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Effects of in vitro radiocobalt irradiation of rabbit ova on subsequent development in vivo with special reference to the irradiation of maternal organism.

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Effects of in Vitro Radiocobalt Irradiation of Rabbit
Ova on Subsequent Development in Vivo with
Special Reference to the Irradiation of
Maternal Organism’
M. C. C H A N G AND D. M. HUNT
Worcester Foundation f o r Experimental Biology, Shrewsbury,
Massachusetts, a n d Department of Biology, Boston University,
Boston, Massaschusetts
The peculiar sensitivity of the embryo
and fetus to ionizing radiations has been
reviewed by O’Brien (’56) and Rugh (’59).
Rugh (’59) stated that this is due to “the
high preponderance of cells in transition
from the primitive to the differentiated
state,” and that “such interference can result from damage to cells clear back to the
original fertilized egg.” In their reviews
most literature cited is on lower vertebrates, rats and mice. There is practically
no work done on rabbits.
Previous studies (Chang et al., ’57, ’58)
have shown that irradiation of rabbit spermatozoa or of unfertilized ova in vitro at
32,000 to 65,000 r has no severe damage
on their capacity of fertilization, that irradiation of unfertilized ova in vitro at
800 r prohibits their subsequent normal
development in vivo following fertilization,
and that irradiation of two- to 4-celled
fertilized ova or of spermatozoa at 800 r reduces the normal development of embryos
to 1 0 % . It is obvious that irradiation of
mature germ cells in vitro apparently only
manifests its harmful effect on the subsequent development of ova and that fertilized ova appear to be more radioresistant
than unfertilized ova. The following experiment was performed to determine (1)
whether or not there is a differential radiosensitivity of fertilized rabbit ova at various stages of development before implantation, ( 2 ) whether abnormal development
could be induced by irradiation of ova at
various stages and ( 3 ) whether there is a
differential effect of irradiation in the ova
alone, recipient females alone (without irradiation of the ova), or in pregnant ani-
mals at the corresponding stages for irradiation of ova and of recipient animals.
METHODS A N D PROCEDURES
Adult female rabbits of mixed breeds
purchased from local farms were kept in
separate cages in the laboratory for about
three weeks before use. Four female rabbits at a time were bred twice by fertile
males or were artificially inseminated.
These served as donors of ova. At the same
time another two rabbits were injected
with sheep pituitary extract intravenously
to induce ovulation and were used as recipients of ova. One, two, 4, or 6 days
after insemination the donor rabbits were
sacrificed and their ova were recovered
either from the Fallopian tubes (one or
two days after insemination) or from uteri
( 4 or 6 days after insemination) by Aushing these organs with 50% fresh rabbit
serum in saline. The ova were divided into
4 groups and placed into small Pyrex tubes,
1 cm in diameter, containing 1 ml of diluted serum. These 4 tubes were then
exposed simultaneously to a radiocobalt
source at distances according to the dosages required. Immediately after irradiation, the ova were transplanted either into
the Fallopian tube or into the uterus of a
recipient animal through a flank incision
under Nembutal anesthesia. As a rule, ova
irradiated at one dose level were transferred into the left side and ova irradiated
at another dose level were transferred into
the right side of the same animal. DelThis study was supported by the Atomic
Energy Commission under contract AT( 30-1)1943.
511
512
M. C . CHANG AND D. M. HUNT
tailed methods for the recovery and transfer of fertilized ova have been described
elsewhere (Chang, '50). The recipient
rabbits were sacrificed 22-28 days after
transplantation and the number of fetuses
were counted, measured, and examined
externally and internally. The maternal
or fetal placentae in the uteri were also
carefully noted to ascertain the degree of
degeneration after implantation.
In the study of the irradiation of recipient animals alone, the whole body of an
animal was exposed to a radiocobalt source
at 400 r two, 4, or 6 days after the induction of ovulation. Four to 5 hours after
irradiation, non-irradiated ova of corresponding post-ovulation age were transplanted into the irradiated recipients.
