Introduction

The impact of male infertility on human reproduction is still a matter of debate. For many years, male fertility has been defined as the ability of sperm to fertilize oocytes that develop into early cleavage stage embryos. In human IVF, the development of two to four cell embryos was considered to be a test for sperm fertilizing ability, with the assumption that all of the resulting embryos had the same developmental potential, irrespective of sperm and oocyte quality. However, it is now admitted that differences in male fertility are not related simply to sperm penetration failure, but also to the ability of the oocyte to support zygote development post fertilization. Attention has therefore been more carefully focused towards the quality of sperm DNA: a range of methods have been developed to evaluate paternal DNA integrity that are independent of all semen parameters including sperm morphology, concentration and motility [1]. The same conclusion is reached irrespective of the methodology used to examine DNA integrity: sperm DNA fragmentation and decays related to reactive oxygen species (ROS) lead to reduced fertility [2, 3], and ROS have a heavily deleterious impact on sperm DNA [4, 5]. A major end product of ROS damage to DNA is the formation of apurinic/apyrimidic (AP) sites [6, 7], but 8 oxo deoxyguanosine (8 OXO dG or 8 OH dG) can also be produced. We recently described the formation of ethenoadenosine and ethenoguanosine (1,N 2-etheno-2′-deoxyguanosine (ɛdGuo), and 1,N 6-etheno-2′-deoxyadenosine (ɛdAdo) in human sperm [8]. Shortened telomeres, in relation to age and sperm DNA fragmentation [9] may lead to inappropriate end to end fusion and the formation of chromosome bridges. On the other hand, paternal DNA always carries a varying level of DNA strand breaks [1–10]. These decays are the result of ROS and/or anomalies of topoisomerase activity during spermatogenesis. Other types of decays may arise, especially in sperm with high teratospermia. Demethylation mediated by DNA repair is also a potent promoter of epigenetic activation [11].

The worldwide use of ICSI raises questions regarding oocyte DNA repair activity. All DNA lesions have profound effects on cell viability, leading to apoptosis and then cell death, which is a recurrent feature in IVF. The most highly detrimental situation is probably when the cell tolerates these unrepaired decays—this leads sooner or later to mutations and oncogenic transformation. Fortunately, mammalian cells, including oocytes, have systems that allow DNA repair [12–15]. DNA repair is probably one of the most important processes taking place during and post-fertilization in the oocyte and zygote, in order to allow complete embryonic development and avoid developmental arrests. The quality of maternal DNA during oocyte maturation at the genetic and epigenetic level is a matter for conjecture, but there is indirect evidence showing a positive effect of antioxidants on oocyte quality [16]. GV oocytes collected during a stimulated IVF cycle are unsuitable for treatment and are therefore surplus; therefore, primarily for ethical reasons as the French law forbids any type of work destroying human embryos, these were selected to examine the expression profile of genes involved in DNA repair activity. However, the mRNA content in GV oocytes is the same as that of MII oocytes, since there is no transcription during the final stages of oocyte maturation, only a remodeling of mRNA that is basically related to polyadenylation [17]. The first steps of preimplantation embryo development, at least until maternal to zygotic transition (i.e. four to eight-cell stage), are carried out with the use of maternal stores of protein and mRNA [18–20].

Materials and methods

The oocytes were collected from two different IVF units, at the time of preparation for ICSI. The current protocol is semi long analog treatment followed by stimulation with recombinant FSH. The mean age of the patient was 33.1 years. After careful denudation of all oocytes retrieved, any GV oocytes present were used for this study, a total of 160 oocytes. The pooled oocytes were divided into six samples: the first four samples had ten oocytes per batch, and the remaining two samples were allocated sixty oocytes each, in order to increase the starting quantity of mRNA.

