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© The Author 2013. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com
Male-driven de novo mutations in haploid germ cells Marie-Chantal Grégoire, Julien Massonneau, Olivier Simard, Anne Gouraud, Marc-André Brazeau, Mélina Arguin, Frédéric Leduc and Guylain Boissonneault Department of Biochemistry, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke (Québec) CANADA, J1E4K8 Correspondence should be addressed to: guylain.boissonneault@usherbrooke.ca
Mol. Hum. Reprod. Advance Access published March 20, 2013
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Abstract At the sequence level, genetic diversity is provided by de novo transmittable mutations
that may act as a substrate for natural selection. The gametogenesis process itself is
considered more likely to induce endogenous mutations and a clear male bias has been
demonstrated from recent next-generation sequencing analyses. As new experimental
evidence accumulates, the post-meiotic events of the male gametogenesis
(spermiogenesis) appear as an ideal context to induce de novo genetic polymorphism
transmittable to the next generation. It may prove to be a major component of the
observed male mutation bias. As spermatids undergo chromatin remodeling, transient
endogenous DNA double-stranded breaks are produced and trigger a DNA damage
response. In these haploid cells, one would expect that the non-templated, DNA end-
joining repair processes may generate a repertoire of sequence alterations in every
sperm cell potentially transmittable to the next generation. This may therefore represent
a novel physiological mechanism contributing to genetic diversity and evolution.
Keywords: DNA damage, DNA replication/repair, spermiogenesis, polymorphism, gene
mutations
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The replication hypothesis for male-biased mutation
Although many genetic disorders are transmitted as pre-existing mutations, a significant
fraction of deleterious germ line mutations are created de novo. Being rare, some
mutations however may be important for the generation of genetic diversity that
contributes to adaptive evolution. Germline mutations may arise from several
endogenous or exogenous mechanisms in both male and female. However, owing to the
many more replications cycles of spermatogonia throughout the male reproductive life
and the constant replenishment of gametes, it has become rather intuitive that male
germ line must have a higher propensity for spontaneous mutations as originally
proposed by J.B.S Haldane in 1947 (Haldane, 1947). Replication was initially suspected
because the male-to-female mutation rate ratio is found correlated to the male-to-female
ratio of the number of cell divisions (Vogel and Motulsky, 1997; Hurst and Ellegren,
1998). Aging is therefore expected to increase this so-called “male bias” as the total
number of cell divisions in oogenesis remains constant while there is a linear increase in
total spermatogonial cell division with age. As the difference in the number of
chromosomal replications between male and female increases, the proportion of
paternally-derived base substitutions would be expected to increase as well. A very
recent study using whole genome sequencing of 78 parent-offspring trios revealed that
the de novo mutation rate reaches 1.2 x 10-8 per nucleotide per generation or about 60
mutations per offspring on average. This study confirmed that 75% of these mutations
arise from the father and that this proportion dramatically increases with paternal age at
conception, rising by about two mutations per year (Kong et al., 2012). Alteration of the
male gamete genetic integrity during aging may therefore contribute to the etiology of
genetic diseases and it is worth noting that over twenty autosomal dominant disorders
have been reported to be associated with advanced paternal age (Glaser and Jabs,
2004; Sayres and Makova, 2011). Base substitution is likely the most prominent type of
male biased mutation to arise from mitotic cell divisions because of the misincorporation
of nucleotides by DNA polymerase (Johnson et al., 2000). Other types of mutations such
as insertions and deletions (indels) were also found to be male-biased from whole
genome studies in rodents (Makova et al., 2004). Since indels can occur from strand–
slippage during replication, this lent further support to the potential contribution of the
higher number of cell divisions in the male mutation bias.
