REVIEW

Spermatogenesis, DNA damage and DNA repairmechanisms in male infertility

Sezgin Gunes a,b, Maha Al-Sadaan c, Ashok Agarwal a,*

a American Center for Reproductive Medicine, Cleveland Clinic, USA; b Faculty of Medicine, Department of MedicalBiology, Ondokuz Mayis University, Samsun, Turkey; c College of Medicine, Alfaisal University, Saudi Arabia* Corresponding author. E-mail address: agarwaa@ccf.org (A Agarwal).

Sezgin Gunes, PhD is an Associate Professor of Medical Biology in the Department of Medical Biology at OndokuzMayis University, Turkey. Sezgin has been a Visiting Research Scientist at the Cleveland Clinic’s Center for Re-productive Medicine. Back in Turkey, she teaches molecular biology and genetics courses to medical studentsand directs a number of MSc and PhD graduate students. She has over 30 research articles and her current re-search is on epigenetics, cytogenetics, and gene expression alterations.

Abstract Spermatogenesis is a complex process of proliferation and differentiation during male germ cell development involvingmitosis, meiosis and spermiogenesis. Endogenous and exogenous physical, chemical and biological sources modify the genome of sper-matozoa. The genomic integrity and stability of the sperm is protected by DNA repair mechanisms. In the male germline cells, DNArepair mechanisms include nucleotide excision repair, base excision repair, DNA mismatch repair, double strand break repair andpost-replication repair. Defects in repair mechanisms cause arrest of spermatogenesis and abnormal recombination, ultimately re-sulting in male infertility. This review focuses on molecular mechanisms of the DNA repair pathways, DNA repair defects and maleinfertility.© 2015 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

KEYWORDS: apoptosis, DNA damage, DNA repair mechanisms, infertility, spermatogenesis

Introduction

Spermatogenesis is the process that involves the develop-ment of spermatozoa in the seminiferous tubules of the testisand epididymis (Chocu et al., 2012; Nussbaum et al., 2007;Xiao et al., 2013). Sperm DNA integrity is essential forproducing normal motile spermatozoa (de Rooij andRussell, 2000; Mahmoud, 2012; Ménézo et al., 2010; Simhadriet al., 2014). Reactive oxygen species (ROS), abnormalsperm chromatin packaging and apoptosis result in DNA

damage within spermatozoa (Agarwal et al., 2012, 2014a,2014b, 2014c; Mahfouz et al., 2010). The five important DNAdamagemechanisms involved in male germline include nucleo-tide excision repair (NER), base excision repair (BER), DNAmis-match repair (MMR), double strand break repair and post-replication repair. In this review article, the process ofspermatogenesis, origin of DNA damage and DNA repairmechanisms are examined closely to gain a better under-standing of the role of DNA repair mechanisms duringspermatogenesis.

http://dx.doi.org/10.1016/j.rbmo.2015.06.0101472-6483/© 2015 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Reproductive BioMedicine Online (2015) 31, 309–319

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Spermatogenesis

Sperm cells that develop from the primordial germ cells movetoward the seminiferous tubule lumen as they mature, aftera series of mitoses followed by meiosis I and II (Nussbaumet al., 2007; Zini and Agarwal, 2012). Spermatogenesis isdivided into three phases: proliferative; spermatogonial (primi-tive) and spermiogenesis phases respectively (Chocu et al.,2012). In the first phase, spermatogonia undergo a series ofDNA replication cycles and mitoses. Initially, the spermato-gonial cells enter the interphase, where cellular growth andDNA synthesis take place. The S phase is the most signifi-cant step in the process for the replication of genetic mate-rial before it advances to the spermatogonial phase of the cellcycle. Germ cells must be protected against high rates of mu-tation to maintain the species survival during DNA replica-tion in S phase of cell cycle (Nussbaum et al., 2007). Themismatched nucleotides of DNA chains are corrected byexonucleolytic proofreading property of DNA polymerase andstrand-directed mismatch repair.