The analyses were performed with microarrays, using the Affymetrix HG U-133 plus 2.0 chips. Amplification was performed via double IVT (in vitro transcription, two-cycle cDNA synthesis kit, Affimetrix). Data were normalised using the algorithm MAS5, in order to generate a signal for each of the 54,613 transcripts of the chips (Probe set ID) that was proportional to the level of expression level. A call signal points out and specifies if the gene is expressed at a level above the background. The expression level is given as high, medium or low according to the signal. Housekeeping and spike controls were used for each determination. Expression was interpreted as low when the signal was above the background (Signal = 15) and up to a signal of 200 in at least three of the samples. The expression was classified as medium for a signal between 200 and 1,000; it was considered as high for a signal repeatedly over 1,000. Our references for the human DNA repair genes were based on Wood et al. [12, 21] and Cline and Hanawalt [22].

Results

Out of 54,613 probes tested, the overall number detected was 15,615 (27.7%, range 23.7–33.7). The number of probes at the limit of detection was between 1.2 and 1.5%, with a maximum of 879 with no clear cut absence or presence. No one of the DNA repair probe was in this borderline zone of expression. Five housekeeping gene probes and 17 spikes were used as internal controls. Absence of Amphiregulin (AREG), epiregulin (EREG) and betacellulin (BTC) that are normally present in cumulus cells, was checked in order to confirm that the GV oocytes were completely denuded.

The list of the genes involved in DNA repair activity [12, 21, 22], with their level of expression is listed in Table 1.

Table 1 Expression levels of mRNAs coding for DNA repair in human oocytes
Full size table

Discussion and conclusion

In all eukaryotic cells, DNA damage must be highlighted by DNA sensors that initiate a cellular response. Three options are then open in response to this signal: DNA repair, cell cycle arrest or apoptosis—events that occur frequently in human IVF. In order to maintain genomic stability, the early embryo must have the capacity to fully repair DNA, in particular damage linked to ROS-related decays in sperm DNA. DNA oxidation leads globally to three different oxidized molecules for each of the bases, the most important being 8 oxo guanine, 8 oxo adenine, FapyA and G (aA and G) and thymine glycol. Most of the genes with a proven or a suspected DNA repair activity for these decays are expressed in the oocyte.

Using BER, a multistep pathway involving several protein complexes, the removal of a single base is performed by a DNA glycosylase in the oocyte. The remaining AP site (apurinic/apyrimidic) site is hydrolyzed by an AP endonuclease. The single nucleotide gap is filled by polymerase beta (highly expressed in the oocyte—see Table 1—DNA polymerase, catalytic subunits) and ligated. BER pathways seem able to remove etheno-adducts and other oxidative products. APEX, capable of initiating the repair of apurinic/apyrimidic (AP) sites, the most common decay in damaged DNA, is highly expressed. APEX-1 also known to play an important role in response to oxidative stress, providing the reducing potential necessary for ref-1 activity in relation to thoredoxin:. [6, 23, 24]. The DNA-binding activity of APEX is modulated by a post translational mechanism involving reduction oxidation, and so is at least partly mediated by ROS: the RNAs coding for thiorexdoxin and Thioredoxin reductase and the transcripts for 6 members of the peroxiredoxin family are also present. This suggests that there is a very active complete pathway for the repair of ROS induced DNA decays. Surprisingly, OGG1, which removes 8 oxo DG, does not seem to have a high level of expression. The mRNA for 8-oxo-GTPase is highly expressed (see modulation of nucleotide pools): this enzyme hydrolyzes the oxidized nucleotide 8-oxo-dGTP to 8-oxo-dGMP, preventing incorporation of 8-oxo-dGTP into DNA.

After DNA replication, mismatch (MMR) repair is one of the pathways that is also associated with the repair of 8-oxoG lesions; MSH2–MSH6, highly expressed here, binds the 8-oxo containing DNA substrates, enhances ATPase and increases ADT–→ ADP exchange activity [25]. The oocyte seems to be well equipped for removal of DNA lesions linked to ROS, but not through the most common pathway, OGG1. We recently described the presence of etheno adducts in human sperm [8]. N-methyl purine DNA glycosylase (MPG) mRNA is highly expressed in the oocyte, and this is also true, to a lesser extent, for TDG (thymine DNA glycosylase): both enzymes remove the etheno adducts from DNA. TDP1 (tyrosyl–DNA phosphodiesterase 1), also highly expressed in the oocyte, removes glycolate from single-stranded DNA containing a 3′-phosphoglycolate, suggesting a role in the repair of free-radical mediated DNA double-strand breaks.

