RADIATION RESEARCH 182, 430–434 (2014)0033-7587/14 $15.00
Ó2014 by Radiation Research Society.
All rights of reproduction in any form reserved.
DOI: 10.1667/RR13776.1

Mutagenic Effects of Ionizing Radiation on Immature Rat Oocytes
Jun-ichi Asakawa,a,1 Yujiroh Kamiguchi,b Kenji Kamiyac and Nori NakamuraaaDepartment of Genetics, Radiation Effects Research Foundation, Hiroshima, Japan; b Department of Biology, Asahikawa Medical University,
Asahikawa, Japan; and c Department of Experimental Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University,
Hiroshima, Japan

been derived from data from specific-locus tests in mice [thedata from the 7 loci used at the Oak Ridge National
Laboratory is the most extensive; for reviews see refs. (1,2)]. In males, spermatogonia cells are considered as thetarget cells, and dose-response data for mutations after acuteor chronic exposure conditions as well as after exposure todifferent qualities of radiation have been obtained. In
females, the target cells are immature oocytes, but in micethese cells are hypersensitive to radiation-induced apoptosis[e.g., 10–20 day-old female mice lose 90% of theirimmature oocytes as a result of an exposure to 0.18 Gy of
gamma rays (3)], while offspring derived from irradiatedimmature oocytes are essentially mutation free. In contrast,
mature or maturing oocytes are not sensitive to radiationinduced apoptosis but their mutation induction rates aretwice as high as those in spermatogonia cells after a dose of
4 Gy. In humans, such high sensitivity to radiation-induced
apoptosis is not observed in immature oocytes. In fact, an
exposure of 1 or 1.5 Gy of X rays delivered to the ovary wasonce a standard medical procedure for treatment ofinfertility (4). These differences among species thus makeit difficult to estimate genetic risks from radiation delivered
to human immature oocytes.
The BEIR III report addresses and fully discusses thisissue (5). Briefly, two alternative scenarios that may apply
to human immature oocytes are presented. First, humanimmature oocytes might be sensitive to radiation-induced
mutagenesis if it is assumed that the high sensitivity toradiation-induced apoptosis observed in mice actuallyfunctions to eliminate damaged gametes and to eliminate
mutations. Alternatively, a second scenario suggests thatimmature mammalian oocytes are inherently insensitive tomutation induction, and human immature oocytes are no
exception. This assumes that the high sensitivity toradiation-induced apoptosis observed in immature mouse
oocytes is irrelevant to mutagenesis. This is a reasonableassumption because immature oocytes in mammals remainquiescent for a long period of time after birth, and hence
they may be equipped to withstand various types of DNA
damage that may occur during their resting period. Anotherreport, which suggests a lack of association between
apoptosis and mutagenesis, comes from a study that showed

Asakawa, J., Kamiguchi, Y., Kamiya, K. and Nakamura, N.
Mutagenic Effects of Ionizing Radiation on Immature Rat
Oocytes. Radiat. Res. 182, 430–434 (2014).

Estimates of genetic risks from radiation delivered tohumans are derived largely from mouse studies. In males, thetarget is spermatogonia and a large amount of information isavailable. In contrast, in females, immature oocytes are thetarget, but extrapolations from mice to humans are not very
definitive because immature mouse oocytes are highly
sensitive to radiation and die by apoptosis, which is not thecase in humans. Since mouse offspring derived fromsurviving immature oocytes have to date not shown any signsof mutation induction, two alternative hypotheses areproposed: 1. Apoptotic death effectively eliminates damaged
oocytes in mice and therefore human immature oocytes may
be highly mutable; and 2. Immature oocytes are inherently
resistant to mutation induction and apoptotic death is not
relevant to mutagenesis. To test these hypotheses, rat
immature oocytes, which are not as sensitive as those in mice
to radiation-induced apoptosis were exposed to 2.5 Gy of
gamma rays and the offspring were examined using a twodimensional DNA analysis method. Screening of a total of
2.26 million DNA fragments, we identified 32 and 18
mutations in the control and exposed groups, respectively.
Of these, in the two groups, 29 and 14 mutations weremicrosatellite mutations, two and one were base changes, andone and three were deletions. Among the four deletions most
relevant to radiation exposure, only one was possibly derived
from the irradiated dam (but not determined) and three werepaternal in origin. Although the number of mutations was
small, the results appear to support the second hypothesis
and indicate that immature oocytes are generally lesssensitive than mature oocytes to mutation induction. Ó 2014by Radiation Research Society

INTRODUCTION

A large amount of information on genetic (i.e., transgenerational) risks from exposure to ionizing radiation has
1 Address for correspondence: Department of Genetics, Radiation
Effects Research Foundation, 5-2 Hijiyama Park, Minami-ku,
Hiroshima, 732-0815, Japan; e-mail: jun@rerf.or.jp.

