**3. Nature of endogenous DNA damages during spermiogenesis**

#### **3.1 Single strand damage and repair**

Depending on the type of damage, specific pathways achieve single strand damage repair (see Table 1). Mispaired DNA bases that primarily arise during replication are corrected by mismatch repair (MMR), while small chemical alterations of DNA bases such as alkylation, deamination and oxidative damage are repaired by base excision repair (BER) (Mukherjee et al., 2010; Robertson et al., 2009). More complex lesions such as those induced by UV (pyrimidine dimers and helix-distorting lesions) are corrected by nucleotide excision repair (NER), a multistep pathway that involves more than 30 proteins (Hoeijmakers, 2009; Nouspikel, 2009). DNA nicks are repaired by single-strand break repair (SSBR). These DNA repair pathways are known to be present and active during spermiogenesis (Olsen et al., 2001; Schultz et al., 2003). To our knowledge, single-strand damages do not present a major threat to spermatids. With the exception of exposures to toxicant that could challenge these pathways, in normal conditions, single-strand DNA damage during spermiogenesis is likely attributed to the massive transcription that is taking place at these steps and is efficiently resolved by spermatids (Olsen et al., 2001). DNA double-strand breaks were reported as part of the normal differentiation program of spermatids during spermiogenesis which may represent an important source of genetic instability and therefore we will focus on these pathways.

#### **3.2 Double-strand breaks in spermatids 3.2.1 Possible origin of DNA breaks**

Several hypotheses have been formulated to elucidate the origin and role of DNA strand breaks in spermatids. Sakkas and colleagues suggested that "abortive apoptosis" may be the cause since abnormal human spermatozoa presented some apoptotic-like features (Sakkas et al., 1999). Further investigation led to the demonstration that other biomarkers of apoptosis in sperm cells were present such as BCL-X, TP53, caspases, in addition to diverse structural defects (Baccetti et al., 1997; Donnelly et al., 2000; Gandini et al., 2000; Sakkas et al., 2002; Weng et al., 2002). Due to technical limitations at the time, DNA breaks were only observed

Post-Meiotic DNA Damage and Response in Male Germ Cells 309

spermatogenesis, including in the nucleus of spermatids (Branciforte and Martin, 1994; Ergün et al., 2004; Gasior et al., 2006). However, topoisomerases have long been considered likely candidates to support chromatin remodeling from bulky histone-bound chromatin to compact and transcriptionally inert protamine-bound DNA because of their ubiquitous role in chromosome dynamics during the somatic cell cycle (McPherson and Longo, 1993).

Change in DNA topology can be achieved by single-strand breaks (SSBs) generated by type I topoisomerase, which modifies the linking number in steps of one. Single-strand breaks would be considered a much smaller threat for the genome's integrity of spermatids than a DSB that could be generated by type II topoisomerases. However, chromatin remodeling in spermatids was clearly shown to be associated with an increase in type II topoisomerase (Chen and Longo, 1996; Laberge and Boissonneault, 2005; Leduc et al., 2008a; McPherson and Longo, 1992, 1993; Meyer-Ficca et al., 2011b; Roca and Mezquita, 1989). A possible link between type II topoisomerases and DNA breaks found in elongating spermatids was suggested by the elimination of DNA breaks in spermatids nuclei incubated with type II topoisomerase inhibitors such as suramin and etoposide (Laberge and Boissonneault, 2005). In mammal cells, the α and β isoforms of topoisomerase share more than 80% of homology and are differentially expressed. Topoisomerase IIα (TOP2A) is mostly found in replicating cells whereas topoisomerase IIβ (TOP2B) predominates in quiescent cells (Morse-Gaudio and Risley, 1994; Turley et al., 1997). Using immunofluorescence on mouse testis sections, we have observed TOP2B foci in nuclei of elongating spermatids whereas TOP2A remained undetected in these cells but highly present in spermatocytes (see Figure 1) (Leduc et al., 2008a). Detection of TOP2B in elongating spermatids is not surprising, as spermatids are non-replicative cells. Recent studies confirmed the involvement of TOP2B in elongating spermatids (Meyer-Ficca et al., 2011b) and also observed its presence further downstream of the male germ cells differentiation program as part of the nuclear matrix of sperm cells, supporting its earlier role in the chromatin remodeling of spermatids (Shaman et al., 2006).

