Aitken 2016 causes and consequences of oxidative stress in spermatozoa
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Aitken 2016 causes and consequences of oxidative stress in spermatozoa
- 2. Causes and consequences of oxidative stress in spermatozoa Robert John AitkenA,D , Zamira GibbA , Mark A. BakerA , Joel DrevetB and Parviz GharagozlooC A Priority Research Centre in Reproductive Science and Hunter Medical Research Institute, Faculty of Science and IT, University of Newcastle, Callaghan, NSW 2308, Australia. B GReD laboratory, CNRS UMR6293-INSERM U1103-Clermont Universite´, 63171 BP80006, Aubie`re cedex, France. C CellOxess LLC, 15 Roszel Street, Princeton, NJ 08540, USA. D Corresponding author. Email: john.aitken@newcastle.edu.au Abstract. Spermatozoa are highly vulnerable to oxidative attack because they lack significant antioxidant protection due 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 abound in significant amounts of polyunsaturated fatty acids. Susceptibility to oxidative attack is further exacerbated by the fact that these cells actively generate reactive oxygen species (ROS) in order to drive the increase in tyrosine phosphorylation 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 ROS generation, loss of mitochondrial membrane potential, caspase activation, phosphatidylserine exposure and oxidative DNA 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 the DNA backbone and generating strand breaks. Because oxidative damage to sperm DNA is associated with both miscarriage 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 fertilisation and deliver the paternal component of the embryonic genome to an MII oocyte. Although defective sperm function has long been recognised as a major cause of human infertility (Hull et al. 1985), this condition has conventionally been equated with the ability of spermatozoa to achieve fertilisation (Aitken et 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 of these cells to fertilise an oocyte; it also needs to incorporate an assessment of their ability to program a normal pattern of embryonic development. Spermatozoa may affect embryonic development via both genetic and a variety of epigenetic mechanisms involving the methylation profile of the DNA, the post-translational modification of nuclear histones and the composition of a variety of coding and non-coding RNA species 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, affected by 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 male germline. Oxidative stress is known to interfere with the fertilising capacity of spermatozoa, to damage sperm nuclear DNA and to affect the epigenetic profile of these cells (Aitken et al. 2014b). Herein, we review the evidence relating to the origins and consequences of such stress and consider potential strategies for its remediation. CSIRO PUBLISHING Reproduction, Fertility and Development, 2016, 28, 1–10 http://dx.doi.org/10.1071/RD15325 Journal compilation Ó IETS 2016 www.publish.csiro.au/journals/rfd
- 3. Oxidative stress and fertilisation potential The notion that sperm function may be compromised by the onset of oxidative stress can be traced back to the early studies of Evans (1947), who observed that heavily irradiated seawater impaired the fertilising capacity of sea urchin spermatozoa. He concluded that the irradiation process had generated hydrogen peroxide (H2O2) and that this powerful oxidising agent was damaging to spermatozoa. Although this conclusion was not subsequently supported by Barron et al. (1949), these authors did generate unequivocal data indicating that H2O2 is extremely damaging to sperm function. Around the same time, Tosic and Walton (1946) demonstrated that the metabolite generated by bovine spermatozoa in the presence of egg yolk-based cryo- preservatives was H2O2 and that this oxidant actively sup- pressed their respiration. Furthermore, these authors identified the source of the H2O2 to be an L-amino acid oxidase with an affinity for aromatic amino acids, particularly phenylalanine, which is abundant in egg yolk (Tosic and Walton 1950). MacLeod (1943) also demonstrated that human spermatozoa lost motility at high oxygen tensions via mechanisms that could be 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 reactive oxygen species (ROS) was later