T H E ACTION O F X-RAYS ON NUCLEATED AND
NON-NUCLEATED EGG FRAGMENTS
PAUL S. HENSHAW, PH.D.
(From the Biophysical Lahoratovy, Memorial Hospital, New Y o r k C i t y )
This report deals with the question of whether the changes produced in
cells by x-rays are due to their action on the nucleus or some other part of the
cell.
I t might be expected that irradiation effects which involve the nucleus, such
as mutations, chromosome abnormalities, changes in the mitotic figure, and
the like,’ are due to modifications in the nucleus produced directly by the
radiation. Since, however, cytoplasm, cell membranes, and surrounding media
are by necessity irradiated at the same time in practically all cases, the nuclear
changes may result indirectly from changes produced in these.
Only a few attempts have been made to determine which part of the cell is
most important from the standpoint of vulnerability to radiation. Among the
more significant of these are the classic experiments of G. and 0. Hertwig (1,
2 ) , who demonstrated the seeming paradox that slightly irradiated sperm
causes more disturbance in the development of certain eggs than severely
irradiated sperm. The explanation lies in the fact that both lightly and
heavily irradiated sperm are capable of participating in fertilization and thus
initiating development, but only the lightly irradiated ones are capable of entering into the nuclear activities of the zygotes. Thus, having undergone some
injury, the lightly irradiated sperm cause more disturbance in development
than the heavily irradiated ones, since the latter take no part in the nuclear
functions and accordingly exert no influence on them.
These experiments were repeated by Dalcq ( 3 ) and similar studies were
made by Packard ( 4 ) on Chaetopterzts eggs, with similar results. Likewise,
we have shown that a certain radiobiological change--cleavage delay in
Arbacia eggs-can be produced by irradiating sperm alone ( 5 ) . Since the
sperm in such cases is composed almost exclusively of nuclear material, these
experiments lead to the presumption that the important irradiation effects are
produced in the nucleus. Vintemberger ( 6 ) irradiated frogs’ eggs through
small holes in a lead plate and obtained important changes only when the
nucleus was in the ‘‘ line of fire.”
Certain work with alpha particles also suggests that biological changes resulting from irradiation are due to effects produced in the nucleus. Zirkle ( 7 )
treated fern spores with these radiations and studied the effects on subsequent
development and growth. He obtained the greatest effect when the cells were
oriented so that the nuclei, which in these forms are eccentric, were within the
range of the alpha particles. I n like manner, we have found Drosophila eggs
to become more sensitive to alpha particles as the nuclei in the developing eggs
1 For references see Biological Effects of Radiation, ed. by B. M. Duggar, McGraw-Hill Book
Co., New York, 1936.
258
ACTION O F X-RAYS O N EGG F R A G M E N T S
259
move toward the periphery and thus into the penetration range of the
particles ( 8 ) .
While these observations suggest that the significant effect of radiation is a
direct one upon the nucleus, they do not preclude the possibility of an indirect
action resulting from extranuclear changes. Since it will be necessary in the
formulation of any adequate explanation of radiobiological reactions to know
where the important modifications occur, precise information along these lines
is important.
During the past year we have been able to study the action of radiation on
cells with and without a nucleus and thus obtain results bearing on the above
question. In previous reports (9, 10, 5 ) it has been shown that x-rays tend
to depress the rate of cell division in the eggs of Arbacia punctulata. When
the eggs are irradiated just prior to fertilization, the cleavage time-that is the
time intervening between the moment of insemination and the moment when
50 per cent have divided-is noticeably prolonged and the delay varies with
the amount of radiation delivered (Fig. 1). From this it is clear that radial
FERTILIZATION
NORMAL CLEAVAGE
TIME
I
NORMAL CLEAVAGE
FIG.1. EFFECTOF IRRADIATION
ON CLEAVAGE
TIMEIN ARBACIA
Eccs
The first line indicates the normal cleavage time, that is, from the moment of insemination to
the moment when 50 per cent of the eggs in a sample have divided. The second line indicates
cleavage delay resulting from exposure to radiation before insemination. The third line shows that
an increase in effect results from an increase in exposure. The fourth line shows that the effect becomes less (recovery) when an interval is allowed between the end of treatment and the moment of
insemination. The fifth line indicates that an increase in this time causes an increase in the amount
of recovery observed.
tion modifies some part of the cell and causes the division process to be slowed.
The question then arises whether this modification is due to changes produced
in the nucleus or in the cytoplasm of the egg.
By means of recently developed experimental technic it is possible to
obtain with ease fragments of eggs which are suitable for such a study.
Harvey (11) in 1932 showed that both nucleated and non-nucleated fragments obtained by the centrifuge method are capable of fertilization and development. The present work has consisted of irradiating the two types of
fragments and observing the amount of delay produced by a given amount of
radiation in each case.