Ten-thousand units of penicillin was given
daily for three days to prevent infection.
They were examined 22 to 28 days after
operation. Whole body irradiation at 400 r
of another group of animals was performed two, 4 or 6 days after artificial insemination. Twenty-two to 28 days later
they were sacrificed and their conceptuses
were similarly examined.
For the cytological study of the irradiated
blastocysts, late blastocysts recovered from
the uterus 6 days after mating were irradiated at 100, 1,000 and 10,000 r. Soon
after irradiation one group of blastocysts
were fixed, spread according to Chang
('54) and then stained by the Feulgen
method and another group of blaystocysts
were cultured in Carrel flasks with 50% of
fresh rabbit serum for 2-3 hours and then
fixed and stained.
RESULTS
Irradiation of ova in vitro at dose levels
f r o m 50 t o 5,000 r
Table 1 summarizes the results when
rabbit ova, recovered one day (at 2-4 cell
stage), two days (at morula stage), 4 days
(at early blastocyst stage) or 6 days (at
late blastocyst stage) after insemination
were irradiated at 50, 100, 1,000 or 5,000
r and then transplanted into the Fallopian
TABLE 1
Effects of in vitro irradiation of rabbit ova on subsequent in vivo development ( 5 0 r to 5,000r)
Dosages
Age of
ova at
irrad.
r
day
No. of
recipient
rabbits
No. of
ova transplanted
fetuses
No,
%
Fetal
placentae
No.
%
Maternal
placentae
No.
Total
implantation
%
No.
%
~
50
100
1'
2
4
6
Total
5
3
3
3
14
61
21
12
21
115
28
45.9
33.4
83.45
28.8
44.4
2
0
0
7
9
3.3
0
0
33.3
7.8
1 1.6
0
0
0
0
4 19.0
5
4.3
31
7
10
6
51
7
10
17
65
50.8
33.3
83.0
80.9
56.5
12
6
3
3
3
15
77
21
16
21
135
18
10
11
6
45
23.4
47.6
68.75
28.8
33.3
2
0
0
5
7
2.6
0
0
23.8
5.2
0
0
0
1
1
0
0
0
4.8
0.7
20
10
11
12
53
26.0
47.6
68.7
57.1
39.2
3
3
3
3
12
41
22
17
25
105
5
3
0
12.2
13.6
0
2
0
0
4.9
0
0
0
0
0
0
8
3
0
3
8
7.6
2
1.9
1 2.4
0
0
0
0
3 12.0
4
3.8
14
19.5
13.6
0
12.0
13.3
3
3
3
3
12
44
24
19
26
113
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
4
6
Total
1000
13
2
4
6
Total
5000
14
2
4
6
Total
0
0
0
0
'At 45 r.
At 90 r.
At 800 r.
4 A t 6,500 r. These data are derived from a previous experiment (Chang et al., '58).
51n these experiments the best blastocysts were selected for transfer, see text.
0
DEVELOPMENT OF IRRADIATED OVA
tubes or uteri of recipient rabbits, The
recipient rabbits, were made pseudopregnant a t a stage corresponding to the
age of the ova at the time of transplantation, by injection of pituitary extract one,
two, 4 or 6 days previously.
