Molecular Human Reproduction vol.2 no.1 pp. 46-51, 1996
DNA repair by oocytes
M.J.Ashwood-Smrth13 and R.G.Edwards2
biology Department, University of Victoria, Victoria, V8W 2Y2 BC, Canada, and 2 Churchill College, University of
Cambridge, Cambridge, CB3 ODS, UK
•*Tb whom correspondence should be addressed as Visiting Fellow (1995), Churchill College, University of Cambridge,
Cambridge, CB3 ODS, UK
Experimental evidence in a number of different in vivo and in vitro systems indicates clearly that the
vertebrate oocyte is capable of repairing endogenous and exogenous DNA damaged as a result of meiotic
recombination, the action of UV and X-irradiation or the effects of mutagenic chemicals. It would appear that
both before and after the dictyate stage of meiosis the oocyte has less repair capacity and/or is more sensitive
to DNA damaging agents. Epigenetic factors associated with the expression of genetic faults arising in
oocytes have been largely ignored in the past. It is probable that attention to such factors, will in the future,
lead to a better appreciation of the capacity of oocytes to repair genetic damage. Non-disjunctional events
are particularly prone to occur in dictyate oocytes. Oxygen deprivation, perturbations of microtubular structure
by temperature and other factors appear to have disastrous cytogenetic consequences at this otherwise
resistant resting stage.
Key words: DNA damage/DNA repair/mammalian oocytes/meiotic recombination/repair mechanisms
DNA repair systems
All cells except viruses and spermatozoa possess a variety of
enzymatic mechanisms that repair damaged DNA. Usually the
DNA is intrinsic to the cell but on occasions it may be
damaged DNA introduced by viruses, plasmids, pieces of
naked nucleic acid or spermatozoa. The mechanisms involved
in DNA repair have been worked out in detail for bacteria
and mammalian cells (Hanawalt, 1977). Often these repair
processes are induced by the presence of changed bases or
chain breaks (SOS repair). Excision of damage, usually with
several normal bases on either side of the targeted lesion, is
followed by small amounts of new DNA synthesis (unscheduled synthesis) in which the undamaged DNA strand is used
as a template. DNA polymerases and ligases play a role in the
concerted repair of the initial lesion(s). Repair is not always
perfect and thus some mechanisms are known as 'error prone
repair pathways'.
DNA repair systems are involved in gene recombination
through the formation of chiasmata and crossing over during
meiosis. Recombination occurs up to 1000 times more frequently in germinal than in somatic cells. This high frequency
is evidently associated with the formation of synaptonemal
complexes during meiosis, since various mutants reduce germinal but not somatic cell recombination in Drosophila (Singer
and Berg, 1991; Kornberg and Baker, 1992). Synaptonemal
complexes form in human oocytes during prophase of meiosis
1 which occurs in the fetal ovary from 11 weeks of pregnancy.
Double stranded breaks arising during recombination are
evidently repaired by those same systems which also repair
DNA damaged by irradiation or chemical injury. Displacement
loops bridge the gap between the broken ends of the chromatids,
and mismatches are removed by repair systems.
46
X-ray and UV sensitive mutants in yeast and bacterial cells
owe their sensitivity to a variety of missing or non-functional
genes (Little, 1994). It is not surprising that a number of
genetic defects in man associated with altered DNA repair are
now recognized, and are in essence, the counterpart of the
prokaryotic models. Xeroderma pigmentosum (XP), Ataxia
telangiectasia, Fanconi's anaemia and Bloom's syndrome are
a few of the human counterparts of the bacterial radiation
sensitive mutants. In many instances, heterozygotes may also
display increased sensitivity as they possess insufficient quantities of repair enzymes. Not surprisingly, given the complexity
of the enzymatic processes, sensitivity to DNA damage may
be due to insufficiency or non-functionality in any of a number
of the multistep pathways. Complementation experiments in
which deficient cells are fused often result in normal function.
This complementation can also be demonstrated in cell free
extracts (Wood et al., 1988). Enzyme components from bacteria
and man can also act in a complementary manner.