Two strands of the parental DNA unwind by enzymes calledDNA helicases during the S phase of the cell cycle. The un-winding sequences of the parental DNA that are rich in ad-enines and thymines are called the replication origins. Thereplication origin forms a Y-shaped replication fork fol-lowed by the binding of single-stranded DNA-binding pro-teins to prevent the reformation of the double helix. Duringthe unwinding process, when a supercoiling problem is en-countered, DNA topoisomerases prevent supercoiling of theDNA by cutting the single or double stranded DNA ahead ofthe replication bubble. The leading strand is copied in the di-rection of the replication fork (5′→3′) and synthesized con-tinuously (Champe et al., 2005), whereas the lagging strandis copied away from the direction of replication fork and syn-thesized as discontinuous short pieces called Okazaki frag-ments. DNA polymerases require a RNA primer to initiate thesynthesis of a complementary strand. DNA polymerase III beginsto synthesize a new DNA strand after recognizing the RNA

primer, and the elongation process will be sustained in thelagging strand until a stretch of RNA is encountered. Finally,the DNA polymerase I begins to fill the gaps and the DNA-ligase seals the remaining nicks. As a result of the DNA rep-lication process, two identical strands are produced (Champeet al., 2005) (Figure 1). The mismatch repair mechanism playsa crucial role in correcting mismatches after DNA replica-tion in all cells, including the spermatozoa. During mitosis,chromosomes are most vulnerable and DNA repair processesare shut down to prevent fusion of telomeres (Orthwein et al.,2015).

In the second phase of spermatogenesis, the spermato-cyte diploid genome is reduced to haploid spermatids. Finally,in the third phase, spermatids differentiate to spermatozoa(Figure 2).

Daughter cell type A spermatogonium remains at the basallamina as a precursor cell and type B spermatogonium movesto the adluminal compartment. Spermatogonium B under-goes two meiotic divisions, meiosis I and meiosis II, with onlyone round of DNA synthesis to produce haploid spermatids thatare genetically different from each other during spermato-cyte stage (Gordon and Lamb, 2006; Nussbaum et al., 2007).Meiosis I is different from meiosis II and mitosis in that it in-volves exchange of genetic material between homologueschromosomes and reduction of the chromosome number byone-half. The exchange of genetic material occurs during pro-phase I (de Vries et al., 1999; Sun et al., 2005). This phaseis subdivided into leptotene, zygotene, pachytene, diplo-tene and diakinesis. Recombination is initiated at leptotenestage by formation of double stranded breaks (DSB), as de-scribed later (Costa et al., 2005). The final process of sper-matogenesis, spermatid differentiation into spermatozoa iscalled spermiogenesis (Nussbaum et al., 2007). This processhas been described in four stages; Golgi phase, the cap phase,formation of the tail and the maturation stage. Golgi phaseis characterized by the head and axoneme formation. Duringthis phase, spermatozoal DNA undergoes packaging with spe-cific nuclear proteins called transition proteins, which will be

Figure 1 DNA replication fork. A view of the arrangement of the replication proteins at the replication fork is shown. Several enzymesand proteins are involved in replication process of genome. Accurate duplication of genetic material is important for both somaticand germ cells and is carried out by polymerase enzymes. SSBP = single-stranded DNA-binding proteins.Reprinted with permission,Cleveland Clinic Center for Medical Art & Photography © 2014. All Rights Reserved.

310 S Gunes et al.

replaced with protamines later. This process is followed bythe elongation of spermatid and acrosomal cap formationduring the cap phase. The next phase is where the elonga-tion of one of the centrioles by manchette occurs in order todifferentiate into the tail. The last stage is the phagocytosisof residual bodies by Sertoli cells (Zini and Agarwal, 2012).

Origin of DNA damage and male infertility

Sperm DNA damage is characterized by abasic sites, base modi-fications, single-strand and double-strand DNA breaks and DNA

proteins cross links. The three main theories that have beenproposed to identify the origin of the sperm DNA damage arereactive oxygen species (ROS), sperm chromatin packagingand apoptosis.

Reactive oxygen species

ROS are highly reactive molecules, containing oxygen (Aitkenet al., 1998). Examples include hydrogen peroxide (H2O2), hy-droxyl radical (HO•), nitric oxide (NO) and hypochlorous acid

Figure 2 Spermatogenesis. Primordial germ cells, proliferative type A spermatogonia, undergo a series of mitotic divisions. One ofthe daughter cells renews the stock of type A spermatogonia, the other becomes a type B spermatogonia with incomplete cytoplas-mic separation. These divide and their daughter cells migrate towards the lumen. Type B spermatogonial cells enter the meioticpathway. Meiosis involves two cell divisions to produce haploid spermatids with only one round of DNA synthesis. Finally, sperma-tids differentiate into spermatozoa and are released into the lumen of tubule. Reprinted with permission, Cleveland Clinic Centerfor Medical Art & Photography © 2014. All Rights Reserved.

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(HOCl). Sources of ROS could be exogenous or endogenous.Exogenous sources of ROS are exposure to radiation (x-rays,ultraviolet light), cigarette smoking, herbicides, alcohol abuse,chronic stress, drugs (acetaminophen) and air pollution. Onthe other hand, the endogenous sources are mitochondrial res-piration and enzymatic systems such as xanthine oxidase andNADPH oxidase (Ménézo et al., 2010).

