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

### **4.1 DNA damage signaling pathways**

310 DNA Repair

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

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

immunofluorescence. Bars = 10 µm (A and B), 20 µm (C) and 5µm (D).

determined and will be discussed further in section 4.

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 et al., 2010).

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 on HR repair processes.

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

Post-Meiotic DNA Damage and Response in Male Germ Cells 313

Although these DSBs are considered the most harmful genetic damage for a cell, we know from experimental data (Marcon and Boissonneault, 2004) that these breaks are repaired by the end of spermiogenesis in fertile animals. The disappearance of γH2AFX in mouse spermatids (step 13 to 16) shown in Figure 1 cannot be associated with completion of DNA repair or dephosphorylation as a majority of histones are expulsed from the nucleus to be replaced by PRMs. However, we obtained other evidences of an active DNA repair system at these steps by demonstrating incorporation of dNTPs *in situ* that is sustained through all the chromatin remodeling steps (see Figure 3) (Leduc et al., 2008a). Furthermore, as seen in Figure 1, the appearance and disappearance of TUNEL labeling is coincident with γH2AFX fluorescence. To confirm that the loss of TUNEL labeling was associated with DNA repair and not with the lack of penetrability of the TdT in the nuclei of condensed spermatids, we decondensed spermatids prior to TUNEL with similar results (Marcon and Boissonneault, 2004) (Acteau et al., unpublished observations). Therefore, DNA breaks are properly repaired by the end of the spermatids differentiation program. As previously stated, mammalian cells can rely on four DNA DSBs repair pathways, each of which having different degree of fidelity. As spermatids differentiate to spermatozoa with fertilizing potential, any errors due to faulty or incomplete DNA repair may be transmitted to the next generation. Severe alteration in the repair process may cause infertility or possibly be

Fig. 3. In situ endogenous DNA polymerase activity assay (Leduc et al., 2008a) on squash preparation of stage IX and XII tubules. DNA was counterstained by DAPI. Bar = 10 µm

Double-strand breaks are processed either by homologous recombination, single-strand annealing (SSA) or non-homologous end-joining (Caldecott, 2008). Two types of NHEJ are available to mammalian cells: the pathway that is dependent of DNA PKcs (referred to as D-

**4.2 Evidences of an active DNA repair system during spermiogenesis** 

incompatible with embryonic development (Leduc et al., 2008b).

**4.3 Towards identification of DNA repair pathways** 

prominent (Chowdhury et al., 2005; Rogakou et al., 1999). Therefore, the implication of γH2AFX in all cases of DSBs makes it a novel biomarker for DSBs detection by immunoflorescence (Mah et al., 2010; Mah et al., 2011). Upon γH2AFX signaling, specific pathways are recruited according to cell type or the cell cycle phase (Shrivastav et al., 2008). The presence of γH2AFX during spermiogenesis has been first shown in rats (Meyer-Ficca et al., 2005) and we confirmed its presence at the corresponding steps during mouse spermiogenesis (Leduc et al., 2008a) (see Figure 1). As shown in Figure 1, the presence of γH2AFX and hyperacetylated histone H4, a biomarker of chromatin remodeling coincides with the presence of TOP2B. These results confirm the previously published strong TUNEL labeling of elongating spermatids during chromatin remodeling (Laberge and Boissonneault, 2005; Marcon and Boissonneault, 2004).

Therefore, spermatids undergo multiple transient DSBs, inducing a classic DDR signaling. In addition, as seen by immunofluorescence in Figure 1, γH2AFX is present in all spermatids throughout chromatin remodeling as part of the normal process of maturation of spermatids. The pattern of γH2AFX in spermatids as seen in Figures 1 and 2 is dependent on fixation and tissue processing; ethanol fixation provides a better context for TUNEL labeling but alters nuclear distribution of proteins. Furthermore, we have also found the presence of γH2AFX and DNA breaks during human spermiogenesis (see Figure 2), while other groups subsequently demonstrated similar DDR signaling in grasshoppers (Cabrero et al., 2007) and even in the algae *Charas vulgaris* (Wojtczak et al., 2008). Moreover, the presence of DNA breaks has also been found during spermiogenesis of drosophila (Rathke et al., 2007). Altogether, these results suggest that the DDR triggered by endogenous breaks in spermatids is evolutionary conserved and could represent a new source of male–driven genetic instability in species where gametogenesis requires condensation of the genetic material.

