J. Embryol. exp. Morph. Vol. 20, 1, pp. 129-40, August 1968
129
Printed in Great Britain
A quantitative approach
to the detection of nuclear activity after
differential damage to nucleus and cytoplasm
in early development
By A. A. NEYFAKH 1 & N. N. ROTT 1
Institute of Developmental Biology of the Academy of Sciences
of the USSR, Moscow
For studying nucleo-cytoplasmic relations during development various selective influences on the nucleus and cytoplasm are widely used as the main method
of experimental analysis. However, the application of such techniques presents
difficulties both in obtaining evidence that shows the specificity of a nuclear or
cytoplasmic effect by a chosen agent and in the quantitative evaluation of the
extent of damage.
In this paper a method is described for differentiating between nuclear and
cytoplasmic sites of action of a given agent as well as for evaluating quantitatively the extent of nuclear damage. The method is based on the determination
of the morphogenetic activity of nuclei at different stages of embryonic development. As has been previously shown, after complete inactivation of nuclei (for
instance, by heavy doses of radiation) development proceeds up to the stages
programmed for by the genetic cell apparatus (Neyfakh, 1959, 1964). Within
a certain range of doses ionizing radiation may be regarded as a factor selectively affecting the nucleus, which is proved for instance by the identity of
androgenetic and gynogenetic embryos obtained after heavy irradiation of ova
and spermatozoa.
On comparing the ability to develop of embryos with nuclei inactivated at
different developmental stages, one can determine both the moment of onset of
morphogenetic nuclear function and its intensity (Fig. 1). Thus in loach embryos
morphogenetic nuclear function does not begin until practically the mid-blastula
stage (6 h at 21 °C). Development from fertilization to the late blastula stage
(9 h) proceeds utilising genetic information obtained during oogenesis. This is
concluded from the cessation of development of embryos exposed to heavy
doses of radiation at different developmental stages up to 6 h. The arrest takes
place at the 9 h stage and doesn't depend on the time of irradiation. When
1
Authors' address: Institute of Developmental Biology, Vavilov Street 26, Moscow
V-133, U.S.S.R.
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130
A. A. NEYFAKH & N. N. ROTT
embryos are irradiated at the stages from 6 to 8-9 h, the arrest of development
does depend on the time of irradiation, and embryos irradiated at the late
blastula stage are able to proceed through the whole of gastrulation (9-18 h).
Thus the morphogenetic nuclear function appearing during the 2 to 3 h from
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r
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16
14
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Duration of
development
provided during
ihofMNF
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Period of MNF
Time at inactivation of nuclei (h)
Fig. 1. Dependence of the time of arrest in the course of development (ordinate) on
the time at inactivation of nuclei (abscissa), a—the angle of the slope of the curve.
M.N.F.—morphogenetic nuclear function.
the 6 h stage to the 8-9 h stage provides the information for the whole process
of gastrulation (from the 9 h stage to the 18 h stage). The intensity of morphogenetic function may be expressed as the number of hours of development
potentially programmed for by nuclei during 1 h of their previous functioning.
On the graph it may be determined by the slope of the curve, or more precisely,
by the tangent of slope angle. Similar data, but somewhat shifted in time, are
obtained when the time (in hours) from the moment of irradiation to the moment
of death of a proportion (for instance, 80 %) of embryos is used as a criterion.
Unlike the stage of arrest, the time of 80 % mortality of embryos may be deter-
Nuclear activity after damage
131
mined with accuracy. This criterion has been used in our investigation. The
activity of nuclei allowing the survival of embryos is denoted conventionally as
'morphogenetic function', although in this case the term is not quite precise.
One may expect that a partial inhibition of the genetic apparatus will reduce
the intensity of later morphogenetic activity and will manifest itself in a decrease
in life span of embryos with nuclei inactivated during the period of morphogenetic
activity.
The percentage survival after irradiation seems to be determined not by the
whole genome but only by a limited number of genes. However, this makes no
difference, as in such a case a decrease in embryonic life span indicates that a
given agent affects the nucleus. Damaging agents which do not affect the genetic
apparatus may induce a decrease in the life span of embryos, but this decrease
does not depend on the developmental stage at which inactivation occurred.