DNA damage induced by ROS involves abasic sites, basemodifications, single-strand and double-strand DNA breaks andDNA proteins cross links. The presence of high ROS concen-tration results in loss of sperm motility, fertilizing potentialand DNA damage (Aitken et al., 1992; Sharma et al., 2013;Tamburrino et al., 2012). Recent reports provide evidence tosupport that 20–88% of subfertile men had high presence ofROS in the semen (Agarwal et al., 2014a, 2014b).

Sperm chromatin packaging

Sperm chromatin packaging is a critical process required toaccommodate the enormous amount of DNA into a small spermcell. Fertilization requires many physiological events, includ-ing movement of sperm cells along the entire female repro-ductive system, attachment to zona pellucida and penetrationinto oocyte. To accomplish all these phases, a regulatorymechanism involving striking modifications that require re-placement of 90–95% histones by protamines becomes effec-tive (Torregrosa et al., 2006). Protamines are small proteinsrich in arginine. They are located in the nucleus and synthe-sized during advanced stages of spermatogenesis.Protamination of sperm chromatin facilitates compaction ofnucleus required for sperm motility, and also protects spermgenome from oxidation, and harmful molecules within thefemale reproductive system (Torregrosa et al., 2006).

Replacement of histones by protamines involves translo-cation of histone by selected histones variants that are ex-pressed during spermatogenesis. Hyper-acetylation of histonetails leads to loosening of chromatin structure and stimu-lates DNA strand breaks caused by topoisomerase enzyme thatfacilitates separation of histones and replacement by tran-sition proteins (Meistrich et al., 2003; Rousseaux et al., 2011).DNA topoisomerase covalently attaches to DNA phosphate,thereby breaking a phosphodiester bond in the single or doublestrands of DNA and the phosphodiester bond re-forms spon-taneously. Some agents may inhibit this ligation, and suchbreaks are usually repaired spontaneously and end up asdamaged only if the repair process is disrupted.

TP 1 and 2 stick to DNA and are totally replaced by prot-amines. Transition proteins play critical role in separation ofhistones and later condensation by protamines of sperm DNA(Meistrich et al., 2003). Non-homologous end-joining (NHEJ)repair is active during replacement of sperm protamines bynucleosomes, as described later (Derijck et al., 2008).

Apoptosis

Apoptosis is a genetically-controlled, programmed form of celldeath and is necessary for the development of spermatogen-esis (Kumar et al., 2012). During spermatogenesis, apoptosislimits the size of germ cell population and aids in optimal

germ–Sertoli cell ratio (Aitken and Baker, 2013; Leduc et al.,2008). Programmed cell death is a physiological mechanismto eliminate excess cells during proliferation, removal ofderived hormone-dependent cells and elimination of poten-tial harmful cells (Aitken and Baker, 2013; Shaha et al., 2010;Tripathi et al., 2009). Sometimes, the cells destined to beeliminated escape the process of apoptosis and find their wayinto the ejaculate. This phenomenon is called abortive apop-tosis (Sakkas et al., 1999a, 1999b). Production of ejacu-lated spermatozoa that possess remnant apoptotic markersexhibiting abnormal morphological forms, irregular biochemi-cal function or nuclear DNA damage may contribute damagedDNA to subsequent generations with the increased use of as-sisted reproduction techique procedures, such as intracyto-plasmic sperm injection. Exposure to radiation or toxic agentsinduces DNA damage, which stimulates synthesis of p53 pro-teins. p53 proteins stimulate cell sensors to activatepro-apoptotic Bax and Bak proteins and induce the synthe-sis of Bcl-2 family of pro-apoptosis proteins to trigger apop-tosis (Aitken and Baker, 2013; Kumar et al., 2012). In addition,DNA damage and other pathological conditions cause misfoldedproteins, cell injury and atrophy (Ménézo et al., 2010).

Apoptosis pathways

Intrinsic pathway

Reactive nitrogen and oxygen species are generated continu-ously and create oxidative stress in spermatozoa, resultingin the triggering of intrinsic apoptotic pathways.

Mitochondrial proteins play a major role in the activa-tion and termination of intrinsic apoptosis. BH3 proteins(interacting-domain death agonist) activate Bax and Bak pro-apoptotic members and inhibit Bcl-2 and Bcl-X1 (Shaha et al.,2010). Activation of pro-apoptotic proteins induce mitochon-drial cytochrome c leakage by dimerization and insertion intomitochondrial membrane. Cytochrome c in turn activatescaspase-9, resulting in activation of the caspase cascade, fol-lowed by nuclear fragmentation (Kumar et al., 2012).

