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Human Molecular Genetics Advance Access originally published online on March 18, 2008
Human Molecular Genetics 2008 17(13):1922-1937; doi:10.1093/hmg/ddn090
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

DNA double-strand break repair in parental chromatin of mouse zygotes, the first cell cycle as an origin of de novo mutation

Alwin Derijck1,{dagger},{ddagger}, Godfried van der Heijden1,{dagger},, Maud Giele1,§, Marielle Philippens2,3 and Peter de Boer1,*

1 Department of Obstetrics and Gynaecology 2 Department of Radiation Oncology, Radboud University Nijmegen Medical Centre, PO Box 9101, 6500 HB, Nijmegen, The Netherlands 3 Department of Radiotherapy, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX, Utrecht, The Netherlands

* To whom correspondence should be addressed. Tel: +31 243610869; Fax: +31 243668597; Email: p.deboer{at}obgyn.umcn.nl

Received January 29, 2008; Accepted March 16, 2008


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
In the human, the contribution of the sexes to the genetic load is dissimilar. Especially for point mutations, expanded simple tandem repeats and structural chromosome mutations, the contribution of the male germline is dominant. Far less is known about the male germ cell stage(s) that are most vulnerable to mutation contraction. For the understanding of de novo mutation induction in the germline, mechanistic insight of DNA repair in the zygote is mandatory. At the onset of embryonic development, the parental chromatin sets occupy one pronucleus (PN) each and DNA repair can be regarded as a maternal trait, depending on proteins and mRNAs provided by the oocyte. Repair of DNA double-strand breaks (DSBs) is executed by non-homologous end joining (NHEJ) and homologous recombination (HR). Differentiated somatic cells often resolve DSBs by NHEJ, whereas embryonic stem cells preferably use HR. We show NHEJ and HR to be both functional during the zygotic cell cycle. NHEJ is already active during replacement of sperm protamines by nucleosomes. The kinetics of G1 repair is influenced by DNA-PKcs hypomorphic activity. Both HR and NHEJ are operative in S-phase, HR being more active in the male PN. DNA-PKcs deficiency upregulates the HR activity. Both after sperm remodeling and at first mitosis, spontaneous levels of {gamma}H2AX foci (marker for DSBs) are high. All immunoflurescent indices of DNA damage and DNA repair point at greater spontaneous damage and induced repair activity in paternal chromatin in the zygote.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Both cell functioning and the environment pose threats to the integrity of the genetic information of the cell. A nucleus can be confronted with a variety of potentially genotoxic damages to which an intricate set of DNA repair mechanisms has evolved (1). A DNA double-strand break (DSB) is the most toxic lesion and is mainly repaired by either non-homologous end joining (NHEJ) or homologous recombination (HR) (2,3).

These pathways are not of equal importance during the cell cycle. NHEJ is operational during the whole cell cycle, while HR functions during late S/G2 (4) ensuing the typical radioresistance found during S-phase (5). DSBs produced by replication stalling are preferably repaired by HR (3,4,6).

Additionally, the cell type and developmental stage influence the balance between DSB repair pathways, making HR relatively more important in pluripotent embryonic stem cells and embryonic stages (7). During adulthood, the contribution of HR to the repair of ionizing radiation (IR)-induced DSBs is only detected when both NHEJ and HR are impaired (7–9).

Analysis of mutant cell lines is often combined with fluorescence microscopy of the DSB marker {gamma}H2AX (4,10–13) and of factors involved in repair and cell cycle regulation. Such proteins show specific nuclear localization patterns after damage induction (14) as they relocalize into IR-induced foci (IRIF) that are implicated in DSB repair. The IRIF forming molecule Rad51, a key protein of HR, is essential for cellular viability (15,16). Rad51 foci are mainly associated with the ssDNA microcompartment (14,17) found within the {gamma}H2AX defined IRIF.

During mammalian spermiogenesis, DNA repair comes to a halt at chromatin remodeling during nuclear elongation, when nucleosomes are replaced by protamins in a stepwise manner (18–20). The length of this repair-deprived period depends on the species and is influenced by time of storage in the epididymis and ejaculation behavior. As a consequence, sperm DNA damage is repaired in the zygote after fusion with the oocyte (21–23).

The zygote has several unique features influencing DNA repair such as: (i) lack of transcription coupled translation, thereby relying on mRNAs and proteins stored in the oocyte (24,25), (ii) Topoisomerase II mediated remodeling of sperm derived chromatin resulting in transient DSBs (26–28), (iii) start of the cell cycle after gamete fusion, lacking a known G1–S checkpoint (29,30), (iv) male and female chromatin is physically separated and epigenetically dissimilar (31–33). Finally, in the genesis of chromosome aberrations (CA), the zygote shows a bias for chromosome type over chromatid-type abnormalities (22,23,34). This is illustrated by UV irradiation, solely inducing chromatid-type abnormalities in somatic cells (35), whereas chromosome and chromatid-type abnormalities were found in the zygote after treatment of sperm (34). Sperm mutagenized by the alkylating agents MMS and EMS, result in only chromosome-type aberrations, strengthening this notion (34). Chromosome-type aberrations point towards NHEJ as the favored repair pathway, a highly active mechanism in zygotes of several model organisms (including mouse) as determined by microinjected DNA fragments (36–38).

In the human, for many mutational endpoints, there is a male bias for induction (39). It has been known for a long time that structural chromosome mutations such as reciprocal translocations are primarily of male descend (40). Another characteristic of the human is its high incidence of de novo reciprocal translocations estimated to affect one in 2000 implantations which is regarded to be an underestimate (41). As assisted reproductive techniques (ART) are becoming habitual in human reproduction, research on zygotic DNA repair has gained a new impetus. Sperm from sub-fertile males has an increased likelihood to contain DNA damage and poses an increased risk for the induction of de novo CA and maybe other mutations as well (42,43).