This difference enables us to differentiate between nuclear and cytoplasmic
damage.
In this work the following specific agents have been used to influence the
nuclear apparatus: (1) moderate doses of ionizing radiation; (2) treatment with
actinomycin; (3) elimination of one of the chromosome sets (haploid development) or its replacement by another set of chromosomes (hybridization). Sodium
desoxycholate and sodium dodecylsulphate solutions, which damage lipoprotein
cell membranes, served as factors assumed to produce a cytoplasmic effect.
MATERIALS AND METHODS
Experiments were carried out on embryos of the loach Misgurnus fossilis L.
Developmental stages were expressed as hours of normal development at 21 °C.
At early developmental stages embryos were subjected to the action of one of
the above agents. The dose was chosen so as to permit the survival of embryos
up to the end of gastrulation. Embryos were then X-irradiated with heavy doses
(10-35 kr, 15 mA, 190 kV without filter) at successive developmental stages at
10-60 min intervals. As has been previously shown, such doses result in a complete inactivation of the nucleus, the effect being constant within a given dose
range (Neyfakh, 1959). 200-400 eggs were taken at each time of irradiation. To
determine the time of 80 % mortality, dead embryos were counted each hour.
Appropriate curves were constructed, the percentage of dead embryos plotted
against time. The time of 80 % mortality of embryos could be calculated precisely (± 10-15 min) from these curves. The results obtained were plotted on
a graph showing the dependence of time of 80 % mortality of embryos on the
time of irradiation. The slope of the curve reflects the intensity of morphogenetic
nuclear function.
9-2
132
A. A. NEYFAKH & N. N. ROTT
RESULTS
1. Haploid embryos
Gynogenetic haploid embryos were obtained by irradiating spermatozoa prior
to fertilization with heavy doses (about 60-70 kr), which caused a 100 % haploidy
of embryos (Bakulina, Pokrovskaga & Romashov, 1962). As to the rate and
character of development, such embryos do not differ from diploid ones at early
developmental stages, their defects being revealed only after gastrulation. Almost
all of them reach the stage of hatching.
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Stage of development (h)
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Time at inactivation of nuclei (h)
Fig. 2. Morphogenetic nuclear function in diploid (2n) and haploid («) embryos.
(a) Dependence of the time of 80 % mortality (ordinate) on the time at radiation
inactivation of nuclei (abscissa). Dose—10 kr.
(b) Dependence of intensity of morphogenetic nuclear function (ordinate) on the
developmental stage (abscissa). The graph was drawn using the data represented in
Fig. 2 a. The intensity of nuclear morphogenetic function was determined by the
increase in the embryonic life span when irradiation was carried out an hour later.
The area (S) contained by this curve gives the summation of activity of nuclei in early
development.
Fig. 2 a shows the changes in life span of haploid and diploid embryos after
irradiation at different developmental stages. One can see that both in diploid
and haploid embryos nuclear function, which ensures later viability of embryos,
appears approximately from the 4 h stage (early blastula) to the 8-9 h stage (late
blastula).
Nuclear activity after damage
133
Although both curves presented in Fig. 2a are very similar, it is clear that
haploid embryos die much earlier than diploid ones when irradiated at the 5-9 h
stages. When nuclei are inactivated at the 9-10 hr stage the curves coincide.
Interesting results may be obtained when the intensity of nuclear function in
haploid and diploid embryos is compared (Fig. 2b). The total values of nuclear
activity during the period of their functioning in haploid and diploid embryos
appear to be approximately the same, indicating that they have the same total
amount of nuclear 'production'. However, in haploids the bulk of this 'production' is elaborated later than in diploids. Compared to diploid nuclei the
less intense activity of haploids at early developmental stages is compensated by
their more intense activity at later stages; the peak of the curve showing the
intensity of nuclear function in haploids is shifted to the right in Fig. 2b. One
can suggest that this late compensation is not able to normalize completely the
development of embryos disturbed by the deficiency of nuclear function in the
initial period. This appears to be one of the likely causes of the haploid syndrome.
2. Hybrid embryos
If loach eggs are fertilized by goldfish sperm, fertilization proceeds normally,
and development does not differ from that of normal diploid loach embryos.