Type II topoisomerase activity may be modulated by post-translational modifications, such as phosphorylation by kinases and poly (ADP-ribosyl)ation by poly (ADPribose) polymerases (PARPs), a well-known family of proteins involved in a multitude of nuclear events, such as DNA repair and chromatin remodeling. This complementary interaction between TOP2B and PARPs may be involved in numerous cellular processes. For example, TOP2B and PARP1 are known to modulate transcription in somatic cells (Ju et al., 2006). Furthermore, these proteins may be important constituents of the nuclear matrix; Zaalishvili and coworkers observed the stimulation of cleavage of nuclear matrix associated DNA loops of neuron and leukocyte nucleoids when incubated in buffer supporting topoisomerase and PARP activity (Zaalishvili et al., 2005). This stimulation was reversed by the addition of thymidine, a PARP inhibitor. The authors suggested that a PARP-modified topoisomerase II may cut efficiently but the (ADP-ribosyl)ation could inhibit the religation. Recently, Meyer-Ficca and colleagues demonstrated a possible modulation of TOP2B activity by PARP and PARG *in vitro* using recombinant proteins as well as *in vivo* during mouse spermiogenesis through the use of inhibitors and knockout mouse models (Meyer-Ficca et al., 2011b). According to their findings, there is a functional relationship between the DNA strand break activity of TOP2B and the DNA strand break-dependent activation of

**3.2.2 Topoisomerases as candidates to supercoiling removal** 

**3.2.3 Topoisomerases and DNA repair** 


Table 1. Summary of the single strand DNA repair pathways in mammalian cells (Ciccia and Elledge, 2010; Hoeijmakers, 2009; Martin et al., 2010; Mukherjee et al., 2010; Nouspikel, 2009; Robertson et al., 2009).

in a subset of the whole population of elongating spermatids and therefore abortive apoptosis could represent a sound explanation. However, some studies demonstrated that round spermatids are radioresistant to apoptosis and may not have the proper machinery and checkpoints to trigger such process (Ahmed et al., 2010; Oakberg and Diminno, 1960). Furthermore, our group have demonstrated that transient DNA breaks were present in the whole population of elongating spermatids of fertile mice and humans during chromatin remodeling and were therefore part of the normal differentiation program of these cells (Marcon and Boissonneault, 2004). The persistence of these breaks beyond the chromatin remodeling steps in pathological conditions may explain the presence of DNA fragmentation found in spermatozoa of infertile men (Leduc et al., 2008b).

Generation of controlled DNA breaks either single- or double-stranded may be important to relieve the torsional stress induced by the withdrawal of histones (Boissonneault, 2002). The simple mechanical stress resulting from the accumulation of free supercoils could induce non-B DNA structures and possibly DNA breaks as the chromatin remodeling is extensive and takes place within many differentiation steps. However, enzymatic induction of DNA strand breaks is more likely, as their free ends can be end-labeled with polymerases that require a 3'OH as substrate, such as the terminal deoxynucleotidyl transferase (TdT) used in TUNEL labeling. Specific nucleases could be involved in this process, and it is not excluded that retrotransposon nucleases could play a role as they are expressed throughout

DNA repair pathways DNA damages Implicated proteins

chemical alteration arising from alkylation, deamination and oxidative damage

Pyrimidine dimer

Single strand break (SSB)

Table 1. Summary of the single strand DNA repair pathways in mammalian cells (Ciccia and Elledge, 2010; Hoeijmakers, 2009; Martin et al., 2010; Mukherjee et al., 2010; Nouspikel, 2009;

in a subset of the whole population of elongating spermatids and therefore abortive apoptosis could represent a sound explanation. However, some studies demonstrated that round spermatids are radioresistant to apoptosis and may not have the proper machinery and checkpoints to trigger such process (Ahmed et al., 2010; Oakberg and Diminno, 1960). Furthermore, our group have demonstrated that transient DNA breaks were present in the whole population of elongating spermatids of fertile mice and humans during chromatin remodeling and were therefore part of the normal differentiation program of these cells (Marcon and Boissonneault, 2004). The persistence of these breaks beyond the chromatin remodeling steps in pathological conditions may explain the presence of DNA

Generation of controlled DNA breaks either single- or double-stranded may be important to relieve the torsional stress induced by the withdrawal of histones (Boissonneault, 2002). The simple mechanical stress resulting from the accumulation of free supercoils could induce non-B DNA structures and possibly DNA breaks as the chromatin remodeling is extensive and takes place within many differentiation steps. However, enzymatic induction of DNA strand breaks is more likely, as their free ends can be end-labeled with polymerases that require a 3'OH as substrate, such as the terminal deoxynucleotidyl transferase (TdT) used in TUNEL labeling. Specific nucleases could be involved in this process, and it is not excluded that retrotransposon nucleases could play a role as they are expressed throughout

Long-patch UNG, APEX1, POL β / δ ,

Short-patch Small DNA bases

MSH1-6, MLH1, MLH3, PMS1, PMS2, EXO1, RPA, PCNA, RFC

UNG, APEX1, POL β , XRCC1, LIG3

FEN1, PCNA, LIG1

XPC complex, DDB complex, ERCC3 (TFIIH), XPA-RPA complex, ERCC5 (XPG), ERCC1-ERCC4 (XPF), LIG3, DNA polymerase δ

APE1, PNKP, APTX, TDP1, POL β/δ/ε, PCNA, XRCC1, LIG1/3, FENI, PARP

Mismatch repair (MMR) Mispaired DNA bases

Transcription -coupled or not

Short-patch or long-patch

fragmentation found in spermatozoa of infertile men (Leduc et al., 2008b).