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Other mechanisms for male-biased mutations
The replication hypothesis however may represent an oversimplification since it is now
well known that DNA lesions may arise from many different mechanisms and the
contribution of some replication-independent factors may be significant. Oxidative
damage is one such replication-independent factor resulting from accumulation of
reactive oxygen species (ROS) that may act in a replication-independent manner to
increase male germline mutations rate. Male germ cells exist in a more ROS-rich
environment than eggs and the latter have a denser cell membrane affording better DNA
protection (Velando et al., 2008). Other replication-independent mutations such as
spontaneous conversion of methylated cytosine into thymine by deamination are also
possible although in this case the male-bias is much weaker than that observed at non-
CpG sites. Site heterogeneity in the male mutation bias arising from this mechanism
may therefore be expected (Taylor et al., 2006; Ellegren, 2007). Male bias is gaining
wider acceptance and was confirmed from genome analyses of many species (Sayres
and Makova, 2011). It is also associated with several human genetic disorders where
substitutions are involved (Arnheim and Calabrese, 2009).
Meiotic recombination is certainly one key step of male gametogenesis allowing for the
generation of genetic diversity in offspring. In some instances however, meiosis may
have the potential to affect nucleotide composition. The recombination rate, which may
vary between male and female of some species, may have a direct influence on the
pattern of nucleotide substitutions because of the so-called GC-biased gene conversion.
GC-biased gene conversion arises when the pairing of homologous chromosomes occur
at polymorphic sites. The base pair mismatch is then repaired by converting one allele
into the other but the repair of the A- or T-containing heteroduplexes is slightly biased
towards conversion to a GC pair (reviewed in Duret and Galtier, 2009). It is not sure
however that a male mutation bias would arise from this mechanism since genomic
regions with high recombination rates seemingly experience more GC-biased gene
conversion independent of sex (Popa et al., 2012).
Chromatin remodeling during spermiogenesis as a source for male-biased mutation
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The post-meiotic events of spermatogenesis, termed spermiogenesis, may represent
one such replication-independent mechanism with a strong potential to contribute to the
male mutation bias. The latter has not received much consideration but recent work from
our group and others have suggested that this crucial process may eventually be
considered as a significant source of male-driven de novo mutations and genetic
diversity. During spermiogenesis, the round haploid spermatids undergo a major
morphological differentiation program characterized by one of the most dramatic change
in chromatin structure known to the eukaryotic world. Most of the histones are replaced
by protamines providing both mechanical and chemical stability to the mature sperm
chromatin (Ward, 2011). The molecular mechanism leading to such a striking nuclear
transition is yet poorly understood but relies on histone variants, key post-translational
modifications, and general degradation of histones (Govin et al., 2006; 2007; Awe and
Renkawitz-Pohl, 2010; Zini and Agarwal, 2011). Most important is the observation that
the chromatin remodeling steps are specifically associated with transient, endogenous
DNA strand breaks that are detected in the whole population of both mouse and human
spermatids (Marcon and Boissonneault, 2004). Comet assays (Collins, 2004) in neutral
conditions showed a clear accumulation of double-stranded breaks (DSBs) specifically in
the nuclear DNA of spermatids throughout elongation which are then repaired at
subsequent steps (Laberge & Boissonneault 2005). These physiological DSBs trigger a
repair response, based on the detection of the phosphorylated histone variant γH2AX
(γH2AFX) and in situ detection of DNA polymerase activity in elongating spermatids
(Leduc et al., 2008; Kruman and Chen, 2012). The origin of these DSBs is not clear but
they may be created enzymatically, for instance by type II topoisomerases (McPherson
and Longo, 1993), from the activity of ROS (Muratori et al., 2006; Sakkas and Alvarez,
2010), or simply from the mechanical stress induced by the change in chromatin
structure (Boissonneault, 2002; Muratori et al., 2006; Sakkas and Alvarez, 2010). We
observed these transient breaks in spermatids of both human and mouse (Marcon and
Boissonneault, 2004; Leduc et al., 2008) but evidence of transient DSBs in spermatids
was also reported in rat (Meyer-Ficca et al., 2005), in fruit-flies (Drosophila
melanogaster) (Rathke et al., 2007), in grasshopper (Eyprepocnemis plorans) (Cabrero
et al., 2007) as well as in algae (Chara vulgaris) (Wojtczak et al., 2008). Taken together,
these observations point to a highly conserved, physiological mechanism that should
deserve further investigation regarding its genetic and evolutionary consequences.