Here we utilize mouse oocytes defective in either NHEJ or HR for the analysis of cell survival, CA and immunofluorescent signals of {gamma}H2AX, the HR key protein Rad51 and Brca1, an IRIF building DSB repair protein with functions in NHEJ and later in the cell cycle in HR (14,44). This way, we determined the balance between the two DSB repair pathways during the zygotic cell cycle. To probe for NHEJ, the scid mouse was employed, which is characterized by homozygosity for a severe hypomorhic DNa-PKcs allele (45). HR was followed by using double homozygotes for knock out alleles of Rad54 and Rad54B (46). Special attention is given to the behavior of paternal versus maternal chromatin and to the presence of {gamma}H2AX foci in first cleavage chromosomes, suggesting transmission of repair complexes to the two-cell stage. NHEJ and HR are both active and cell biological features of the zygote uniquely influence DNA repair, determining the fate of damaged sperm DNA. When combined with suboptimal oocytes, this could have implications for the genetic outcome of ART.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
A maternal effect on misrepair and non-repair of sperm DSBs at the first cleavage division
Historically, the outcome of DSB DNA repair is measured by chromosome analysis. Zygotic DSB repair can therefore be analyzed at metaphase of the first mitotic cleavage division. To address the relative contributions of NHEJ and HR in the repair or misrepair of sperm DSBs leading to CA, genetic dissection of zygote cytoplasm was applied by use of respectively scid and mRad54–/–mRad54B–/– (henceforth mRad54/54B–/–) oocytes (45,46). C.B17 has a similar genetic background as scid and serves as the appropriate control for the scid DNA-PKcs hypomorphic phenotype (47). C.B17 contains a hypomorphic allele for the NHEJ key enzyme DNA-PKcs not found in most commonly used mouse strains (i.e. C57BL/6, DBA) (48). Functionally, this DNA-PKcs variant acts as a wild-type allele, in contrast to the severely compromised scid DNA-PKcs allele (47). The HR compromised double mutant mRad54/54B–/– was compared with its respective control B6.129, that originated from the same founder population.

According to the expectation (23), 3 Gy irradiation of cauda epididymal sperm (CBA/B6 F1) readily induced chromosome-type and a small minority of chromatid-type CA in the paternal chromosome complement at first metaphase (Table 1, Supplementary Material, Fig. S1). Zygotes of NHEJ proficient strains show similar radiosensitivities (Table 1). Scid zygotes are more frequently affected by a paternal CA (factor 2–2.4). The CA frequency is up by a factor 3.5 relative to C.B17. Maternal deficiency for Rad54/54B had little effect on the outcome of repair of sperm DSBs. However, a significant shift towards chromatid-type abnormalities was found when compared to scid derived zygotes (ratio 0.11 versus 0.45, Table 1). These data demonstrate a role for ooplasmic DNA-PKcs, hence the NHEJ pathway, in the repair of sperm derived DSBs.


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  		Table 1. Chromosome abnormalities at first mitotic cleavage in the male chromosome complement after 3 Gy sperm irradiation

 
The number of {gamma}H2AX positive chromatin domains at sperm chromatin remodeling is affected by the oocyte
After sperm entry in the oocyte, paternal chromatin starts its transition to a nucleosome-based state and forms a pronucleus (PN) that enters zygotic S-phase 5–6 h post-fusion (pf) (Fig. 1A). DSBs, marked by {gamma}H2AX, are frequently found in remodeled paternal chromatin and are more numerous after sperm irradiation or chemical insult (28,49). To determine the influence of genetic background, including NHEJ disruption by scid, on the presence of DSBs at sperm chromatin remodeling, control and irradiated CBA/B6 F1 cauda epididymal sperm was used to fertilize oocytes from four different mouse strains. {gamma}H2AX foci were found in paternal chromatin, ~80 min pf (Fig. 1B and C and Table 2). Maternal chromatin showed no DSB-related {gamma}H2AX foci, as has been reported previously (28). The average number of {gamma}H2AX foci found in control sperm chromatin ranged from 0.9 to 3.8 depending on oocyte genotype (Fig. 1D and Table 2). Oocytes from repair proficient strains (B6/CBA and B6.129) showed similar numbers of {gamma}H2AX foci. Both C.B17 and scid showed an elevated number of DSB-related {gamma}H2AX foci at sperm chromatin remodeling (Fig. 1D and Table 2).


Figure 1
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  		Figure 1. Number of DNA double-strand breaks arising during sperm chromatin remodeling is influenced by ooplasm. (A) Schematic overview zygotic cell cycle. S- and M-phase are indicated (light gray area indicates variation in inter zygotic S-phase timing). Lightning symbol stands for ionizing radiation (IR) used to introduce DSBs in cauda epididymal sperm. (B) Two examples of {gamma}H2AX foci (in fully remodeled sperm chromatin (H3S10ph) 80 min pf of CBA/B6 control sperm with scid oocytes). (C) Two examples of {gamma}H2AX foci 80 min pf of CBA/B6 control sperm with B6.129 oocyte. (D) Quantitative data on {gamma}H2AX foci in sperm chromatin 80 min pf. Oocytes from the indicated genetic background were fused with either control or 3 Gy irradiated CBA/B6 sperm. Triangles represent the amount of {gamma}H2AX foci in remodeled male chromatin from a single zygote. Horizontal bar indicates mean. Numbers are listed in Table 1. Scale bar: 10 µm. B6/CBA data were taken from (28).

 


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  		Table 2. DSBs measured by {gamma}H2AX in remodeled male chromatin 80 min pf of various oocyte genotypes

 
Irradiation of cauda epididymal sperm introduces DSBs that are detected by the oocyte (28). In oocytes from all four genotypes, a significant increase of {gamma}H2AX foci in remodeled paternal chromatin was observed (3 Gy, Fig. 1D and Table 2). The induction level was highest in scid oocytes and intermediate in C.B17 (Fig. 1D and Table 2).

Processing of {gamma}H2AX foci during zygotic G1 shows two classes of DSB repair kinetics
In a somatic G1 nucleus repair of DSBs induced by irradiation or chemical treatment can be observed by measuring the number of {gamma}H2AX foci at different intervals after DSB induction (4,10,11,13). In the B6/CBA hybrid background, processing of {gamma}H2AX foci in paternal chromatin derived from (non-)irradiated sperm showed {gamma}H2AX kinetics comparable to a somatic G1 nucleus (28). To address whether differences in CA frequency at first mitotic cleavage (Table 1) could be explained by altered G1 repair kinetics, {gamma}H2AX foci in early (~210 min pf) and late (~285 min pf) G1 male pronuclei (PN) were determined. B6/CBA oocytes fertilized with non-irradiated sperm showed a low incidence of DSB-related {gamma}H2AX foci throughout G1 (male PN, Fig. 2C and Table 3). The increased frequency of {gamma}H2AX foci in remodeling paternal chromatin pf with C.B17 and scid oocytes persisted into the G1 male PN stages (Fig. 2A and C and Table 3). In addition, at late G1, a significant increase in the number of {gamma}H2AX foci was observed in scid zygotes (Fig. 2C and Table 3). After fusion with 3 Gy irradiated sperm, both B6/CBA and B6.129 showed a reduction in {gamma}H2AX foci during G1 (Fig. 2B and D and Table 3). Although the reduction in number of {gamma}H2AX foci is initially faster in the B6/CBA oocytes, both B6/CBA and B6.129 had similar levels of {gamma}H2AX foci in late G1 (Fig. 2D and Table 3). The DNA-PKcs hypomorphic C.B17 and NHEJ severely hypomorphic scid oocytes showed a reduction in early G1 followed by a significant rise at late G1 (Fig. 2D and Table 3).