With the exception of enzymatic photorepair, which is
functionally absent in most higher vertebrate cells and which
involves the in-situ reversal of covalently linked pyrimidine
dimers (TT, CC and CT) produced by UV radiation (257 nm),
nearly all mammalian DNA repair is non-specific. Single and
double strand breaks, the major result of X-rays, monoadducts
and cross links produced by chemicals (benzo(a)pyrene,
nitrogen mustards, and numerous carcinogenic and chemotherapeutic agents and UV radiation are all repaired by mammalian
cells with differing degrees of efficiency (Little, 1994). Relatively recent papers on DNA repair are discussed by several
groups (Wood et al., 1988; Hansson et al., 1989; Wiebauer
and Jiricny, 1990; Thomas et al., 1991).
© European Society for Human Reproduction and Embryology
DNA repair by oocytes
There is a dearth of information concerning DNA repair in
oocytes but it would be surprising indeed if a cell that remains
for many years in an arrested state of the cell cycle (the
dictyate stage of meiosis) does not have enzymatic repair to
counteract the effects of ionizing radiation and chemical
modification to its DNA. Little states 'Cells stimulated to
divide immediately after irradiation would have to replicate
their DNA on a damaged template' (Little, 1994). DNA repair
systems are effective over relatively short time periods. Normal
human diploid cells when X-irradiated with 4 Gy (400 rads)
recover from a low survival of 0.1 to 0.25% after 6 h at 37*C.
It is therefore ironic (or perhaps to be expected) that repair
systems are least effective in dealing with the chromosome
imbalance in oocytes, which is perhaps the major cause of
anomalies in these gametes.
DNA and chromosome damage and repair in
oocytes
A consideration of chromosomal imbalance in oocytes leads
to a salutary lesson in cellular homeostasis and epigenetics.
The causes of these anomalies will be discussed before
discussing some of the molecular repair mechanisms that have
been investigated either in oocytes or their extracts.
The most common anomaly originating in oocytes is probably trisomy or monosomy for various human chromosomes.
Most human trisomies arise during the first meiotic division
in oocytes (Ishikiriyama and Nikawa, 1984). Chandley (1991)
comments on this fact and concludes that 'Temporal disturbance
of meiotic progression seems likely to underlay aneuploidy
production in the female mouse, and this could equally well
be true in women, most especially as they approach the
menopause when irregular cyclicity sets in'. The occurrence
of trisomy 21 in human fetuses is correlated with maternal
age, as known for many years. The high frequency of this and
other trisomies in fetuses of older women may be related to a
lower frequency of chiasma formation in meiotic prophase I
in the last-formed oocytes. Chromosome pairing could be
impaired, to cause a high frequency of meiotic non-disjunction
with increasing maternal age, as in mice (Henderson and
Edwards, 1968; Polani and Crolla, 1991). The low chiasma
frequency was provisionally ascribed to an impaired recombination frequency possibly caused by a low oxygen tension in
the fetal ovary, which affected the last-formed oocytes.
Gaulden (1992) advances another interesting hypothesis on
the origin of Down's syndrome children, namely that the 95%
of them arising during the first or second meiotic division in
the oocyte are caused by a lack of oxygen in the developing
follicle. There is no internal blood circulation in follicles, and
hormonal imbalances (see Alberitni, 1992) result in imperfect
microvasculature of the theca in maturing and matured follicles.
The resultant anoxia, acidosis and hypercapnia cause a smaller
than normal spindle to be formed, leading eventually, to nondisjunction. Gaulden states: "The compromised microcirculation hypothesis explains the occurrence of aneuploidy in
primary and secondary oocytes, sperm precursor cells, tumor
and embryonic cells. It also explains why women of all
reproductive ages may have a Down's syndrome child'. This
hypothesis resembles earlier theories on the consequences of
low oxygen tension on chiasma formation, and it might also
clarify recent concepts on premature chromatid segregation at
telophase I as a major cause of human monosomies and
trisomies (Angell et al., 1993).