The eggs and sperm were collected in the usual way ( 9 ) by removal from
adult Arbacia to sea water in separate containers. After proper washing, the
eggs were centrifuged. The method in general was that used by Harvey but
differed in certain respects, chiefly in the type of centrifuge employed. Sev-
2 60
PAUL S. HENSHAW
era1 were tried but the Beams air-turbine type (12) was found most satisfactory. A special rotor was arranged to accommodate two small glass centrifuge
tubes 1 2 mm. deep and 2.2 mm. in diameter (inside dimensions).
When egg fragments were to be obtained the tubes were half filled with
0.95 molal sugar solution. Above this was placed the sea water and egg suspension-enough practically to fill the tubes. These were then placed in the
rotor and centrifuged with a force about 100,000 times gravity.
Under these conditions the eggs move through the sea water into the interphase mixture of sea water and sugar solution to a point where the density of
the medium is equal to that of the eggs. Lying suspended at this point and
still being subjected to the centrifugal force, the egg substances become stratified into a series of layers (Fig. 2). As indicated by Harvey, these are, in the
order of their increasing density :
1. Oil drop layer.
2 . Clear layer containing the nucleus.
3. Granular layer.
4. Yolk layer.
5. Red pigment layer.
When the egg becomes stratified there is a pull of the lighter (centripetal)
pole against the heavier (centrifugal) pole. Thus, if the centrifugal force is
continued after the stratification has occurred, there is a tendency for the two
poles to pull apart. This actually happens if the amount of force is great
enough, the time required for separation depending on the force applied. Division in such a case is usually through the yolk layer, although it may occur
at other levels and in more than one place, producing more than two fragments. In the majority of cases there is a single separation, the lighter nucleated part containing oil drops, clear protoplasmic substances, granular material and some yolk, while the heavier, non-nucleated part contains yolk and
pigment .
The rapidity of stratification will vary with conditions in the egg such as
viscosity of the protoplasm, density of granules, etc. (which are different in
different batches of eggs) and also with the centrifugal force applied. In the
less viscous material, for example, the stratification is more rapid and in the
more viscous it is slower for a given force. Likewise, for a given viscosity the
higher the centrifugal force the faster the stratification. Thus more complete
stratification occurs when the pull on the two poles of the egg is not sufficient
to cause it to divide before stratification is completed-a point to which we
wish to call special attention. If only a moderate amount of force ( a few
thousand times gravity) is applied, the pull is not sufficient to bring about
separation unless the stratification is practically complete. On the other hand,
if a greater amount of force is applied division will occur with less stratification
than would otherwise be required. Hence eggs of a given viscosity will yield
fragments by the centrifuge method with a greater or less complete separation
of component parts depending on the centrifugal force applied.
This is important because the measurement of irradiation effect depends on
cleavage, and cleavage of the heavy, non-nucleated fragment, in turn, is affected by the degree of separation of the egg materials. When stratification
2
FIG. 2. PHOTOMICROGRAPHS
OF WHOLEAND FRAGMENTED
EGGS
A 1. Normal whole egg. B 1. Centrifuged whole egg showing stratification; the clear transparent nucleus may be seen in faint outline in the clear layer just beneath the fat drops. C 1.
Lighter half containing nucleus. C 2. Heavier pigmented half without nucleus. D 1. Cleavage of
lighter half. D 2. Cleavage of heavier half (fairly normal). E 1. Same as D 1. E 2. Same as D 2
but with incomplete cleavage.
261
262
PAUL S. ISENSHAW
approaches completion before division, the heavy fragment contains chiefly
densely packed pigment granules. In such cases the egg has difficulty in
driving the cleavage furrow through the heavy pigment material, and the cleavages are indistinct and irregular, making it difficult to determine when 50 per
cent have divided. If stratification is less complete before division, and the
pigment is not so densely packed in the heavy fragment, cleavage occurs in a
more normal fashion. With the air-turbine centrifuge, delivering a force of
about 100,000 times gravity, the eggs are in most instances fragmented in one
minute or less. When such a procedure is used, heavy parts are occasionally
obtained that cleave very nearly like normal controls, both in respect to the
percentage dividing and the distinctness of cleavage. Never was this observed when centrifugation required several minutes or longer; in such cases
few or no eggs divided and the cleavage furrows, when present, were indistinct.
Since there was no way of telling before hand whether the heavy part would
cleave badly or well, experiments were conducted on both kinds of materials.
While the clearest results were naturally obtained with materials that manifested good cleavage, both types yielded information that pointed toward the
same conclusion.
TABLE
I : Effect of Irradiatioia on Nccleated and Non-nacleated Egg Fragments
-
Whole
egg
Nucleated Non-nucleated
part
part
NON-IRRADIATED
Time of fertilization. . . . . . . . . . . . . . . . . . . . . . . . . .
9:35
Time of 50% cleavage. . . . . . . . . . . . . . . . . . . . . . . .
10:22
Minutes required for cleavage. . . . . . . . . . . . . . . . . 47
9 :35
10:24
49
9 :35
11:oo
95
9:35
1056
91
9 :35
1I :oo
95
IKRADI.~TED
Time of fertilization. . . . . . . . . . . . . . . . . . . . . . . . . .
9:35
Time of 50% cleavage. . . . . . . . . . . . . . . . . . . . . . . .
