage (9 h of development). The experiments
501
Nuclear control of protein synthesis
2500
2500
Control
S. 1500
E 1500
4
500
3 2
A
500
0
3
Clcavaee
6
Blastula
12
15h
Gast filiation
5
Blastula
9
J3
Gastrulation
4
J7
20 h
Fig. 6. (A). Effect of radiation inactivation of the nuclei on protein synthesis at
early developmental stages of the loach. Portions of loach eggs were irradiated at a
dose of 20 kr for 15 min (a), 2 h (b), 4 h (c) and 5-5 h (d) after fertilization. The intensity
of labelled amino acid incorporation into protein was investigated in all the versions
for 9 h. 1, Control; 2, total curve for all the irradiation times. The arrows indicate the
moments of irradiation.
(B). Protein synthesis in loach embryos irradiated at the stages of mid-blastula.
Egg portions were irradiated at a dose of 20 kr at 5-5 h (4), 6-5 h (3), 7-5 h (2) after
fertilization. (1) Control. Beginning with the stages of late blastula the intensity of
amino acid incorporation into protein was determined in all the versions during the
incubation of blastoderms with labelled amino acids. The arrows indicate the
moments of irradiation.
of irradiation applied in early development and on 'enucleated' embryos and
androgenetic haploid hybrids loach? x goldfishc? indicated that up to the stage
of mid-blastula the genetic apparatus of the cell was not virtually involved in
protein synthesis. Hence it is possible to believe that up to the late blastula
stage protein synthesis was programmed in oogenesis and was independent of the
nuclei.
A totally different picture was observed when the inactivation of the nuclei
was performed from 5-5 to 7-5 h of development (Fig. 6B). The later radiation
was applied within this period the higher was the level of the specific activity of
protein in irradiated embryos. While the development of the embryos irradiated
5-5 h after fertilization with the dose which totally blocked the nuclear function
was arrested and they were at the late blastula stage by the level of protein
synthesis (9 h of development), the inactivation of the nuclei performed 1 h
later allowed the embryos to begin the gastrulation. Reaching the developmental
stage of 12 h, they perished 15 h after fertilization. The activity of protein synthesis in these embryos approximately corresponded to that of 12 h control embryos.
502
M. R. KRIGSGABER AND A. A. NEYFAKH
4800
3200
1600
10
14
Gastrulation
18
22
26 h
Organogenesis
Fig. 7. Protein synthesis in loach embryos irradiated at the stages of late blastula/
early gastrula. Loach eggs were irradiated at a dose of 20 kr at 9, 11 and 13 h after
fertilization. The intensity of [14C]amino acid incorporation into blastoderm protein
was studied during gastrulation and at the onset of organogenesis. 1, Control; 2,
irradiation for 9 h, 3 for 11 h, 4 for 13 h after fertilization. The arrows indicate the
moments of irradiation.
The synthesis was found to be more intensive in embryos irradiated 7-5 h after
fertilization. These embryos lived longer and developed up to the stage of 15-16 h
(mid-gastrula), the level of labelling being approximately the same as in 15-16 h
control embryos. Consequently, at the stages of mid-blastula, protein synthesis
was controlled by the nuclei and each hour of their functioning made protein
synthesis more prolonged and intense. Evidently, the realization of the nuclear
function during blastulation is required for the specific activation of protein
synthesis during gastrulation. This period, which can be characterized as an
active one with regard to the control over protein synthesis, coincided with the
first manifestation of the morphogenetic function of nuclei (Neyfakh, 1959,
1964) and with an intensification of template activity of the genome (Kafiani,
Timofeeva, Melnikova & Neyfakh, 1968; Kafiani, Timofeeva, Neyfakh, Melnikova &Rachkus, 1969). The partial elimination of chromosomes in loach androgenetic haploids also demonstrated the dependence of protein synthesis, in loach
during gastrulation, on morphogenetic function of the nuclei and on the synthesis
of RNA. The latter was capable of serving as a template for protein synthesis in
vivo. Protein synthesis in androgenetic haploid loach embryos with partially
503
Nuclear control of protein synthesis
2500
3
o
D.
'o 1500
oo
"E"
400
12
24
18
30
Hours
Fig. 8. Protein synthesis in the embryos irradiated at the stages of mid-late gastrula.