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Mechanisms of DNA mutation during spermiogenesis
The presence of DSBs in this haploid context would necessarily prevent homologous
recombination to be used as a reliable, templated DNA repair mechanism that depends
on sister chromatids as this is the case during the S phase in somatic cells. Non–
homologous end joining repair processes (NHEJ) must therefore be used in order to
repair DSBs in spermatids but, based on studies in somatic cells, these mechanisms are
associated with limited insertions or deletions at the repair site which alter the DNA
sequence although the structural integrity of the DNA is restored. Even from an
homogeneous set of starting DNA ends as substrate, NHEJ creates important variations
in the non-templated addition at the two DNA ends (Lieber, 2010). There are two
different NHEJ pathways known to date that nevertheless display a similar potential to
induce mutations. The canonical pathway, known as DNA-PKcs-dependent NHEJ (D-
NHEJ) uses DNA ligase IV, KU70, KU80 and XRCC4 to complete the DNA repair. NHEJ
may proceed without some of the canonical factors using PARP1, DNA ligase III and
XRCC1 as the alternative “back-up” mechanisms still known as B-NHEJ (Iliakis, 2009).
In addition to having to rely on error-prone DNA repair systems, the chromatin
remodeling context is likely to create an impediment to the repair process and, not
surprisingly, the overall DNA repair capacity was found to decrease as spermatids
progress through their differentiation program (Olsen et al., 2005; Marchetti and
Wyrobek, 2008). At the moment, the extent and distribution of DSBs is unknown but a
random distribution would be expected to produce a different set of mutations in each
spermatid leading to a wide repertoire of genetic polymorphism given the large
population of spermatozoa produced over time. In addition to the chromosome
reshuffling provided by meiosis, each offspring would also inherit from a given set of
mutations induced by the chromatin remodeling in the spermatid of origin. The particular
nature of spermatid chromatin does not allow any useful prediction with respect to
whether randomly distributed versus clustered DNA strand breaks (hotspots) can be
found. It is known however that structural heterogeneity in spermatidal chromatin
establishes domains of general sensitivity to endogenous and exogenous nucleases that
could be generated by the transient open chromatin structure at given loci (Barratt et al.,
2010; Johnson et al., 2011). It stands to reason therefore that heterogeneity exists in
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spermatidal chromatin structure and that some strand breaks possibly arise at specific
loci.
Chromatin remodeling in spermatids involves massive withdrawal and degradation of
histones that should leave transient free DNA supercoils (Boissonneault, 2002) (Figure
1). Such a high degree of free superhelical density is likely to generate non B-DNA
structures which can be responsible for breakpoint hotspot and chromosomal
rearrangements (Wang et al., 2008). For instance, Z-DNA, characterized by a left-
handed instead of a typical right-handed double helical structure, is generated within
regions of high negative supercoiling and may serve as a recognition signal for DSBs
formation (Kha et al., 2010). High density of free supercoils independent of replication
can produce cruciform extrusion that may also act to signal breakpoints involved in
translocations (Inagaki et al., 2009). Interestingly, the break points of a well-known
recurrent non-Robertsonian translocation, t(11;22)(q23;q11), are concentrated within
regions harboring palindromic AT-rich repeats. All of eight studied cases of such de novo
translocations were found to be of paternal origin and linked to gametogenesis (Ohye et
al., 2010). Analyses of sperm samples from 10 donors indicated that there was no age-
dependent increase in the frequency of this de novo translocation and no increase in
translocation frequency was observed in follow-up studies (Kato et al., 2007). Since
aging is associated with a greater number of cell divisions, this suggests that these
translocations are independent of replication. Thus, the possibility of gross chromosomal
rearrangements in spermatids deserve further consideration as it may represent one
additional mechanism by which the chromatin remodeling process contributes to
diversity.