However, before hatching embryos acquire such features of the goldfish as early
eye pigmentation and typical yolk form. After hatching, the development of
hybrid larvae is delayed and they gradually die. It has been shown (Neyfakh &
Radzievskaya, 1967) that in hybrids a major part (up to £) of the paternal
chromosome set is eliminated. Thus it might be expected that the paternal genotype would show a certain morphogenetic activity, but it should be less than
that of the maternal genotype due to the elimination of a portion of chromosomes and/or incompatibility of paternal chromosomes with maternal cytoplasm.
Consequently the intensity of nuclear function in hybrid embryos should be
higher than in haploid loach embryos but less than in diploid ones. In Fig. 3 it is
seen that the survival curve for hybrid embryos after radiation inactivation of
nuclei lies between the corresponding curves for haploid and diploid loach
embryos. Of particular interest is the fact that at the late blastula stage, when
the intensity of morphogenetic function in loach embryos is temporarily declining, no decrease is observed in hybrid embryos. These experiments show that
hybrids follow the paternal species in this respect. This suggests that the time
pattern of morphogenetic nuclear function is under genetic control.
3. Irradiation with moderate doses of ionizing radiation
Diploid embryos were irradiated with doses from 500 to 2000 r at the 1-5 h
developmental stages. The irradiation didn't markedly affect early developmental
stages, although it did cause mortality of some embryos at later stages. After this
preliminary irradiation, nuclei were completely inactivated by 35 kr irradiation
at different times of development. Irradiation is known to produce the same
134
A. A. NEYFAKH & N. N. ROTT
effect within the dose range from 10 to 40 kr. Thus it appears that summation
of the dose of preliminary irradiation with that of inactivation is unlikely to
affect the survival of embryos.
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Time at inactivation of nuclei (h)
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Fig. 4
Fig. 3. Morphogenetic nuclear function in diploid ( •
• ) and haploid
(O
O) loach embryos and hybrids goldfish x loach (x — — x). Dose, 15 kr.
Fig. 4. Morphogenetic nuclear function in normal ( •
• ) and previously
irradiated ( O
O) embryos. Dose, 35 kr.
Fig. 4 shows the changes in life span of normal and early irradiated embryos,
with nuclei inactivated at different later developmental stages. For embryos
irradiated with 750 r the life expectancy is decreased, and the summed intensity
of nuclear function is also reduced. Thus, irradiation-induced partial loss of
genetic material causes a decrease in intensity of morphogenetic nuclear function
at early developmental stages as well as anomalies of development at later times.
4. Action of actinomycin
In this work actinomycin prepared in Moscow University (chrysomaline) was
used. Actinomycin hardly penetrates fresh-water fish embryos. On intact loach
embryos a marked effect of actinomycin treatment can be obtained only if
the latter is applied during the first moments after fertilization in as high concentrations as 100-200 /^g/ml. The development of isolated blastoderms is
Nuclear activity after damage
135
blocked at concentrations 10-20 /«g/ml. It means that cells show their usual
sensitivity to actinomycin, but only a small amount can penetrate the embryo
from the medium.
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Time at inactivation of nuclei (h)
Fig. 5. Morphogenetic nuclear function in normal ( •
pre-treated embryos. Actinomycin concentrations, 25 (O
100 (O • • • O) and 200 ( • x x • ) /ig/ml. Dose, 15 kr.
1
8
• ) and actinomycin
O), 50 ( x - - - x ) ,
In our experiments fertilized eggs were placed in aqueous actinomycin solution
for 1 h and then washed. The following concentrations were used: 25, 50, 100
and 200 /*g/ml. Development proceeded quite normally to the late blastula
stage. Further development at a concentration of 200 /*g/ml was completely
arrested. Treatment with 100/^g/ml resulted in a partial arrest at the same
developmental stage: a proportion of embryos proceeded through the first steps
of gastrulation. After treatment with 50/£g/ml, embryos gastrulated and developed
further, although some abnormalities and delay were observed in their develop-
136
A. A. NEYFAKH & N. N. ROTT
ment. After the action of 25 /^g/ml the first half of embryonic development
proceeded normally.