Loach eggs were irradiated at a dose of 20 kr at 12 h (4), 15 h (3), 17 h (2) after
fertilization. (1) Control. During gastrulation and organogenesis [14C]amino acid
incorporation into protein of blastoderms isolated from each experimental version
was determined.
eliminated haploid chromosomes remained at the level of' enucleated' embryos
deprived of chromosomes, despite an intensive RNA synthesis. But this RNA
seems not to realize its morphogenetic function either, for the development of
the embryos was arrested at the late blastula stage while normal haploids of
the loach pass all the stages of early development.
The study of the changes of protein synthesis of loach embryos irradiated at
the late blastula mid-gastrula stages (9,11,13 h) gave similar results (Fig. 7). The
activation of protein synthesis in such embryos proceeded for several hours and
stopped 16-18 h after fertilization. But apparently the level of protein synthesis
was enough for the completion of gastrulation.
The pattern of the curves showing the intensity of labelling after irradiation
during mid-late gastrula (12, 15, 17 h of development) differed little from the
control ones, which demonstrates the absence of a direct relationship with the
nuclear function. At the same time protein synthesis proceeds rather intensively
for a long time from the moment of the inactivation of the nuclei to the death of
the embryos (Fig. 8). The curves expressing protein synthesis of the embryos
with inactivated nuclei almost repeat the shape of the control curve. A similar
picture was obtained on embryos irradiated at the developmental stages from
8 to 17 h.
504I
M . R. KRIGSGABER AND A. A . NEYFAKH
1
A
200
Moment of
irradiation
100
-•6
-D5
3 f 5
- 2 L^Kt 1
100
•
/
—
D
A
A4
1
^-^r* -3
i
Control /
/
/
/
1
Moment of
actinomycin
addition
Control/
1
I
I
0
6
12
18
21
Cleavage Blastula
Mesenehvme Gastrula
Hatching blastula
1
27h
N^xJ*"-* 4
i
i
i
0
6
12
IS h
Cleavage Blastula
Mesenchyme (jastrula
Hatching blastula
Fig. 9. Comparison of protein synthesis intensity in the sea urchin S. nudus early
development after irradiation (A) and actinomycin (B) inactivation of the nuclei,
at different times after fertilization.
(A) 2,4, 6, 8 or 10 h after fertilization the eggs were irradiated at the dose of 15 kr.
104 eggs were incubated in 1 ml of sea water with 0-5 /id of [14C]lysine at each of
successive developmental stages. The specific activity was expressed in cpm/mg of
protein. (1), Control; (2), irradiation 2 h after fertilization; (3), 4 h; (4), 6 h; (5), 8 h;
(6), 10 h.
(B) 2, 4, 6, 8 or 10 h after fertilization the eggs were placed into actinomycin
solution (25/*g/ml) and protein synthesis intensity was determined. (1), Control;
(2), the embryos placed into actinomycin 2 h after fertilization; (3), 4 h; (4), 6 h:
(5), 8h; (6), 10 h.
It is known, however, that the so-called inactivation of the nuclear function
induced by irradiation with high doses does not completely inhibit the synthesis
of high-polymeric non-ribosome RNA (Belitsina, Gavrilova, Neyfakh & Spirin,
1963; Kafiani, Timofeeva, Neyfakh, Rachkus & Melnikova, 1966). While it is
readily apparent from the data obtained on 'enucleated' embryos virtually deprived of chromosomes that protein synthesis is independent of transcription
simultaneously proceeding at blastula stage, the independence of protein synthesis
of simultaneous RNA synthesis during early differentiation is less obvious. The
effect of irradiation and actinomycin D treatment on protein synthesis at different developmental stages was also studied on sea-urchin embryos, which are
freely permeable for the solutions of inhibitors and other substances.
Study of protein synthesis in sea-urchin embryos
The rate of protein synthesis in normal development of StrongyJocentrotus
nudus embryos increased from the beginning of development up to the stage of
mid-blastula, somewhat decreased during hatching and then increased again at
the stages of mesenchyme blastula and gastrula. These results are in good
agreement with the data on the change of protein synthesis obtained for early
Nuclear control of protein synthesis
505
sea-urchin embryos of other species (Smith, 1963; Gross, Malkin & Moyer,
1964; Berg, 1965, 1968). Fig. 9 summarizes several experiments on protein
synthesis in S. nudus. In all cases the specific activity of protein 6 h after fertilization (at the mid-blastula) at the end of the first period of the activation of protein
synthesis was taken as 100 % and the incorporation into protein at other stages
of normal development was expressed as a percentage. The data on the intensity
of protein synthesis after irradiation and actinomycin D treatment are also
presented as a percentage of the control at corresponding developmental stages.