Fig. 2. Detection of γH2AFX during spermiogenesis of human (upper panel), and mouse (lower panel). DNA was counterstained by TO-PRO3. Bars = 5µm. Immunofluoresence on paraformaldehyde-fixed testis sections was done as previously described (Leduc et al., 2008a).

prominent (Chowdhury et al., 2005; Rogakou et al., 1999). Therefore, the implication of γH2AFX in all cases of DSBs makes it a novel biomarker for DSBs detection by immunoflorescence (Mah et al., 2010; Mah et al., 2011). Upon γH2AFX signaling, specific pathways are recruited according to cell type or the cell cycle phase (Shrivastav et al., 2008). The presence of γH2AFX during spermiogenesis has been first shown in rats (Meyer-Ficca et al., 2005) and we confirmed its presence at the corresponding steps during mouse spermiogenesis (Leduc et al., 2008a) (see Figure 1). As shown in Figure 1, the presence of γH2AFX and hyperacetylated histone H4, a biomarker of chromatin remodeling coincides with the presence of TOP2B. These results confirm the previously published strong TUNEL labeling of elongating spermatids during chromatin remodeling (Laberge and

Therefore, spermatids undergo multiple transient DSBs, inducing a classic DDR signaling. In addition, as seen by immunofluorescence in Figure 1, γH2AFX is present in all spermatids throughout chromatin remodeling as part of the normal process of maturation of spermatids. The pattern of γH2AFX in spermatids as seen in Figures 1 and 2 is dependent on fixation and tissue processing; ethanol fixation provides a better context for TUNEL labeling but alters nuclear distribution of proteins. Furthermore, we have also found the presence of γH2AFX and DNA breaks during human spermiogenesis (see Figure 2), while other groups subsequently demonstrated similar DDR signaling in grasshoppers (Cabrero et al., 2007) and even in the algae *Charas vulgaris* (Wojtczak et al., 2008). Moreover, the presence of DNA breaks has also been found during spermiogenesis of drosophila (Rathke et al., 2007). Altogether, these results suggest that the DDR triggered by endogenous breaks in spermatids is evolutionary conserved and could represent a new source of male–driven genetic instability in species where gametogenesis

Fig. 2. Detection of γH2AFX during spermiogenesis of human (upper panel), and mouse (lower panel). DNA was counterstained by TO-PRO3. Bars = 5µm. Immunofluoresence on paraformaldehyde-fixed testis sections was done as previously described (Leduc et al.,

Boissonneault, 2005; Marcon and Boissonneault, 2004).

requires condensation of the genetic material.

2008a).

#### **4.2 Evidences of an active DNA repair system during spermiogenesis**

Although these DSBs are considered the most harmful genetic damage for a cell, we know from experimental data (Marcon and Boissonneault, 2004) that these breaks are repaired by the end of spermiogenesis in fertile animals. The disappearance of γH2AFX in mouse spermatids (step 13 to 16) shown in Figure 1 cannot be associated with completion of DNA repair or dephosphorylation as a majority of histones are expulsed from the nucleus to be replaced by PRMs. However, we obtained other evidences of an active DNA repair system at these steps by demonstrating incorporation of dNTPs *in situ* that is sustained through all the chromatin remodeling steps (see Figure 3) (Leduc et al., 2008a). Furthermore, as seen in Figure 1, the appearance and disappearance of TUNEL labeling is coincident with γH2AFX fluorescence. To confirm that the loss of TUNEL labeling was associated with DNA repair and not with the lack of penetrability of the TdT in the nuclei of condensed spermatids, we decondensed spermatids prior to TUNEL with similar results (Marcon and Boissonneault, 2004) (Acteau et al., unpublished observations). Therefore, DNA breaks are properly repaired by the end of the spermatids differentiation program. As previously stated, mammalian cells can rely on four DNA DSBs repair pathways, each of which having different degree of fidelity. As spermatids differentiate to spermatozoa with fertilizing potential, any errors due to faulty or incomplete DNA repair may be transmitted to the next generation. Severe alteration in the repair process may cause infertility or possibly be incompatible with embryonic development (Leduc et al., 2008b).