Chromosomal non-disjunction is not amenable to molecular
analyses or to any form of regulation such as interfering with
gene recombination systems in fetal ovaries. Attention has
shifted to oocyte cytoarchitecture and the origin of monosomy
and trisomy. Albertini (1992) discusses the regulation of the
transitional states of the nucleation ability of centrosomes, and
the tubulin pool, for the formation of functional microtubules
during meiosis I and II. The hormonal follicular environment
is regarded as very important in this respect. He considers 11
pre- and periovulatory stages as subject to perturbation and
leading to various detrimental consequences, and states: 'The
structural transitions that have been detected coincide chronologically with key endocrine events in the developing follicles.
These events include the generation of oestrogens through the
aromatase system at the preantral to antral stages of follicular
differentiation, the changes in the steroidal milieu of the
preovulatory follicle elicited by gonadotrophins and environmental changes associated with transfer of the cumulus-oocyte
complex from the follicle to the oviduct'.
Albertini and Gaulden stress the need for a complete
understanding of all aspects of ovarian physiology and biochemistry as well as molecular and cytological genetics to
understand the complex interplay of xenotoxic agents. A
simple change in circulation, hormone balances or microtubule
assembly may set in motion a disastrous series of events
leading to a trisomic child, and have no relationship to point
mutations or DNA repair. Chiasma formation and DNA repair
may be fundamental causes of some trisomies, especially if they
influence the association of homologous chromosome pairs.
Indirect evidence of repair systems in oocytes
DNA repair systems are known to be present in oocytes, and
their activity can be influenced in various ways. Some of
these are genetic, and others are environmental. The distinct
possibility that DNA repair in oocytes may be 'turned on' by
radiation damage is hinted at in a paper by Fritz-Niggli and
Schaeppi-Buech (1991). An adaptive response to the effects
of low doses of X-rays (0.02 Gy prior to 2 Gy) was observed
in Drosophila melanogaster oocytes and suggested the existence of 'a repair stimulating effect' of low doses for both the
repair-deficient strains as well as for the highly radiosensitive
mature oocytes. This may not be unlike the mechanism in
bacteria where DNA damage is responsible for the inactivation
of DNA repair repressor molecules.
Russell and Russell (1992) have reviewed, in considerable
detail and depth, the effects of radiation and chemicals on
mutation frequency in female mice using the specific locus
test This measures genetic changes or partial loss of function
in seven genes. The information is exceptionally useful as it
provides a measure of 'final' mutation rates after normal repair
and, no doubt, the results of 'error prone' repair. They gave a
value of six out of 536 207 as the spontaneous rate in females
47
M.J.Ashwood-Smrth and R.G.Edwards
which was considerably lower than the spontaneous rate in
males (43 out of 801406). This is an highly significant
difference; although whether it is due to repair or selection is
unknown.
The same authors also analysed the effects of both fractionated and acute X-rays and a number of mutagenic chemicals
(mitomycin, triethylenemelamine, procarbazine hydrochloride,
ethylnitrosourea and chlorambucil) on the specific-locus mutation rate in oocytes. There are clear indications of stagespecific variations in the mutagenic sensitivity of oocytes.
Diplotene oocytes proved to be relatively insensitive, and also
less sensitive than spermatogonia. Efficient repair in oocytes
is one postulated possibility for these differences between
male and female gametes. An early example of variation
between oocyte stages showed that dictyate oocytes and
pronucleate mouse eggs were less sensitive than maturing
oocytes at metaphase and anaphase I and at metaphase II
(Edwards and Searle, 1963). In treated female mice, mutations
induced at any time after 12 days following conception (close
to the onset of meiotic prophase) were associated with nondividing cells (Russell and Russell, 1992).
The dose rate of the applied X-rays exerted significant
effects on mutation frequency. In mature and maturing oocytes,
high dose rates of X-rays proved to be more mutagenic than
in spermatogonia. However, as dose rates were reduced,
mutation rates declined considerably. Among chemical mutagens, ethylnitrosourea and triethylenemelamine were the only
two shown to be mutagenic to mouse oocytes. Again, spermatogonia were more sensitive than dictyate oocytes to these
chemicals. The relatively high proportion of mosaic mutants
seen after mutagenesis with chemicals in oocytes may be the
result of only one DNA strand being damaged.
