Origin of damage

Most damage originates in the male gamete. Unfortunately little is known about the quality of DNA in human oocytes presumably due to the lack of material available for study. A summary of the major causes of DNA damage is shown in Figure 2.

Figure 2 A summary of major causes of DNA damage.

Oxidation of bases is probably the most important source. This damage may be of environmental origin such as UV-A and B, ionizing radiations, or of endogenous origin. The oxidases xanthine, glycolate, amine (EC1.4.3.6), membrane NADPH oxidases and mitochondrial metabolic activity (OXPHOS) generate ROS. The most common damage observed in DNA oxidation is the formation of 8-oxo-deoxyguanosine (8-OHdG), however other oxidation products may also be detected (Table 1). ROS induces the formation of AP (apurinic/apyrimidic) sites, the most common decay in damaged DNA. ROS can also induce the formation of adducts (Table 1). Peroxidation of membrane polyunsaturated fatty acid leads to malondialdehyde (MDA), 4-hydroxynonenal (4-HNE) and 2,3-epoxy-4-HNE degradation products which form MDA guanine, 4-HNE guanine and 1,N ²-ϵguanine and 1,N ⁶-ϵadenine (Table 1). The formation of these adducts and sperm DNA fragmentation appear independent, even if both originate from the action of ROS (Montjean et al., Reference Montjean, Ménézo, Benkhalifa, Cohen and Belloc2010). Oxidation processes can also lead to de-amination of the bases (Table 1), with replication of damaged bases leading to wrong opposing bases. Exogenous agents others than those originating from lipid peroxidation can form DNA adducts: e.g. vinyl chloride or benzopyrene in heavy smokers. Adducts can also lead to the formation of bridges between DNA strands.

Table 1 Degradation of bases by oxidation.

*Methyl cytosine forms thymine after deamination

MDA G, malonaldehyde guanosine; 4-HNE, G adduct derived from hydroxynonenal; Fappy; formamidopyrimidine; 1,N ⁶ϵ ethenoA, 1,N ²ϵ G, derived from epoxy-4-hydroxy-2-nonenal. 8-OHdG

Replication errors are also an important cause of AP site formation or mismatched pairing. In fact we may have primary damage, AP site, modification of a base, i.e. oxidation or deamination, that affects the primary structure of DNA. Damage can also affect the secondary structure, single-strand or double-strand breaks and the tertiary structure, i.e., the spatial configuration of the helix: bridges (adducts cancelled). The mechanism of action of DNA repair will depend on the type of decay. There may be direct reversal for lesions in the primary structure, or more complex processes for single-strand damage and double-strand breaks, the most toxic lesions. Decay may be recognized immediately, but more often at the time of replication and/or transcription. As there is none or very little transcription during the first stages of division (Ao et al., Reference Ao, Erickson, Winston and Handyside1994; that anyway only concerns the sex chromosomes), the transcription block DNA repair activity is probably weakly represented. Shortening of telomeres is a specific DNA decay linked to senescence, which affects both secondary and tertiary structures. Telomeres (TTAGGG) in repeats are moreover very sensitive to oxidation due to the high G content.

Direct reversal of damage (DDR)

In the oocyte and early embryo, this type of repair mechanism is involved in anomalies in the methylation of bases. As oocytes have high methylation activity and imprinting is mainly the result of methylation, this process is of extreme importance (Ménézo et al. Reference Ménézo, Khatchadourian, Gharib, Hamidi, Greenland and Sarda1989; Benkhalifa et al., 2009). A rather high expression of the enzymes ABH2 (60 times background) and ABH3 (20 times background) involved in DDR has been observed in human oocytes. These di-oxygenases remove 1-methyladenine and 3-methylcytosine from methylated polynucleotides and then un-modified bases are replaced. Methylguanine methyltransferase (MGMT) that reverses 6-O-methylguanine is also expressed in human oocytes at 30 times background signal. It can remove alkyl substituent up to benzyl (C⁶ aromatic). The correct duplication of the genome is particularly important at this time in development and it is not surprising that several mechanisms are employed for repair.

Single-strand damage repair (SSR)

This pathway is used when one strand only has been damaged. Although less important than double-strand breaks (DSB), SSR needs to be carried out rapidly in order to avoid the creation of DSB at replication. Damage can occur at the level of the sugar moiety as well as at the base (see earlier). Topoisomerase may be a source of single-strand breaks (SSB). First the damage needs to be recognised, followed by an incision and then an excision. The complementary strand is used as a pattern to fix the repair. When the repair involves large pieces of strand, following the removal of the damaged zone, polymerase(s) fills the hole and ligase(s) complete the repair.