Figure 2
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  		Figure 2. Dynamics of {gamma}H2AX foci during zygotic G1. (A) {gamma}H2AX foci persist in male chromatin of zygotes derived from scid oocytes at 285 min pf. Male and female PN can be distinguished by DAPI morphology and H3K9me2. Male PN depicted on the left side. (B) DNA-PKcs proficient B6.129 zygotes have only few {gamma}H2AX foci left at 285 min pf in male PN derived from 3 Gy irradiated sperm. DNA-PKcs deficient scid zygotes have an increased amount of residual {gamma}H2AX foci in male PN at 285 min pf. *Second polar body. (C) Amount of {gamma}H2AX foci in male chromatin at 80, 210 and 285 min pf in zygotes derived from control CBA/B6 sperm fused to indicated oocyte strain. (D) Same {gamma}H2AX kinetics plot for 3 Gy irradiated CBA/B6 sperm. Triangles represent the amount of {gamma}H2AX foci in male chromatin from a single zygote. Horizontal bar indicates mean. Numbers are listed in Table 3. Scale bar: 10 µm. Eighty and 210 min pf B6/CBA data were taken from (28). Data for 80 min were imported from Figure 1.

 


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  		Table 3. {gamma}H2AX foci in paternal pronuclei at G1

 
Thus, we were able to distinguish two types of zygotes. One type (B6/CBA and B6.129) was able to reduce {gamma}H2AX foci in number by late G1 (~285 min pf). The other type (C.B17 and scid), initially reduced numbers, followed by an increase to an equal or higher level when S-phase approached.

The homologous recombination mediating protein Rad51 localizes to male and female chromatin during zygotic S-phase
To determine HR activity during the zygotic cell cycle, localization of Rad51 was analyzed in control zygotes. In late G1, ample cytoplasmic but little pronuclear Rad51 staining was found (Fig. 3A). The male PN starts S-phase prior to the female PN (50). After the onset of S-phase, bright focal {gamma}H2AX staining was visible in the male PN, coinciding with nuclear transport of Rad51 (Fig. 3B). In mid S-phase, a punctate Rad51 signal was found in both PNs (Fig. 3C). The female PN exits S-phase first, showing diminished {gamma}H2AX staining. No difference in overall Rad51 staining was found at this stage (Fig. 3D). Upon chromosome condensation Rad51 starts to leave the PNs (Fig. 3E) and at M-phase was no longer visible, whereas mitotic chromosomes displayed an overall non-focal {gamma}H2AX staining as has been reported earlier for somatic nuclei (Fig. 3F, 51). Zygotes treated with 2 Gy IR during S-phase (8.3 h pf) were analyzed 1.5 h post treatment, to investigate whether a functional HR response is present in zygotes. Both Rad51 and Brca1 IRIF were found and especially for Rad51, showed colocalization with {gamma}H2AX foci, indicative for an active HR response (Supplementary Material, Fig. S2A and B). Involvement of Brca1 at this stage may also signal NHEJ activity at S-phase (52).


Figure 3
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  		Figure 3. Cell-cycle dynamics of {gamma}H2AX and the HR protein Rad51. Male PN depicted on the left side. Zygotes are derived from control CBA/B6 sperm fertilizing B6/CBA oocytes. (A) 3.5 h pf. Asterisk represents non-fertilizing sperm nucleus. (B) 4.8 h pf. (C) 7.8 h pf. (D) 10.8 h pf. (E) 13.8 h pf. (F) 15.8 h pf. Scale bar=10 µm.

 
Pronuclear stalling and residual {gamma}H2AX foci at first mitotic cleavage as tools to follow DNA repair
Zygotes from B6.126, mRad54/54B–/–, C.B17 and scid oocytes were treated with either the chemical mutagen 4-Nitroquinoline 1-oxide (4NQO) or IR and allowed to progress to first mitotic cleavage that was inhibited by Vinblastine (Fig. 4A). At 20 h pf, PFA spreads were made. In these preparations, CA often are unambiguously present (Fig. 4D, G and I–K) but cannot safely be quantified (in contrast to acetic acid/methanol spread mitotic chromosomes, Supplementary Material, Fig. S1). Staining of {gamma}H2AX depicted a fine punctate chromosome-wide signal on both parental chromosomes as was noted earlier (Fig. 3F). Additional staining for the histone modification H4K20me3 allows the unambiguous distinction between male and female-derived chromosomes (53) (Fig. 4). In these preparations, DSB-related DNA processing was visible by {gamma}H2AX foci (Fig. 4D and F–H). Zygotes that were delayed or blocked in their development showed PNs with high amounts of {gamma}H2AX, Rad51 and Brca1 foci (Fig. 4E and data not shown). At mitosis, bright {gamma}H2AX foci usually were positioned in a single chromatid (Fig. 4D and F). Occasionally, these chromatid foci were found adjacent to a chromatid gap (Fig. 4G). At a much lower frequency, isochromatid {gamma}H2AX foci that are at seemingly homologous locations on single chromosomes, depict chromosome-type lesions (Fig. 4H). Usually the chromosome abnormalities found in these preparations were not associated with {gamma}H2AX foci (Fig. 4D, G, and I–K).