emphasised in studies revealing that catalase, but not superoxide dismutase, was able to relieve the detrimental effect of ROS generated by the xanthine oxidase free radical-generating system on human sperm motility in vitro (Aitken et al. 1993a). The possibility that excess ROS generation may be associated with defective sperm function in vivo was highlighted by two papers that appeared in 1987 and demonstrated that the sperma- tozoa of infertile males were characterised by high levels of ROS generation and the induction of lipid peroxidation (Aitken and Clarkson 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 that these cells contain exceptionally high levels of polyunsaturated fatty acids (PUFA; particularly docosahexanoic acid), which are vulnerable to free radical attack, generating lipid peroxides and aldehydes that have a direct inhibitory action on sperm move- ment. These early studies have subsequently been confirmed in many independent laboratories, all of which agree on the fundamental tenet that defective sperm function is frequently induced by oxidative stress, affecting the motility of these cells, their DNA integrity and their competence for sperm–oocyte fusion (e.g. Aitken et al. 1991, 2010; Zalata et al. 1995; Sanocka et 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 motility appears to be directly related to the induction of lipid per- oxidation. When ROS attack the PUFA that abound in human spermatozoa, a variety of lipid metabolites is generated, including lipid peroxyl radicals, alkoxyl radicals and various aldehydes, such as malondialdehyde, 4-hydroxynonenal (4HNE) and acrolein (Jones et al. 1978; Moazamian et al. 2015). The addition of both lipid peroxides and lipid aldehydes to popula- tions of human spermatozoa results in the rapid immobilisation of these cells via different mechanisms. Lipid peroxyl radicals destabilise the sperm plasma membrane by virtue of their ten- dency to abstract hydrogen atoms from adjacent PUFA to achieve a measure of stabilisation as the corresponding lipid hydroperoxide. 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 to stabilise, generating additional lipid radicals and promoting the propagation of the lipid peroxidation chain reaction (Fig. 1a). The lipid peroxides generated in this process destabilise the plasma 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, generates lysophospholipids that destabilise the sperm plasma membrane, affecting the microarchitecture of this structure and changing the functions of integral membrane proteins that are critical to the maintenance of sperm motility, such as ATP-dependent ion pumps and voltage-regulated ion channels (Nishikawa et al. 1989; Lundbaek and Andersen 1994). The disruptive effect of peroxidative damage on lipid membrane architecture also affects the ability of spermatozoa to participate in the membrane 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 aldehyde by-products that include alkanals, such as malondialdehyde, and alkenals, such as 4HNE and acrolein. These compounds, partic- ularly 4HNE and acrolein, are powerful electrophiles that form adducts with several proteins within the spermatozoa that, in turn, affect sperm function. For example, the formation of adducts with 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, 4HNE has been shown to bind to mitochondrial proteins in human spermatozoa, triggering electron leakage and the formation of ROS (Fig. 1b). The oxidative stress associated with the latter then forces the spermatozoa to enter the intrinsic apoptotic cascade, beginning with a loss of mitochondrial membrane potential 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 possible causes of this condition is critical. In considering this matter, it is important to emphasise that spermatozoa are not only vulnerable to oxidative stress because of the targets they offer for free radical attack in the form of PUFA, proteins and nucleic acids, but they are also lacking significant intracellular antioxidant protection, including ROS-metabolising enzymes, such as catalase and glutathione peroxidase, by virtue of the limited volume and restricted distribution of cytoplasmic space in which to house such mediators of cell survival. Furthermore, these 2 Reproduction, Fertility and Development R. J. Aitken et al.
