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Causesandconsequencesofoxidativestressinspermatozoa

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Causes and consequences of oxidative stressin spermatozoa

Robert John AitkenA,D, Zamira GibbA, Mark A. BakerA, Joel DrevetB

and Parviz GharagozlooC

APriority Research Centre in Reproductive Science and Hunter Medical Research Institute,

Faculty of Science and IT, University of Newcastle, Callaghan, NSW 2308, Australia.BGReD laboratory, CNRS UMR6293-INSERM U1103-Clermont Universite, 63171 BP80006,

Aubiere cedex, France.CCellOxess LLC, 15 Roszel Street, Princeton, NJ 08540, USA.DCorresponding author. Email: john.aitken@newcastle.edu.au

Abstract. Spermatozoa are highly vulnerable to oxidative attack because they lack significant antioxidant protectiondue to the limited volume and restricted distribution of cytoplasmic space in which to house an appropriate armoury of

defensive enzymes. In particular, sperm membrane lipids are susceptible to oxidative stress because they aboundin significant amounts of polyunsaturated fatty acids. Susceptibility to oxidative attack is further exacerbated by thefact that these cells actively generate reactive oxygen species (ROS) in order to drive the increase in tyrosinephosphorylation associated with sperm capacitation. However, this positive role for ROS is reversed when spermatozoa

are stressed. Under these conditions, they default to an intrinsic apoptotic pathway characterised by mitochondrial ROSgeneration, loss of mitochondrial membrane potential, caspase activation, phosphatidylserine exposure and oxidativeDNA damage. In responding to oxidative stress, spermatozoa only possess the first enzyme in the base excision repair

pathway, 8-oxoguanine DNA glycosylase. This enzyme catalyses the formation of abasic sites, thereby destabilising theDNA backbone and generating strand breaks. Because oxidative damage to sperm DNA is associated with bothmiscarriage and developmental abnormalities in the offspring, strategies for the amelioration of such stress, including

the development of effective antioxidant formulations, are becoming increasingly urgent.

Additional keywords: apoptosis, fertilizing potential, lipid peroxidation, male germ line, oxidative DNA damage,ROS generation.

Introduction

Traditionally, spermatozoa are regarded as highly specialised

cells that have but one function in life: to achieve fertilisationand deliver the paternal component of the embryonic genometo an MII oocyte. Although defective sperm function has longbeen recognised as a major cause of human infertility (Hull

et al. 1985), this condition has conventionally been equatedwith the ability of spermatozoa to achieve fertilisation (Aitkenet al. 1987; Aitken 2006). With the passage of time, we have

come to understand that the functional competence of sper-matozoa cannot be defined merely in terms of the ability ofthese cells to fertilise an oocyte; it also needs to incorporate an

assessment of their ability to program a normal pattern ofembryonic development. Spermatozoa may affect embryonicdevelopment via both genetic and a variety of epigeneticmechanisms involving the methylation profile of the DNA,

the post-translational modification of nuclear histones and thecomposition of a variety of coding and non-coding RNAspecies that are integrated into these cells, to find ultimate

expression in the zygote and early embryo (Aitken 1999;Ostermeier et al. 2005; Prescott et al. 2012; Hosken and

Hodgson 2014; Metzler-Guillemain et al. 2015; Soubry 2015).These sperm-borne epigenetic marks are, in turn, affectedby a variety of paternal factors, including genotype, age,obesity, smoking and exposure to environmental contaminants

(Aitken 2014).The mechanisms by which such environmental and lifestyle

factors affect mammalian spermatozoa, to define both their

potential for fertilisation and the subsequent initiation of embry-onic development, are poorly understood. The central hypothe-sis outlined in this article is that all such factors ultimately

converge to induce a high level of oxidative stress in the malegermline. Oxidative stress is known to interfere with thefertilising capacity of spermatozoa, to damage sperm nuclearDNA and to affect the epigenetic profile of these cells (Aitken

et al. 2014b). Herein, we review the evidence relating to theorigins and consequences of such stress and consider potentialstrategies for its remediation.