In Fig. 5 the data are summarized on the life span of normal and actinomycintreated embryos after nuclear inactivation with 15 kr at different developmental
stages. One can see that actinomycin, at all concentrations used, markedly
diminished the life span of embryos. This was the consequence of a decrease in
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Fig. 6. Morphogenetic nuclear function in normal (<
>) and sodium dodecylsulphate (SDS) treated embryos. Embryos were treated for 1 h with 25 /tg/ml SDS
before radiation inactivation of nuclei (O
O) or with 5 /*g/ml after irradiation
( x — x). Dose, 30 kr.
the intensity of nuclear function on the whole proportional to the antibiotic
concentration. Hence actinomycin even at concentrations which don't induce
morphological changes affects the intensity of nuclear function.
5. Sodium desoxycholate and sodium dodecylsulphate action
Embryos were treated with sodium dodecylsulphate solution (SDS) (25 /^g/ml)
for 1 h and morphogenetic nuclear function was investigated thereafter. In
another experiment embryos were placed in SDS (5/^g/ml) after radiation
Nuclear activity after damage
137
inactivation of nuclei and kept there until death. In non-irradiated control
embryos, treatment with these concentrations didn't cause death up to the end
of gastrulation. Fig. 6 shows that treatment with SDS accelerated death of
irradiated embryos. However, unlike the action of the nuclear factors referred
to above, SDS accelerated the death of embryos with inactivated nuclei whether
inactivation occurred during the period of morphogenetic function or before it.
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Fig. 7. Morphogenetic nuclear function in normal ( •
• ) and desoxycholate
(DOC) treated embryos. Embryos were treated for 1 h with 0-75 mg/ml desoxycholate
(O
O) before radiation inactivation of nuclei or with 0-5 mg/ml after irradiation
( x - - - x ) . Dose, 30 kr.
The angle of the slope of the survival curve practically didn't change during the
period of morphogenetic activity. In other words, SDS didn't change intensity
of morphogenetic nuclear function, but only accelerated the realization of
nuclear, irradiation-induced, damage. It is of interest that SDS produced the
same effect when applied after radiation inactivation of nuclei, while a nuclear
agent applied after radiation inactivation of nuclei could not be expected to
produce any effect.
Treatment with sodium desoxycholate (DOC) at a concentration 0-75 mg/ml
138
A. A. NEYFAKH & N. N. ROTT
for 1 h prior to irradiation and with 0-5 mg/ml (continuously) after irradiation
affected nuclei in a similar way to that observed for SDS. In this case also the
cytoplasmic character of action of this agent is revealed by the fact that it
accelerates embryonic death independent of the moment of irradiation, and
remains effective when appplied after radiation inactivation of nuclei (Fig. 7).
DISCUSSION
All the factors investigated which affect the genetic apparatus of the cell
(haploidy, hybridization, ionizing radiation and actinomycin treatment) significantly influence the character and intensity of morphogenetic nuclear function
in early development. The method described in this paper allows the detection
of the effect of these factors at stages and doses which do not reveal this action
morphologically.
Agents affecting cytoplasmic cell structures (sodium dodecylsulphate and
desoxycholate) do not influence morphogenetic nuclear function. They accelerate
embryonic death to the same extent independent of the moment of inactivation
of the genetic apparatus of the cell, either during the period of morphogenetic
nuclear activity or before it. This would enable us to differentiate nuclear and
cytoplasmic action by damaging agents of an unknown nature and to evaluate
quantitatively the extent of nuclear damage.
It seems of interest to compare the data described in the present paper with
such direct measures of nuclear activity as the intensity of RNA synthesis. In
haploid loach embryos nuclear RNA synthesis proceeds twice as slowly at the
mid-blastula stage (6-8 h) as similar synthesis in diploid embryos. However,,
by the mid-gastrula stage (10 h) the intensity of RNA synthesis in diploid and
haploid embryos becomes practically the same (Timofeeva, Neyfakh & Kafiani,.