- 4. cells actively generate physiological levels of ROS in order to drive the tyrosine phosphorylation events associated with sperm capacitation (Aitken and Nixon 2013). The involvement of ROS in the capacitation of mammalian spermatozoa has been appreciated since the pioneering studies of Claude Gagnon in the 1990s (de Lamirande and Gagnon 1993a). The ROS responsible 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) and the 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, the interconversion of these various ROS and reactive nitrogen species is very rapid and it is probable that several different redox entities are involved in various aspects of the capacitation process, 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 achieve capacitation 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 are the mitochondria. Human sperm mitochondria are particularly active in the generation of ROS via mechanisms that are not dependent on a loss of mitochondrial membrane potential (Koppers et al. 2008). Activation of ROS generation at Complex III was found to stimulate the rapid release of H2O2 into the (a) PUFA R Free radical attack H O2 Initiation OO • R Lipid radical Mitochondrial ROS generation Lipid radical PUFA Peroxyl radical Lipid hydroperoxide R R H R Hydrogen abstraction R OOH OO • R Peroxyl radical Lipid aldehydes 4HNE/acrolein (c) H2O2 (b) ϩ H2O • OH • • Nucleus Mitochondria Adduction of mitochondrial proteins Lipid peroxidation ϩ 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 combine with 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 spermatozoa means that nucleases activated in the midpiece 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
- 5. 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 in peroxidative damage to the midpiece and a loss of sperm movement 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 ROS with dihydroethidium indicated that 68% of the variability in such measurements could be explained by differences in mito- chondrial ROS production (Koppers et al. 2008). Another potential source of ROS are the NADPH oxidase enzymes (NOX), including the calcium-dependent NOX5, which are known to be present in the spermatozoa of certain species, including human (Ba´nfi et al. 2001), although other species, 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 by diphenylene iodonium (DPI), a flavoprotein inhibitor (Aitken et 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 P450 reductase (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 a ROS 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 potentially reflective of an involvement of NOX5 in the aetiology of defective 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 patients exhibiting 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 the calcium-dependent signals observed with unfractionated sperm suspensions are the result of low-level leucocyte contamination (Aitken and Clarkson 1987; Aitken et al. 1992). The ability of the NOX inhibitor apocynin to suppress the ROS signals generated by human sperm suspensions (Dona` et al. 2011) could also be accounted for by leucocyte contamination because this reagent prevents assembly of the key cytosolic components of 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 currently lacking and the role of these enzymes in the creation of oxidative stress within the germline remains unresolved. One possibility that cannot be excluded is that the NOX enzymes present in mammalian spermatozoa play no role at all in the regulation of sperm 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 and ram spermatozoa (Tosic and Walton 1946; Aitken et al. 2015a; Houston et al. 2015). In the case of equine spermatozoa, which are heavily dependent on oxidative phosphorylation (Gibb et al. 2014), the primary role for this amino acid oxidase may be to support the energy metabolism of these cells through the oxidative deamination of aromatic amino acids, generating keto acids 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 metabolism because these cells are largely dependent of glycolysis to meet their 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 supplying the 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 (Fraga et al. 1991, 1996), obesity (Fariello et al. 2012) exposure to infection (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 iron and 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 (Delbe`s et al. 2010), air pollution, plasticisers, pesticides (Evenson and Wixon 2005; O’Flaherty 2014) and chloracetanilide herbicides such 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 oxidative stress. Such stress results in the generation of oxidised DNA base 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 mitochondria and can actively excise 8OHdG, releasing this base adduct into the extracellular space. Remarkably, spermatozoa do not pos- sess the downstream components of the BER pathway, namely apurinic endonuclease 1 (APE1) and X-ray repair complement- ing defective repair in Chinese hamster cells 1 (XRCC1). The net result of this truncated DNA repair capacity is to generate abasic sites at locations that have been affected by 8OHdG formation. Such abasic sites destabilise the ribose–phosphate backbone, leading to a b-elimination or a ring opening reaction of the ribose unit and a consequential strand break. This type of DNA chemistry has been identified as being central to the initiation 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 mammalian spermatozoa has been observed in relation to infertility (Shen and 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.