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Reproduction, Fertility and Development, 2016, 28, 1–10

http://dx.doi.org/10.1071/RD15325

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Oxidative stress and fertilisation potential

The notion that sperm function may be compromised by theonset of oxidative stress can be traced back to the early studies ofEvans (1947), who observed that heavily irradiated seawater

impaired the fertilising capacity of sea urchin spermatozoa. Heconcluded that the irradiation process had generated hydrogenperoxide (H2O2) and that this powerful oxidising agent wasdamaging to spermatozoa. Although this conclusion was not

subsequently supported by Barron et al. (1949), these authorsdid generate unequivocal data indicating that H2O2 is extremelydamaging to sperm function. Around the same time, Tosic and

Walton (1946) demonstrated that the metabolite generated bybovine spermatozoa in the presence of egg yolk-based cryo-preservatives was H2O2 and that this oxidant actively sup-

pressed their respiration. Furthermore, these authors identifiedthe source of the H2O2 to be an L-amino acid oxidase with anaffinity for aromatic amino acids, particularly phenylalanine,

which is abundant in egg yolk (Tosic and Walton 1950).MacLeod (1943) also demonstrated that human spermatozoalost motility at high oxygen tensions via mechanisms that couldbe reversed by catalase, again suggesting that H2O2 generation

was causally involved in the loss of sperm function. The par-ticular destructive power of H2O2 relative to any other reactiveoxygen species (ROS) was later emphasised in studies revealing

that catalase, but not superoxide dismutase, was able to relievethe detrimental effect of ROS generated by the xanthine oxidasefree radical-generating system on human spermmotility in vitro

(Aitken et al. 1993a).The possibility that excess ROS generationmay be associated

with defective sperm function in vivo was highlighted by two

papers that appeared in 1987 and demonstrated that the sperma-tozoa of infertilemales were characterised by high levels of ROSgeneration and the induction of lipid peroxidation (Aitken andClarkson 1987; Alvarez et al. 1987). The susceptibility of human

spermatozoa to lipid peroxidation had previously been highlight-ed by Thaddeus Mann (Jones et al. 1979), who pointed out thatthese cells contain exceptionally high levels of polyunsaturated

fatty acids (PUFA; particularly docosahexanoic acid), which arevulnerable to free radical attack, generating lipid peroxides andaldehydes that have a direct inhibitory action on sperm move-

ment. These early studies have subsequently been confirmedin many independent laboratories, all of which agree on thefundamental tenet that defective sperm function is frequentlyinduced by oxidative stress, affecting the motility of these cells,

their DNA integrity and their competence for sperm–oocytefusion (e.g. Aitken et al. 1991, 2010; Zalata et al. 1995; Sanockaet al. 1996; Sharma and Agarwal 1996; Nakamura et al. 2002;

Kao et al. 2008; Sakamoto et al. 2008; Bejarano et al. 2014;Morielli and O’Flaherty 2015).

Oxidative stress and lipid peroxidation

The way in which oxidative stress suppresses sperm motilityappears to be directly related to the induction of lipid per-

oxidation. When ROS attack the PUFA that abound in humanspermatozoa, a variety of lipid metabolites is generated,including lipid peroxyl radicals, alkoxyl radicals and variousaldehydes, such as malondialdehyde, 4-hydroxynonenal (4HNE)

and acrolein (Jones et al. 1978; Moazamian et al. 2015). Theaddition of both lipid peroxides and lipid aldehydes to popula-

tions of human spermatozoa results in the rapid immobilisationof these cells via different mechanisms. Lipid peroxyl radicalsdestabilise the sperm plasma membrane by virtue of their ten-

dency to abstract hydrogen atoms from adjacent PUFA toachieve a measure of stabilisation as the corresponding lipidhydroperoxide. This process creates carbon-centred lipid radi-

cals that combine with oxygen to generate more peroxyl radi-cals, which, in turn, abstract hydrogen from adjacent PUFA tostabilise, generating additional lipid radicals and promoting thepropagation of the lipid peroxidation chain reaction (Fig. 1a).

The lipid peroxides generated in this process destabilise theplasma membrane by becoming targets for phospholipase A2,which moves into the plasma membranes to cleave out the lipid

peroxides so they can be further processed by glutathione per-oxidase (van Kuijk et al. 1985). This process, in turn, generateslysophospholipids that destabilise the sperm plasma membrane,

affecting the microarchitecture of this structure and changingthe functions of integral membrane proteins that are critical tothe maintenance of sperm motility, such as ATP-dependent ionpumps and voltage-regulated ion channels (Nishikawa et al.