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that dominant lethal mutations were less frequent inimmature oocytes in both guinea pigs and golden hamsters,
although the frequency of apoptotic death was less frequentin immature oocytes than in maturing oocytes in guinea
pigs, whereas the converse was true in golden hamsters (6).
An ongoing problem in the field of radiation genetics has
been to decide which of the two scenarios described above
is more likely to apply to humans (the BEIR III report
indicated that the latter hypothesis is more likely). Althoughimmature rat oocytes are known to be more resistant toradiation-induced apoptosis than those of mice, conducting
specific-loci tests in rats was not thought to be feasible since
it requires a large number of offspring that requiresignificantly more space to house than is needed for mice.
In recent years, various new molecular technologies havebeen developed, one of which is the Restriction Landmark
Genome Scanning method (i.e., two-dimensional gel
electrophoresis of radio-labeled DNA fragments or 2DE
of DNA), making it possible to examine over 1,000
genomic sites for each individual. We previously reported
on radiation effects on spermatogonia in mice using the2DE method to analyze DNA (7, 8), and in this current
study, we present data on immature oocytes in rats.

431

of alleles that are either from the X chromosome or specific to either
parent). The intensity of each spot was quantified with the value
generated by the density 3 area.
Cloning and sequencing of DNA fragments that had undergone
mutations was done as previously described (8). Multiple bacterial
clones were isolated and sequenced for each mutation, and aconsensus sequence was mapped on the rat reference sequence,
Baylor 3.4/rn4, to obtain chromosomal locations and upstream anddownstream sequence information.
Microsatellite sequences were PCR amplified with florescencelabeled primers flanking the repeats, and the amplicon size of eachmicrosatellite mutation was estimated by capillary electrophoresis
with size markers to determine the number of repeat units (9).

RESULTS AND DISCUSSION

Menstrual Cycle

If the menstrual cycle is prolonged after a radiation
exposure, collecting offspring derived from fertilization
80 days after irradiation does not guarantee that they were
derived from irradiated immature oocytes. To ensure thatthe cycles were not disturbed, five female rats irradiatedwith 4 Gy were examined for regularity of cycles with avaginal smear technique for up to one year after exposure.
No evidence of prolongation or perturbation of the cycle
was observed (results not shown).

MATERIALS AND METHODS
All animal experiments were carried out at Asahikawa Medical
College under approval of the in-house Animal Care Committee.
Female Sprague-Dawley (SD) rats (7 weeks old) were purchased from
CLEA Japan (Sapporo, Japan). After an acclimation period of 1 week,
an i.p. injection of Somnopentyl (Pitman-Moore, Ontario, Canada)
was administered at a concentration of 25–28 mg (as sodium
pentobarbital) per kilogram of body weight to anesthetize the rats,
after which the animals were placed in a lead block to shield theirheads and upper trunks and exposed to 2.5 Gy of 137Cs gamma rays
(0.83 Gy/min). Shielding the head from radiation was done to avoid
perturbation of hormone production by the radiation exposure, andshielding the upper trunk was done to avoid development ofradiogenic thymic lymphomas. To collect offspring derived fromimmature oocytes at the time of the radiation exposure, irradiatedfemales were mated with nonirradiated males [Brown Norway (BN)strain] starting at 80 days after irradiation. The irradiated females haddeliveries three to four times and up to 56 offspring per dam. F1 rats
were humanely sacrificed at 3 weeks of age, and the spleen, kidney
and liver were collected and stored at 808C until use. Fifty-two SD
females (dams) and 16 BN males (sires) were used, a total of 1,500 F1
rats were examined, of which 750 were derived from 21 nonirradiatedSD females and 750 from 31 irradiated SD females.
Detailed methods for 2DE of 32P-labeled genomic DNA fragments
have been previously described in refs. (7, 8). Briefly, DNA isolated
from the spleen was digested with NotI and EcoRV restriction
enzymes. Subsequently, 5 0 overhangs were filled with 32P-labeleddGTP and dCTP, and the DNA was subjected to first dimensional gel
electrophoresis to separate the fragments by size. Gels containing 1–4
kb DNA fragments and 4–10 kb fragments were subjected to digestion
with a third enzyme HinfI in situ and were placed on top ofpolyacrylamide gels to perpendicularly separate the DNA in thesecond dimension. Autoradiograms were prepared after exposure of
X-ray films, and 32P-labeled DNA fragments were visualized as spots.
The autoradiograms were scanned and the images were screened forthe presence of spots with an intensity reduced by 50% (the loss of oneof the two alleles in an autosome) or the loss of an entire spot (the loss