The doses producing a complete effect were chosen. It means that the damage
did not increase with the further increase of the dose. For X-rays it was a dose of
10-15 kr; for actinomycin D, 20 /*g/ml. The data presented show that irradiation
and actinomycin treatment of the eggs produce a similar effect on development
and protein synthesis.
The difference between the effect of X-irradiation and actinomycin treatment
is that the development of irradiated embryos was arrested but that they
survived, while the actinomycin-treated embryos whose development was
arrested at the same stages perished much earlier. It has been shown elsewhere
that irradiation does not block mRNA synthesis but inhibits its increase in the
course of development (Kafiani et ah 1966). At the same time actinomycin D
almost completely inhibited mRNA synthesis. Evidently the high level of RNA
synthesis into RNA does not reflect the ability of this rapidly labelled RNA to
serve as a template for protein synthesis. A similar conclusion was drawn from
the studies on mRNA and protein synthesis in haploid androgenetic hybrids
loach $x goldfish $.
The inactivation of the nuclei in irradiated or actinomycin-treated sea-urchin
embryos in the period from fertilization to the stage of 32-64 blastomeres
produced no appreciable effect on protein synthesis up to the early blastula
stage. However, the rate of protein synthesis decreased more abruptly in
experimental embryos 5-7 h after fertilization than in the control. The development of such embryos had been arrested before they reached the stage of
hatching. Both factors applied at early blastula stage inhibited protein synthesis,
which manifested itself virtually at once. A decrease of labelling in embryos
irradiated or treated with actinomycin at the stages of early /mid-blastula (4-6 h
after fertilization) was more intensive at the stages of hatching and mesenchyme
blastula and prevented the onset of gastrulation. Evidently the nuclear function
at the early blastula, when high polymer non-ribosomal RNA was actively
synthesized by the nuclei (Timofeeva, Ivanchik & Neyfakh, 1969) provided for
the activation of protein synthesis at the stages of mesenchyme blastula. On the
contrary, when X-rays and actinomycin D were applied during hatching and
passage to the mesenchyme blastula, protein synthesis and development were
considerably less affected. Actually such embryos morphologically did not differ
from the control. Protein synthesis was also nearer to the control. Protein
synthesis in these embryos was activated during mesenchyme blastula stage,
506
M. R. KRIGSGABER AND A. A. NEYFAKH
i.e. simultaneously with the control embryos. The pattern of protein synthesis
after actinomycin treatment resembled that in the control embryos though on a
lower level.
DISCUSSION
Three aspects of the data obtained can be considered. The first one is concerned with the relationship between nuclear morphogenetic function and
nuclear control over protein synthesis. This relationship is expressed by the fact
that the intensity of protein synthesis up to the early blastula in the sea urchin
and to the late blastula stage in the loach is independent of the embryo's nuclei,
when their morphogenetic function does not manifest itself. However, at this
early developmental period when protein synthesis and development are independent of the embryo's genome, the morphogenetic function of the nuclei providing early differentiation occurs. The inactivation of the nuclei at the early
blastula in the sea urchin and mid-blastula stage in the loach allows the dependence of protein synthesis on nuclear control to be revealed. One can see that the
morphogenetic nuclear function provides a specific activation of protein synthesis
concerned with mesenchyme blastula in the sea urchin and gastrulation in the
loach. Finally, at the stages of hatching and mesenchyme blastula in the sea
urchin and gastrula in the loach, when the morphogenetic function of the
nuclei is weaker, the nuclear control over protein synthesis is also less pronounced. This time the coincidence between the morphogenetic activity of the
nuclei and nuclear control over protein synthesis is supposed to be due to the
fact that the control over protein synthesis is the first manifestation of the
nuclear morphogenetic function. It is but natural that the nuclear control over
protein synthesis is realized more directly than over morphogenetic processes.