1989; Lundbaek and Andersen 1994). The disruptive effect ofperoxidative damage on lipid membrane architecture alsoaffects the ability of spermatozoa to participate in themembrane

fusion events associated with fertilisation (Aitken et al. 1989,1993b, 1993c).

The lipid peroxidation chain reactions initiated in spermato-

zoa may also result in the formation of a cascade of aldehydeby-products that include alkanals, such asmalondialdehyde, andalkenals, such as 4HNE and acrolein. These compounds, partic-

ularly 4HNE and acrolein, are powerful electrophiles that formadducts with several proteins within the spermatozoa that, inturn, affect sperm function. For example, the formation ofadductswith the flagellar axonemal protein, dynein heavy chain,

may explain the effect of these aldehydes on sperm movement(Baker et al. 2015; Moazamian et al. 2015). In addition, 4HNEhas been shown to bind to mitochondrial proteins in human

spermatozoa, triggering electron leakage and the formation ofROS (Fig. 1b). The oxidative stress associated with the latterthen forces the spermatozoa to enter the intrinsic apoptotic

cascade, beginning with a loss of mitochondrial membranepotential and terminating in oxidative DNA adduct formation,DNA strand breakage and cell death (Aitken et al. 2012).

Sources of ROS and oxidative stress in spermatozoa

With oxidative stress being such a major factor in the aetiology

of defective human sperm function, resolving the possiblecauses of this condition is critical. In considering thismatter, it isimportant to emphasise that spermatozoa are not only vulnerable

to oxidative stress because of the targets they offer for freeradical attack in the form of PUFA, proteins and nucleic acids,but they are also lacking significant intracellular antioxidant

protection, including ROS-metabolising enzymes, such ascatalase and glutathione peroxidase, by virtue of the limitedvolume and restricted distribution of cytoplasmic space inwhichto house such mediators of cell survival. Furthermore, these

2 Reproduction, Fertility and Development R. J. Aitken et al.

cells actively generate physiological levels of ROS in order to

drive the tyrosine phosphorylation events associated with spermcapacitation (Aitken and Nixon 2013). The involvement of ROSin the capacitation of mammalian spermatozoa has been

appreciated since the pioneering studies of Claude Gagnon inthe 1990s (de Lamirande and Gagnon 1993a). The ROSresponsible for sperm capacitation have been variously reported

as H2O2 (Bise et al. 1991; Aitken et al. 1995, 1996; Rivlin et al.2004) superoxide anion (de Lamirande and Gagnon 1993b) andthe peroxynitrite radical generated by the reaction of superoxide

anion with another free radical species, namely nitric oxide(Herrero et al. 2001; Rodriguez and Beconi 2009). In reality, theinterconversion of these various ROS and reactive nitrogenspecies is very rapid and it is probable that several different

redox entities are involved in various aspects of the capacitationprocess, including the suppression of tyrosine phosphatase

activity and the stimulation of cAMP generation (Aitken and

Nixon 2013). It has recently been hypothesised that the con-tinued generation of ROS, particularly peroxynitrite, to achievecapacitation ultimately overwhelms the limited antioxidant

defences of these cells and precipitates a state of apoptosis.According to this concept, capacitation and the intrinsic apo-ptotic cascade are the opposite ends of a metabolic continuum

driven by ROS (Aitken et al. 2015b).If ROS are so important for sperm function, what is the

subcellular source of these molecules? In mammalian sperma-

tozoa there can be little doubt that the major sources of ROS arethe mitochondria. Human sperm mitochondria are particularlyactive in the generation of ROS via mechanisms that are notdependent on a loss of mitochondrial membrane potential

(Koppers et al. 2008). Activation of ROS generation at ComplexIII was found to stimulate the rapid release of H2O2 into the

(a) PUFA

R

Freeradicalattack

H

O2

Initiation

OO •

R

Lipid radical

Mitochondrial ROS generation

Lipid radical

PUFA Peroxyl radical Lipid hydroperoxide

R

R

H

R

Hydrogenabstraction

R

OOHOO •

R

Peroxyl radical

Lipid aldehydes4HNE/acrolein

(c)H2O2

(b)

