DNA Repair in Human Germ Cells

DNA Repair in Human Germ Cells: History

Please note this is an old version of this entry, which may differ significantly from the current revision.

Subjects: Genetics & Heredity

Contributor: Guylain Boissonneault

DNA repair
haploid
diploid
gametes

1. Introduction

Maintenance of genome integrity is a permanent cell challenge as both intra- and extracellular conditions can lead to chemical alterations of nucleotides or their sequence. Proper repair mechanisms have evolved so as to maintain a balance between maintenance of cellular function and adaptative processes improving fitness. For obvious reasons, germline cells must be especially proficient at this task as diversity must be transmitted while maintaining the gametes’ integrity through the many differentiation steps. In addition, anisogamy, whereby the two sexes produced highly different and specialized haploid gametes, enhances diversity. Indeed, while diploidy allows gene versions compensation and error-free repair, haploidy is expected to create more variation as there is no templated repair for homologous recombination. Ploidy is therefore a key element to consider for genome plasticity or stability.

2. General Consideration Regarding Ploidy States

Since the turn of the century, knowledge regarding the advantages of different ploidy states has been increasing thanks to the study of lower eukaryotes. Thus, fission and budding yeasts have proved to be valuable models because they, respectively, represent the impact of haploid and diploid genomes on evolution and genetic integrity. The ploidy choice for lifespan appears to be strongly related to the evolution and maintenance of genetic integrity [1,2,3,4]. It is well known that diploidy confers greater fidelity to replication of both genomic and mitochondrial DNA, in particular to attenuate single-nucleotide mutations. However, large chromosomal rearrangements or deletions occur more frequently in the diploid budding yeast impacting cellular fitness. Although there is twice the amount of genetic material in diploid cells, therefore statistically twice as many possible mutations [5,6], observations show that there are only 1.4 times more mutations in diploid cells than in haploid cells [7]. Such a bias is perhaps linked to the faster replication in the haploid state allowing a much shorter repair time. Errors mainly involve AT or CG transversions in late replicating genes [8]. In addition to this mutation bias, acquisition of a recessive mutation that would clearly alter the cell survival or mutation load is reduced by diploidy because two such similar recessive mutations must happen over both alleles to alter the phenotype. However, such recessive mutations will be eliminated in the population, a process coined “heterozygote advantage”.

On the other hand, this would prevent diploid cells from positively adapting to a changing environment [9]. Diploid cells can nevertheless accumulate heterozygous mutations, creating a pool of sequence variants that can be useful in reaction to a changing environment, whereas haploid cells either die or survive. Larger chromosome rearrangements in the diploid cells should be explained by the homologous recombination between chromosomes, which represents the main difference from haploid cells. However, the deletion of RDH54, a protein necessary for recombination between homologous chromosomes, does not alter the mutation rate of diploid cells [7]. This observation may be reconciled when considering the fission yeast S. Pombe, which maintains a haploid life cycle. S. pombe seems to be protected against the instability of the haploid life cycle by spending most of its time in the G2 phase. Indeed, in the G2 phase, sister chromatids are present allowing error-free repair of Double Strand Break (DSB) by Homologous Recombination (HR). Although the alternative to HR is the error-prone Non-Homologous End-Joining (NHEJ), S. pombe promotes HR through stalling at the G2 phase. Deletion of NHEJ repair protein (PKu70^Sp or Lig4^Sp) displays low sensitivity to DSB-inducing genotoxins, indicating that even outside the G2 phases, S. pombe may not rely on NHEJ for damage repair. However, these observations are in contrast to the higher mutation rate observed in haploid cells compared to diploids. For example, DNA microsatellites are 100 times more stable in diploid cells [10]. Another example is the higher rate of mutation found in mitochondrial DNA of haploid cells [7].

One potential explanation is the higher number of mitochondria in diploid cells while mitochondria often mate, mixing alleles versions, rapidly reducing heteroplasmia. In addition, polyploid cells evolve faster than haploids, in relation to chromosomal rearrangement and the aberrant condition of polyploid cells [11]. It, therefore, appears that the difference in the amount of DNA between the ploidy states only partially explains the difference in the mutation rate. Although haploidy allows a faster evolution, this condition is protected by some elements preventing excessive genome instability. Similarly, anisogamy in mammals results in very numerous haploids spermatozoa versus a single diploid ovum before fertilization. As stated above, genetic stability of the ovum is provided by diploidy and a complete array of repair systems, while the haploid character of sperm and the condensed chromatin may provide the proper context for DNA repair errors at the origin of the well-known male bias in the transmission of de novo mutations. In that sense, the ovum stands as the guardian of heredity while spermatozoa are providers of diversity.