1054
Minutes required for cleavage . . . . . . . . . . . . . . . . . 79
The experiments were carried out at the Marine Biological Laboratory in
Woods Hole, Mass., with the irradiation equipment available there. The
conditions of irradiation were as follows: 195 kv., 150 pv., 30 ma., 4 mm.
Bakelite filter, 9.5 cm. from target to material. With these factors the intensity of irradiation at the point of exposure was approximately 7,600 roentgens
per minute. A duration of exposure was selected which would produce a delay in cleavage of thirty minutes or more in normal whole eggs. Usually
forty-five seconds, or a dose of about 5,700 roentgens, was sufficient.
For the actual tests three types of material were used, some of which in
each instance was irradiated and some retained as non-irradiated controls.
These were: (1) normal whole eggs, ( 2 ) nucleated light parts, and ( 3 ) nonnucleated heavy parts. Following treatment the irradiated material and the
non-irradiated material of all three types was arranged in Syracuse dishes in
fresh sea water and all samples inseminated practically simultaneously. They
were then observed for the moment when 50 per cent had divided.
Table I shows the results obtained in a typical experiment, ten of such
ACTION O F X-RAYS O N EGG FRAGMENTS
263
having been performed. From this table it may be seen, first of all, that normal whole eggs and nucleated light parts have approximately the same cleavage time, whereas, the non-nucleated heavy parts have a longer cleavage time.
These observations are in accordance with those of Harvey. On comparing
the cleavage time of irradiated whole eggs, it is seen that a delay of thirty-two
minutes was produced by the radiation. Thus the cleavage time for the
treated material was more than half again as much as for the non-treated.
Similarly it will be seen that delay in this case is slightly greater than in the
case of whole eggs. This, however, is probably insignificant, since similar
differences were not observed in the other tests.
On comparing the non-nucleated heavy parts, it will be seen that the cleavage time was the same irrespective of irradiation; i.e., when no nucleus was
present the cleavage time was not affected by radiation. In testing the validity of this finding, special attention was given to non-nucleated material.
As mentioned above, cleavage in the heavy part was sometimes less distinct
and in certain batches only part of the eggs divided. In view of this fact not
only was a record kept of the time when 50 per cent had divided, but the moment when the first signs of cleavage occurred was also noted. Thus, if 50
per cent cleavage did not occur, we still had a means of comparison. Invariably it was found that as soon as any evidence of cleavage appeared in the
non-irradiated material, similar activity could be observed in the irradiated.
Furthermore, in those samples where the percentage of cleavage was low, the
few heavy parts that did divide were watched for subsequent development.
I t was found that the four-cell, eight-cell, and other distinguishable stages in
early development were reached a t the same time in irradiated and non-irradiated material. All evidence, therefore, points to a direct correlation between
the presence of a nucleus at the time of irradiation and the manifestation of an
irradiation effect.
DISCUSSION
It happens that the conditions of this experiment were especially fortunate. Eggs with and without nuclei were treated and an irradiation effect
was observed only when a nucleus was present at the time of treatment. This
observation makes it clear that the nucleus was involved in the process of
bringing about a slowing of cell division following irradiation but it alone does
not show that the direct action of the radiation was on the nucleus.
The nuclear injury sustained when a nucleus was present during treatment
might well have come indirectly from modifications produced in the cytoplasm
or surrounding medium, so far as the evidence on this point is concerned. It
will be remembered, however, that the process of detecting irradiation effect
involved the addition of a normal non-irradiated nucleus in the form of a sperm
to each egg or egg fragment. I t would thus be expected that the sperm nucleus would become injured upon entry into the treated cytoplasm if the observed effect were due to an indirect action on the nucleus by way of changes
produced in the cytoplasm. The fact that no injury was manifest and the
observation that injury is produced when sperm alone is irradiated, make it
clear that the radiation must act directly on the nucleus in order to bring about
delay in cell division in these forms as observed.
2 64
PAUL S. HENSHAW
SUMMARY
1. Experiments have been carried out to determine whether a certain radiobiological effect results from changes produced in the nucleus or some other
part of the cells irradiated.
2. The test material used was eggs of Arbacia punctulata and the effect
observed was delay in cell division caused by x-rays.
3. Nucleated and non-nucleated egg fragments were obtained by the centrifuge method, both of which will undergo fertilization and development.
These were irradiated and inseminated, and the moment when 50 per cent had
divided was determined.
4. I t was found that for a given dose of radiation an appreciable delay of
about the same extent was produced in whole eggs and nucleated fragments,
whereas no delay was produced in the non-nucleated fragments.
5 . Thus, since there is a direct correlation between the presence of a nucleus and the manifestation of an irradiation effect and, since irradiation
changes occurring in parts exclusive of the nucleus produce no observable
effect, it appears that the slowing in cell division brought about in Arbacia eggs
by x-rays is due to changes produced directly in the nucleus by the radiation.
ACKNOWLEDGMENT:
The writer wishes to acknowledge indebtedness to Dr. G. Failla
for his interest and valuable suggestions in connection with the work.
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