**2. Spermatogenesis**

In most mammals, testes are found in the scrotum and are maintained at lower temperature (2-8°C) than the core body (Harrison and Weiner, 1949; Setchell, 1998). In fact, spermatogenesis is known to work better at lower temperature and it was shown that fertility declines with scrotal hyperthermia. For example, higher scrotal temperature due to fever or lifestyle correlates with decreased semen quality in humans (reviewed in Jung and Schuppe, 2007).

To support germ cells in their development, Sertoli cells are located at the basal lamina, throughout the seminiferous tubules (Russell, 1990). They provide nutrients and essential

<sup>\*</sup> Frédéric Leduc, Geneviève Acteau, Marie-Chantal Grégoire, Olivier Simard, Jessica Leroux, Audrey Carrier-Leclerc and Mélina Arguin

*Department of biochemistry, Faculty of medicine and health sciences, Université de Sherbrooke, Canada* 

Post-Meiotic DNA Damage and Response in Male Germ Cells 307

2009; Krishnamoorthy et al., 2006; Leduc et al., 2008a; Meyer-Ficca et al., 2005) are known to initiate and participate in the exchange from histones to the more basic proteins such as TPs and PRMs during the transition from the round to the elongated spermatids. These modifications are known to modulate the affinity of histones for DNA, but also the affinity of other proteins for histones, such as chromatin remodelers, DNA repair proteins or the transcription machinery. After spermiation, this unique protamine-based chromatin is further stabilized by the creation of intraprotamine disulfide bonds during the transit through the epididymis (Golan et al., 1996). Therefore, protamination provides both chemical and mechanical protection to the male haploid genome. Interestingly, protamination of the male genome is not complete and varies across species. In the mouse spermatozoon, about 1-2% of the genome remains organized by histones (Balhorn et al., 1977), whereas up to 15% of histones are still found in humans spermatozoa (Gatewood et al., 1990; Gusse et al., 1986; Tanphaichitr et al., 1978). This observation lead to hypothesize that these nucleosomes could serve as epigenetic markers for embryonic development (Arpanahi et al., 2009; Zalenskaya et al., 2000) (for a more detailed review on the sperm

chromatin organization, see Johnson et al., 2011)

**3.1 Single strand damage and repair** 

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

pathways.

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

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

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

molecules to the differentiating germ cells and regulate the seminiferous tubular fluid (Griswold, 1998; Rato et al., 2010). The Sertoli cells are interconnected by different junctions, creating a unique barrier between surrounding blood vessels and differentiating germ cells that is known as the "blood-testis barrier" (Cheng and Mruk, 2002; Dym and Fawcett, 1970; Setchell, 1969; Vogl et al., 2008). This barrier restricts molecules to enter or exit the adluminal compartment, creating a microenvironment with diverse transporters and preventing immunological response against germ cells (reviewed in Mital et al., 2011).

Spermatogenesis is the cellular differentiation pathway leading to the production of male gametes. This process takes place in seminiferous tubules in the testis, which is a unique environment regulated by follicle-stimulating and luteinizing hormones, secreted by the pituitary gland (Russell, 1990). From birth to puberty, seminiferous tubules are composed of spermatogonia, the precursor stem cell of the germinal cells, and Sertoli cells. At the onset of puberty, spermatogonia undergo mitosis and commit to the differentiation pathway leading to male germ cells. As the germ cells differentiate, they migrate towards the lumen of the tubule, creating an organized stratified structure. Spermatogenesis can be divided in two phases, spermatocytogenesis and spermiogenesis.

Spermatocytogenesis is the process by which a spermatogonium differentiates into primary spermatocytes, which duplicate their DNA to undergo meiosis and become haploid spermatids. This meiotic division is important to create genetic variations by meiotic crossovers and random segregation of parental chromosomes. Spermiogenesis is characterized by the radical metamorphosis of the haploid spermatid into spermatozoa, requiring the reorganization of their organelles. The acrosome, a cap-like structure needed for enzymatic digestion of the oocyte outer membrane, is formed from the Golgi apparatus. At the opposite nuclear pole, the flagellum begins to grow from the centrioles and mitochondria groups at the mid-piece of the emerging flagellum to produce the required energy for its later motion. Finally, the spermatid is stripped of most of its cytoplasm and ultimately released in the lumen of the seminiferous tubule. Most interestingly, the nucleus of spermatids is also remodeled and condensed to protect the paternal genome as well as providing a more hydrodynamic shape. However, this nuclear reorganization is characterized by transient DNA strand breaks that may be necessary to relieve the torsional stress as outlined below.