The efficacy of ova transplantation in
the rabbit has been determined previously
(Chang, ’ 5 0 ) and it is known that about
54% of one-day ova and about 42% of
4-day or 6-day ova will develop into normal young when transplanted into the
proper female tract a t the corresponding
stage of pseudopregnancy. From the results presented in table 1, the percentage
of normal development following the irradiation of ova at 50 r was 46 in the case
of one-day ova and 29 in the case of 6-day
ova. The percentage of normal development following the irradiation at 100 r was
23 in the case of one-day ova and 29 in
the case of 6-day ova. From these figures
it appears that there may be a harmful
effect even after the irradiation of ova in
uitro at 50 r. Following irradiation at 100
r the percentage of normal development
for one-day ova was lower (23% ) than at
50 r (46% ) but the percentages of normal development in the cases of ova of
other ages were irregular; thus whether
there is a differential effect between 50 r
and 100 r is uncertain. As for the extremely high percentage of normal development in the case of irradiation of 4-day
ova at 50 r or at 100 r, this is probably due
to the fact that most of the 4-day ova
shrunk during manipulation and only the
best and the largest blastocysts were transferred following irradiation in this study.
Following irradiation at 1,000 r , only
12 to 14% of one-day or two-day ova, but
none of the 4-day or 6-day ova, developed
into normal fetuses, indicating that at
1,000 r, the younger ova are slightly more
radioresistant than the older ones. Degeneration after implantation, however, was
observed in the irradiation of 6-day ova as
shown by the presence of maternal
placentae. At a dose level of 5,000 r
neither normal development nor evidence
of implantation was observed in any case,
showing that a dose of 5,000 r is definitely
lethal to rabbit ova at all stages before
implantation.
O/O
80
-0
0
6 0 -
513
514
M. C . CHANG A N D D. M. HUNT
after insemination were irradiated at 200,
400, 600 or 800 r and then transplanted
into the female tracts of rabbits pseudopregnant at the appropriate stage. Table 2
and figure 2 present the results. From the
results in table 2 it can be seen that follow-
ing the irradiation of ova at 200 r, there
is no significant difference in the proportion of normal fetal development between
ova at various stages of development. At
a dose level of 400 r the proportion of normal fetal developemnt in the case of 6-day
TABLE 2
Effects of in vitro irradiation of rabbit ovu o n subsequent in vivo development (200 r to 800 r )
-
Dosages
~
Age of
ova at
irrad.
Fetal
placentae
9
6
8
23
62
31
39
132
22
9
14
45
35.5
29.0
35.9
34.1
7 11.3
2 6.5
3 7.7
12 9.1
2 3.2
5 16.1
9 23.1
16 12.1
31
16
26
73
7
6
9
22
51
33
47
131
15
8
24
47
29.4
24.2
51.1
35.9
2 3.9
3 9.1
9 19.2
14 10.6
3
5.9
8 24.2
3 6.4
14 10.6
20
8
5
8
21
58
28
41
127
ia 31.0
2
7.1
4 9.8
24 18.9
5 8.6
3 10.7
6 14.6
14 11.0
1
1.7
5 17.8
8 19.5
14 11.0
24
10
18
52
41.4
35.7
43.9
40.9
8
5
8
21
57
29
42
128
5 8.8
2 6.9
6 14.3
13 10.1
14
9
21
44
24.6
31.0
50.0
34.4
2DAY OVA
80
fetuses
No.
%
~-
No.
2
4
6
400
2
4
6
Total
Total
2
4
6
Total
aoo
No.
%
-.
%
No.
%
day
200
600
Total
imp1ant ation
No. of
ova transplanted
.___.
7
Maternal
placentae
No. of
recipient
rabbits
2
4
6
Total
5
2
2
9
8.8
6.9
4.8
7.0
4
7.0
17.2
13 30.9
22 17.2
5
%
6 DAY OVA
70
60-
40-
60y
I
4 DAY OVA
/ O \
x-x
I
zoo
I
400
normal fptuses
GOO
DOSAGE r.
aoo
I
zoo
I
400 600
DOSAGE r.
I
aoo
Fig. 2 Effects of radiation dosage in vitro on subsequent development of ova.