- 6. 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) and chemotherapeutic agents (Ghosh et al. 2002). Indeed, it would appear that most DNA damage in mammalian spermatozoa is the result of an oxidative insult generated as a result of either impaired antioxidant protection because of endogenous (e.g. age) or exogenous (e.g. phthalate esters) factors or changes in the redox 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 that nuclease-mediated DNA fragmentation must occur as a result of spermatozoa defaulting to an apoptotic state rather than oxidative stress (Muratori et al. 2015). This interesting hypothesis is difficult to reconcile with the fact that apoptosis invariably involves the induction of oxidative stress (Koppers et al. 2011), so it isdifficult to imagine how these phenomena can beseparated in 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 undergo apoptosis as part of a physiological process designed to optimise germ cell : Sertoli cell ratios and to bring a measure of quality control to the spermatogenic process, ensuring that no defective germ cells are allowed to differentiate into spermatozoa (Shukla et al. 2012). Apoptosis may also occur during spermatogenesis in 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 have been implicated as key modulators of this process (Boekelheide 2005; Lagos-Cabre´ and Moreno 2012). However, the focus of this discussion is the spermatozoa. Spermatozoa are highly differentiated, transcriptionally silent cells that, by virtue of their inert nuclear constitution and highly specialised architecture, cannot undergo apoptosis in the conventional sense. Nevertheless, they can undergo a truncated version of this process. One of the key features of sperm cell biology is that we do not have to expend energy searching for factors that will induce these cells to undergo apoptosis. 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 die a lonely apoptotic death in the male or female tract. The fortunate exceptions to this rule are the handful of individual gametes that manage to fertilise an oocyte and, in so doing, achieve potential immortality for the genotype they carry. When apoptosis does eventually occur, it is generally the intrinsic apoptotic cascade that is induced, mediated by the sperm mitochondria. Although receptor-mediated extrinsic apoptosis remains a theoretical possibility in spermatozoa, no ligands have been 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 elicit apoptosis 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 validated and 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 little capacity 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 activity of phosphatidylinositol 3-kinase (PI3K; Koppers et al. 2011). If PI3K is inhibited, then the spermatozoa default to an apoptotic cascade characterised by rapid loss of motility, generation of ROS, caspase activation in the cytosol, annexin V binding to the cell surface, cytoplasmic vacuolisation and oxidative DNA damage. The anti-apoptotic action of PI3K appears to depend on 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 BAD to remain associated with its cytoplasmic keeper protein, 14-3-3. However, dephosphorylation allows BAD to orchestrate an apoptotic process that has many similarities with the intrinsic apoptotic cascade observed in somatic cells. There are two major points of difference between apoptosis in spermatozoa and somatic cells, as follows: 1. Mammalian spermatozoa are structurally different from somatic cells in that all the mitochondria and most of the cytoplasm are compartmentalised in the mid-piece of the cell, physically separated from the DNA in the sperm nucleus. As a result, even if apoptosis is activated in these cells, the endonucleases released from the mitochondria (e.g. endonuclease G) or activated in the cytoplasm (e.g. caspase-activated DNAse) are physically impeded from attacking the sperm nucleus (Koppers et al. 2011). The only element of the apoptotic cascade that can exit from the sperm midpiece and penetrate the nuclear compartment is H2O2. It is for this reason that most of the DNA damage present in human 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 to be further processed by the oocyte following fertilisation. One consequence of spermatozoa lacking the next enzyme in this pathway, namely APE1, is that these cells cannot create the 30 -OH termini that are required by the terminal deoxyribonucleotidyl transferase-mediated dUTP–digoxigenin nick end-labelling (TUNEL) assay. As a result, TUNEL is a very insensitive methodology for assessing DNA damage in spermatozoa. Under circumstances where DNA damage is induced by, for example, exposure to H2O2, intracellular and extracellular 8OHdG can be clearly detected in the affected sperm suspension and DNA strand breakage can be detected with 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