#### **2.1 Chromatin remodeling process**

Through the chromatin remodeling process of spermatids, the paternal genome is condensed tenfold compared to somatic cells, forming a nucleus with an hydrodynamicshape (Balhorn et al., 1984). To achieve such a high degree of compaction, chromatin must first rely on a set of abundant transition proteins (TPs) subsequently replaced by the protamines (PRMs) (Balhorn et al., 1984; Braun, 2001). The arginine-rich PRMs bind DNA and neutralize the phosphodiester backbone of the double helix (Balhorn, 1982), allowing for a tight compaction of the DNA into torroids (Ward, 1993). Although the onset of chromatin remodeling is poorly understood, incorporation of testis-specific histone variants (Churikov et al., 2004; Govin et al., 2007; Lu et al., 2009; Martianov et al., 2005; Yan et al., 2003) and regulated post-translational modifications of histones, such as acetylation (Christensen et al., 1984; Grimes and Henderson, 1984; Marcon and Boissonneault, 2004; Meistrich et al., 1992), ubiquitination (Baarends et al., 1999; Chen et al., 1998), methylation (Godmann et al., 2007; van der Heijden et al., 2006) and phosphorylation (Blanco-Rodríguez,

molecules to the differentiating germ cells and regulate the seminiferous tubular fluid (Griswold, 1998; Rato et al., 2010). The Sertoli cells are interconnected by different junctions, creating a unique barrier between surrounding blood vessels and differentiating germ cells that is known as the "blood-testis barrier" (Cheng and Mruk, 2002; Dym and Fawcett, 1970; Setchell, 1969; Vogl et al., 2008). This barrier restricts molecules to enter or exit the adluminal compartment, creating a microenvironment with diverse transporters and preventing

Spermatogenesis is the cellular differentiation pathway leading to the production of male gametes. This process takes place in seminiferous tubules in the testis, which is a unique environment regulated by follicle-stimulating and luteinizing hormones, secreted by the pituitary gland (Russell, 1990). From birth to puberty, seminiferous tubules are composed of spermatogonia, the precursor stem cell of the germinal cells, and Sertoli cells. At the onset of puberty, spermatogonia undergo mitosis and commit to the differentiation pathway leading to male germ cells. As the germ cells differentiate, they migrate towards the lumen of the tubule, creating an organized stratified structure. Spermatogenesis can be divided in two

Spermatocytogenesis is the process by which a spermatogonium differentiates into primary spermatocytes, which duplicate their DNA to undergo meiosis and become haploid spermatids. This meiotic division is important to create genetic variations by meiotic crossovers and random segregation of parental chromosomes. Spermiogenesis is characterized by the radical metamorphosis of the haploid spermatid into spermatozoa, requiring the reorganization of their organelles. The acrosome, a cap-like structure needed for enzymatic digestion of the oocyte outer membrane, is formed from the Golgi apparatus. At the opposite nuclear pole, the flagellum begins to grow from the centrioles and mitochondria groups at the mid-piece of the emerging flagellum to produce the required energy for its later motion. Finally, the spermatid is stripped of most of its cytoplasm and ultimately released in the lumen of the seminiferous tubule. Most interestingly, the nucleus of spermatids is also remodeled and condensed to protect the paternal genome as well as providing a more hydrodynamic shape. However, this nuclear reorganization is characterized by transient DNA strand breaks that may be necessary to relieve the torsional

Through the chromatin remodeling process of spermatids, the paternal genome is condensed tenfold compared to somatic cells, forming a nucleus with an hydrodynamicshape (Balhorn et al., 1984). To achieve such a high degree of compaction, chromatin must first rely on a set of abundant transition proteins (TPs) subsequently replaced by the protamines (PRMs) (Balhorn et al., 1984; Braun, 2001). The arginine-rich PRMs bind DNA and neutralize the phosphodiester backbone of the double helix (Balhorn, 1982), allowing for a tight compaction of the DNA into torroids (Ward, 1993). Although the onset of chromatin remodeling is poorly understood, incorporation of testis-specific histone variants (Churikov et al., 2004; Govin et al., 2007; Lu et al., 2009; Martianov et al., 2005; Yan et al., 2003) and regulated post-translational modifications of histones, such as acetylation (Christensen et al., 1984; Grimes and Henderson, 1984; Marcon and Boissonneault, 2004; Meistrich et al., 1992), ubiquitination (Baarends et al., 1999; Chen et al., 1998), methylation (Godmann et al., 2007; van der Heijden et al., 2006) and phosphorylation (Blanco-Rodríguez,

immunological response against germ cells (reviewed in Mital et al., 2011).

phases, spermatocytogenesis and spermiogenesis.

stress as outlined below.

**2.1 Chromatin remodeling process** 

2009; Krishnamoorthy et al., 2006; Leduc et al., 2008a; Meyer-Ficca et al., 2005) are known to initiate and participate in the exchange from histones to the more basic proteins such as TPs and PRMs during the transition from the round to the elongated spermatids. These modifications are known to modulate the affinity of histones for DNA, but also the affinity of other proteins for histones, such as chromatin remodelers, DNA repair proteins or the transcription machinery. After spermiation, this unique protamine-based chromatin is further stabilized by the creation of intraprotamine disulfide bonds during the transit through the epididymis (Golan et al., 1996). Therefore, protamination provides both chemical and mechanical protection to the male haploid genome. Interestingly, protamination of the male genome is not complete and varies across species. In the mouse spermatozoon, about 1-2% of the genome remains organized by histones (Balhorn et al., 1977), whereas up to 15% of histones are still found in humans spermatozoa (Gatewood et al., 1990; Gusse et al., 1986; Tanphaichitr et al., 1978). This observation lead to hypothesize that these nucleosomes could serve as epigenetic markers for embryonic development (Arpanahi et al., 2009; Zalenskaya et al., 2000) (for a more detailed review on the sperm chromatin organization, see Johnson et al., 2011)