Base excision repair (BER)

Removes the damage at a single base level (primary structure oxidation, adduct. . .). After recognition of specific lesions in the DNA, excision is performed near the damage site. As seen in Table 1, uracil can be formed after deamination of cytosine. Uracil-DNA glycosylase UNGs recognize the U damages. The mitochondrial protein, UNG1 is found at 200 times background in human oocytes, while the nuclear protein UNG2 is expressed 100 times background in human oocytes (Ménézo et al., Reference Ménézo, Russo, Tosti, Mouatassim and Benkhalifa2007a). N-methylpurine-DNA glycosylase (MPG), that recognizes ethenoadenosine, hypoxanthine and 3-Me-A, is highly expressed at 100 times background, while OGG1 (8-oxoguanine DNA glycosylase), involved in 8-OHdG and Fappy G damage recognition is moderately expressed and found at 15 times background. For other recognition proteins see (Ménézo et al., Reference Ménézo, Russo, Tosti, Mouatassim and Benkhalifa2007a). After damage identification, a scaffold is built. AP1 (or APEX/ref1) the major endonuclease initiates the repair, in association with XRCC1, the ligase accessory factor having a main ligation function for ligases 1 and 3, that manage sealing after polymerase beta action. All these components are expressed at a medium intensity at 15 times background in the oocyte except for polymerase beta, which is expressed at 350 times background.

Nucleotide excision repair (NER)

The NER pathway repairs bulky zones of damage that distort the shape of the helix. Large excisions of up to 30 nucleotides including the damaged zone may be repaired. In human oocytes two pathways are active: global genomic NER (GG-NER) and transcription coupled NER (TC-NER), which differ only in their recognition of helix distortion. These pathways are important as severe human diseases result from in-born genetic mutations of NER proteins. Pyrimidine dimers are the main targets removed by NER. In GG-NER, a complex between RAD23, an excision repair protein and xeroderma pigmentosum, complementation group C (XPC), a protein binding to damaged DNA is formed for distortion recognition. Both are highly expressed in the oocyte. Damage-specific DNA binding proteins 1 and 2 (DDB1 and DDB2) can also recognize certain types of damage. DDB1 is expressed at 50 times background; DDB2 is absent. This pathway is preferentially used for non-transcribed strands and transcriptionally inactive DNA. DNA-damage binding (DDB) and XPC–Rad23B complexes permanently check helix distortions in the genome.

The TC-NER pathway repairs the transcribed strands of transcriptionally active genes; it is more rapid than the GG-NER pathway. During pronucleus formation only sexual chromosomes are transcriptionally active. TC-NER and GG-NER pathways differ only in their initial steps of DNA damage recognition. TC-NER starts when RNA polymerase is blocked at the DNA lesion, without any further recognition signal. Next, a complex of human repair protein XPA (xeroderma pigmentosum, complementation group A) and replication protein A (RPA) is formed: surprisingly its expression is very high (45 times and 500 times background), much higher than the XPC–RAD23 complex. The repair patch size for mammalian cells in vivo is around 30 nucleotides.

For both GG-NER and TC-NER, DNA polymerase delta or epsilon fills the gap using the other strand as a template by copying the undamaged strand. Proliferating cell nuclear antigen (PCNA) assists DNA polymerase in the reaction and RPA protects the other DNA strand from degradation during NER. Finally, DNA ligase seals the nicks to finish NER. Whereas expression of PCNA is high (800 times background), the expression of both polymerase delta or epsilon is of medium intensity (10 to 30 times background).

Mismatch repair (MMR)

Mismatch repair fixes errors in DNA replication (mispairing including G/T or A/C pairing). Mismatches are commonly due to tautomerization of bases during synthesis. During this process, large regions (of up to 1000 base pairs) of DNA containing the damage are involved and can be removed: much in excess of the mismatched nucleotides only. This system repairs abnormal insertion, mis-pairing, but also O ⁶-methylG, O ⁴-methylG, 8-oxoG, interstrand crosslinks, thymidine dimerization and deletions. The damage is recognised in relation to the deformation and anomalies of the helical tertiary (spatial) conformation. An important difference with NER is that the removal process may involve from a few nucleotides up to 100s and even a 1000 base nucleotides. The damage has to be first recognised. MSH2–MSH6 (MutSα), for base substitution and MSH2–MSH3 (MutSβ), for loop repair, are protein complexes that recognise the error on the daughter strand and bind the failed DNA. MSH 2, 3 and 6 are highly expressed at 50 times background for each. The DNA clamp that increases the efficiency of DNA synthesis, PCNA (proliferating cell nuclear antigen) is very intensively expressed (>800-fold the background signal). PCNA is the sliding clamp for DNA polymerase delta or epsilon in this process. This process minimizes the risk of induction of the apoptosis pathway. The exonuclease 1 (40 times background), the flap structure-specific endonuclease FEN1 (200 times background), the replication protein, RPA1, (500 times background), replication factors (RFCs) and DNA ligase 1, ATP dependant (LIG1, moderately expressed at 15 times background), are all active in this process. Removal of the main oxidation product 8-OHdG is performed by this process rather than the OGG1 base excision repair process.