50.0
51.6
66.8
55.3
39.2
19 57.5
36 76.4
75 57.3
515
DEVELOPMENT O F IRRADIATED OVA
ova (51% ) is higher than in the case of
two- to 4-day ova (24-29% ) . At a dose
level of 600 r, the proportion of normal
development in the case of two-day ova
( 3 1% ) is higher than in the case of 4- and
6-day ova (7-10% ) . But due to the small
number of ova used and the wide variation
of results between recipient animals it is
uncertain whether there is a differential
sensitivity of ova at a particular stage of
development to a specific dose level of irradiation. Following irradiation at 800 r,
the proportion of ova developing into normal fetuses ranged from 7 to 14% in all
ova at various stages.
Taking the results as a whole and by
inspection of figure 2 it appears that there
is no difference between ova at various
stages of development following irradiation at dose levels from 200 r to 800 r;
and that the proportion of irradiated ova
developing into normal fetuses and the
proportion of total ova implanted decreased
together a s the dosage increased. However, the curves of percentage of total implantation and that of the percentage of
normal development are relatively more
close to each other in the case of two-da-y
ova than in the case of 4-day and 6-day
ova (fig. 2 ) . This may indicate that after
irradiation younger ova would develop into
normal fetuses if they were implanted
while older ones might survive after im-
plantation but fail to develop into normal
fetuses in the end. In general the proportion of ova degenerating after implantation increased as the dose level increased.
Although following a certain dose of irradiation, ova at blastocyst stage may survive for a longer time than a segmenting
ova but the cellular components that determine their future development into normal fetuses are equally radiosensitive in
the two-day ovum as in the 6-day ovum.
Irradiation of recipient and pregnant animals as compared with the irradiation of
ova alone on subsequent embryonic development
In order to determine whether the irradiation of recipient females would play
a role in the subsequent development of
embryos, ova recovered two, 4 or 6 days
after insemination were transplanted into
Fallopian tubes or uteri of recipient animals that had been irradiated at 400 r on
the second, 4th, or 6th day after a n ovulation-inducing injection. Another group of
animals were irradiated at 400 r two, 4 or
6 days after insemination, to determine the
effect of irradiation of ova in situ on their
subsequent development A third group of
animals, insemfnated but without irradiation served as controls. Table 3 and figure
3 present the results alongside previous
data on the irradiation of ova in vitro at
various stages of development at 400 r.
TABLE 3
Development after irradiation at 400~:
comparison o f the effects o f radiation applied to the ova in
vitro, to the recipient does, and to the does while pregnant
Irradiation
of
Age
Of
NO.of
rabbits
No'
Of
?%?$'
Fetal
placenta
Maternal
placenta
Total
implantation
~~
No.
-
%
No.
%
No.
%
No.
%
day
Ova
2
4
6
Total
Recirients
2
4
6
Total
Pregnant
2
4
6
Total
Control
7
6
9
22
51
33
47
131
15
8
24
47
29.4
24.2
51.1
35.9
2
3
9
14
3.9
9.1
19.2
10.6
3
8
3
14
5.9
24.2
6.4
10.6
20
19
36
75
39.2
57.5
76.4
57.3
4
4
4
12
35
47
37
119
15
22
11
48
42.8
46.8
29.7
40.4
4
2
2
8
11.4
4.3
5.4
6.7
2
5.7
5 10.6
11 29.7
18 15.1
21
29
24
74
60.0
61.7
64.9
62.2
3
4
4
11
47
41
48
136
7
8
8
23
14.9
19.5
16.7
16.9
4
0
4
8
8.5
0
8.3
5.9
1
0
0
1
2.1
0
0
0.7
12
8
12
32
25.6
19.5
25.0
23.6
10
115
74
64.4
2
1.7
0
0
76
66.0
516
M. C. CHANG AND D. M. H U N T
80
%
1
QO
40
20
2 DAY
4 DAY
6 DAY
ALL STAGES
Fig. 3 Total implantations (whole column) and degeneration after implantations (unshaded area of column) as of ova treated by irradiation a t 400 r at two, 4, and 6 days.