1967). Similar results have been obtained in our experiments. At very early
stages the morphogenetic nuclear activity in haploids is sharply decreased as
compared with that of diploids, but at subsequent developmental stages (8-10 h)
the decrease has been compensated. As a result, the total amount of nuclear
function appears to be approximately the same in haploids and diploids. In
haploid embryos the number of nuclei at this time is approximately 1-5 times
higher than in diploids (Rott & Sheveleva, 1968). Hence the total amount of
genetic material in haploids is less than in diploids. One can suggest that each
chromosome of a haploid cell is capable of intensifying its function by revealing
higher genetic activity and/or synthesizing more RNA than a homologous
chromosome of the diploid cell. Such compensation of gene dosage has been
found for the X-chromosome of Drosophila males for both genetic activity
(Miiller, League & Offermann, 1932) and RNA synthesis (Mukherjee & Beermann, 1965).
Actinomycin treatment (100 /*g/ml) causes approximately a 50 % decrease in
RNA synthesis (Timofeeva, personal communication). According to our data,
Nuclear activity after damage
139
the same treatment decreases nuclear function to about half intensity. One
would not expect a complete coincidence of the data on intensity of RNA
synthesis and nuclear function determined by the method described, but the
preceding examples show that phenomena of the same type underlie both
inhibitions.
Thus the use of the method described for comparing haploid and diploid
embryos permits clarification of the mechanism of compensation of developmental processes at the change of ploidy and understanding of the causes
of the haploid syndrome. The determination of changes in the intensity of
nuclear function as an index of nuclear damage may be used to solve certain
radiobiological problems as well. The high sensitivity of the method as well as
its quantitative character suggests that it can be used to detect the action of
various chemical and physical agents on the cell nucleus.
SUMMARY
1. A method is suggested for distinguishing nuclear and cytoplasmic action
by various harmful agents permitting quantitative evaluation of the extent of
nuclear damage. The method is based on the determination of morphogenetic
nuclear activity at different developmental stages.
2. The elimination of or damage to some portion of the genetic material
(haploidy, distant hybridization, irradiation and actinomycin treatment) results
in a decrease of nuclear activity in the period of morphogenetic function. In
haploid embryos the absence of one of the chromosome sets may be partially
compensated by intensified functioning of a single chromosome set.
3. Agents affecting cytoplasmic cell structures (sodium dodecylsulphate,
sodium desoxycholate) do not influence directly morphogenetic nuclear function,
but cause a decrease of embryonic life span independent of the time of nuclear
inactivation.
4. There exists a certain quantitative correlation between morphogenetic
nuclear activity and RNA synthesis.
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140
A. A. NEYFAKH & N. N. ROTT
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REFERENCES
E. D., POKROVSKAYA, G. L. & ROMASHOV, D. D. (1962). On the radiosensitivity
of loach spermatozoa. Radiobiologia 2, 92-100 (in Russian).
MUKHERJEE, A. & BEERMANN, W. (1965). Synthesis of ribonucleic acid by the X-chromosomes of Dwsophila melanogaster and the problem of dosage compensation. Nature, Lond.
207, 785-6.
MULLER, H. J., LEAGUE, B. B. & OFFERMAN, C. A. (1932). Effects of dosage changes of sexlinked genes and the compensatory effects of other gene differences between male and
female. Anat. Rec. 51, 110.
NEYFAKH, A. A. (1959). X-ray inactivation of nuclei as a method for studying their function
in early development of fishes. /. Embryol. exp. Morph. 7, 173-92.
NEYFAKH, A. A. (1964). Radiation inactivation of nucleo-cytoplasmic interrelation in morphogenesis and biochemical differentiation. Nature, Lond. 201, 880-4.
NEYFAKH, A. A. & RADZIEVSKAYA, V. V. (1967). On morphogenetic nuclear function in goldfish and loach hybrids. Genetika (in Russian) 12, 80-88
ROTT, N. N. & SHEVELEVA, G. A. (1968). Changes in rate of cell division in the course of early
development of diploid and haploid loach embryos. /. Embryol. exp. Morph. (in the Press).
TIMOFEEVA, M. J., NEYFAKH, A. A. & KAFIANI, K. A. (1967). The change of RNA-synthesizing nuclear function in early embryonic development. In Structure and Function of
Cell Nucleus, pp. 200-5 (in Russian). Moscow: Nauka.
BAKULINA,
(Manuscript received 27 April 1967, revised 2 February 1968)