Selection of SpotAn example of an autoradiogram of an F1 hybrid from anirradiated SD strain (dam) and a nonirradiated BN strain(sire) is shown in Fig. 1A and B. Spots for screening
purposes were selected based on conditions requiring good
reproducibility and the absence of nearby spots that mightoverlap the spot of interest. After examination of scanned
autoradiograms from four families (50 offspring and four
pairs of parents), 162 spots unique to the SD strain, 179
spots unique to the BN strain and 1,387 spots common toboth strains were selected (i.e., 1,549 spots for the SD
genome and 1,566 spots for the BN genome).
Detection of Mutations

Two autoradiograms (first dimension sizes of 1–4 kb and4–10 kb) were obtained for each rat and 1,500 offspring(750 from the control and 750 from irradiated groups) werescreened along with the parents. In total, 32 and 18
mutations were detected in the control and exposed groups,
respectively, among about 1.13 3 106 spots screened in eachgroup (Table 1). Theoretically, the total number of spots
screened should be 1,549 3 750 ¼ 1.16 3 106 spots for the
SD strain and 1,566 3 750 ¼ 1.17 3 106 for the BN strain.
However, autoradiograms were not always ideal andportions of a gel were occasionally unsuitable or unusable,
resulting in a 3–4% loss of spot information. Thesemutations were all confirmed using DNA from the liverand kidney, indicating that they are germ cell mutations inorigin (if the mutation occurred after fertilization, the zygote

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ASAKAWA ET AL.

FIG. 1. Two-dimensional autoradiograms of 32P-labeled rat DNA fragments. Panel A: First dimension size of
1–4 kb. Panel B: First dimension size of 4–10 kb. Panels C–E: Examples of mutations, right panels represent
normal patterns and left panels include mutations. Mutant spots are indicated with white circles andcorresponding normal spots are indicated with black circles. Panel C: One spot was lost; panel D: one spot
divided into two spots; and panel E: one spot shifted downward.

will have a mosaic structure and consequently may show
discordant results depending on the tissues examined).
Characterization of the Mutations

The majority of mutations detected had lost spot intensity
by nearly one half, and were accompanied by a new onecopy spot near the original position (29 out of 32 in thecontrol and 14 out of 18 in the exposed groups) (examples
are shown in Fig. 1C–E). Our past experience strongly
suggested that these were likely to be mutations at
microsatellites (repeats of 2–8 core sequences), and indeed
they were found to be microsatellite mutations after cloning
and sequencing the normal and the affected spots. The
contribution of the paternal and maternal origins seemedroughly equal: in the control group, 11 were paternal, 11were maternal and 7 were undetermined; in the exposedgroup, 7 were paternal, 3 were maternal and 4 wereundetermined (Table 2). Thus, the results do not indicate
any radiation-induced effect and are consistent withpreviously published results showing that microsatellite
mutations were not induced in germ cells (9, 10). Lastly, it
should be noted that several highly mutable spots weredetected, the most mutable being the BN-specific spot
RS158 that was mutated 15 times. The second most mutable
was the SD-specific spot RS163 that was mutated 10 times,
and the third most mutable was the spot RL1388, common

to both the SD and BN strains, which was mutated 4 times.
Sequence data showed that RS158 and RS163 are BN- andSD-specific alleles of the same microsatellite locus that
contain 25 and 20 repeats of GCGGGGC sequences,respectively (Table 2). It is possible that long GC-richrepeat sequences may tend to undergo slippage after
replication stress as observed in the FMR1 locus of fragile
X syndrome (11).
Other Mutations

In the irradiated group, there was one base-change
mutation (the parental origin could not be determined) andthree deletions (two were paternal and one could not be
determined). In the control group, there were two basechange mutations (both were maternal) and one deletion
mutation (paternal). Therefore, there was only one deletion
mutation that was possibly derived from irradiated immature oocytes.
Uncertainty