Extrinsic apoptosis pathway

Sperm cell extrinsic apoptosis is mediated by activation of Fasprotein receptors (Agarwal, 2003; Lee et al., 1997). Fas re-ceptors are activated by binding to Fas ligand, which is ex-pressed on T lymphocytes. The FasL binding will induce Fastrimerization to recruit activated caspase-8. Apoptosis willbe stimulated by its activation in two parallel cascades: cleav-age and activation of caspase-3 directly, or cleavage of theBcl-2 family’s pro-apoptotic protein (Bid) resulting in acti-vation of the mitochondrial pathway (Kumar et al., 2012). Thecombination of the intrinsic and extrinsic pathway result instrong lethal cell response. Both pathways will activatecaspase-9, which will in turn activate executioner caspases.The activation of caspases results in fragmentation of cells(Martin et al., 1995). A link has been suggested between spermquality and Fas receptor presence indicating that the pres-ence of the Fas receptor occurs in less than 10% of ejaculated

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healthy spermatozoa and more than 50% of ejaculated semenwith oligozoospermia (Sakkas et al., 1999b). Pro- and anti-apoptotic molecules involved in the various stages of spermdifferentiation are indicated in Table 1. Apoptotic sperm cellsinitiate signals to macrophages in order to be phagocytized(Kumar et al., 2012). These signals could be a result of ex-ternal expression of phosphatidylserine on the cell mem-brane by a protein known as scramblase or by expressingadhesive glycoproteins that are recognized by phagocytes (Saidet al., 2010).

Apoptosis manifestations in spermatozoa andits association with infertility

Several early and late stage markers can be used to identifyapoptosis in the human sperm. The activation of phos-phatidylserine mark by caspases is one such early apoptoticmarker. DNA fragmentation is one of the well-characterizedlate-stage markers of apoptosis. Low motile sperm reflectshigh presence of apoptosis markers compared to the highlymotile sperm cells. These findings suggest a possible rela-tionship between apoptosis markers and male infertility(Brugnon et al., 2006). Low percentage of Fas-positive sper-matozoa in men indicate that they have normal semencharacteristics. On the other hand, the percentage of Fas-positive spermatozoa can be as high as 50% in men with ab-normal semen parameters (Sakkas et al., 1999b). Thisobservation suggests that the correct clearance of sperma-tozoa in infertile men via apoptosis is not occurring. Thepresence of spermatozoa with apoptotic markers, such as Fas-positivity and DNA fragmentation, thereby, prove vital to com-pellingly conclude that, in men with abnormal semenparameters, abortive apoptosis occurs (Sakkas et al.,1999a).

DNA repair mechanisms

In the mammalian genome, it is estimated that about 105 DNAlesions are produced per cell/day as a result of spontaneousreplication errors and cellular metabolism (Hoeijmakers, 2009).

To compensate for the DNA damage in spermatozoa, fiverepair mechanisms have been evolved to maintain genomicintegrity: nucleotide excision repair; base excision repair; mis-match repair; double-strand break repair and post-replication

repair (Figure 3). These mechanisms detect and repair DNAdamage regardless of the cause.

Nucleotide excision repair

The nucleotide excision repair (NER) mechanism acts upon sig-nificant lesions such as pyrimidine dimers caused by the ul-traviolet, mismatched bases or bulky adducts, oxidativedamage and DNA intra-strand cross links (Lyama and Wilson,2013). Thesemodifications cause distortion of the helical struc-ture of DNA (Gillet and Scharer, 2006). The DNA damage isscanned and detected by roughly 30 different proteins in theNERmechanism. This mechanism consists of two sub-pathways,termed global genome NER (GG-NER) and transcription-coupled NER (TC-NER). Each pathway is responsible for rec-ognition of different lesions (Ménézo et al., 2010). Throughoutthe genome, GG-NER is responsible for DNA damage (Fagbemiet al., 2011) and TC-NER is responsible for detecting lesionsin the coding strand of actively transcribed genes (Lyama andWilson, 2013). In GG-NER, the DNA damage is scanned and de-tected by XPC/RAD23B proteins (Fagbemi et al., 2011; Fousteriand Mullenders, 2008; Lyama and Wilson, 2013; Sugasawaet al., 1998). TC-NER is activated by DNA distortions that blockthe elongating RNA polymerase II complex (Fousteri andMullenders, 2008). Following damage recognition, both path-ways use the repair machinery. DNA helix unwinds to permitXPA binding by replication protein A to DNA strand for sec-ondary DNA damage recognition. Furthermore, endonuclei XPGand XPF/ERCC1 cleave the DNA, leading to the removal oflesions (Dexheimer, 2013). Lastly, DNA polymerase fills thegap and the nick is sealed by DNA ligase. Defects in the NERmechanism are found in autosomal recessive disorders suchas xeroderma pigmentosum, Cockayne syndrome andtrichothiodystrophy. These disorders are characterized bymarked hypersensitivity to sunlight, predisposition to skincancer, neurological abnormalities and immature sexual de-velopment in adults. Xeroderma pigmentosum group A gene-deficient male mice become subfertile after 24 months, withimpaired spermatogenesis and diminished testis (Nakane et al.,2008). A case study of 620 infertile men and 385 controls re-ported a significant association between XPA (–4) G/A poly-morphism and sperm DNA damage (Gu et al., 2010).Endogenous and exogenous agents produce a variety of DNAlesions during spermatogenesis. To overcome the DNA damage,spermatogenic cells benefit from DNA repair mechanisms, in-cluding NER; however, knowledge about nucleotide exci-sion in spermatogonial stem cells is limited.