Future studies
Compared to somatic cells, a general attenuation in spontaneous mutation frequency
was previously observed in spermatogenic cells of young mice based on the functional
analysis of the lacI reporter transgene used as a retrievable mutational target (Kohler et
al., 1991). Although this strongly supports the concept that germ cells are in a
“protected” state relative to somatic cells, some key steps of spermatogenesis must
nevertheless remain responsible for the transmission of a significant number of de novo
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transgenerational mutations and the clear male bias reported so far. The lacI reporter
transgene system, such as that of the Big Blue® mouse used in these studies, rely on a
multicopy concatemer of the lacI gene on a single chromosome (Hill et al., 1999). The
mutation potential of the chromatin remodeling in spermatids will have to be established
on a much larger genome-wide scale or in the vicinity of the DSBs, providing a more
systematic approach. In this context, the use of such a reporter transgene system will be
limited because of the weaker probability that these hotspots be found within the
inserted transgene.
The capture of DNA at strand breaks followed by next generation sequencing should
allow genome-wide mapping of DNA strand breaks in elongating spermatids (Leduc et
al., 2011). The distribution of strand breaks between coding and non-coding regions will
be of special interest for evolutionary perspective especially if one can establish the
intrinsic mutagenic activity of the chromatin remodeling at these hotspots. Because of
the end-joining repair process likely involved, search for indels should probably be
emphasized since only substitutions were the focus of the whole genome
transgenerational studies published so far.
Conclusions
Overall, there is now substantial evidence that the chromatin remodeling steps in
spermatids may not be genetically inert but could represent an evolutionary conserved,
replication-independent, process that may act more specifically to introduce de novo
mutations, indels or even chromosomal rearrangements such as translocations. Its
contribution to the genetic landscape, diseases and evolution will now be possible with
improvement in the sensitivity of next generation sequencing that allows the monitoring
of mutations occurring at low frequency.
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ACKNOWLEDGEMENTS
The experimental work related to this article was made possible by grants from the
Canadian Institute of Health Research (grant #MOP-93781) and from the Natural
Sciences and Engineering Council of Canada (grant # 155182). M.-C.G is the recipient
of a scholarship from FRQ-S (Québec).
AUTHORS CONTRIBUTION
M.-C.G. and G.B. wrote the manuscript. J.M., O.S., A.G., M.-A.B., M.A. & F.L. provided
editorial comments.
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Arnheim N, and Calabrese P. Understanding what determines the frequency and pattern of human germline mutations. Nat Rev Genet 2009;10:478–488.
Awe S, and Renkawitz-Pohl R. Histone H4 acetylation is essential to proceed from a histone- to a protamine-based chromatin structure in spermatid nuclei of Drosophila melanogaster. Syst Biol Reprod Med 2010;56:44–61.
Barratt CLR, Aitken RJ, Björndahl L, Carrell DT, de Boer P, Kvist U, Lewis SEM, Perreault SD, Perry MJ, Ramos L, et al. Sperm DNA: organization, protection and vulnerability: from basic science to clinical applications--a position report. 2010;pp.824–838.
Boissonneault G. Chromatin remodeling during spermiogenesis: a possible role for the transition proteins in DNA strand break repair. FEBS Lett 2002;514:111–114.
Cabrero J, Palomino-Morales RJ, and Camacho JPM. The DNA-repair Ku70 protein is located in the nucleus and tail of elongating spermatids in grasshoppers. Chromosome Res 2007;15:1093–1100.
Collins AR. The comet assay for DNA damage and repair: principles, applications, and limitations. Mol Biotechnol 2004;26:249–261.
Duret L, and Galtier N. Biased gene conversion and the evolution of mammalian genomic landscapes. Annual Review of Genomics and Human Genetics 2009;10:285–311.