When considering the percentages of combination of harmful effect on embrytotal implantation and normal fetal devel- onic development. Here again the results
opment, the results show that following show that there is no striking differential
irradiation on the second or fourth day radiosensitivity between the ova at differthese percentages are higher in the case of ent stages of development before implantathe recipient animals than in the case of tion, either when isolated and irradiated
ova alone or in the pregnant animals. This in vitro or when irradiated in situ.
shows that the harmful effect of irradiation
Eight dead fetuses, (so-called neonatal
is mainly on the ova rather than on the death), were found at the time of examinamaternal system. Following irradiation on tion among 56 “normal” fetuses following
the 6th day, the percentage of total im- irradiation of two-, 4-, and 6-day ova in
plantation and normal development are vitro at 100 or at 400 r. Neonatal death,
higher in the case of ova alone than in the however, was not found in other cases, incase of irradiation of recipient or pregnant cluding the irradiation of recipient and
animals. This indicates that on the 6th pregnant animals. There was no obvious
day the harmful effect of irradiation may correlation of neonatal death with the age
be greater in the maternal system rather of ova at irradiation or the dose level emthan in the embryos. In general, there is ployed.
no striking difference whether the ova,
Cytology of irradiated blastocysts
recipients, or pregnant mothers were irradiated on the second, fourth, or on the
Irradiated blastocysts were studied cytosixth day in the determination of future logically. Soon after irradiation mitosis at
development of the ova; but the propor- various stages was observed in whole
tions of total implantation, degeneration blastocysts - even after a dose of 10,000
after implantation, and normal develop- r. However, effects on mitotic activity bement showed a marked reduction follow- came apparent when the blastocysts were
ing the irradiation of pregnant animals. cultured for 2-3 hours. Non-irradiated
When the results are combined and com- blastocysts after culture showed normal
pared with the non-irradiated mothers (fig. mitotic figures and no obvious fragmenta3 ) , it is obvious that the disturbance of tion of chromatin (fig. 7). After irradianormal development can be induced by ir- tion at 100 r, various phases of mitosis
radiation of maternal environment without were seen, but there were many chromothe irradiation of embryo and that the ir- somal bridges (fig. 5) and a few scattered
radiation of both mother and ova exert a chromatin fragments, mainly in the germ
D E V E L O P M E N T O F IRRADIATED OVA
517
Figures 4-7 are Feulgen preparations of spread rabbit blastocysts photographed with a n
oil immersion objective and a 1OX ocular, approx. x 750.
Fig. 4 Germ disk area of a blastocyst irradiated at 1,000 r and cultured for three hours.
Showing the abundance of fragmentation and condensation of chromatin without any mitotic figures.
Fig. 5 Trophoblastic area of a blastocyst irradiated at 100 r and cultured for three hours.
Showing chromosome bridges.
Fig. 6 Germ disk area of a blastocyst irradiated at 100 r and cultured for three hours.
Showing the presence of equatorial plates, and a few chromatin fragments.
Fig. 7 Germ disk area of a non-irradiated blastocyst.
disc area (fig. 6). In blastocysts irradiated
at 1,000 and 10,000 r there were no mitotic
figures, but there were many condensed
chromatin fragments which gave a n intense Feulgen reaction (fig. 4 ) .