� H2O•OH

•

•

Nucleus

Mitochondria

Adduction ofmitochondrial

proteins

Lipidperoxidation

�

Fig. 1. Oxidative stress in mammalian spermatozoa. (a) Spermatozoa are susceptible to oxidative stress because they contain high

concentrations of polyunsaturated fatty acids (PUFA). Free radical attack leads to the formation of lipid radicals that then combinewith the

universal electron acceptor, oxygen, to generate a lipid peroxyl radical. In order to stabilise as a hydroperoxide, the latter extracts hydrogen

atoms from adjacent lipids, generating lipid radicals that then perpetuate the peroxidation cascade. (b) Lipid aldehydes generated as a

consequence of lipid peroxidation, such as 4-hydroxynonenal (4HNE), bind to mitochondrial proteins, including succinic acid

dehydrogenase and stimulate yet more free radical generation, further enhancing lipid peroxidation in a self-propagating cycle that

propels spermatozoa towards an apoptotic fate. (c) The unusual architecture of spermatozoameans that nucleases activated in themidpiece

cytoplasm, or released from the mitochondria, cannot enter the nuclear compartment. The only product of apoptosis that can pass from the

midpiece to the sperm head to damage the DNA is H2O2; this is why most DNA damage in spermatozoa is oxidative.

Oxidative stress in spermatozoa Reproduction, Fertility and Development 3

extracellular space, but no detectable peroxidative damage.Conversely, the induction of ROS on the matrix side of

the inner mitochondrial membrane at Complex I resulted inperoxidative damage to the midpiece and a loss of spermmovement that could be prevented by the concomitant presence

of a-tocopherol (Koppers et al. 2008). Defective human sper-matozoa spontaneously generate mitochondrial ROS in a man-ner that is negatively correlated with motility (Koppers et al.

2008). Indeed, simultaneous measurement of total cellular ROSwith dihydroethidium indicated that 68% of the variability insuch measurements could be explained by differences in mito-chondrial ROS production (Koppers et al. 2008).

Another potential source of ROS are the NADPH oxidaseenzymes (NOX), including the calcium-dependent NOX5,which are known to be present in the spermatozoa of certain

species, including human (Banfi et al. 2001), although otherspecies, such as the mouse, do not possess this enzyme. Expos-ing spermatozoa to NADPH can trigger a redox response that is

detectable with the redox probe lucigenin and inhibitable bydiphenylene iodonium (DPI), a flavoprotein inhibitor (Aitkenet al. 1997; Vernet et al. 2001). However, this lucigenin-dependent activity was subsequently shown to be due to the

direct enzymatic reduction of the probe by cytochrome P450reductase (Baker et al. 2004) and cytochrome b5 reductase(Baker et al. 2005) when the electron donors were NADPH

and NADH, respectively. In contrast, using luminol as aROS probe, clear evidence has been obtained for a calcium-dependent increase in ROS generation, which is particularly

marked in the spermatozoa of infertile patients and potentiallyreflective of an involvement of NOX5 in the aetiology ofdefective sperm function (Aitken and Clarkson 1987). There

is even some evidence to suggest that NOX5 may be overrepre-sented in the defective spermatozoa recovered from patientsexhibiting teratozoospermia (Ghani et al. 2013). However,definitive proof that NOX5 is the source of ROS under such

circumstances is currently lacking. There is a possibility that thecalcium-dependent signals observed with unfractionated spermsuspensions are the result of low-level leucocyte contamination

(Aitken and Clarkson 1987; Aitken et al. 1992). The abilityof the NOX inhibitor apocynin to suppress the ROS signalsgenerated by human sperm suspensions (Dona et al. 2011) could

also be accounted for by leucocyte contamination because thisreagent prevents assembly of the key cytosolic componentsof the NADPH oxidase system (p40phox, p47phox and p67phox),which is not necessary for NOX5 to be active. A detailed study

of the NOX species present in human spermatozoa is currentlylacking and the role of these enzymes in the creation of oxidativestress within the germline remains unresolved. One possibility

that cannot be excluded is that the NOX enzymes present inmammalian spermatozoa play no role at all in the regulation ofsperm function, but rather function much earlier in germ cell

production, controlling spermatogonial stem cell proliferation(Morimoto et al. 2013).