A total of over 1 million spots were screened in both thecontrol and irradiated groups, however only one deletion
mutation was detected in the irradiated group (but theparental origin could not be determined). The paucity ofdeletion mutations does not necessarily mean that immature

TABLE 1
Summary of Mutations Derived from Immature Rat Oocytes Irradiated with 2.5 Gy of Gamma Rays
Number of mutations
5

Group

Total number of spots screened (310 )Total

Microsatellite mutations

0 Gy
2.5 Gy

11.3
11.3

32
18

29
14

Base change mutations

Deletion mutations

2 (maternal)
1 (undetermined)

1 (paternal)
2 (paternal)
1 (undetermined)

GENETIC RISK ESTIMATE OF RADIATION IN FEMALE RATSTABLE 2
Parental Origins and Sequence Changes in
Microsatellite Mutations
Dose
0 Gy

Spot ID Parental origin*

RL1388RL1388RL1388RL1388
RL184
RL650
RS488
RS007
RS007
RS1028
RS158
RS158
RS158
RS158
RS158
RS158
RS158
RS158
RS163
RS163
RS163
RS163
RS163
RS163
RS163
RS163
RS379
RS379
RS488
RS488
2.5 Gy RL174
RL357
RL953
RS214
RS158
RS158
RS158
RS158
RS158
RS158
RS158
RS163
RS163
RS1028





P

M



P
P
P
P
P
P
P
P
M
M
M
M
M
M
M
M
P
P
M
M


M

P
P
P
P
P
P
P
M
M


Sequence change(CGA) 8 ! 7(CGA) 8 ! 7(GGA) 8 ! 7(GC)4(AC)17 ! (GC)6(AC)17(CA)29 ! 25
(TAG)22 ! 21
(TAG)22 ! 21
Not cloned(GT)26 ! 25
(GCGGGGC)20 ! 21
(GCGGGGC)20 ! 19
(GCGGGGC)20 ! 22
(GCGGGGC)20 ! 21
(GCGGGGC)20 ! 21
(GCGGGGC)20 ! 21
(GCGGGGC)20 ! 22
(GCGGGGC)20 ! 22
(GCGGGGC)25 ! 23
(GCGGGGC)25 ! 24
(GCGGGGC)25 ! 24
(GCGGGGC)25 ! 26
(GCGGGGC)25 ! 23
(GCGGGGC)25 ! 26
(GCGGGGC)25 ! 23
(GCGGGGC)25 ! 22
(TAG)25 ! 24
(TAG)25 ! 24
(TAG)22 ! 21
(TAG)22 ! 21(GT)23 ! 20
(ATTT)11 ! 10(CA)27 ! 12
(AGGGGC) 29 ! 30
(GCGGGGC)20 ! 21
(GCGGGGC)20 ! 22
(GCGGGGC)20 ! 22
(GCGGGGC)20 ! 21
(GCGGGGC)20 ! 22
(GCGGGGC)20 ! 21
(GCGGGGC)20 ! 22
(GCGGGGC)25 ! 23
(GCGGGGC)25 ! 27(GT)31 ! 29

* P ¼ paternal; M ¼ maternal; dash () ¼ undetermined parental
origin.

oocytes are refractory to recording radiation damage
because it may well be that the number of spots screened
was not large enough. In this context, it may be useful tocompare these results with those previously obtained after
irradiation of mouse spermatogonia [for a summary of theseresults, see Table 3 in ref. (8)] with the same 2DE method.
Briefly, about 1,800 spots were screened per offspring, and
5 deletion mutations, most likely caused by radiation
exposure, were observed among 500 offspring derived from
spermatogonia irradiated with 4 Gy. In contrast, only one
deletion mutation was observed in the offspring of the433

control group consisting of a similar number of mice.
Although the number of mutations is rather small and hence
the confidence intervals are wide, taking the results at facevalue it appears that one deletion mutation may occur
among 100 offspring after males were irradiated with 4 Gy.
From this, it is possible to calculate the expected number ofdeletion mutations in the current study under the assumptions of equal radiation sensitivity between mouse spermatogonia and rat immature oocytes. Compared with themouse data, the current rat data is derived from a 40% lower
radiation dose (2.5 vs. 4 Gy), and utilized 15% fewer spots
screened per individual (1,500 vs. 1,800). Thus, theexpected number of deletion mutations is calculated as:
750 3 (1/100) deletion mutation 3 (2.5 Gy/4 Gy) 3 (1,500
spots/1,800 spots) ¼ 3.9. This number does not seem large
enough to have a high probability of being detected, whichleads us to conclude that compared with mouse spermatogonia, rat immature oocytes do not seem to be moresensitive to the induction of deletion mutations.
CONCLUSION