Double-strand breaks (DSBs)

The precise replication of the genome during S-phase is of fundamental importance especially in the 1-cell embryo. Double-strand breakage is probably the most important type of DNA damage as it induces chromosomal instability and failed rearrangements. DSB repair is performed by two sub-pathways. Non-homologous end joining (NHEJ) and homologous recombination (HR). These pathways have different relative importance. HR is active during the late part (S/G₂) of the cycle, when sister chromatids are available as a template for repair (Rothkamm et al., Reference Rothkamm, Krüger, Thompson and Löbrich2003), whereas NHEJ, predominantly active in G₁, is also active throughout the cell cycle. Thus, the phase of the cell cycle determines by and large what type of repair mechanism is operative. HR seems to be the most active system in mammalian cells, when replication fails or blocks (replication fork or replication stalling, see Arnaudeau et al., Reference Arnaudeau, Lundin and Helleday2001). Finally, NHEJ appears a less reliable mechanism with deletions appearing at the site of the break leading to translocations.

Homologous recombination (HR)

The cell cycle arrest checkpoint at G₁/S or G₂/M allows for correction of error or damage before cell division, especially when stored mRNAs can be a limiting factor. Ataxia telangiectasia mutated (ATM) is a sensor activated by DNA double-strand breaks. It initiates activation of the DNA damage checkpoint Chek2, which is highly expressed. RAD51C, also highly expressed, interacts with BRCA2 or 1, to form complexes for homologous pairing (Yoshida et al., Reference Yoshida and Miki2004). The complex formed uses the undamaged strand as a template for repair. HR passes also through the formation of a complex including MRE11A (3′ exonuclease, highly expressed), Rad50 (ATPase, moderately expressed) and NSB1. Failed damage repair may lead to cell cycle arrest or apoptosis (p53 linked). Another recruitment for HR passes through interaction of BRCA1 and FANC1/FANCD2.

Non-homologous end joining (NHEJ)

The sensor is the heterodimer KU70/80 (helicase) having a low (ku80, less than 10 times background) or nil (Ku70) expression in the oocyte. The DNA cross-link repair ‘Artemis’ (DCLRE1C, 15 times): DNA-PKcs endonuclease, which cleaves DNA loops, flaps and gaps, has an intermediate expression level. The flap structure-specific endonuclease FEN1 is highly expressed as well (200 times background). The recruited ligase LIG4 (20 times background) and its accessory factor XRRCC4 (80 times background) are well expressed, but their interacting factor (NHEJ1) is not expressed. This pathway is probably not very active as some ‘pieces of the puzzle’ appear to be missing. This situation is in contrast to the mouse model in which sperm-derived DSB are mainly repaired by NHEJ (Derijck et al., 2009). KU70 expression increases significantly at the blastocyst stage in the human (Jaroudi et al., Reference Jaroudi, Kakourou, Cawood, Doshi, Ranieri, Serhal, Harper and Sengupta2009) probably indicating a switch in DNA repair pathways at this time in development.

Cell cycle checkpoints are unlikely to function in the early preimplantation embryo (Delanthy et al., Reference Delhanty and Handyside1995; Harrisson et al., Reference Harrison, Kuo, Scriven, Handyside and Ogilvie2000) as the mRNA turnover is ineluctable and there is a race against time in order to avoid loss of mRNAs. Furthermore, the mechanism may induce too much delay at the G₁/S and G₂/M checkpoints and cell division is already slow. The cell cycle checkpoints required for cell cycle arrest are rather well expressed, Chek1 at 100 times background signal and Chek2 at 10 times background signal.

Repair of DNA- protein crosslink

TDP1 (tyrosyl-DNA phosphodiesterase) hydrolyses the phosphodiester bound at 3′. It is found at stalled topoisomerase I (Top1)-DNA covalent complexes. It requires phosphorylation by ATM for optimal function (Das et al., Reference Das, Antony, Gupta, Dexheimer, Redon, Garfield, Shiloh and Pommier2009). TDP is present at 150 times background.

Reorganization of chromatin structure

After DNA repair (especially DSB) the tertiary structure of the nucleus has to be re-established. H2AFX contributes to histone formation and therefore the structure of DNA. Surprisingly, other key DNA damage response genes 5ATM, ChK1 are found on the same region of chromosome 11. It is highly expressed in oocytes at >100 times background. CAF-1 is another complex thought to mediate chromatin assembly in DNA replication and DNA repair with a high expression of 300 times background.