Base excision repair (BER)

Nucleotide excision repair (NER)

Single strand break repair (SSBR)

Robertson et al., 2009).

spermatogenesis, including in the nucleus of spermatids (Branciforte and Martin, 1994; Ergün et al., 2004; Gasior et al., 2006). However, topoisomerases have long been considered likely candidates to support chromatin remodeling from bulky histone-bound chromatin to compact and transcriptionally inert protamine-bound DNA because of their ubiquitous role in chromosome dynamics during the somatic cell cycle (McPherson and Longo, 1993).

### **3.2.2 Topoisomerases as candidates to supercoiling removal**

Change in DNA topology can be achieved by single-strand breaks (SSBs) generated by type I topoisomerase, which modifies the linking number in steps of one. Single-strand breaks would be considered a much smaller threat for the genome's integrity of spermatids than a DSB that could be generated by type II topoisomerases. However, chromatin remodeling in spermatids was clearly shown to be associated with an increase in type II topoisomerase (Chen and Longo, 1996; Laberge and Boissonneault, 2005; Leduc et al., 2008a; McPherson and Longo, 1992, 1993; Meyer-Ficca et al., 2011b; Roca and Mezquita, 1989). A possible link between type II topoisomerases and DNA breaks found in elongating spermatids was suggested by the elimination of DNA breaks in spermatids nuclei incubated with type II topoisomerase inhibitors such as suramin and etoposide (Laberge and Boissonneault, 2005). In mammal cells, the α and β isoforms of topoisomerase share more than 80% of homology and are differentially expressed. Topoisomerase IIα (TOP2A) is mostly found in replicating cells whereas topoisomerase IIβ (TOP2B) predominates in quiescent cells (Morse-Gaudio and Risley, 1994; Turley et al., 1997). Using immunofluorescence on mouse testis sections, we have observed TOP2B foci in nuclei of elongating spermatids whereas TOP2A remained undetected in these cells but highly present in spermatocytes (see Figure 1) (Leduc et al., 2008a). Detection of TOP2B in elongating spermatids is not surprising, as spermatids are non-replicative cells. Recent studies confirmed the involvement of TOP2B in elongating spermatids (Meyer-Ficca et al., 2011b) and also observed its presence further downstream of the male germ cells differentiation program as part of the nuclear matrix of sperm cells, supporting its earlier role in the chromatin remodeling of spermatids (Shaman et al., 2006).

#### **3.2.3 Topoisomerases and DNA repair**

Type II topoisomerase activity may be modulated by post-translational modifications, such as phosphorylation by kinases and poly (ADP-ribosyl)ation by poly (ADPribose) polymerases (PARPs), a well-known family of proteins involved in a multitude of nuclear events, such as DNA repair and chromatin remodeling. This complementary interaction between TOP2B and PARPs may be involved in numerous cellular processes. For example, TOP2B and PARP1 are known to modulate transcription in somatic cells (Ju et al., 2006). Furthermore, these proteins may be important constituents of the nuclear matrix; Zaalishvili and coworkers observed the stimulation of cleavage of nuclear matrix associated DNA loops of neuron and leukocyte nucleoids when incubated in buffer supporting topoisomerase and PARP activity (Zaalishvili et al., 2005). This stimulation was reversed by the addition of thymidine, a PARP inhibitor. The authors suggested that a PARP-modified topoisomerase II may cut efficiently but the (ADP-ribosyl)ation could inhibit the religation. Recently, Meyer-Ficca and colleagues demonstrated a possible modulation of TOP2B activity by PARP and PARG *in vitro* using recombinant proteins as well as *in vivo* during mouse spermiogenesis through the use of inhibitors and knockout mouse models (Meyer-Ficca et al., 2011b). According to their findings, there is a functional relationship between the DNA strand break activity of TOP2B and the DNA strand break-dependent activation of

Post-Meiotic DNA Damage and Response in Male Germ Cells 311

The first step following a DSB is the detection of the DNA damage by sensors (Lamarche et al., 2010). At least four independent sensors can detect DSBs: PARPs in all cases of SSBs and, to a lesser extent, DSBs, Ku70/80 in D-NHEJ, MRE11-RAD50-NBS1 (MRN) complex in all cases of DSBs and replication protein A1 (RPA) in HR (Ciccia and Elledge, 2010; Lamarche