Figure 4
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  		Figure 4. S-phase DNA damage results in residual {gamma}H2AX and stalled PN at first mitotic cleavage. (A) Schematic overview zygotic cell cycle with two types of DNA insult: 1 h 4NQO (or mitomycin C) prior to S-phase and IR during S-phase. ‘PFA spread’ indicates harvest technique (see Materials and Methods). (B–K) Zygotes were derived from control CBA/B6 sperm. Oocyte genotypes and treatments are indicated. (B–E) Paternal chromosomes/PN depicted on left side. Maternal pericentric heterochromatin is positive for H4K20me3. Single-headed arrows indicate {gamma}H2AX foci and double-headed arrows indicate CA. (B–C) Examples of spreads showing no residual {gamma}H2AX foci. (D) Example of spread with residual {gamma}H2AX foci. (E) Stalled PN stained for {gamma}H2AX and Rad51. (F) Chromatid type {gamma}H2AX focus without CA. (G) Chromatid type {gamma}H2AX focus with chromatid gap. (H) Chromosome type {gamma}H2AX focus. (I–K) CA’s were mainly devoid of {gamma}H2AX staining, (I) fragment, (J) dicentric and (K) chromatid quadriradial. Scale bars: 10 µm.

 
The mitotic index and mitotic {gamma}H2AX foci data are presented in Figure 5 and Table 4 including the effect of sperm irradiation as an extra comparison.


Figure 5
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  		Figure 5. Maternal effects on zygotic DNA repair initiated by pre-S-phase and S-phase DNA damage. (A) Survival curves expressing the percentage of zygotes at first mitotic cleavage after IR during S-phase.‘+’ and '*' indicate mitotic index of zygotes derived from 3 Gy IR-treated sperm fused with C.B17 and scid oocytes, respectively. (B) Plots of the frequencies of chromosome and chromatid-type {gamma}H2AX foci at first mitotic cleavage in male- and female-derived chromosomes after IR of sperm (3 Gy) or S-phase (1 Gy), oocyte genotype indicated. (C) Survival curves for 4NQO. (D) Similar to B for 4NQO. Standard error is plotted. Details are listed in Table 4 and Supplementary Material, Table S1.

 


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  		Table 4. Mitotic indexes of zygotes after sperm/S-phase irradiation and 4NQO pre-S-phase treatment

 
C.B17 and scid zygotes derived from 3 Gy irradiated sperm showed a reduction in mitotic index (Fig. 5A and Table 4). Male mitotic chromosomes in these zygotes were characterized by an increased frequency of chromatid-type {gamma}H2AX foci, especially for C.B17 (Fig. 5B). B6.129 and mRad54/54B–/– oocytes fertilized with 3 Gy irradiated sperm neither showed a reduction in mitotic index, nor an effect on the frequency of persisting {gamma}H2AX foci (Table 4 and Fig. 5B).

After 1 Gy irradiation of zygotes during mid-S phase (~8.3 h pf, Fig. 4A), a significant reduction in mitotic index was found for mRad54/54B–/– zygotes compared to B6.129. No significant reduction was found for either C.B17 or scid zygotes (Fig. 5A, Table 4). A dose of 2 Gy reduced the mitotic index of C.B17, scid and mRad54/54B–/– zygotes alike (Fig. 5A and Table 4). In B6.129 zygotes this effect was not found. Persisting focal DNA damage at first mitotic cleavage was analyzed for 1 Gy irradiated zygotes as the lowered mitotic index at 2 Gy (Table 4) likely involves selection against more heavily damaged zygotes. After 1 Gy, both C.B17 and B6.129 zygotes showed no effect (Fig. 5B). For scid zygotes, a minor effect was observed, mainly for chromatid-type foci in male derived chromosomes. The HR deficient mRad54/54B–/– zygotes showed a distinct increase in frequency of both chromosome and chromatid-type {gamma}H2AX foci, especially in paternal but also in maternal chromosomes (Fig. 5B).

HR deficient zygotes are highly sensitive to 4-nitroquinoline 1-oxide (4NQO)
To further determine the importance of HR for zygote development, two genotoxic compounds were used. The first one, mitomycin C (MMC) (54), introduces interstrand cross-links that are mainly repaired by HR (46). At a dose that induced 55–65% cell death in double ko ES cells (46), MMC-treated mRad54/54B–/– zygotes did not show any sensitivity as measured by mitotic index at first cleavage division (Table 4).

Topoisomerase I mediates DNA replication by reducing torsion of the double helix. Topoisomerase I-linked single-strand DNA breaks are short-lived catalytic intermediates commonly referred to as TopoI cleavage complexes (TopIcc). DNA damage such as ssDNA breaks, base adducts and UV photoproducts, can stabilize TopIcc and generate DSBs at replication forks (55 and references therein). HR plays an important role in repair of replication fork associated DSBs (6). DNA damage generated by 4NQO partially mimics UV induced damage and both agents stabilize TopIcc (56,57).

B6.129 derived zygotes are capable of coping with 4NQO-type damage and only show a reduced mitotic index in the higher nanomolar range (440–880 nM) (Fig. 5C and Table 4). However, a distinct induction of chromatid type (440 and 880 nM) and chromosome-type (880 nM) {gamma}H2AX foci was observed in paternally and maternally derived chromosomes (Fig. 5D). At dose levels of 27.5 and 220 nM, mRad54/54B–/–, C.B17 and scid derived zygotes showed a reduction in mitotic index. HR deficient zygotes were most severely affected with hardly any zygotes progressing to first mitotic division (Fig. 5C and Table 4). Heterozygosity for both Rad54 paralogues restored sensitivity for 4NQO to control level (Supplementary Material, Table S2). By mitotic index, both dose levels produced the same trend in sensitivity: mRad54/54B–/–>C.B17>scid>B6.129. (confirmed by trend in proportions test) (Fig. 5C and Table 4).

At 27.5 nM, a small effect on the frequency of foci at first mitotic division was found for C.B17 and mRad54/54B–/–, the latter showing a greater induction of especially chromosome-type foci (Fig. 5D).

Persistence of Rad51 foci after irradiation at the onset of S-phase
In the previous sections, we observed zygotic S-phase to play an important role in the repair of newly formed DNA damage in the zygote. Effects for both NHEJ and HR were found.

To address the balance between NHEJ and HR, zygotes derived from oocytes of both repair deficient genotypes and their controls were irradiated with 2 Gy at early S-phase (~5.8 h pf) and Rad51 foci were counted at two time points at the end of S-phase (t1: ~10.3–10.8, t2: 11.3–11.8 h pf) (Fig. 6A). C.B17, scid and B6.129 showed no statistical difference in Rad51 foci between time points and data were pooled. When comparing residual Rad51 foci in C.B17 (Fig. 6B) with scid (Fig. 6C), a significantly higher number was found for both paternal and maternal PN of the latter (Fig. 7 and Table 5). B6.129 zygotes showed a lower level of Rad51 foci and no difference between male and female PN (Figs 6D, 7 and 8 and Table 5). Zygotes derived from mRad54/54B–/– oocytes showed a clear difference between the two time points. At t1, the male PN contained a very high number of Rad51 foci (Figs 6E and 7 and Table 5) but also in the female PN, the amount of Rad51 foci was elevated when compared with controls. One hour later, Rad51 levels had returned to the control value in the female PN. In the male PN, Rad51 levels remained elevated (Figs 6F and 7 and Table 5).