Table 1 Pro- and anti-apoptotic molecules involved in the stages of sperm differentia-tion (Shaha et al., 2010).

Stages of spermdifferentiation

Pro-apoptotic molecule Anti-apoptotic molecule

Primordial germ cells Bax, Bad, Bim, Bak Bcl-xSpermatogonia Bax, p53 c-kitSpermatocyte Bax, Fas/FasL Bcl-xL, Bcl-xS

Round spermatid Fas/FasL Bcl-xL, Bcl-xS

Elongated spermatid Fas/FasL Bcl-xSpermatozoa Fas/FasL Bcl-x

313DNA damage, repair and male infertility

Base excision repair

Base excision repair (BER) is a highly coordinated pathway re-sponsible for the removal of non-helix-distorting base lesions(Boiteux and Radicella, 1999). These specific base altera-tions can be increased by deamination or oxidation. Many stepsinvolving various enzymes and proteins, conducted by the BERpathway, protect against the harmful consequences of DNAdamage (Lyama and Wilson, 2013). The most significant andwell-characterized base lesion is 8-Oxo-2′-deoxyguanosine (8-oxo-dG) that pairs with adenine or cytosine during DNA rep-lication and cause G:C to T:A transversion mutations. Inaddition, it gives rise to mutant RNA transcripts (Said et al.,2004). The BER pathway is initiated by recognition and ex-cision of damage by DNA glycosylases, which detect alteredsingle DNA base, and cleave the N-glycosidic bond that joinsthe damaged base to deoxyribose (Champe et al., 2005). Thisresults in a missing base in the DNA sugar-phosphate back-bone known as apurinic or apyrimidinic site, which will becleaved by apurinic or apyrimidinic (AP) endonuclease. Theresulting single-strand nick is then processed through eithershort-patch by DNA ligase III or long-patch through DNA ligaseI (Said et al., 2004). 8-hydroxy 2′oxoguanine (8OHdG) is a baseadduct generated during oxidative stress in spermatozoal DNA.8-oxoguanine glycosylase 1 (OGG1) cuts the 8OHdG residueand generates abasic sites in spermatozoa (Aitken et al., 2014;Maki and Sekiguchi, 1992). Then, apyrimidinic endonucle-ase incises the phosphate backbone of DNA to insert an un-modified nucleotide in somatic cells and oocytes. Inspermatozoa, apyrimidinic endonuclease 1 is absent andapyrimidinic sites created by OGG1 in damaged spermato-zoa DNA are repaired in the S phase of the first mitotic divi-sion in the zygote (Aitken et al., 2014; Shimura et al., 2002).

Mismatch repair mechanism

Mismatches occur as a result of tautomerization of the DNAstrand bases during DNA replication owing to inefficient proof-reading by DNA polymerase (Champe et al., 2005). Mis-matches are base–base mismatches such as G-T or A-C, andinsertion-deletion loops (Jiricny, 2006). Mismatch repairmechanism (MMR) increases the fidelity of DNA replication byabout 100 times and suppresses genomic instability. Themechanism is well-conserved evolutionarily to prevent genomicinstability (Shimura et al., 2002) in all living organisms. In orderto repair the mismatch, MMR proliferating cell nuclear antigenproteins first identify mispaired nucleotides. The differen-tiation of parental and new synthesized strand is throughmeth-ylation, in which the parental strand is methylated and thenewly synthesized strand is not methylated in prokaryotes.In eukaryotes, however, MMR is associated with DNA repli-cation machinery that facilitates discrimination via bindingof proliferating cell nuclear antigen in leading strand and free5′ ends of Okazaki fragments at the lagging strand (Champeet al., 2005; Lyama and Wilson, 2013). The MutS protein isresponsible for the recognition and binding to the mis-matched base of the newly synthesized DNA strand. MutL isa latent clamp-structuredmolecule that binds at unmethylatedsites along the newly synthesized strand to induce exonucle-ase activity of MutH in prokaryotes (Lyama and Wilson, 2013).