As previously stated in section 3.2.3, the presence and activity of PARP1 and PARP2 have been recently investigated during spermiogenesis of mouse and rat (Ahmed et al., 2010; Dantzer et al., 2006; Meyer-Ficca et al., 2005; Meyer-Ficca et al., 2011a; Meyer-Ficca et al., 2009; Meyer-Ficca et al., 2011b). Although the individual absence of these proteins leads only to subfertility in male, it is believed that they play a key role in the maintenance of genomic integrity of spermatids. As discussed previously, PARPs may be involved in DNA repair and signaling, in the drastic chromatin remodeling of spermatids and even in the repackaging of their genome with protamines (Quénet et al., 2009). However, the embryonic lethal phenotype of double knockout mouse prevent a better assessment of their critical role during spermiogenesis, as the absence of one can be compensated for by the other. The Ku heterodimer binds to DSB ends and is required for the non-homologous end-joining pathway (NHEJ). In addition to its role in DNA repair, Ku proteins are also required for the maintenance of telomeres and subtelomeric gene silencing (Celli et al., 2006; Lamarche et al., 2010). KU70 is present during the spermiogenesis of mouse (Goedecke et al., 1999; Hamer et al., 2003), human (Leduc *et al.*, unpublished observations), and grasshoppers (Cabrero et al., 2007), but seems to decrease as spermiogenesis proceeds, most notably after the expulsion of histones. Although initial analyses of the implication of MRN complex as sensor in non-homologous end-joining pathways produced conflicting results (Di Virgilio and Gautier, 2005; Huang and Dynan, 2002), recent studies showed that siRNA mediated knockdown of Mre11 results in reduced end-joining efficiency in both D-NHEJ and B-NHEJ pathways (Rass et al., 2009; Xie et al., 2009) and should be considered a good candidate for DNA breaks detection and signaling in spermatids. As for Ku proteins, Mre11 is also present during spermiogenesis (Goedecke et al., 1999). Contrary to these DNA break sensor proteins, RPA may not play such an important role during spermiogenesis as spermatids, being haploid, cannot rely

The detection of DNA damage by sensors activates proteins of the phosphatidylinositol 3 kinase-like protein kinase (PIKKs) family such as ATM, ATR, and DNA PKcs and members of the PARP family. These proteins post-translationnally modify key protein targets triggering a signal transduction cascades that forms the DNA damage response (DDR) (Lamarche et al., 2010). During mouse spermiogenesis, ATM and DNA PKcs are present and active (Ahmed et al., 2010; Scherthan et al., 2000). These kinases are responsible for the phosphorylation of the histone H2A variant, H2AFX, at serine 139 (γH2AFX, previously referred to as γH2AX), which quickly occurs after a DSB. This modification can spread up to a 2 Mbp region flanking all DSBs (Kinner et al., 2008) and it could help the recruitment of other proteins of the DDR (Celeste et al., 2003). Within minutes following DNA damage, γH2AFX appears at discrete nuclear foci that dissolve after the completion of DNA repair. It remains unclear whether γH2AFX is replaced completely with new H2AFX histones, or simply dephosphorylated, but strong evidences suggest that the latter mechanism is

**4. DNA damage response and DNA repair of double-strand breaks** 

**4.1 DNA damage signaling pathways** 

et al., 2010).

on HR repair processes.

Fig. 1. Presence of type II topoisomerases, hyperacetylated histone H4, γH2AFX and DNA breaks during mouse spermiogenesis. (A) Overlay of TOP2B immunofluorescence (green) and DAPI nuclear staining (blue) of a stage IX tubule demonstrating the presence of TOP2B in nuclei of elongating spermatids (ES) at the onset of chromatin remodeling. (B) Overlay of TOP2B immunofluorescence (green) and DAPI (blue) nuclear staining of stages VII and X tubules. (C) Overlay of immunofluorescence of TOP2A (green) and TO-PRO3 (red) nuclear staining of stages XII and II-III demonstrating the nuclear presence of TOP2A in zygotene and pachytene spermatocytes but complete absence in spermatids. (D) Detection of hyperacetylated histone H4 and γH2AFX by immunofluorescence and DNA breaks by TUNEL during mouse spermiogenesis. DNA was counterstained by TO-PRO3. (A-C) Immunofluoresence on Bouin-fixed testis sections was done as previously described(Leduc et al., 2008a). (D) Squash preparation were done as previously described (Kotaja et al., 2004; Leduc et al., 2008a), fixed with ice-cold ethanol and processed for TUNEL and immunofluorescence. Bars = 10 µm (A and B), 20 µm (C) and 5µm (D).

PARP enzymes. Moreover, alteration in the PAR metabolism leads to a greater retention of histones in spermatozoa (Meyer-Ficca et al., 2011a). Whether PARP1 is involved directly in chromatin remodeling, DNA repair or combination of both in spermatids remains to be determined and will be discussed further in section 4.