The effect of ethylnitrosourea as a mutagen on mature
mouse oocytes was also investigated by Lewis et at (1992).
As in the results reported by the Russells, the effect of
the chemical mutagens was less with oocytes than with
spermatogonia. Increased susceptibility to mutation caused by
X-rays and chemicals is clearly stage specific in oocytes and
probably starts about 6 weeks prior to the first conception, at
a stage claimed to be correlated with the commencement of
oocyte growth and maturation.
Several reports have indicated that female germ cells are
uniquely sensitive to certain chemical mutagens such as
bleomycin, adriamycin, planitol and hycanthone sulphate
(Tease, 1992). In a review of the literature on the chromosomal
damage in mouse oocytes following chemicals and radiation,
he commented on how exceptionally difficult it was to make
comparisons from one study to another. He believed that
insufficient evidence existed to make any claim about specific
mutagenesis in female germ cells. It should be pointed out,
however, that Tease's review concentrated on cytological rather
than genetic evidence. In fact, bleomycin was clearly shown
to be a female-specific mutagenic-inducing agent in mice by
Sudman et al. (1992). In male mice, no dominant lethals or
cytotoxic effects were seen with this chemical, even at the
highest doses tolerated. With females, a dose 0.25 times smaller
gave a high yield of mutations and also killed a number of
oocytes. The authors postulated that the diffuse state of the
48
oocyte dictyate chromosome makes the DNA more susceptible
to intercalation, i.e. the action of chemicals which bind
monocovalently to the DNA double helix during DNA replication causing frame shift mutagenesis to occur. Bleomycin is
thought to be an intercalating agent but this is not absolutely
certain. The possibility that gene expression in the maturing
oocyte might be responsible was also discussed along with
ideas concerning cellular barriers and differential histology.
Mutational risks in females and the possibilities of effects
on genomic imprinting have been addressed in a short review
by Wilson (1992). As he pointed out, the obvious point, often
forgotten perhaps, is that 'Eggs contribute the majority of the
zygote cytoplasm so that the maternal genome has the dual
role of supporting zygote development and contributing half
of the zygotic DNA'. Wilson is one of the few biologists who
reviews the implications of mutations that affect mitochondrial DNA.
Direct evidence of DNA repair in oocytes
There are relatively few papers that clearly demonstrate DNA
repair in oocytes. They may be divided into those that show
enzymatic repair activity in oocyte extracts in vitro, and others
that demonstrate in-vivo repair of damaged spermatozoa or
specific damaged to introduced or injected DNAs. Repair
systems also persist after fertilization.
In-vitro experiments with oocyte extracts
Many of the reports concerning the ability of DNA in vertebrate
oocytes were stimulated, at least to some extent, by experiments
in which either recombination events were studied or because
of interest in the possible integration of injected foreign DNAs.
Most work has been carried out with extracts of varying
complexities obtained from Xenopus oocytes.
Rather than discuss in detail a number of papers, several
have been chosen as they represent the type of molecular
approach which is current. An excellent paper by Matsumoto
and Bogenhagen (1992) illustrates the methodology. Extracts of
Xenopus laevis oocytes were prepared in which the endogenous
nucleotide pools were deliberately depleted. To these extracts
were then added synthetically constructed segments of DNA
that mimicked DNA after damage produced from the formation
of apyrimidine (AP) sites. In other words, the authors presented
the oocyte extracts with segments of abasic DNA (3-hydroxy2-hydroxymethyltetrahydrofuran residues), common in normal
base excision repair. The constructed, deliberately damaged
DNAs were chemically synthesized and added to the oocyte
extracts as covalently closed circular DNA (tetrahydrofuran
cccDNA).