We propose that death of immature mouse oocytes from
apoptosis after radiation exposure is likely to be irrelevant in
the elimination of germ cells that have acquired mutational
DNA damage. This proposal is also supported by a report
indicating that the target of radiogenic apoptosis in mouse
immature oocytes is likely to be plasma membrane (12).
Currently, screening of deletion mutations using microarrays (array-based CGH studies) is underway, and thepreliminary data from these studies also support our
proposal (unpublished results).
ACKNOWLEDGMENTS
The authors thank Dr. L. Kapp for his careful reading of the manuscript,
Dr. Hiroshi Katayama at the Information Technology Department of
RERF for his help in designing an image analysis system in the laboratory,
and Y. Nakamoto, S. Kaneoka, T. Tsuji, A. Miura, S. Mishima, J. Kaneko,
M. Imanaka and H. Haba for their technical assistance. The Radiation
Effects Research Foundation (RERF), Hiroshima and Nagasaki, Japan is a
private, nonprofit foundation funded by the Japanese Ministry of Health,
Labor and Welfare and the U.S. Department of Energy (DOE). The
research was also funded in part through DOE Award DE-HS0000031 to
the National Academy of Sciences. This publication was supported by
RERF Research Protocol RP 1–07. A part of this work was supported by
JSPS KAKENHI Grant Number 20312033 to JA. The views of the authors
do not necessarily reflect those of the two governments.
Received: April 22, 2014; accepted: June 5, 2014; published online:
September 17, 2014

REFERENCES
1. Searle AG. Mutation induction in mice. Adv Radiat Biol 1974;
4:131–207.
2. Hereditary effects of radiation. 2001 Report to the General
Assembly with Scientific Annex. New York: United Nations
Scientific Committee on the Effects of Atomic Radiation(UNSCEAR); 2001.

434

ASAKAWA ET AL.

3. Dobson RL, Kwan TC. The tritium RBE at low-level exposure –variation with dose, dose rate, and exposure duration. Curr Top
Radiat Res Q 1978; 12:44–62.
4. Ron E, Auvinen A, Alfandary E, Stovall M, Modan B, Werner A.
Cancer risk following radiotherapy for infertility or menstrual
disorders. Int J Cancer 1999; 82:795–8.
5. Committee on the Biological Effects of Ionizing Radiations. The
effects on populations of exposure to low levels of ionizing
radiation: 1980. BEIR III Report. Washington D.C.: National
Academy Press; 1980.
6. Cox BD, Lyon MF. X-ray induced dominant lethal mutations inmature and immature oocytes of guinea-pigs and golden hamsters.
Mutat Res 1975; 28:421–36.
7. Asakawa J, Kuick R, Kodaira M, Nakamura N, Katayama H,
Pierce D, et al. A genome scanning approach to assess the genetic
effects of radiation in mice and humans. Radiat Res 2004;
161:380–90.

8. Asakawa J, Kodaira M, Cullings HM, Katayama H, Nakamura N.
The genetic risk in mice from radiation: an estimate of the mutationinduction rate per genome. Radiat Res 2013; 179:293–303.
9. Kodaira M, Ryo H, Kamada N, Furukawa K, Takahashi N,
Nakajima H, et al. No evidence of increased mutation rates at
microsatellite loci in offspring of A-bomb survivors. Radiat Res
2010; 173:205–13.
10. Furitsu K, Ryo H, Yeliseeva KG, Thuy le TT, Kawabata H,
Krupnova EV. Microsatellite mutations show no increases in thechildren of the Chernobyl liquidators. Mutat Res 2005; 581:69–82.
11. Yudkin D, Hayward BE, Aladjem MI, Kumari D, Usdin K.
Chromosome fragility and the abnormal replication of the FMR1
locus in fragile X syndrome. Hum Mol Genet 2014; 23:2940–52.
12. Straume T, Dobson RL, Kwan TC. Neutron RBEs and theradiosensitive target for mouse immature oocyte killing. Radiat
Res 1987; 111:47–57.