- 7. The later do eventually appear when the cells are close to death. At this point, it is possible that a DNAse does become activated in the spermatozoa (Sotolongo et al. 2005). For the reasons given above, such a nuclease would have to be incorporated into the sperm chromatin, not released from the mitochondria or activated in the cytoplasm. The nature of this DNAse (topoisomerase? DNAse 1?) and its mechanisms of activation are not known. It is possible such nuclease activity may be activated by a rise in intracellular calcium as cells die, because calcium-dependent nuclease activity has been described in this cell type on several independent occasions 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 of different intrinsic and extrinsic factors converge to generate a state of oxidative stress in the germline. Once such stress has been initiated, it tends to become accentuated because the lipid aldehydes generated during the peroxidative process bind to proteins in the mitochondrial electron transport chain, particu- larly succinic acid dehydrogenase, stimulating the generation of yet 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 association between oxidative DNA damage in the male germline and poor chromatin protamination during spermiogenesis (De Iuliis et al. 2009b). This relationship may reflect a certain vulnerability towards oxidative stress as a consequence of the failure of sperm nuclear 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 as sacrificial antioxidants and by chelating redox active metals such as copper (Liang et al. 1999). The relationship between oxidative stress and apoptosis is complex. Clearly, spermatozoa do express the classical markers of apoptosis, such as ROS generation, phosphatidylserine expo- sure, caspase activation and DNA fragmentation (Koppers et al. 2011). If PI3K activity is inhibited with wortmannin, then human spermatozoa rapidly default to the intrinsic apoptotic cascade, displaying all of the above features, including high levels of mitochondrial ROS generation (Koppers et al. 2011). Therefore, entry of spermatozoa into the senescence-driven apoptotic pathway as a consequence of compromised PI3K activity inevitably results in an apoptotic cascade involving the 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 as alachlor, the induction of apoptosis is inextricably linked with the induction of oxidative stress (Grizard et al. 2007). Within the infertile population, DNA damage is again associated with the simultaneous appearance of oxidative stress and apoptosis (Wang et al. 2003; Aitken et al. 2010) via mechanisms that can be reversed by the sustained administration of antioxidants such as melatonin (Bejarano et al. 2014). Similarly, cryostorage leads to the induction of oxidatively driven DNA damage and apoptosis that can be reversed by the presence of the antioxidant quercetin (Zribi et al. 2012). However, there are occasional circumstances where DNA damage can be visualised in the absence of any evidence that the cells have been subjected to oxidative 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 somehow becomes activated when these cells are under stress. Developmental consequences of oxidative damage in the germ line Whatever the causes of oxidative stress in the male germline, there can be no doubt that this pathophysiological mechanism leads to both impaired fertility and disrupted embryonic development. At high levels of oxidative stress, fertilisation is prevented because the damage to the sperm plasma membrane impairs both the motility of these cells and their competence for fusion with the oocyte. At lower levels of oxidative stress the spermatozoa can retain their capacity for fertilisation while the DNA in their nuclei is still oxidatively damaged (Aitken et al. 1998). The developmental consequences of fertilising eggs with spermatozoa exhibiting oxidative DNA damage has been explored using the glutathione peroxidase 5 (GPx5)-knockout mouse. 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 spermatozoa does not impair their fertilising capacity, but does induce high levels of oxidative DNA damage in the sperm nuclei. The consequence of this damage can be seen in the developmental status of the embryos when Gpx5-null males are mated with wild-type females, because such unions are accompanied by a significant increase in the incidence of miscarriage and devel- opmental abnormalities (Chabory et al. 2009). In related studies in which mouse spermatozoa were oxidatively damaged by exposing them to H2O2, several developmental abnormalities were observed in the offspring, including a delay in embryonic development 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 gestation was also reduced in offspring produced from H2O2-treated spermatozoa. Female offspring from peroxide-treated sperma- tozoa were smaller, became glucose intolerant and accumulated increased levels of adipose tissue compared with control female offspring. Interestingly, the male offspring phenotype was less severe, 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 (Simo˜es et al. 2013) have confirmed the effect of oxidative DNA damage in spermatozoa on the developmental potential of fertilised ova. Given the developmental significance of this oxidative sperm DNA damage, it is important that strategies are developed to reduce such pathological changes as a matter of good practice in 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.
- 8. oxidative damage in spermatozoa associated with obesity, for example, can clearly be reversed by a combination of diet and exercise (Palmer et al. 2012). The use of antioxidants has also met with some success in treating idiopathic oxidative stress in spermatozoa (Gharagozloo and Aitken 2011; Showell et al. 2014). The advantages of using an effective oral antioxidant treatment are that, in cases where oxidative stress has been diagnosed in the male germline, it should result in increases in both the fertilising potential of human spermatozoa and their genetic integrity. The disadvantage of using antioxidants, par- ticularly as an empirical treatment for patients where there is no evidence of oxidative stress, is that it may create a reductive stress (Chen et al. 2013). 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