Finally, L-amino acid oxidases with a particular affinity for

phenylalanine have been identified in bull, horse, human andram spermatozoa (Tosic and Walton 1946; Aitken et al. 2015a;Houston et al. 2015). In the case of equine spermatozoa, whichare heavily dependent on oxidative phosphorylation (Gibb et al.

2014), the primary role for this amino acid oxidase may be tosupport the energy metabolism of these cells through the

oxidative deamination of aromatic amino acids, generating ketoacids that are then processed by the sperm mitochondria.However, in the case of human spermatozoa, oxidative phos-

phorylation appears to play a minor role in sperm metabolismbecause these cells are largely dependent of glycolysis to meettheir energy needs (du Plessis et al. 2015). In these cells, the

L-amino acid oxidase (interleukin 4 induced protein 1, IL4I1)seems to have acquired a new biological function in supplyingthe redox drive to sperm capacitation (Houston et al. 2015).

Role of oxidative stress in DNA damage

One of the major complications associated with male infertility

is the presence of high levels of DNA damage in the sperma-tozoa. Such damage can arise as a consequence of infertility(Irvine et al. 2000; Aitken and Curry 2011) age (Singh et al.

2003) smoking (Fraga et al. 1996) antioxidant deficiency (Fragaet al. 1991, 1996), obesity (Fariello et al. 2012) exposure toinfection (Reichart et al. 2000; Burrello et al. 2004), heat(De Iuliis et al. 2009a; Santiso et al. 2012) acidic pH (Santiso

et al. 2012), metals, particularly transition metals such as ironand copper (Aitken et al. 2014a), radiofrequency electromag-netic radiation (De Iuliis et al. 2009a), ionising radiation (Singh

and Stephens 1998), environmental toxicants such as acrylam-ide (Katen and Roman 2015), chemotherapeutic agents (Delbeset al. 2010), air pollution, plasticisers, pesticides (Evenson and

Wixon 2005; O’Flaherty 2014) and chloracetanilide herbicidessuch as alachlor (Grizard et al. 2007).

There can be little doubt that most of these factors affect the

integrity of sperm chromatin through the induction of oxidativestress. Such stress results in the generation of oxidised DNAbase adducts such as 8-hydroxy-20-deoxyguanosine (8OHdG),particularly in areas of the genome that are not heavily prota-

minated (De Iuliis et al. 2009b; Noblanc et al. 2013). Spermato-zoa only possess one enzyme in the base excision repair (BER)pathway, 8-oxoguanine DNA glycosylase (OGG1). This glyco-

sylase is associated with the sperm nucleus and mitochondriaand can actively excise 8OHdG, releasing this base adduct intothe extracellular space. Remarkably, spermatozoa do not pos-

sess the downstream components of the BER pathway, namelyapurinic endonuclease 1 (APE1) and X-ray repair complement-ing defective repair in Chinese hamster cells 1 (XRCC1). Thenet result of this truncated DNA repair capacity is to generate

abasic sites at locations that have been affected by 8OHdGformation. Such abasic sites destabilise the ribose–phosphatebackbone, leading to a b-elimination or a ring opening reaction

of the ribose unit and a consequential strand break. This type ofDNA chemistry has been identified as being central to theinitiation of cancer in other cell types. Therefore, oxidative

DNA base lesions are not only potentially mutagenic but,importantly, also contribute indirectly to the DNA fragmenta-tion observed in the patient population (Ohno et al. 2014).

An oxidative involvement in DNA damage to mammalianspermatozoa has been observed in relation to infertility (Shenand Ong 2000; Aitken et al. 2010), heat (De Iuliis et al. 2009a),antioxidant deficiency (Fraga et al. 1991), age (Weir and

4 Reproduction, Fertility and Development R. J. Aitken et al.

Robaire 2007; Smith et al. 2013a), smoking (Fraga et al. 1991),obesity (Bakos et al. 2011), radiofrequency electromagnetic

radiation (De Iuliis et al. 2009a), herbicides (Grizard et al.