3. Repair in Human Germ Cells

3.1. Gametes

Most vertebrates are anisogametic whereby gametes from the opposite sex are specialized and differ in morphology and number. They are therefore expected to respond differently to DNA damage inducers. This is especially the case for mammalian gametes where spermatozoa are ex-soma cells produced in large numbers whereas the oocyte is a resident cell. One additional major difference is the much greater level of DNA compaction provided by protamines in mature sperm following the eviction of most histones whereas oocytes maintain histone-based chromatin.

In addition to the greater number of cell divisions required to produce mature sperm, the peculiar steps of the sperm differentiation program may be more vulnerable to both exogenous and endogenous DNA damage inducers. For instance, endogenous DNA breaks may arise from the change in chromatin structure in post-meiotic spermatids coincident with a decreased DNA repair activity of these cells, whereas oocytes possess a far greater DNA repair capacity and harbour much fewer DNA breaks.

3.1.1. Spermatozoa

Male gametogenesis is a complex differentiation program producing mature spermatozoa from spermatogonia. During meiosis, HR takes place during the late phase of prophase I. At the beginning of prophase I, programmed DSBs allow parental alleles to recombine [204]. After meiosis, spermiogenesis undergoes a Golgi phase whereby spermatozoa head and axoneme are formed. Then chromatin is remodelled with Transition Proteins (TP1 and TP2) followed by protamination where histones are replaced by protamines (PRM1 and PRM2) to achieve the highest level of DNA compaction known to the eukaryotic world.

In spermatozoa, DNA damages, strand breakage, or base alteration may result from (1) Faulty compaction/protamination of DNA [205]; (2) Abortive apoptosis, or “anastasis”, in late spermiogenesis [206]; (3) Oxidative stress [207]; (4) Persistence of enzymatic DNA breaks induce during chromatin remodelling. The contribution of each to sperm DNA damages is still unknown but it becomes clear that an elevated number of DNA breaks is associated with male infertility [208] or with impaired development of the early embryo. Chromatin remodelling in spermatids leads to the replacement of 90–95% of histones by protamines [209]. During the eviction of histones, it is assumed that free DNA supercoils are formed that can be eliminated by the action of topoisomerases [210]. Potential hindrance of topoisomerases catalytic cycle in this context may lead to SSBs in the case of topoisomerase I, or DSBs in the case of a topoisomerase II activity. A possibility also exists that mechanical breaks resulting from the major chromatin remodelling can also be created. Protamination itself can induce damage [211,212] as the PRM1/PRM2 ratio can impact proper DNA packaging [209,211,213]. We, however, generated evidence that DSBs were indeed generated during this process in the whole cell population and harbour a 3′ OH so is consistent with endonuclease digestion. In this haploid context, only direct end ligation repair mechanisms such as NHEJ are expected [214]. Although apoptosis was shown to maintain the germ cell vs Sertoli cell ratio [215,216], canonical apoptosis may not be responsible for the transient DNA breaks shown in spermatids as these are observed in 100% of spermatids. This could represent an “apoptosis-like” reversible mechanism that is reminiscent of “anastasis”, or the recovery from apoptosis described recently [217]. Various degrees of recovery, or repairs, would then modulate the persistence of DNA breaks in the mature sperm. At other spermatogenesis steps, DSBs could be linked to the altered balance of anti and pro-apoptotic factors through different steps of spermatogenesis [218] and to the compartmentalisation of the mitochondria which prevents nucleus-organelles exchanges. Reactive oxygen species (ROS) are a common source of DNA damage. ROS create abasic sites, base modifications, inter-strand crosslinking, and both single and double-strand breaks. However, ROS are essential for some sperm functions including capacitation or acrosome formation and are produced in the sperm mitochondria [219,220]. Sperm and white blood cells are the main sources of ROS in semen. Sperm cells are very sensitive to ROS because of their high content in unsaturated fatty acids and their weak DNA repair activity [207,219]. Antioxidants appear to balance a high level of ROS [221], for this purpose, seminal fluid possesses catalases, superoxide dismutases, and glutathione peroxidases and is rich in vitamins C, E, A, lactoferrin, and Q10. Ultimately, prostasomes can decrease the release of ROS from leukocytes [222]. The relevance of ROS in male infertility is emphasized as 20 to 88% of sub-fertile men show an elevated level of ROS [223].

So far, evidence of NER, BER, SSA, MMR, HR, and NHEJ DNA repair processes has been reported during spermatogenesis.