Eukaryotes have several homologues of the proteins MutSand MutL. MutS homologs such as MSH1-6 and MutL ho-mologs include MLH1-MLH3, PMS1 and PMS2, which formheterodimers (Gordon and Lamb, 2006; Jun et al., 2006).Maduro et al. (2003), showed genomic instability and defectsof MLH1 or MSH2 in non-obstructive azoospermia. MSH4 andMSH5 are essential in themeiotic recombination process. There

Figure 3 DNA damage and DNA repair mechanisms. Common DNA damaging agents (left), DNA lesion induced by these agents (middle),and the most relevant DNA repair mechanisms responsible for the removal of the lesions (right).Reprinted with permission, Cleve-land Clinic Center for Medical Art & Photography © 2014. All Rights Reserved.

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are two types of MutS homologue heterodimers. The first typeis MutSa (MSH2 /MSH6), which functions in DNA base–pairmis-match. The second is MutSb (MSH2 / MSH3), which functionsin insertion–deletion loopmismatch repair (Jun et al., 2006).The connection of MutL with MutS-DNA complex activates theMutH,whichnicks thedaughter strandand recruitsDNAhelicaseII to disconnect the DNA double strands (Brierley and Martin,2013). Germ line mutations of these proteins are associatedwith hereditary non-polyposis colorectal cancer. Aberrantmethylation ormutations of these genes are relatedwith spo-radic cases (Barnetsonet al., 2006; Peltomaki andVasen, 2004).Exonucleases are recruited to digest the single stranded DNAtail followed by the formation of a gap. The gap is filled byDNA polymerase and sealed by an unidentified DNA ligase.

Four important proteins involved in MMR have been re-ported (MLH1, MLH2, MSH4 and RAD51) to be involved in maleinfertility. MLH3 gene encodes a DNA repair protein that in-teracts with MLH1. MLH1 and MLH3 are essential to facili-tate recombination and chiasmata separation during pachyteneand diplotene. Deficiency or absence of these genes are as-sociated with gametogenesis failure as a result of meioticarrest at pachytene resulting in reduced level of chiasmata(Gordon and Lamb, 2006). Mukherjee et al. (2010) reportedthat deletion of MLH1 gene displays microsatellite instabil-ity and male mice infertility. The summary of their findingon the consequences of deletion of MLH1 gene and other DNA

repair proteins involved in infertility in mouse models is pre-sented in Table 2. Xu et al. (2010) reported an associationbetween polymorphism C85T inMSH5 or C2531T inMLH3 genesand spermatogenesis impairment. Similarly, tagged polymor-phisms in MLH1 and PMS2 genes were shown to be associ-ated with male infertility and DNA damage in spermatozoa(Ji et al., 2012). Non-obstructive azoospermia is character-ized by primary spermatids deficiencies at the pachytene (Jiet al., 2012).

MSH4-MSH5 forms a complex with RAD51 and interacts withthe synaptonemal complex (SC) to stabilize the interactionsbetween homologous chromosomes during recombination. In-activation of these genes during meiosis results in meioticarrest at zygotene stage and decreased synapsis (Mukherjeeet al., 2010). The meiotic arrest will enable the formationof a gametogenesis-specific phenotype. To date, only a fewstudies (Ridgeway et al., 2014; Xu et al., 2010) have re-ported that MSH5 is associated with azoospermia. These studiesare not conclusive and more studies are needed to explainthe link betweenmismatch repair proteins andmale infertility.

DNA double-strand repair

Several factors may cause DNA double-strand breaks.These include ROS, failed DNA replication and DNA repair,

Table 2 DNA repair and DNA repair associated proteins involved in infertility in mouse models and in humans.