Figure 6
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  		Figure 6. Irradiation of zygotes at the onset of S-phase results in residual Rad51 foci at late S. (A) Schematic overview zygotic cell cycle with 2 Gy irradiation and t1 and t2 fixation times. (B–F) Rad51 foci level in zygotes derived from indicated genotype showing effect of HR and time of harvest. Male PN on left side. Scale bar: 10 mm.

 


Figure 7
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  		Figure 7. Residual Rad51 foci after 2 Gy irradiation at the onset of S-phase. Triangles represent the number of Rad51 foci in a single PN from male or female origin. Horizontal bar indicates mean. Details listed in Table 5.

 


Figure 8
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  		Figure 8. Ratio between male and female residual Rad51 foci after 2 Gy irradiation at the onset of S-phase. Triangles represent the ratio between male and female Rad51 foci in a single zygote. Horizontal bar indicates mean. Details listed in Table 6.

 


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  		Table 5. Residual Rad51 foci 5–6 h after 2 Gy IR at onset of S-phase

 
The ratio of Rad51 foci between male and female PN gives an indication of the difference in repair capacity of both pronuclei. Because the average male/female ratio was above 1 in all strains (Fig. 8 and Table 6), we determined the number of zygotes with an amount of Rad51 foci in the male PN equal or higher than in the female one (Table 6). The repair proficient B6.129 derived zygotes showed an equal distribution of residual Rad51 foci between male and female PN (Table 6). C.B17 had a slight increase in the number of zygotes with a higher residual number of male Rad51 foci (Table 6), increasing in the order scid, mRad54/54B–/–. This analysis shows that a repair deficient ooplasm more severely affects the male PN than the female PN.


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  		Table 6. Male–female ratio analysis of residual Rad51 foci 5–6 h after 2 Gy IR at onset of S-phase

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Sperm derived DSBs are mainly restored by NHEJ
The analysis of zygote chromosomes derived from 3 Gy irradiated sperm shows an increase in chromosome-type aberrations after fertilization of scid oocytes. The scid maternal effect was found both at the level of the affected percentage of zygotes (2.4-fold) and the frequency of aberrations (3.5-fold) (Table 1). An effect of 4 Gy sperm irradiation in the scid strain has recently been reported by (58). After in vivo fertilization, the frequency of zygotes with chromosome abnormalities was about doubled.

The NHEJ proficient strains, C.B17, B6.129 and mRad54/54B–/– showed similar rates of chromosome-type abnormalities. Therefore, DNA-PKcs stored in the oocyte plays an important role in the repair of sperm DSBs after the onset of fertilization. When comparing {gamma}H2AX foci in remodeling male chromatin with those in the early male PN, zygotes with a maternal wildtype DNA.PKcs allele showed a relative reduction of 0.5–0.8. Both C.B17 and scid derived zygotes showed impaired kinetics, with a reduction of 0.3–0.4. The G1 foci kinetics in wildtype and DNA-PKcs deficient zygotes resembles that of somatic G1 cell counterparts (4,10,11,13). C.B17 is exceptional in that it is different from scid in somatic cells (47) but not so much in zygotes. C.B17 contains a hypomorphic variant of the DNA-PKcs gene, Prkdc (48). This hypomorphic allele does not severely affect DSB repair (47,59). Therefore, the similarity in {gamma}H2AX foci kinetics suggests that repair during chromatin remodeling and early PN development is sensitive for variation in DNA-PKcs activity. However, the hypomorphic allele has no long term effect on genetic stability as shown by chromosome analysis of C.B17 derived zygotes (Table 1). Zygotes derived from either C.B17 or scid oocytes fused with 3 Gy irradiated sperm show a comparable reduction in mitotic index (Fig. 5A and Table 4) and an increase in residual {gamma}H2AX foci at first mitotic cleavage (Fig. 5B and Supplementary Material, Table S1). As no G1 S checkpoint is known in the mouse zygote (29,30), the reduction in mitotic index likely occurs at S-phase as does the repair of the late G1 {gamma}H2AX foci in especially C.B17 (CA frequency is back to control level, Table 1). Somehow, the balance between NHEJ and HR in C.B17 is such that at S-phase, repair suffices to avoid the extra generation of chromosome type CA, at the expense of an increase in chromatid-type yH2AX foci (Fig. 5B).

The reason why induction of HR in scid (see later in this discussion) cannot compensate for impaired NHEJ in G1 is not clear from our data.

We have adopted literature data on CA type and frequency (22) to construct a relationship between yH2AX foci in remodeling sperm and the number of DSBs as indicated by CA at first cleavage, suggesting linearity (Fig. 9). Hence, the fraction of {gamma}H2AX that leads to a CA is constant for the different dose levels. In comparison with data on lymphocytes, for the zygote a greater yield of CA per focus is obtained.


Figure 9
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  		Figure 9. Comparison of {gamma}H2AX foci with number of breaks. The number of breaks was calculated from supernumerary fragments (one break) and dicentrics (two breaks). (A) {gamma}H2AX foci in recondensing male chromatin (zygote) and G1 somatic nuclei (generally accepted induction 1 Gy = 32 foci corrected to 1 Gy = 20 foci based on (83) were plotted against the number of breaks at first cleavage division (zygote) or mitosis (mouse lymphocytes). Mouse lymphocyte data were taken from (84). Linear regression was performed on both data sets. Regression coefficients suggest that a {gamma}H2AX focus in recondensing male chromatin is approximately two times more likely to result in a lasting chromosome type abnormality compared with mouse lymphocytes (B) Blowup of zygotic data set. (*) B6/CBA {gamma}H2AX foci data were taken from (28), the number of breaks was taken from (22). Linear regression indicates that {gamma}H2AX foci in recondensing male chromatin have predictive value for the amount of lasting chromosome-type abnormalities at first cleavage division.