2007), plasticisers (Erkekoglu et al. 2010; Zhou et al. 2010) andchemotherapeutic agents (Ghosh et al. 2002). Indeed, it would

appear that most DNA damage in mammalian spermatozoais the result of an oxidative insult generated as a result of eitherimpaired antioxidant protection because of endogenous (e.g. age)

or exogenous (e.g. phthalate esters) factors or changes in theredox status of spermatozoa because of internal (e.g. mitochon-drial electron leakage) or external (e.g. radiation or alachlor)influences. However, it is also undeniable that not every

spermatozoon afflicted with DNA damage shows signs of oxida-tive stress. Under these conditions, it has been suggested thatnuclease-mediated DNA fragmentation must occur as a result of

spermatozoa defaulting to an apoptotic state rather than oxidativestress (Muratori et al. 2015). This interesting hypothesis isdifficult to reconcile with the fact that apoptosis invariably

involves the induction of oxidative stress (Koppers et al. 2011),so it is difficult to imagine how these phenomena can be separatedin vivo. A possible resolution of this dilemma is set out below.

Role of apoptosis in DNA damage

It is well known that testicular precursor germ cells can undergoapoptosis as part of a physiological process designed to optimisegerm cell : Sertoli cell ratios and to bring a measure of qualitycontrol to the spermatogenic process, ensuring that no defective

germ cells are allowed to differentiate into spermatozoa (Shuklaet al. 2012). Apoptosis may also occur during spermatogenesisin response to adverse circumstances, including heat shock,

ionising radiation, growth factor deprivation and chemothera-peutic agents. The apoptotic process is largely, but not exclu-sively, targeted to spermatocytes and both the intrinsic

mitochondrial pathway and the extrinsic p53/Fas system havebeen implicated as key modulators of this process (Boekelheide2005; Lagos-Cabre and Moreno 2012). However, the focus ofthis discussion is the spermatozoa.

Spermatozoa are highly differentiated, transcriptionallysilent cells that, by virtue of their inert nuclear constitutionand highly specialised architecture, cannot undergo apoptosis in

the conventional sense. Nevertheless, they can undergo atruncated version of this process. One of the key features ofsperm cell biology is that we do not have to expend energy

searching for factors that will induce these cells to undergoapoptosis. Rather, these cells are designed to undergo apoptosis;it is their default position. Spermatozoa are the ultimate symbol

of disposable cell types; indeed, all these cells are destined to diea lonely apoptotic death in the male or female tract. Thefortunate exceptions to this rule are the handful of individualgametes that manage to fertilise an oocyte and, in so doing,

achieve potential immortality for the genotype they carry.Whenapoptosis does eventually occur, it is generally the intrinsicapoptotic cascade that is induced, mediated by the sperm

mitochondria. Although receptor-mediated extrinsic apoptosisremains a theoretical possibility in spermatozoa, no ligands havebeen convincingly described to date that are capable of eliciting

such a response in the fully differentiated gamete. There has

been a claim that bacterial lipopolysaccharide (LPS) can elicitapoptosis in spermatozoa by interacting with Toll-like receptor

(TLR) 2 and TLR4 on the sperm surface (Fujita et al. 2011).However these data have not yet been independently validatedand our research group has not yet been able to achieve apoptosis

using commercially available LPS (R. J. Aitken, unpubl. obs.).As a result, our view is that the mature gamete has very littlecapacity to activate the extrinsic apoptotic cascade, but is

extremely vulnerable to its intrinsic counterpart.Spermatozoa are normally prevented from entering the

intrinsic apoptotic pathway by virtue of the continuing activityof phosphatidylinositol 3-kinase (PI3K; Koppers et al. 2011). If

PI3K is inhibited, then the spermatozoa default to an apoptoticcascade characterised by rapid loss of motility, generation ofROS, caspase activation in the cytosol, annexin V binding to the

cell surface, cytoplasmic vacuolisation and oxidative DNAdamage. The anti-apoptotic action of PI3K appears to dependon its ability to promote the phosphorylation of another kinase,

AKT, which, in turn, is responsible for phosphorylating anti-apoptotic effector proteins such as Bcl-2-associated death pro-moter (BAD). Phosphorylation of the latter is essential for BADto remain associatedwith its cytoplasmic keeper protein, 14-3-3.