Fig. 3. In situ endogenous DNA polymerase activity assay (Leduc et al., 2008a) on squash preparation of stage IX and XII tubules. DNA was counterstained by DAPI. Bar = 10 µm

#### **4.3 Towards identification of DNA repair pathways**

Double-strand breaks are processed either by homologous recombination, single-strand annealing (SSA) or non-homologous end-joining (Caldecott, 2008). Two types of NHEJ are available to mammalian cells: the pathway that is dependent of DNA PKcs (referred to as D-

Post-Meiotic DNA Damage and Response in Male Germ Cells 315

because HR cannot proceed during that time (Daley et al., 2005). Spermatids provide a similar cellular context as G1 phase of somatic cells. However, dynamics of DNA repair by NHEJ pathways, as illustrated in irradiated round spermatids, are much slower (Ahmed et al., 2010). According to Ahmed and colleagues both pathways are present and active during mouse spermiogenesis: spermatids of SCID mice, lacking the D-NHEJ because of the absence of DNA PKcs, displayed slower repair than those from wild type mice (Ahmed et al., 2010). Further studies on the end-joining pathways in elongating spermatids will be required as these are known to be error-prone in somatic cells. This may also be the case in spermatids. Although an attenuation of the frequency of mutations may be found in the germ line (Walter et al., 1998), the chromatin remodeling in spermatids may still be the key differentiation steps where most of the new mutations repertoire is being produced for the

High level of sperm DNA fragmentation, sperm DNA damages and chromatin alterations decrease pregnancy rates in natural fertilization, intrauterine insemination and *in vitro* fertilization (Bungum et al., 2007; Duran, 2002; Evenson et al., 1999; Evenson and Wixon, 2006; Spano et al., 2000; Zini, 2011). Moreover, pregnancy loss following *in vitro* fertilization (IVF) or intracytoplasmic sperm injection (ICSI) treatments has also been attributed to poor sperm DNA integrity (Zini et al., 2008). Although sperm DNA fragmentation is more frequent in infertile men, sperm of fertile men display DNA fragmentation but to a lesser extent (Bellver et al., 2010; Brahem et al., 2011; Perrin et al., 2009; Rybar et al., 2009; Venkatesh et al., 2011; Watanabe et al., 2011). After fertilization, the oocyte can efficiently repair some paternal DNA damages (Brandriff and Pedersen, 1981; Marchetti et al., 2007), but in the case of highly damaged sperm DNA, this could exceed the DNA repair capacity of the oocyte leading to some genetic aberrations, developmental arrest or pregnancy loss.

Chromosomal structural aberrations such as translocations, deletions and inversions, may originate from meiotic recombination involving non-allelic repeated DNA sequences (Heyer et al., 2010). However, since about 80% of chromosomal rearrangements are reported to be of paternal origin (Buwe et al., 2005; Thomas et al., 2006) and that male and female meiosis involves similar genetic mechanisms (Gu et al., 2008; Thomas et al., 2006)**,** one can surmise that yet another process unique to male gametogenesis may be involved. We therefore hypothesize that the chromatin remodeling process in spermatids, generating transient double-strand breaks, may provide the proper context for faulty repair and induction of transgenerational polymorphism. In addition, it is tempting to speculate that, because chromatin condensation occurs, free DNA ends are brought in juxtaposition, increasing the chance of NHEJ repair involving two different chromosomes, which may lead to translocations. Interestingly, chromosomes possess their own territory within the nucleus of somatic cells and in sperm cells (Hazzouri et al., 2000; Manvelyan et al., 2008; Mudrak et al., 2005; Zalenskaya et al., 2000). Moreover, chromosomes known to have higher translocation rates have close chromosomal territories in somatic cells (Branco and Pombo, 2006; Brianna

transmission to the next generation.

**5.1 Incomplete DNA repair** 

**5.2 Faulty repair Structural aberrations** 

**5. Possible consequences on fertility and genetic integrity** 

NHEJ) and the alternative (or "back-up") pathway (referred to as B-NHEJ), which is also known as microhomology-mediated end-joining (MMEJ) (Ciccia and Elledge, 2010; West, 2003). Therefore, we will discuss known somatic DNA repair pathways and their potential role in spermatids when supported by published data.