Ellegren H. Characteristics, causes and evolutionary consequences of male-biased mutation. Proc Biol Sci 2007;274:1–10.
Glaser RL, and Jabs EW. Dear old dad. Sci Aging Knowledge Environ 2004;2004: re1.
Govin J, Escoffier E, Rousseaux S, Kuhn L, Ferro M, Thévenon J, Catena R, Davidson I, Garin J, Khochbin S, et al. Pericentric heterochromatin reprogramming by new histone variants during mouse spermiogenesis. J Cell Biol 2007;176:283–294.
Govin J, Lestrat C, Caron C, Pivot-Pajot C, Rousseaux S, and Khochbin S. Histone acetylation-mediated chromatin compaction during mouse spermatogenesis. Ernst Schering Res Found Workshop 2006:155–172.
Haldane JBS. The mutation rate of the gene for haemophilia, and its segregation ratios in males and females. Ann Eugen 1947;13:262–271.
Hill KA, Nishino H, Buettner VL, Halangoda A, Li W, and Sommer SS. The Big Blue(R) transgenic mouse mutation detection assay: the mutation pattern of sectored mutant plaques. Mutat Res 1999;425:47–54.
Hurst LD, and Ellegren H. Sex biases in the mutation rate. Trends Genet 1998;14:446–452.
Iliakis G. Backup pathways of NHEJ in cells of higher eukaryotes: cell cycle dependence. Radiother Oncol 2009;92:310–315.
Inagaki H, Ohye T, Kogo H, Kato T, Bolor H, Taniguchi M, Shaikh TH, Emanuel BS, and Kurahashi H. Chromosomal instability mediated by non-B DNA: cruciform conformation and not DNA sequence is responsible for recurrent translocation in humans. Genome Res 2009;19:191–198.
11
Johnson GD, Lalancette C, Linnemann AK, Leduc F, Boissonneault G, and Krawetz SA. The sperm nucleus: chromatin, RNA, and the nuclear matrix. Reproduction 2011;141:21–36.
Johnson RE, Washington MT, Prakash S, and Prakash L. Fidelity of human DNA polymerase eta. J Biol Chem 2000;275:7447–7450.
Kato T, Yamada K, Inagaki H, Kogo H, Ohye T, Emanuel BS, and Kurahashi H. Age has no effect on de novo constitutional t(11;22) translocation frequency in sperm. Fertil Steril 2007;88:1446–1448.
Kha DT, Wang G, Natrajan N, Harrison L, and Vasquez KM. Pathways for double-strand break repair in genetically unstable Z-DNA-forming sequences. J Mol Biol 2010;398:471–480.
Kohler SW, Provost GS, Fieck A, Kretz PL, Bullock WO, Sorge JA, Putman DL, and Short JM. Spectra of spontaneous and mutagen-induced mutations in the lacI gene in transgenic mice. Proc Natl Acad Sci USA 1991;88:7958–7962.
Kong A, Frigge ML, Masson G, Besenbacher S, Sulem P, Magnusson G, Gudjonsson SA, Sigurdsson A, Jonasdottir A, Jonasdottir A, et al. Rate of de novo mutations and the importance of father’s age to disease risk. Nature 2012;488:471–475.
Kruman DI, and Chen G. DNA Repair. D.I. Kruman, ed. 2012
Leduc F, Faucher D, Bikond Nkoma G, Grégoire M-C, Arguin M, Wellinger RJ, and Boissonneault G. Genome-wide mapping of DNA strand breaks. PLoS ONE 2011;6:e17353.
Leduc F, Maquennehan V, Nkoma GB, and Boissonneault G. DNA damage response during chromatin remodeling in elongating spermatids of mice. Biol Reprod 2008;78:324–332.
Lieber MR. The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway. Annu Rev Biochem 2010;79:181–211.
Makova KD, Yang S, and Chiaromonte F. Insertions and deletions are male biased too: a whole-genome analysis in rodents. Genome Res 2004;14: 567–573.