 
Our data show that the role of HR at S-phase in the repair of sperm DNA damage is only small, provided normal alleles for DNA-PKcs are present, although an indication for a small rise in chromatid aberrations was obtained.

The ooplasm effects the fidelity of sperm chromatin remodeling and interacts with sperm DNA damage
A dose response relation between IR treatment of cauda epididymal sperm and {gamma}H2AX foci at chromatin remodeling after gamete fusion has been described before (28). Here we show that the source of the ooplasm influences the level of {gamma}H2AX foci found after chromatin remodeling (Table 2). IR treatment (13,28) and fertilization (60) are stochastic events. Three Gy IR treatment of sperm from different males of the same CBA/B6 hybrid would therefore theoretically result in a comparable increase in the number of {gamma}H2AX foci found at sperm chromatin remodeling in repeated fertilization experiments. In scid oocytes, the difference between control and induced numbers of {gamma}H2AX foci is high compared to the other strains (Table 2). This suggests that in the virtual absence of DNA-PKcs activity, IR induced DNA damage more often results in dsDNA break signaling. A role for DNA-PKcs in safeguarding DNA integrity during the topological changes necessary to facilitate the protamine to histone transition is implied. DNA breaks are a normal phenomenon in remodeling male chromatin after gamete fusion and have been shown to be etoposide sensitive at this stage, indicating the involvement of TopoII at male chromatin remodeling (27,28) Conclusively, there is a maternal effect on the level of DSBs as measured by {gamma}H2AX focus formation at the end of male chromatin remodeling.

Spontaneous DSBs during G1 PN development
Repair proficient zygotes generated with 3 Gy irradiated sperm show a reduction in {gamma}H2AX foci from 80 min pf to early G1 with comparable kinetics to IR treated G1 somatic cells (4,10,11,13). However, in sperm-irradiated C.B17 and scid zygotes and scid control zygotes, a rise was observed at late G1 (Fig. 2C and D and Table 3). This would argue for the de novo formation of male DSBs during G1. In line with this observation, radiosensitivity of mouse zygotes increases from sperm fusion to the pronuclear stage (23) with highest sensitivity at the late G1 PN stage (61,62).

Unfortunately, the S-phase related {gamma}H2AX signal conceals the fate of residual G1 {gamma}H2AX foci (Fig. 3B and C).

When too heavily damaged, the sperm nucleus either disintegrates at chromatin remodeling (63) after sperm entry or chromatin collapses at the onset of replication (64), which could be the consequence of the absence of a DNA damage checkpoint at this stage (29,30).

Unrepaired DNA damage likely persists into next stage of pre-implantation development
IR-induced DSBs in condensed chromosomes are marked by {gamma}H2AX in unfertilized oocytes and somatic cells (28,65). Such {gamma}H2AX foci have been found on chromosomal fragments and in intact metaphase chromosomes. Somatic cells irradiated in S/G2 have been shown to progress through mitosis, with {gamma}H2AX foci located at ends of chromosome fragments (4). In addition, {gamma}H2AX foci have been observed on metaphase chromosomes at fragile sites after replication stress (66). If not inhibited to allow these observations to be made, these cells would have progressed into G1. We regularly found mitotic {gamma}H2AX foci of especially the chromatid type (as also portrayed in 66) on intact chromosomes in control zygotes (Fig. 5). When mouse sperm is irradiated in doses up to 100 Gy, developmental capacity up to the two-cell stage is not impaired but development to the blastocyst stage is (60), mainly due to the heavily unbalanced karyotypes in the blastomeres, that originate from chromosome abnormalities at first mitosis as is summarized in (67) for a series of mutagens in the mouse. Here we found a rise of mitotic {gamma}H2AX foci after genotoxic treatment that was in majority chromatid type on usually intact chromosomes (Fig. 5). The concept of DNA repair that is delayed to the next cell cycle is known from a study that employs in vivo fluorescence of PCNA in UV induced repair (68). Consequently, depending on the fidelity and quality of repair, mutations can arise in the next cell cycle.

NHEJ and HR function in response to S-phase DSBs after IR and chemical insult
Radioresistance during S-phase is attributed to HR repair (5) and, like somatic cells, zygotes show a decrease in radiosensitivity during S-phase (61,62). This was clearly demonstrated in B6.129, which showed no reduction in mitotic index after 2 Gy irradiation (Fig. 5A and Table 4). At this stage, both Rad51 and Brca1 show clear IRIF that partially colocalize with {gamma}H2AX, indicative for active repair of DSBs by HR after IR treatment during S-phase (Supplementary Material, Fig. S2). Further evidence for a functional role of HR in S-phase radioresistence is provided by a reduced mitotic index of mRad54/54B–/– zygotes after 1 and 2 Gy IR (Fig. 5A and Table 4). Analysis of Rad51 foci at the end of S-phase, induced by 2 Gy IR at its onset, confirmed that both NHEJ and HR play a role in DSB repair as the number of Rad51 foci is influenced by deficiencies in both repair pathways (Fig. 7 and Table 5). When we attribute the stronger increase in Rad51 foci for scid relative to C.B17 to a greater activity of HR (that supposedly is better induced with lower NHEJ performance), the reported compensating interplay between NHEJ and HR (4,52,69,70) also exists in zygotes. The contribution of NHEJ after IR in mid-S-phase maybe lower, as a clear reduction of mitotic index was not found at 1 Gy IR in scid and C.B17 (Fig. 5A and Table 4). At 2 Gy, however, all repair-deprived genotypes, including C.B17, showed increased arrest. An increase in the frequency of {gamma}H2AX foci at first mitotic cleavage is especially clear in irradiated mRad54/54B–/– zygotes, another signal for the involvement of HR at S-phase (Fig. 5B).

The lack of sensitivity of mRad54/54B–/– zygotes for mitomycin C (Table 4) was unexpected and needs further investigation. Conclusively, both NHEJ and HR protect the zygotic genome of IR damage at S-phase and strengthen the results found by injection of DNA substrates that have underscored the importance of HR for the zygote (36).