DISCUSSION
Fellner and Neumann ('06) observed
the arrest of gestation with resorption of
the embryos in rabbits irradiated during
the first half of pregnancy. In the rat,
Hicks ('54) reported that no damage of
nervous system was observed when irradiated at 0 to 8 days but on 9th to 13th day,
various malformations of the nervous
system were observed. Wilson ('54) also
reported a high incidence of malformations when rats were irradiated on the 9th
to 1l t h day of pregnancy. In the mouse,
Russell and Russell ('54) reported a high
incidence of prenatal death following irradiation during the pre-implantation
period, various malformations after irradiation during the major period of organo-
518
M. C . CHANG AND D. M. HUNT
genesis (days 6.5 to 12.5), high incidence
of neonatal death after irradiation on day
9.5 and 10.5; and the absence of gross
abnormalities when mice were irradiated
on day 0.5 to 5.5 or on days 13.5. Recently, however, Rugh and Grupp (’59)
reported that exencephaly (cerebral hernia) may be produced by irradiation of the
newly fertilized egg before any cleavages
or by damage to neuroblasts at 8.5 days in
the mouse. The present study revealed
that in the rabbit no abnormality, or any
kind of malformations externally or internally, was observed in fetuses obtained by
irradiation either of ova alone or of recipient and pregnant animals, during the
pre-implantation period. Certainly this
does not imply that the ovum before implantation is more radioresistant than at
other stages. It only shows that at this
stage the ova were either killed by irradiation or developed into apparently normal
fetuses. Whether or not these fetuses are
physiologically normal is still to be determined.
According to Rugh (’54) “the newly
fertilized egg and the blastula are the most
radiosensitive stages in early embryonic
development of the frog, whereas the ovarian egg, the neurula and the older tadpole
are the most radioresistant.” From his list
of radiosensitivity, the fertilized egg is the
first, the blastula is the second, the first
cleavage the third, the uterine egg is the
fourth, etc. It is rather surprising from
the present study that the radiosensitivity
of a two-celled rabbit ovum is very much
the same as that of a late blastocyst which
contains thousands of cells, and whose
cells are differentiated into embryonic and
trophoblastic portions. It has been postulated that radiation induces chromosome
breaks or aberration, and thus leads to the
failure of mitosis and the death of cells
(Russell and Russell, ’54). From cytological examination of the irradiated blastocysts in the present study it is obvious that
irradiation did not induce an instantaneous
chromosomal breakage; but chromosomal
abnormalities, fragmentation and condensation of chromatin show up later during
culture. It seems that the effect of irradiation is on a more fundamental biological
system, probably at a molecular level,
which is revealed during cell division. This
is especially so in the case of spermatozoa
whose nucleus is at a resting stage without
visible chromosomes. After irradiation the
motility and fertilizing capacity of spermatozoa are not damaged but the cleavage
of ova fertilized by irradiated spermatozoa
is inhibited (Amoroso and Parkes, ’47;
Chang et al., ’57).
According to Rugh (’54) “the differentiating cell, even prior to its mitosis, is
more radiosensitive than is the differentiated cell in the process of mitosis.” It is
really hard to say, especially during embryonic development, which group of cells
is differentiating and which group of cells
is differentiated. One may say that the
cells in a blastocyst are more in a differentiating stage than two blastomeres in a
segmenting ovum; but their radiosensitivity
in determining future development is very
much the same. Furthermore, mature
spermatozoa are highly differentiated cells
and yet irradiation of sperm will lead to
the degeneration of fetrilized ova (Chang
et al., ’57).
It is known that irradiation of unfertilized rabbit ova at 800 r results in a total
failure of development of such ova fertilized by non-irradiated sperm (Chang et
al., ’58) and that irradiation of sperm at
800 r reduces the proportion of embryonic
development to 11% when non-irradiated
ova were fertilized by such sperm (Chang
et al.,’57). The present study has revealed
that irradiation of fertilized ova at 800 r
before implantation reduces the proportion
of embryonic development also to 1 0 % . It
seems that unfertilized ova are more radiosensitive than sperm, but once fertilized the
ova at various stages before implantation
are as radioresistant as sperm although
their nuclear activities are quite different.
One wonders whether the radiation damage
to the gametes and zygotes affects the same
biological system.
SUMMARY
Fertilized rabbit ova recovered one to 6
days after mating were irradiated in vitro
from a radiocobalt source and then transplanted into recipient animals. When examined 22-28 days later 44, 33, 8 and
0% of ova irradiated respectively at 50,
100, 1,000 and 5,000 r developed into apparently normal fetuses without external or
DEVELOPMENT OF IRRADIATED OVA
internal malformation. No significant differential sensitivity was apparent in ova
irradiated at different ages.