Gene/proteinPhenotype Reference

MLH1 Microsatellite instability (Mukherjee et al., 2010)Failure of crossing over and prematuredesynapsis of homologous chromosomesInfertility

MSH2 Loss of some germ cells (Mukherjee et al., 2010)Mismatch repair (MMR) deficiency in somaticcells

MSH3 MMR deficiency in somatic cells (Lipkin et al., 2002)Fertile

MSH4 Failure of spermatogonial maturation beyondzygonemaInfertility

(Gordon and Lamb, 2006)

MSH5 Incomplete and non-homologous chromosomalpairing

(de Vries et al., 1999)

InfertilityMSH6 MMR deficiency in somatic cells (Gordon and Lamb, 2006)

FertileMLH3 Infertile (Lipkin et al., 2000, 2002)PMS2 Genomic instability (Lipkin et al., 2002)

Disruption of normal chromosomal synapsisInfertility

EXO1 Infertile (Wei et al., 2003)Rb Essential for maturation of male germ cells (Nalam et al., 2009)Ubiquitin-proteasome

pathway proteinsSperm malformation (Hou and Yang, 2013)Infertility

FANCN Reduced fertility (Simhadri et al., 2014)Impaired meiosisIncreased apoptosis in germ cells

ATM Gonadal atrophy (Barlow et al., 1996;Xu and Baltimore, 1996)Azoospermia

BRCA1 Spermatogenic arrest (Gordon and Lamb, 2006)

315DNA damage, repair and male infertility

recombination, meiosis, ionizing radiation and chemothera-peutic agents. Unrepaired double strand breaks can lead totranslocations, DNA fusions and cell death. Homologous re-combination and NHEJ repair mechanisms function to repairDNA double-strand breaks (Lyama and Wilson, 2013).

Homologous recombination

Homologous recombination repair mechanism is an error-free mechanism that operates mainly during S phase of in-terphase and G2 phases (Lyama and Wilson, 2013; Ménézoet al., 2010). In this process, double strand breaks are pro-tected from exonuclease activity, by the binding of Rad51protein, homolog of RecA protein in Escherichia coli, to thestrands. Ataxia telangiectasia mutated (ATM) and MRE11–RAD50–NBS1 complex are activated by DNA double strandbreaks (Stracker and Petrini, 2011) and generate 3′-ssDNA byresecting the broken DNA ends through interactions withcarboxy-terminal binding protein (You and Bailis, 2010). Thetail of the single stranded DNA is coated by replicationprotein-A to remove secondary disruptive structures; repli-cation protein-A are replaced with RAD51 homologous se-quence on the sister chromatid (Forget and Kowalczykowski,2010). RAD51C interacts with BRCA2 to form complexes forhomologous pairing (Forget and Kowalczykowski, 2010; Yoshidaand Miki, 2004). Two studies have suggested a link betweenalterations of homologous recombination mechanism and in-fertility. A study conducted by Xu and Baltimore (1996) in-dicated that men with ataxia telangiectasia have azoospermiaand gonadal atrophy, owing to the failure of primary sper-matocytes at the leptotene-zygotene transition (Xu andBaltimore, 1996). Mutations of MRE11 blocks the meiotic re-combination (Gordon and Lamb, 2006).

Non-homologous end-joining

The Ku70 and Ku80 heterodimers recognize and bind to doublestrand breaks in DNA and then recruit DNA-dependent proteinkinase (Gordon and Lamb, 2006). Binding of Ku70 and Ku80heterodimers leads to recruitment of the MRE11 complex,which induces the removal of non-ligatable termini by aninward translocation followed by replication by DNA poly-merases and ligation to create compatible ends. Defects inthis repair system, whether in NHEJ or homologous recom-bination predisposes to cancer and immune deficiency syn-dromes (Kohl and Sekelsky, 2013; Lyama and Wilson, 2013).

DNA repair-associated proteins and infertility

Retinoblastoma

Retinoblastoma gene (RB1) encodes proteins involved in regu-lation of the cell cycle and differentiation (Nalam et al., 2009).Defects in RB1 causes osteogenic sarcoma, retinoblastoma andbladder cancer (Weinberg, 1995). In addition, few caseshave been reported in which RB1 gene inactivation is asso-ciated with infertility. In case of RB1 gene inactivation,

hypermethylation of O6-methylguanine-DNAmethyltransferase(MGMT) takes place (Choy et al., 2002) resulting in the si-lencing and reduction of MGMT. Patients suffering from thedepletion of MGMT display sperm microsatellite instability aswell as reduction in DNA damage repair (Choy et al., 2002,2004).