However, dephosphorylation allows BAD to orchestrate anapoptotic process that has many similarities with the intrinsicapoptotic cascade observed in somatic cells. There are two

major points of difference between apoptosis in spermatozoaand somatic cells, as follows:

1. Mammalian spermatozoa are structurally different fromsomatic cells in that all the mitochondria and most of thecytoplasm are compartmentalised in the mid-piece of the

cell, physically separated from the DNA in the spermnucleus. As a result, even if apoptosis is activated in thesecells, the endonucleases released from the mitochondria

(e.g. endonuclease G) or activated in the cytoplasm(e.g. caspase-activated DNAse) are physically impeded fromattacking the sperm nucleus (Koppers et al. 2011). The onlyelement of the apoptotic cascade that can exit from the sperm

midpiece and penetrate the nuclear compartment is H2O2. Itis for this reason that most of the DNA damage present inhuman spermatozoa appears to be oxidatively induced

(Fig. 1c; Aitken et al. 2010).2. Spermatozoa are also characterised by a severely truncated

BER pathway, as discussed above, that stalls after OGG1 has

removed the oxidised base to create abasic sites that have tobe further processed by the oocyte following fertilisation.One consequence of spermatozoa lacking the next enzyme

in this pathway, namely APE1, is that these cells cannotcreate the 30-OH termini that are required by the terminaldeoxyribonucleotidyl transferase-mediated dUTP–digoxigeninnick end-labelling (TUNEL) assay. As a result, TUNEL is a

very insensitive methodology for assessing DNA damage inspermatozoa. Under circumstances where DNA damage isinduced by, for example, exposure to H2O2, intracellular and

extracellular 8OHdG can be clearly detected in the affectedsperm suspension and DNA strand breakage can be detectedwith the sperm chromatin structure assay (SCSA); however,

TUNEL signals are not apparent (Smith et al. 2013b).

Oxidative stress in spermatozoa Reproduction, Fertility and Development 5

The later do eventually appear when the cells are close todeath. At this point, it is possible that a DNAse does become

activated in the spermatozoa (Sotolongo et al. 2005). For thereasons given above, such a nuclease would have to beincorporated into the sperm chromatin, not released from

themitochondria or activated in the cytoplasm. The nature ofthis DNAse (topoisomerase? DNAse 1?) and its mechanismsof activation are not known. It is possible such nuclease

activity may be activated by a rise in intracellular calcium ascells die, because calcium-dependent nuclease activity hasbeen described in this cell type on several independentoccasions by independent groups (Sotolongo et al. 2005;

Sibirtsev et al. 2011).

Apoptosis or oxidative stress causes DNA damage

We can conclude from the foregoing discussion that there are

two mechanisms for damaging DNA in mammalian spermato-zoa. It is now generally acknowledged that a wide variety ofdifferent intrinsic and extrinsic factors converge to generate a

state of oxidative stress in the germline. Once such stress hasbeen initiated, it tends to become accentuated because the lipidaldehydes generated during the peroxidative process bind to

proteins in the mitochondrial electron transport chain, particu-larly succinic acid dehydrogenase, stimulating the generation ofyet more free radicals, more DNA damage and more lipid per-oxidation to continue the downward spiral towards apoptosis

(Fig. 1b; Aitken et al. 2012). There is also an associationbetween oxidative DNA damage in the male germline and poorchromatin protamination during spermiogenesis (De Iuliis et al.

2009b). This relationship may reflect a certain vulnerabilitytowards oxidative stress as a consequence of the failure of spermnuclear DNA to adequately compact. However, it may also be a

consequence of inadequate protamination, because these small,basic proteins are thought to protect the DNA by acting assacrificial antioxidants and by chelating redox active metalssuch as copper (Liang et al. 1999).

The relationship between oxidative stress and apoptosis iscomplex. Clearly, spermatozoa do express the classical markersof apoptosis, such as ROS generation, phosphatidylserine expo-

sure, caspase activation and DNA fragmentation (Koppers et al.2011). If PI3K activity is inhibited with wortmannin, thenhuman spermatozoa rapidly default to the intrinsic apoptotic

cascade, displaying all of the above features, including highlevels of mitochondrial ROS generation (Koppers et al. 2011).Therefore, entry of spermatozoa into the senescence-driven

apoptotic pathway as a consequence of compromised PI3Kactivity inevitably results in an apoptotic cascade involvingthe stimulation of mitochondrial ROS generation and the induc-tion of oxidative DNA damage. Under these circumstances,

apoptosis and oxidative DNA damage are inextricably linked.Similarly, when spermatozoa are exposed to xenobiotics such asalachlor, the induction of apoptosis is inextricably linked with

the induction of oxidative stress (Grizard et al. 2007).Within theinfertile population, DNA damage is again associated with thesimultaneous appearance of oxidative stress and apoptosis