The second problem to be discussed is concerned with the relationships
between synthesis of mRNA and protein. The difference between normal
intensity of protein synthesis in the control embryos and protein synthesis in the
embryos after the inactivation of the nuclei is determined by blocking the
synthesis of new mRNA, breakdown of already synthesized short-living mRNA,
and by the changes of protein synthesis related to non-gene mechanisms of
regulation. We have shown that if the activity of genome is suppressed, protein
synthesis is inhibited from the stage early/mid-blastula in the sea urchin and
late blastula in the loach. It seems to be due to the fact that the supply of mRNA
stored in the egg is virtually utilized by these developmental stages (Glisin,
Glisin & Doty, 1966; Rachkus, Kuprianova, Timofeeva & Kafiani, 1969). The
intensification of protein synthesis during early differentiation which begins
after that proceeds only when the mRNA synthesis occurs at early blastula
stages in the sea urchin and mid-blastula in the loach. Although the labelling of
mRNA is revealed at the stages of 2-4 blastomeres in the sea urchin, the activation of transcription takes place at early blastula stage (Timofeeva et al. 1969).
Similar activation of transcription in the loach proceeds at mid-blastula stage
Nuclear control of protein synthesis
507
(Kafiani et al. 1969). The dependence of intensification of protein synthesis at
the stage of mesenchyme blastula in the sea urchin and gastrula in the loach on
the nuclear function at early blastula in the sea urchin and mid-blastula in the
loach seems to be caused by the activation of transcription at the developmental
stages indicated.
At the same time the dependence of protein synthesis on synthesis of highpolymeric non-ribosomal RNA is not so simple. In gynogenetic haploid loach
embryos the intensity of protein synthesis is the same as in diploid ones, although
the intensity of RNA synthesis is about two times lower (Kafiani et al. 1968). On
the contrary, protein synthesis in haploid hybrid loach ? x goldfish <J embryos
remains at the level of'enucleated' embryos although RNA synthesis is activated
and proceeds with the same rate as in loach haploids (Neyfakh et al. 1968).
Hence it can be suggested that not only is the intensity of RNA transcription of
importance but also the ability of newly synthesized RNA to be a template for
protein synthesis.
The role of' non-gene' mechanisms in the control over protein synthesis is the
third aspect of interest. Their role can be revealed when the genetic control is
blocked. Our results on the independence of the activation of protein synthesis
in the sea urchin after fertilization when the nuclear activity is suppressed
agree with the data on the absence of direct genetic control over protein
synthesis at these early developmental stages (Gross & Cousineau, 1963; Denny
& Tyler, 1964). A decrease of protein synthesis during hatching in the sea urchin
when mRNA synthesis is most active and the activation of protein synthesis at
the stages of mesenchyme blastula which precedes when transcription is blocked
with actinomycin seems to be controlled at polysome level (Tnfante & Nemer,
1967;Terman, 1970).
It is shown in the experiments on loach 'enucleated' embryos and after the
inactivation of nuclei at the stages from fertilization to mid-blastula that duration and intensity of protein synthesis in all these experimental versions is the
same and protein synthesis reaches the level characteristic of control embryos at
the late blastula stage. Protein synthesis in the loach up to the onset of gastrulation seems to proceed on the templates stored in oogenesis. In spite of the nuclei
having been inactivated at the stages of late blastula/mid-gastrula the inactivation of protein synthesis in the loach during gastrulation takes place. It seems to
be due to the fact that active polysome complexes are formed at the gastrula
stages with the participation of mRNA which have been synthesized as early as
the blastula stages, while a good deal of templates involved in synthesizing
machinery is formed at the expense of informosome degradation (Belitsina,
Aitkhozhin, Gavrilova & Spirin, 1964; Spirin, Belitsina & Aitkhozhin, 1964).
Hence protein synthesis during cleavage and blastulation (sea urchin, loach) at
the mesenchyme blastula (sea urchin) and during gastrulation (loach) also turns
to be programmed by the transcription of the genome during preceding developmental stages such as oogenesis and early (sea urchin) and mid-blastula (loach)
508
M. R. KRIGSGABER AND A. A. NEYFAKH
stages. Thus a time gap between the occurrence (mRNA synthesis) and the first
manifestation (protein synthesis) of morphogenetic function of nuclei is observed. It is provided by long-living mRNA whose template activity is temporarily
inhibited (informosomes, inactive polysome complexes), and by 'non-gene'
mechanisms which directly regulate the changes in the intensity of translation.
These and some other mechanisms provide a relative independence of protein
synthesis of the simultaneous mRNA synthesis as well as its dependence on the
activity of the nuclei at preceding developmental stages.
The authors wish to thank Professor G. V. Lopashov for his helpful suggestions and
criticism in the course of preparation of the manuscript for publication.
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(Manuscript received 23 December 1971, revised 13 March 1972)