DNA damage generated by 4NQO partially mimics UV induced damage and results in chromatid-type CA (35,36). DSBs are produced at replication forks (55,56). Cells defective for RecQ helicases (like Werner and Bloom syndrome proteins), enzymes facilitating replisome progression and re-initiation of replication after replication fork demise (71), are sensitive for 4NQO (72–74). This sensitivity manifests in reduced survival after prolonged exposure to 260–300 nM (73,74) or 1 h at 525 nM 4NQO (72). Here we find that DSB repair-compromised zygotes are extremely sensitive to 4NQO (Fig. 5C and D and Table 4). The repair proficient B6.129 zygote showed a reduced mitotic index at 440 nM 4NQO followed by the presence of residual {gamma}H2AX foci at mitotic cleavage (Fig. 5C and D and Table 4). HR deficient zygotes were unable to cope with 4NQO dose levels above 27.5 nM, indicative for the crucial role of HR in the repair of these lesions (Fig. 5A and Table 4). Quadriradials are typical for RecQ defective cells (75). HR deficient zygotes already displayed this CA at a dose of 2.75 nM (~20% of the male chromosome complements affected, data not shown). Up to five quadriradials could be observed per male haploid set. In agreement with (58), we found oocytes deficient in both Rad54 and Rad54B to resemble Rad54 deficiency by attracting chromatid-type aberrations that in the present study were most clearly visible after pre S-phase 4NQO treatment. By segregation at mitosis, these quadriradials can resolve as a reciprocal translocation in one daughter blastomere.

In contrast to B6.129 zygotes, both DNA.PKcs hypomorphic C.B17 and NHEJ deficient scid are sensitive to 4NQO, C.B17 more so than scid (Fig. 5C and D and Table 4). Thus, better survival for scid compared with C.B17 (Fig. 5C) suggests improved induction of HR, as was found after irradiation (Fig. 7 and Table 5). Possibly, a combination of partial impairment of NHEJ and insufficient compensatory HR activity in C.B17 governs the enhanced sensitivity for 4NQO compared to scid. Indications for interplay between NHEJ and HR in the zygote, translating into modification of the probability of expanded simple tandem repeat mutations later in life, were found by us before (76).

Conclusively, the data show that zygotic S-phase is extremely sensitive for replication fork demise and highly relies on HR to resolve such damage.

Male and female DNA damage-related differences and S-phase in the genesis of de novo chromosome translocations
A number of observations made in this study point at a difference between male and female chromosome complements in attracting DNA damage at the onset of life:

  1. Extending on our earlier observations (28), we compared {gamma}H2AX foci at male chromatin remodeling with the number of residual breaks [calculated from supernumerary fragments (1 break) and dicentrics (2 breaks for the dicentric and accompanying fragment) at the first cleavage division (Fig. 9)]. A very tight association was observed.
  2. Whether derived from non-irradiated or 3 Gy irradiated sperm, {gamma}H2AX foci during G1 were only found in the male PN. Residual {gamma}H2AX foci at first mitotic cleavage originating from S-phase irradiation and pre-S-phase 4NQO treatment were also more often found in paternal chromosomes (Fig. 5B and D and Supplementary Material, Table S1).
  3. In repair deficient zygotes, radiation-induced residual Rad51 foci at late S-phase were generally higher in the male PN (Fig. 8 and Table 6). A skewed male/female ratio can be explained by the existence of a subset of oocytes that has difficulties in handling male DNA damage at this stage of the cell cycle.
  4. Quadriradial induction at an ultra-low 4NQO dose in male pronuclei of HR-compromised zygotes indicates that reciprocal translocations can efficiently be induced by replication stalling, shedding a different light on the translocation induction found in certain forms of ART (42). At first cleavage, chromatid exchanges potentially segregate into a reciprocal translocation in one of two blastomeres. A 9-fold increase of reciprocal translocation carriers was observed when a large cohort of ICSI pregnancies was followed by amniocentesis (42) (compared with the de novo rate as obtained by 41). Support for their emergence at S-phase is provided by the variance in length of the first cell cycle, with longer cycles related to a lower chance of pregnancy (77), that is observed in IVF clinics the world over. The insight provided here, that an interaction between paternal DNA (pre)-lesions and sub-optimal maternal DNA repair provokes germline mutation induction, has recently been suggested to explain the genesis of bilateral Retinoblastoma from unaffected parents in which cases the de novo mutation was found in the inherited paternal allele. In these families, both parents showed sub-optimal DNA repair. A role for maternal DNA repair could only be explained by postulating a post-gamete fusion maternal effect (78). The vulnerability of the first embryonic cell cycles for replication fork stalling could at sequences prone to copy number variation lead to genetic mosaicism as recently has been observed in concordant and discordant monozygotic twins (79).

The data promote insight into the relation between the fist cell cycle and mutation induction via the characteristics of DSB repair at the onset of mouse development, highlighting the role of stalled replication fork management in especially the male PN. Because of the similarities between mammals in the principles of genetic and cellular transmission at early fertilization and early embryonic development, the results presented here should stimulate comparable mutation research in the human.


   MATERIALS AND METHODS
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Mice
Male CBA/B6 F1 (Charles River) mice served as sperm donors. Oocytes came from the following mouse hybrids and strains: B6/CBA F1 (Charles River), C.B17 (Taconic), scid (Charles River), B6.129 and mRad54–/–mRad54B–/–. The latter two were bred from heterozygous founder mice kindly provided by Dr R. Kanaar (Department of Cell Biology and Genetics, Erasmus MC, Rotterdam, The Netherlands) (46). Mice were housed with adjusted light hours set at 9.00–21.00.

PCR primers genotyping B6.129 and mRad54–/–mRad54b–/– mice
Primers for mRad54 Wt and KO alleles; F1-Rad54 (5'-TGGGACATGTAAGATCGTTGG-3'), R1-Rad54 (5'-CAAGTTCCAGGCCATCTAGG-3'), 54KO2 (5'-TTTGCTTCCTCTTGCAAACCA-3'). Primers for mRad54B Wt and KO alleles; 54B(F1) (5'-TATGATATCGATTGGAACCCAGCTA-3'), 54B-n-(R1) (5'-CACAACTGTCATCCCCAGTG-3') and 54KO2 (5'-TTTGCTTCCTCTTGCAAACCA-3').

Gamete collection and IVF
Gametes were obtained for in vitro fertilization as described before (33). Zygotes were cultured in IVF medium [HTF, 0.5% (w/v) BSA] covered with light mineral oil (Irvine Scientific, no. 9305). Zygotes were transferred to Yamada medium (80) at 5 h pf. For arresting zygotes at first cleavage, Vinblastine-sulphate (Sigma, V-1377) was added to Yamada medium at 0.020 µg/ml. Harvest followed at 20 h pf.