Important variations in NER activity were reported throughout spermatogenesis. For instance, spermatogonia were shown to be more sensitive to UV than meiotic or post-meiotic cells [224]. 6–4 photoproducts repair activity is also reduced significantly in post-meiotic round spermatids and during aging [224]. This may be linked to a decrease in NER protein expression [225]. Various levels of GG- or TC-NER also exist as 16.8% of CPD are removed from transcribed Scp1 gene within 16 h in rat spermatocytes [226], while 50% of CPD are removed from Dhfr and Dazl transcribed genes in spermatogonia [224]. However, spermatogonia can repair both active and inactive genes on both strands, in contrast to meiotic cells where transcribed genes are repaired. TC-NER is lower in round spermatids than in spermatogonia. These differences may reflect the greater mitotic activity of spermatogonia, and the fact that they are lying outside of the blood-testis barrier and so are potentially more exposed to genotoxins. Chromatin compaction during spermiogenesis may also adversely impact NER [227] as chromatin access is known to hinder NER. NER is therefore present at all stages of the male germ cells differentiation albeit with various efficiency.

OGG1^Hs glycosylase is the only BER factor found in spermatozoa and recognizes 8-oxoG [219]. However, as none of the AP endonucleases exist in spermatozoa, AP sites will likely be processed during the first zygotic mitosis [219].

HR is known for its primary role during meiosis but is also important for DSBs and inter-strand lesions repair in spermatogonia. Outside of meiosis, HR acts in the S and G2 phases of the cell cycle, when sister chromatids are present. HR is obviously absent in haploid spermatids but resumes in the diploid zygote.

In male germ cells, some canonical components of NHEJ are missing resulting in an alternative NHEJ pathway [228]. For instance, Ku70^Hs and 53BP1^Hs are not expressed therefore preventing the DNA-PKc^Hs pathway. PARP-1^Hs and XRCC1^Hs are however expressed in elongating spermatids supporting alt-NHEJ pathway as described by Ahmed et al. [228]. PARP-1^Hs may bind to DSB as a catalytic homodimer bringing together both DNA ends. Then, PARP-1^Hs may recruit the XRCC1^Hsc/DNA ligase III^Hs complex at the DSB site to complete the end-joining.

3.1.2. Oocyte

Since a limited number of oocytes can be retrieved, the DNA damage response in this gamete is studied to a lesser extent. DNA damages arise mainly during the active life of oocytes in prenatal stages, before prophase I arrest, and during recovery from pre-ovulatory meiosis [229].

From the primary oocyte stage to meiosis II and ovulation, oocytes can actively repair DNA [230]. During oogenesis, repair genes are overexpressed, and their mRNA are accumulated to be used in the zygote [231]. NER, BER, MMR, HR or NHEJ repair pathways have been described in oocytes from humans to mice [231,232,233,234]. Proper DNA repair machinery must operate as oocytes undergo chromatin remodelling during fertilisation and early steps of a zygote. Oocytes are diploid cells, that possess homologous chromosomes until meiosis II that arises after fertilisation. Thus, the oocyte is virtually never haploid. The oocyte becomes transiently haploid between meiosis II end and the fusion of parental genomes (syngamy). Thus, following meiosis II, only NHEJ should operate as shown in mice [235], although variations exist between species as rodent oocytes have better DNA repair abilities than primate ones [232]. The repair capacity of the oocyte and mRNA levels for repair proteins also decreases with female age [236]. After fertilisation, syngamy is a critical step since (1) Chromatids from each parent exist in different cellular compartments; (2) Chromatin undergoes major remodelling associated with demethylation; (3) Male derived chromatid is repaired by the oocyte DNA repair machinery [237].

As for spermatogenesis, ROS are produced during oogenesis. ROS production is necessary for folliculogenesis, oocyte maturation, ovulation, and luteal function [238]. Maturation and ovulation are linked to inflammatory processes, characterized by high ROS production [239]. Unlike sperm, oocytes are more resistant to ROS due to an environment rich in antioxidants in the follicular fluid [240]. However, ROS imbalance in the female tract decreases fertility and oocyte quality [241]. In addition to its impact on genetic integrity, oxidative stress is known to alter microtubule function and spindle morphology leading to aneuploidy [242].

In contrast to spermatozoa, OGG1^Hs glycosylase is only weakly expressed in oocytes, as 8-oxoG damages are not present in spermatozoa after fertilization [243]. In many aspects, the early zygote may still be considered as an oocyte since only mRNAs from the oocyte are used up until the 4-cell stage [244]. This “zygotic oocyte” is in charge of repairing DNA damages in spermatozoa but may be overloaded if DNA damages occur in more than 8% of the nucleotide genome amount [245,246].

Both the number and the nature of DNA breaks are of concern for fertility. Whereas SSBs are easily repaired as in somatic cells, DSBs can overload the DNA repair capacity of the oocyte. After fertilisation, the persistence of DSBs in spermatozoa will delay DNA replication or can even be lethal for the zygote [247]. Finally, DSBs are mainly repaired by NHEJ during zygote [214], as for somatic cells.

This entry is adapted from the peer-reviewed paper 10.3390/ijms222212418

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