The key findings of this particular study were that repair
was very dependent on suitable amounts of ATP; if concentrations were too low the authors claim that DNA repair was
relatively insignificant and could easily be missed. The
sequence of repair events was very reminiscent of that in other
systems. An AP endonuclease is involved first in recognition,
repair complex formation, and strand breakage (single strand).
DNA synthesis, excision and ligation then follow in a normal,
DNA repair by oocytes
sequential manner. Base-pair mismatches may occur naturally
by recombinational events.
The presence of different repair systems in extracts of
Xenopus oocytes has been inferred by experiments by Varlet
et al. (1990) in which heteroduplex substrates, derivatives
of M13 bacteriophage DNA containing mismatches, were
analysed. C/A and T/C mismatches were repaired more actively
than others. It was suggested that different repair systems were
operational or, perhaps, that there were 'different modes of
mismatch recognition'.
Matsumoto et al. (1994) have subsequently isolated and
characterized a number of the enzymatic steps involved in the
repair of abasic DNA by Xenopus extracts. Five fractions were
isolated and three were essentially purified. The purified
fractions were proliferating cell nuclear antigen (PCNA), AP
endonuclease, and DNA polymerase delta. Both natural AP
sites and the artificially presented tetrahydrofuran cccDNA
was repaired. Further evidence suggested that two independent
pathways exist in Xenopus oocytes for the repair of damaged
DNA. One is a PCNA-dependent pathway and the other is
DNA polymerase beta-dependent. The authors also comment
on the other interesting functions now ascribed to PCNA, a
molecule of considerable biological importance.
A technique for the isolation of large quantities of Xenopus
oocyte nuclei has recently been described (Lehman and Carroll,
1993) and this should be a considerable help to researchers in
the field of DNA repair and enzymology. A cytoplasm-free
extract, obtained in considerable yield, was shown to permit
'the complete recombination of linear, terminally homologous
DNA, as observed in injected oocytes'. A number of other
biochemical functions were also supported by this oocyte
extract including transcription, repair type DNA synthesis and
chromatin assembly, amongst several other functions. In the
authors' words, 'it should expedite the purification of components found in oocyte germinal vesicles, including proteins
required for homologous recombination'.
Oocyte repair of damaged DNA in vivo
Research under this heading includes studies in which oocytes
themselves have been subjected to chemical or radiation
damage or have received damaged spermatozoa or injected
nucleic acids. The induction of specific locus mutations in
mice in which the observed biological effects are the net result
of damage and repair have been already considered (FritzNiggli et al., 1991; Russell and Russell, 1992; Lewis et al.,
1992; Tease, 1992; Studman et al., 1992).
Unscheduled DNA synthesis in mouse oocytes during meiotic maturation in tissue culture has been demonstrated by the
incorporation of radioactive thymidine following UV radiation
(Masui and Pedersen, 1975). The damage, mostly pyrimidine
dimers, was considered to be excised and repaired by standard
repair mechanisms. Evidence suggested that repair was greater
at the germinal vesicle stage than at either metaphase I or
II. Manglia and Pedersen (1978) were able to demonstrate
increased, dose-dependent unscheduled DNA synthesis after
UV irradiation in resting oocytes (from 1-2 day old mice) and
growing oocytes (12-13 days). The ratio of DNA synthesis,
as measured by grain counts after the incorporation of tritiated
thymidine, was about 14 times greater in growing oocytes in
comparison with resting oocytes.
Although these experiments were only indirect evidence of
oocyte DNA repair, later work has fully substantiated them.
Guli and Smyth (1989) isolated mouse dictyate stage oocytes
from both young (8—14 weeks) and old mice (12-15 months).
They were irradiated with UV and cultured in the presence of
tritiated thymidine. Grain counts for unscheduled DNA synthesis confirmed repair which was essentially independent of
maternal age. They commented 'Thus in the female mouse, the
oocytes' capacity to repair UV-induced damage is apparently
maintained at a high level throughout reproductive life'. In
another paper, Guli and Smyth (1988) in similar experiments
detected no UV induced response of oocytes at leptotene,
zygotene or pachytene meiotic stages. In D. melanogaster, a
specific deficiency for nucleotide excision repair following the
treatment of spermatozoa with methyl bromide potentiates
mutagenicity (Ballering et al., 1994).