(Wang et al. 2003; Aitken et al. 2010) via mechanisms thatcan be reversed by the sustained administration of antioxidants

such as melatonin (Bejarano et al. 2014). Similarly, cryostorageleads to the induction of oxidatively driven DNA damage and

apoptosis that can be reversed by the presence of the antioxidantquercetin (Zribi et al. 2012). However, there are occasionalcircumstances where DNA damage can be visualised in the

absence of any evidence that the cells have been subjected tooxidative stress (Muratori et al. 2015). Under these circum-stances, it is possible that the apoptosis involves stimulation of a

DNAse that is integrated into the sperm chromatin and somehowbecomes activated when these cells are under stress.

Developmental consequences of oxidative damagein the germ line

Whatever the causes of oxidative stress in the male germline,there can be no doubt that this pathophysiological mechanismleads to both impaired fertility and disrupted embryonic

development. At high levels of oxidative stress, fertilisation isprevented because the damage to the sperm plasma membraneimpairs both the motility of these cells and their competence for

fusion with the oocyte. At lower levels of oxidative stress thespermatozoa can retain their capacity for fertilisation while theDNA in their nuclei is still oxidatively damaged (Aitken et al.

1998). The developmental consequences of fertilising eggs withspermatozoa exhibiting oxidative DNA damage has beenexplored using the glutathione peroxidase 5 (GPx5)-knockoutmouse. In this mouse model, the spermatozoa suffer from

oxidative areas as they descend the epididymis (Chabory et al.

2009). The level of stress experienced by these spermatozoadoes not impair their fertilising capacity, but does induce high

levels of oxidative DNA damage in the sperm nuclei. Theconsequence of this damage can be seen in the developmentalstatus of the embryos when Gpx5-null males are mated with

wild-type females, because such unions are accompanied by asignificant increase in the incidence of miscarriage and devel-opmental abnormalities (Chabory et al. 2009). In related studiesin which mouse spermatozoa were oxidatively damaged by

exposing them to H2O2, several developmental abnormalitieswere observed in the offspring, including a delay in embryonicdevelopment rates, a decrease in the ratio of inner cell mass cells

in the resulting blastocyst and a reduction in implantation rates(Lane et al. 2014). Crown–rump length at Day 18 of gestationwas also reduced in offspring produced from H2O2-treated

spermatozoa. Female offspring from peroxide-treated sperma-tozoa were smaller, became glucose intolerant and accumulatedincreased levels of adipose tissue compared with control female

offspring. Interestingly, the male offspring phenotype was lesssevere, with increases in fat depots only seen at 4 weeks of age,which returned to control levels later in life (Lane et al. 2014).Studies in primates (Burruel et al. 2013) and cattle (Simoes et al.

2013) have confirmed the effect of oxidative DNA damage inspermatozoa on the developmental potential of fertilised ova.Given the developmental significance of this oxidative sperm

DNA damage, it is important that strategies are developed toreduce such pathological changes as a matter of good practicein assisted conception programs and as a matter of good con-

science in couples contemplating parenthood by natural means.The data accumulated to date are encouraging in that the

6 Reproduction, Fertility and Development R. J. Aitken et al.

oxidative damage in spermatozoa associated with obesity, forexample, can clearly be reversed by a combination of diet and

exercise (Palmer et al. 2012). The use of antioxidants has alsomet with some success in treating idiopathic oxidative stress inspermatozoa (Gharagozloo and Aitken 2011; Showell et al.

2014). The advantages of using an effective oral antioxidanttreatment are that, in cases where oxidative stress has beendiagnosed in the male germline, it should result in increases in

both the fertilising potential of human spermatozoa and theirgenetic integrity. The disadvantage of using antioxidants, par-ticularly as an empirical treatment for patients where there is noevidence of oxidative stress, is that it may create a reductive

stress (Chen et al. 2013).We still await the results of randomiseddouble-blind cross-over trials to definitively establish thetherapeutic value of different antioxidant formulations in the

treatment of male patients exhibiting high levels of oxidativeDNA damage in their spermatozoa. Such studies are eagerlyanticipated.

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