Telomeres

Telomeres are TTAGGG repeats, capping the chromosome ends and therefore preventing end-to-end fusion. As mentioned earlier, the high number of G moieties renders them highly sensitive to damage from ROS. Defence against ROS decreases with age, which could be a reason for chromosome telomere shortening with age. According to Liu et al. (Reference Liu, Trimarchi, Smith and Koefed2002) they shorten at each round of DNA replication as mitochondrial dysfunction leads to telomere attrition probably again via insult from ROS. However, oocytes are ‘generated’ during fetal life. Telomerase, a reverse transcriptase prevents their shortening. Telomeres are short in the oocyte and telomerase activity and mRNA expression not found in the oocyte, start at blastocyst formation. An alternative lengthening of telomeres (ALT) involves telomere–telomere recombination (Keefe & Liu, Reference Keefe and Liu2009). Damage repair proteins i.e. RAD50, MRE11 (MRE11 meiotic recombination 11), Bloom and WRN are partners in this process in association with extensive telomere sister-chromatid exchange (T-SCE). They are all expressed in the oocyte (RAD, 100 times background; MRE11, 65 times background; Bloom, 30 times background; WRN, 30 times background). According to Keefe & Liu (Reference Keefe and Liu2009) and Liu et al. (Reference Liu, Bailey, Okuka, Muñoz, Li, Zhou, Wu, Czerwiec, Sandler, Seyfang, Blasco and Keefe2007), the age-associated meiotic defects observed in oocytes from older patients might be linked to oxidative stress, aberrant DNA damage response and telomere recombination, leading to miscarriages and infertility.

In vivo and in vitro protection against ROS in early human preimplantation development

Even under physiological, in vivo, conditions, the human genome is continuously subjected to aggression. In IVF/ICSI, in vitro manipulations may increase these insults by generating ROS (Pabon et al., Reference Pabon, Findley and Gibbons1989; Muratori et al., Reference Muratori, Maggi, Spinelli, Filimberti, Forti and Baldi2003). For example, spermatozoa are not protected when the seminal plasma is removed. The early embryo has to be protected against newly generated damage, especially as it also generates ROS as a byproduct of its own metabolism. Enzymatic and non-enzymatic defences are present, for example hypotaurine, pyruvate and vitamin E are found in the environment surrounding the male and female gametes. Albumin is also thought to protect the gametes. The oocyte has endogenous protection; mRNAs coding for Cu–Zn (in the cytosol) and Mn SOD (mitochondria) are present in the oocyte, as is glutathione peroxidase. We did not find any transcripts for catalase in human oocytes (El Mouatassim et al., Reference El Mouatassim, Guérin and Ménézo1999). NADPH is furnished by LDH to regenerate reduced glutathione. Thioredoxins (TRXs) are also highly expressed. TRX increases cell growth and resistance to programmed cell death (Powis et al., Reference Powis, Mustacich and Coon2000). In the tubal fluid, hypotaurine, taurine, catalase, SODs, glutathione synthase and glutathione peroxidase are also actively present, probably released by tubal epithelial cells.

In vitro, the constitution of the culture medium is of paramount importance. Protection against damage may be afforded by addition of antioxidants such as hypotaurine, vitamin C and albumin. As the oxygen tension in vivo is less than 10%, thought should be given to this parameter in vitro even though it does not appear to be important before genomic activation. The sulphur amino acids are important for regeneration of glutathione, especially as cystathionine beta synthase (the pathway regenerating homocysteine and forming cysteine) is absent in the human oocyte. It has been reported that ‘essential amino acids’ including cysteine and methionine, are ‘toxic’ for early embryos (Lane et al., Reference Lane, Hooper and Gardner2001) and many commercial media are produced without methionine. We believe this factor to be an error in human in vitro culture medium. For example, lack of methionine prevents formation of endogenous anti-ROS protectants by the embryo and induces caspase-dependent or -independent apoptosis (Liu et al., Reference Liu, Hoestje, Choo and Epner2003). Absence of methionine prevents a normal imprinting process, which could lead to genetic problems (Ménézo et al., Reference Ménézo, Elder, Benkhalifa and Dale2010). Finally, deficiency in folic acid (vitamin B9) in culture medium may induce thymidine starvation (O'Neill, Reference O'Neill1998) and a consequent decrease in gene expression. Equally important is the role of folic acid in homocysteine (Hcy) recycling. In addition to defective methylation, Hcy and folate deficiency are involved in cellular dysfunction and ROS linked apoptosis.