The importance of 'structural proteins', in a sense functioning not unlike microtubules in organizing and maintaining
chromosome structure and movement, is illustrated in an
interesting experiment reported by Pfeiffer et al. (1994). They
have suggested that 'alignment proteins' are involved in nonhomologous DNA end joining in extracts of Xenopus eggs
during the processes of illegitimate recombination. Thus
enzymes alone are not sufficient for recombinational events.
These proteins are 'postulated to structurally support overlap
heteroduplexes during junction formation'.
Several experiments reporting the specific repair of damaged
DNA by oocytes are of considerable interest. Carroll et al.
(1994) injected a recombinational linear DNA substrate with
terminal direct repeats into Xenopus oocyte nuclei. Most of
the recovered DNA recombination products resulted from
simple exchanges in that there were sharp transitions in
sequences derived from the host and injected DNA. The
authors conclude that 'Because of the considerable evidence
supporting a non-conservative, resection-annealing mechanism
for recombination in oocytes, we interpret the distribution of
exchanges as resulting from long-patch repair of extensive
heteroduplex intermediates'. In another series of virtually
identical experiments the same group of researchers (Lehman
et al., 1994) found that nicks (strand breaks) within the injected
DNA can direct DNA repair. When absent, repair is governed
by the recognition of specific mismatches.
Non-replicating DNA plasmids which had been irradiated
with UV in vitro were rapidly repaired when injected into
Xenopus oocytes (Hays et al., 1990). Most of the repair
occurred during the first 2 h at a rate that was estimated to be
more than 100 times greater than that normally observed with
repair-proficient human cells. Nearly all the repair occurred in
the absence of light and thus enzymatic photoreactivation
could be excluded as having any major role.
Saxena et al. (1990) have also examined the excision repair
of DNA plasmids irradiated with UV. This treatment essentially
produces pyrimidine dimers only (TT, CC and mixed dimers,
CT) in the target nucleic acid. Irradiated plasmids were injected
into Xenopus oocytes before analysis in bacteriophage T4. The
49
M.J.Ashwood-Smrth and R.G.Edwards
oocyte was shown to have 'abundant repair activity'. This
particular study was interesting in that specific enzyme antibodies in conjunction with metabolic inhibitors were used to
establish the enzymology of the repair. It was concluded that
DNA polymerase a was active and this was inferred from
antibody experiments and the use of the specific inhibitor,
aphidicolin. Other inhibitors such as hydroxyurea, cytosine pV
D-arabinofuranoside, and specific inhibitors of topoisomerase
II such as novobiocin, did not inhibit pyrimidine dimer
excision. Neither protein synthesis nor photoreactivation was
involved in the DNA repair of dimers.
Lehman et al. (1993) have studied the recombination of
exogenously injected DNA upon injection into Xenopus oocyte
nuclei and developing eggs. They describe the recombination
as proceeding by a homologous resection-annealing mechanism
depending on the presence and activity of a 5'—»3'exonuclease.
In this instance, a specific enzyme was involved in the ligation
or rejoining processes.
Xenopus oocytes injected with X-irradiated plasmid DNA
are capable of extensive repair of X-ray induced DNA strand
breaks and oxidative-type DNA base damage (Sweigert and
Carroll, 1990). When circular DNA is X-irradiated, its recombination potential with host DNA is stimulated and this is Xray dose dependent. It was concluded that, 'oocytes have
considerable capacity to repair X-ray-induced damage and
some X-ray lesions stimulate homologous recombination in
these cells'.