DNA methylation is a major epigenetic process, with a particular role in imprinting. SAM synthetase activity is required for DNA methylation activity through DMT (DNA methyl transferase), and has a high level of expression in both mouse and human oocytes and early preimplantation embryos [26]. The mechanism for direct reversal of damage, repairing alkylation damage in one step, is also expressed. This system is of major importance in the control of hypermethylation, i.e. the regulation of imprinting processes.

The oocyte has a full panel of mRNAs coding for Nucleotide excision repair (NER or global genome repair), a multistep pathway that involves more than 30 proteins. TCR, transcription-coupled-repair, is a more discrete pathway, responsible for removal of DNA insults that block transcription. Since there is no transcription before the 8-cell stage, this pathway is redundant at the earlier stages. However, depending on their mechanism of storage, these mRNAs can be translated at later stages post-fertilization. Other genes involved in this NER/TCR pathway are highly expressed: ERCC6(CSB), GTF2H1,2 and 5, and MMS19. Shortening of telomeres has a strong association with sperm DNA fragmentation [9], and this leads to aberrant DNA repair responses. The tumor suppressor gene TP53 plays a crucial role in controlling this inappropriate response to damage, by determining whether the DNA will be repaired, or whether the cell will undergo programmed cell death. Any decrease in TP53 activity will allow a permissive environment, leading to chromosome fusion and the formation of chromosome bridges. We found a surprisingly moderate level of TP53 transcription. TDP1 is a ubiquitous factor involved in repairing covalent topoisomerase I-DNA complexes (DNA-protein cross-links). DCLE1A and B, with DNA cross-link repair activity, are highly expressed (see Table 1: other identified genes with suspected DNA repair activity).

Maintaining a correct three-dimensional chromatin structure is mandatory, as the 3D structure is critical during chromosome segregation. This is especially true in human IVF, where a high incidence of mosaicism is a common feature. Histones are a central feature of transcription regulation, DNA repair, DNA replication and chromosomal stability; BRCA1 and 2, highly expressed in the oocyte, play a central role in maintaining genomic stability (See also Wells et al. 2005 [20]). We also found a very high level of expression for H2AFX (H2A histone family member X) and CHAF1 (chromatin assembly factor 1), both involved in chromatin structure regulation during and after DNA repair. It must be pointed out that several types of regulation, in particular polyadenylation [17–27], will allow these mRNAs to be translated: portions of the mRNA can be lost both pre and post translation. Poly(A) binding proteins and the cytoplasmic polyadenylation elements are highly expressed in the oocyte (up to 500 times the background level). However, mRNA expression usually indicates that the corresponding proteins are being produced and further utilized, especially in the case of the early embryo prior to genomic activation [20].

In conclusion, high permissivity and tolerance for DNA decays is a highly detrimental situation for oocytes and early preimplantation embryos. Although not all of the mRNAs detected here are fully translated, and we have not considered the level of functional protein repair, the pathways identified are highly expressed, and are somewhat redundant. In human IVF, developmental arrest and implantation failure may be linked to one of the classical responses to DNA decays, i.e. apoptosis [28]. However, when DNA decays reach a very high level in gametes, as is the case in heavy smokers [29], the limits of DNA repair activity might be overreached, with an increase in childhood cancer due to DNA mutations as a potential result. This aspect should not be overlooked in Assisted reproductive technology, especially for IVF/ICSI using poor quality sperm with high levels of ROS-related decays and a high degree of sperm DNA fragmentation [30].

On a final note, growth hormone (GH) up regulates DNA repair capacity in liver cells [31], human naked oocytes and cumulus cells have receptors for GH [32], and co-stimulation with GH has been shown to be efficient in human IVF, especially in patients over 40 years of age where mRNA expression is decreasing. An attempt to increase the DNA repair capacity of the oocyte, perhaps with the use of GH would be an interesting approach to examine.