Marchetti F, and Wyrobek AJ. DNA repair decline during mouse spermiogenesis results in the accumulation of heritable DNA damage. DNA Repair (Amst.) 2008;7:572–581.
Marcon L, and Boissonneault G. Transient DNA strand breaks during mouse and human spermiogenesis new insights in stage specificity and link to chromatin remodeling. Biol Reprod 2004;70:910–918.
McPherson SM, and Longo FJ. Nicking of rat spermatid and spermatozoa DNA: possible involvement of DNA topoisomerase II. Dev Biol 1993;158:122–130.
Meyer-Ficca ML, Scherthan H, Bürkle A, and Meyer RG. Poly(ADP-ribosyl)ation during chromatin remodeling steps in rat spermiogenesis. Chromosoma 2005;114:67–74.
Muratori M, Marchiani S, Maggi M, Forti G, and Baldi E. Origin and biological significance of DNA fragmentation in human spermatozoa. Front Biosci 2006;11:1491–1499.
Ohye T, Inagaki H, Kogo H, Tsutsumi M, Kato T, Tong M, Macville MVE, Medne L, Zackai EH, Emanuel BS, et al. Paternal origin of the de novo constitutional t(11;22)(q23;q11). Eur J Hum Genet 2010;18:783–787.
12
Olsen A-K, Lindeman B, Wiger R, Duale N, and Brunborg G. How do male germ cells handle DNA damage? Toxicol Appl Pharmacol 2005;207:521–531.
Popa A, Samollow P, Gautier C, and Mouchiroud D. The sex-specific impact of meiotic recombination on nucleotide composition. Genome Biol Evol 2012;4:412–422.
Rathke C, Baarends WM, Jayaramaiah-Raja S, Bartkuhn M, Renkawitz R, and Renkawitz-Pohl R. Transition from a nucleosome-based to a protamine-based chromatin configuration during spermiogenesis in Drosophila. J Cell Sci 2007;120:1689–1700.
Sakkas D, and Alvarez JG. Sperm DNA fragmentation: mechanisms of origin, impact on reproductive outcome, and analysis. Fertil Steril 2010;93:1027–1036.
Sayres MAW, and Makova KD. Genome analyses substantiate male mutation bias in many species. Bioessays 2011;33:938–945.
Taylor J, Tyekucheva S, Zody M, Chiaromonte F, and Makova KD. Strong and weak male mutation bias at different sites in the primate genomes: insights from the human-chimpanzee comparison. Mol Biol Evol 2006;23:565–573.
Velando A, Torres R, and Alonso-Alvarez C. Avoiding bad genes: oxidatively damaged DNA in germ line and mate choice. Bioessays 2008;30:1212–1219.
Vogel F, and Motulsky AG. Human Genetics (Springer Verlag). 1997.
Wang G, Carbajal S, Vijg J, DiGiovanni J, and Vasquez KM. DNA structure-induced genomic instability in vivo. J Natl Cancer Inst 2008;100:1815–1817.
Ward WS. Regulating DNA Supercoiling: Sperm Points the Way. Biol Reprod 2011;84:841–843.
Wojtczak A, Popłońska K, and Kwiatkowska M. Phosphorylation of H2AX histone as indirect evidence for double-stranded DNA breaks related to the exchange of nuclear proteins and chromatin remodeling in Chara vulgaris spermiogenesis. Protoplasma 2008;233:263–267.
Zini A, and Agarwal A. Sperm Chromatin (Springer). 2011.
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Figure caption: The three major differentiation states of the haploid spermatids (mouse model). Round spermatids harboring somatic-like chromatin (left). The chromatin remodeling in elongating spermatids involves withdrawals of most of the nucleosomes (curved arrow) and DNA strand breaks (thick arrows, middle). In the late steps and following chromatin remodeling, the non-templated end-joining repair of double stranded breaks is expected to create short sequence alteration (red).
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