Furthermore, studies were conducted on knockout miceto identify the role of RB1 in the terminal differentiation ofSertoli cells using cre-loxP system. Nalam et al. (2009) con-ducted the study using a transgenic mouse expressing Crerecombinase and intercrossed Rbflox/floxmice with Rbwt/ − Amh-Cre + mice to achieve a complete deletion (Nalam et al., 2009;Vooijs et al., 1998). This was followed by collecting the testesfrom mice between 5 and 11 weeks of age to investigate theonset and progression of the Rb cKO phenotype. Mice that havelost retinoblastoma function expressed different pheno-types according to their age. Rb cKO phenotype mice at6 weeks of age are fertile with normal testes; in contrast, micebetween 10 and 14 weeks of age demonstrate severe Sertolicell atrophy, dysfunction and infertile sperm development.The study concluded that retinoblastoma is essential for properterminal differentiation of Sertoli cells (Nalam et al., 2009).

The ubiquitin–proteasome pathway

The ubiquitin–proteasome pathway (UPP) mechanism is es-sential for protein degradation in the mammalian cytosol andnucleus. According to the studies conducted by Ciechanoveron cultured mammalian cells, UPP is involved in DNA repair,protein translocation, circadian rhythm, protein folding, tran-scription and apoptosis (Ciechanover, 1996; Orlowski, 1999).Ubiquitination requires three essential enzymes: ubiquitin-activating enzyme, ubiquitin-conjugating and ubiquitin ligase(E3). Furthermore, Rajapurohitam et al. (2002) conducted astudy in which they concluded that the maximal mamma-lian UPP activity is observed in the testis. A recent investi-gation showed that UPP is involved in different stages ofspermatogenesis, such as meiosis and acrosome biogenesis.Deficient or abnormal ubiquitin proteasome pathway leads tospermmalformation and increases risk of male infertility (Houand Yang, 2013).

Fanconi anaemia

Fanconi anaemia is an autosomal recessive disorder withgenomic instability, impaired DNA repair, chromosome fra-gility, bone marrow failure and high cancer risk. Fanconianaemia genes are essential for the DNA inter strand crosslinksrepair. Proteins that belong to this DNA repair pathway arecrucial to protect genetic information. The FA pathway is com-posed of a total of 16 proteins (Nalepa and Clapp, 2014). Eightof them are essential for inter-strand crosslinks repair and in-volved in mono-ubiquitination of FANCD2 and FANCI (Gordonand Lamb, 2006; Koczorowska et al., 2014). Fanconi anaemiais caused by mutation of one of many Fanconi anaemia genessuch as FANCA, FANCE, FANCC, FANCD1 (BRCA2), FANCD2,FANCG, FANCB, FANCI, FANCJ, FANCF, FANCM, FANCL, FANCP,XPF, FANCN (PALB2) and RAD51C. Ninety per cent of Fanconianaemia cases are caused by FANCG, FANCC and FANCA

316 S Gunes et al.

mutations. Mutations or deletions of these genes result in im-paired repair pathway. Furthermore, the DNA-damaged regionsthat remain unrepaired will ultimately result in uncon-trolled cell growth or death (Koczorowska et al., 2014). FANCNinteracts with BRCA1 and BRCA2 in homologous recombina-tional repair of DSB. Studies conducted by Simhadri et al.(2014) indicated that FANCN mutant male mice showedreduced fertility owing to impaired meiosis and increasedapoptosis in germ cells.

In conclusion, infertility is a complex disorder with geneticand environmental causes. Although some specific muta-tions have been identified, other factors responsible for spermdefects remain unknown. Spermatogonia need proper ex-pression of genes for the regulation of germinal mitosis,meiosis, and apoptosis and maintenance of genomic integ-rity during these processes. Spermatogenesis involves a seriesof cell divisions throughout the life of men. Sperm DNA un-dergoes far more replication cycles then does the DNA inoocyte. Spermatogenic germ cells must be protected againstDNA damage and mutations to maintain reproduction andspecies survival. In addition, spermatogenic cells are as-saulted with exogenous agents that produce DNA damage.Maintenance of genetic stability requires both vitally accu-rate DNA replication processes andmechanisms for the removalof a variety of DNA lesions.

Despite the significant gaps in our knowledge, it is clearthat defects in DNA repair mechanisms result in abnormalsperm and ultimately, male infertility. In order to have a com-plete understanding about the link between DNA-repair mecha-nisms and male infertility in humans specifically, extensivestudies on mice models must be diligently followed in humanstoo. Studies of knockout mouse models of DNA repair genesto understand the role of these genes and their proteins maybe helpful to explain the cause of male infertility. A com-plete characterization of the varied mechanisms of DNAdamage repair will allow the development of customized treat-ments to repair the damage.

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Declaration: The authors report no financial or commercial con-flicts of interest.

Received 6 November 2014; refereed 3 June 2015; accepted 10 June2015.

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