Timing of zygote development
We previously determined (33) that the majority of secondary oocytes was penetrated around 70 min post-insemination (pi). The described stages are timed pf, hence compensated for this lag phase (28).

Carnoy fixation of chromosome complements and karyotype analysis
Chromosome preparations were made from single zygotes by a variant of the Tarkowski (81) air-dry technique that minimized breakage of chromosome complements. Centric heterochromatin was differentiated by C-banding (82) followed by DAPI staining to distinguish between dicentric chromosomes and acentric fragments. The Y chromosome, often longer male chromosomes, and the supposition that sperm irradiation only induces CA in male-derived chromosomes (22) were used in discriminating the two chromosome complements.

PFA-based fixation of zygotes
Whole mount preparations for immunofluorescence (IF) were made as describe before (28).

For paraformaldehyde fixation of sedimented nuclei and chromosome complements (‘PFA spread’, Fig. 4), the zona pellucida was removed with pronase [HTF-HEPES (Cambrex, BE02-022 F), 0.5% BSA 1% Pronase (NBS Biologicals, 9036-06-0)] and zygotes were placed on a slide covered by a film of fixative (1% PFA; 0.1 mM DTT; 0.1% Triton-X100, pH 9.2, in milliQ) in a humid container for 30 min. Thereafter, slides were air dried, washed twice in 0.08% Photoflow (Kodak), air dried once more and stored at –20°C.

Sperm and zygote DNA damaging treatments
DNA-damaged sperm was generated by whole body irradiation of male CBA/B6 F1 mice 1 day prior to sperm isolation for in vitro fertilization. Zygote irradiation was performed using a custom made mobile thermostated CO2 incubator. After irradiation, zygotes were returned to a regular incubator. External beam radiation was used, 6-MV photons of an Elekta linear accelerator (Crawley, UK), with a dose rate of 0.5 Gy/min in doses of up to 3 Gy. The administered dose was verified using TLD (thermo luminescent dosimetry) measurements. Timing of zygote irradiation (pf) is indicated in the figures.

Mitomycin C (Sigma, M-0503) and 4-nitroquinoline-1-oxide (4NQO) (Sigma, N-8141) were dissolved in culture medium at specified concentration. Zygotes were treated for 1 h, starting at 3.5 h pf, washed and placed in a new culture dish.

Antibodies
Mouse anti-{gamma}H2AX, 1:10 000 (Upstate No. 05-636, clone JBW301); rabbit anti-H3S10ph, 1:1000 (Upstate No. 06-570); rabbit anti-H3K9me2, 1:500 (Dr T. Jenuwein); rabbit anti-H4K20me3, 1:500 (Dr T. Jenuwein); rabbit anti-hRAD51, 1:1200 (Dr R. Kanaar); rabbit anti-Brca1(exon 11), 1:250 (Dr C.X. Deng).

Secondary antibodies were applied as following: Molecular Probes, OR, USA: A11001 fluor 488 goat anti-mouse IgG (H+L), A11012 [GenBank] fluor 594 goat anti-rabbit IgG (H+L), both in a 1:500 dilution.

IF and foci quantification
Immunofluorescent staining was performed as previously described (28) on whole mount and pfa spread preparations. At the end of chromatin remodeling (80 min pf), as judged by staining with phosphorylated histone H3S10 (H3S10ph), {gamma}H2AX foci are present in remodeled nucleosomal male chromatin in two varieties: small, not DSB related and large DSB related. The latter were shown to correlate with sperm IR and treatment of early zygotes with DNA damaging compounds (28). The male PN (210 and 285 min pf) is distinguished from the female PN by DAPI morphology and female PN specific histone H3 lysine 9 dimethylation (H3K9me2) (28). Pericentric heterochromatin stained for histone H4 lysine 20 trimethylation (H4K20me3) specifically labels female chromosomes (53). Quantification of foci was performed on coded samples by one observer.

Collection of images
Images were collected with a Zeiss axioplan fluorescence microscope. Pictures were captured by a Zeiss AxioCam MR camera with Axiovision 3.1 software (Carl Zeiss). All images shown are either a single plane derived from stacks with z-axis intervals of 0.4 µm or deconvoluted projections created with Metamorph software version 6, using the nearest neighbor mode. Photoshop (Adobe) was used for correcting background when necessary.

Statistics
Statistical analysis was performed using Prism (Graphpad) and R 2.3.1 (The R foundation for statistical computing, http://www.r-project.org/) software. Details are indicated in the figure legends.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
FUNDING
 REFERENCES
 
Supplementary Material is available at HMG Online.


   FUNDING
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
REFERENCES
 
The Dutch ministry of Health, Welfare and Sport and the EU financed this research.


   ACKNOWLEDGEMENTS
 
We first would like to thank Dr R. Kanaar (Erasmus MC, Rotterdam, The Netherlands) for founder mRad54–/– and mRad54B–/– ko mice and the Rad51 antibody. We would like to acknowledge, Dr T. Jenuwein (IMP, Vienna, Austria), Dr H. Stunnenberg (NCMLS, The Netherlands) and Dr C.X. Deng (National Institutes of Health, Bethesda, USA) for the gift of antibodies; L. van Bolderen (Radboud University Nijmegen MC, The Netherlands) for animal and zygote irradiation; T. Arnoldussen, J. Evers and G. Toenders (Radboud University Nijmegen MC, The Netherlands) for developing the custom incubator for zygote irradiation; P. Borst (Wageningen University, The Netherlands) and E. Commandeur (Tilburg University, The Netherlands) for help with statistics. Dr J. Essers (Erasmus MC, Rotterdam, The Netherlands) and Dr A.H.F.M. Peters (FMI, Basel, Switzerland) are thanked for helpful comments on the manuscript.

Conflict of Interest statement. None declared.


   FOOTNOTES
 
{dagger} Both authors contributed equally. Back

{ddagger} Present address: Department of Neuroscience and Pharmacology, Rudolf Magnus Institute of Neuroscience, University Medical Center Utrecht, Universiteitweg 100, 3584 CG Utrecht, The Netherlands. Back

Present address: Department of Embryology, Carnegie Institution of Washington, 3520 San Martin Drive, Baltimore, MD 21218, USA. Back

§ Present address: Department of Orthopaedics, Radboud University Nijmegen Medical Centre, PO Box 9101, 6500 HB Nijmegen, The Netherlands. Back


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 INTRODUCTION
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 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
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