It was found further that 34, 36, 19 and
10% of two-, 4-, and 6-day ova irradiated
respectively in vitro at 200, 400, 600 and
800 r developed into “normal” fetuses.
Again no malformation of fetuses and no
differential radiosensitivity between ova of
different ages were observed.
Following whole body irradiation at 400
r, it was found that 40% of non-irradiated
ova developed into normal fetuses when
transplanted into recipient animals that
had been irradiated (vs. 36% in the irradiation of ova alone). However, only
17% of estimated ova developed into
“normal” fetuses when pregnant rabbits
were irradiated two, 4 or 6 days after insemination (vs. 64% in the control). It
appears that irradiation of the maternal
organism influences embryonic development and that irradiation of pregnant animals exerts a combination of ill effects,
on the ova and on their environment.
Cytological study of irradiated blastocysts revealed no chromosomal breakage
immediately after irradiation. Chromosomal abnormalities, fragmentation and
condensation of chromatin were observed
during the cuIture of irradiated blastocysts in accordance with the dosages applied.
From this study it is concluded that ( 1 )
although 50 r may affect embryonic development, there seems to be no differential
effect up to 400 r, above which greater
prenatal death occurs, ( 2 ) before implantation, irradiated ova either die or develop
into apparently normal fetuses and there
is no evidence of differential radiosensitivity at various stages of development,
519
( 3 ) irradiation of the maternal organism
alone also affects embryonic development,
and (4) radiation damage affects a fundamental biological system which leads to
the nuclear damage and failure of mitosis,
and the death of ova.
LITERATURE CITED
Amoroso, E. C., and A. S. Parkes 1947 Effects
on embrvonic development of X-irradiation of
rabbit spermatozoa in vitto. Proc. Roy. SOC.B,
134: 57-78.
Chang, M. C. 1950 DeveIopment and fate of
transferred rabbit ova or blastocyst in relation
to the ovulation time of recipients. J. Exp.
ZOO^., 114: 197-226.
1954 Development of parthenogenetic
rabbit blastocysts induced by low temperature
storage of unfertilized ova. Ibid., 125: 127-150.
Chang, M. C., D. M. Hunt and E. B. Romanoff
1957 Effect of radiocobalt irradiation of rabbit spermatozoa in vicTo on fertilization and
early development. Anat. Rec., 129: 211-230.
1958 Effects of radiocobalt irradiation
of unfertilized or fertilized rabbit ova in vit70
on subsequent fertilization and development
in viuo. Ibid., 132: 161-180.
Fellner, 0. O., and F. Neumann 1906 Ueber
Rontgen bestrahlung der Ovarien in der Schwangerschaft. Zentralbl. f. Gyniik. 30: 630-633.
Hicks, S. P. 1954 The effects of ionizing radiation, certain hormones and radiomimetic drugs
on the developing nervous system. J. Cell. and
Comp. Physiol., 43: (suppl.) 151-178.
O’Brien, J. P. 1956 Vertebrate radiobiology:
Embryology. Ann. Rev. Nuclear Sci., 6: 423453.
Rugh, R. 1954 The effect of ionizing radiations
on amphibian development. J. Cell. and Comp.
Physiol., 43: (suppl.) 39-76.
1959 Vertebrate radiobiology (Embryology). Ann. Rev. Nuclear Sci., 9: 493-522.
Rugh, R., and E. Grupp 1959 Exencephalia
following X-irradiation of the pre-implantation
mammalian embryo. J. Neuropath. Exp. Neur.,
18: 468-481.
Russell, L. B., and W. L. Russell 1954 An
analysis of the changing radiation response of
the developing mouse embryo. J. Cell. and
Comp. Physiol., 43: (suppl.) 103-150.
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