DNA repair in oocytes and fertilized eggs
DNA repair enzymes are highly active in mouse oocytes
and preimplantation embryos. Inseminated hamster oocytes
have been used to investigate their repair capacity to
damaged human spermatozoa (Genesca et al., 1992). An
individual who had been treated with chemotherapy 3 years
previously was the donor. The DNA repair inhibitory effects
of caffeine were utilized to show that both chromatid and
chromosome aberrations were increased in the inseminated
spermatozoa. The authors stated, 'Since both chromatid-type
and chromosome-type aberrations increase after treatment
with caffeine, damage to human spermatozoa can probably
be repaired inside the hamster egg cytoplasm by pre- and
post-replication repair mechanisms'.
Martin et al. (1988) microinjected human spermatozoa
into golden hamster oocytes in order to analyse pronuclear
chromosomes. Although only a small sample size was
examined, the authors found that microinjection increased
the the number of chromosome anomalies. They sugested
caution and stated that 'The microinjection of spermatozoa
into eggs should not be recommended for clinical use until
further evaluated'. However, a later study that investigated
the microinjection of spermatozoa into the perivitelline space
of human oocytes (Kola et al., 1990) revealed no increase
in chromosome abnormalities in pre-embryos. "These findings
demonstrate that sperm microinjection does not increase the
incidence of chromosomally abnormal eggs and provide
support for the clinical implementation of a technique that
50
appears to be effective for the treatment of certain forms
of male infertility'.
A low frequency of sister chromatid exchanges has been
found after micromanipulative fertilization in the mouse
(partial zona dissection and perivitelline sperm insertion).
This evidence implies that any damage caused to sperm or
oocyte chromosomes is repaired in the egg or embryo
(Hirayama et al., 1994). Today, the intracytoplasmic injection
of a single spermatozoon into an oocyte alleviates virtually
all known forms of very severe male infertility (Silber,
1995). It is obviously welcome to know that DNA breaks
arising in either gamete during the procedure are repaired.
An experiment (Matsuda and Tobari, 1988) in which
mouse spermatozoa were either irradiated with UV or treated
with the alkylating agents methyl and ethyl methanesulphonate before being used to fertilize oocytes showed how the
newly fertilized eggs were capable of repairing some of the
deliberately damaged DNA. The UV effects were enhanced
in the presence of either caffeine or ara-C (arabinofuransoyl
cytosine) inhibitors of enzymatic DNA repair. Alkylation
was less affected, and the authors commented 'The results
indicate a possibility that UV damage induced in mouse
sperm DNA is repairable in eggs during the period between
the entry of the sperm into the egg cytoplasm and the first
cleavage metaphase'.
Further experiments by Matsuda and Tobari (1989)
involving both X-irradiation of either spermatozoa or mature
mouse oocytes and utilizing three inhibitors of DNA repair
indicated the possibility that X-ray damage to both oocytes
and spermatozoa is subject to a variety of different repair
processes in the fertilized egg. The complex changes in Xray sensitivity of mouse oocytes just after fertilization and
before pronuclear formation are probably related to changes
in chromatin configuration and in repair ability. The
considerable differences in radiosensitivity of the male and
female genomes during the formation of the two pronuclei
were not related to differential repair (Matsuda et al., 1989).
Mutagens may cause irreparable genetic or epigenetic
damage to pronucleate mammalian eggs, despite the activity
of their active repair processes. The exposure of pronucleate
and first-cleavage embryos in vivo to certain mutagens can
lead to anomalies later in development. Unlike earlier and
later stages, this time period has been referred to as 'a
window of susceptibility'. It is suspected that in some cases
epigenetic factors are involved. The mutagens fell into two
distinct classes, namely those that induced mid- and lategestational deaths and those that produce hydrops. A number
of the observed defects were due to defects in the normal
sequence of embryogenesis (closure of the neural tube, nonseparation of the cardiac septum, failure of visceral
migrations), and biochemical or cytological causes whose
effect were not obvious. The 'extraordinary susceptibility to
mutagens' of the mouse zygote may, in the opinion of
Rutledge et al. (1992) be related to the formation of
human congenital abnormalities and premature death. These
observations raise questions about the preimplantation period
being relatively free from the action of teratogenic agents.
DNA repair by oocytes
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Received on June 16, 1995; accepted on August 22, 1995
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