*4.1. Compromised Sperm Membrane Integrity*

The peroxidation of membrane lipids leads to the catabolism of phospholipids and liberation of PUFAs, thus directly contributing to increased membrane fluidity and permeability to ions, which can elicit downstream effects in terms of inactivating membrane enzymes and receptors [20,79]. The loss of sperm membrane integrity is also tightly correlated with reduced sperm motility [83]. Indeed, the compromise of both of these parameters has been well documented in the context of cryopreservation [84–87] in which post-thaw sperm samples typically experience a burst of ROS and high levels of OS [85,86,88]. Sperm motility is also highly sensitive to pH and ion concentration (reviewed in [89]). The disruption of sperm membrane integrity alters the diffusion of ions across the membrane and dysregulates the function of ion pumps and channels, especially as those ion channels that are regulated by PUFAs (reviewed in [90]). Although there is currently limited direct evidence linking sperm membrane integrity and ion channel function, the peroxidation of lipids and the downregulation of some ion channels in low-motility spermatozoa has been documented [83,91–94]. In this context, Baker et al. [22] reported two sperm ion channels, voltage-dependent anion-selective channel protein 2 (VDAC2) and sodium/potassium-transporting ATPase subunit alpha-4 (ATP1A4) that are susceptible to the formation of potentially deleterious adducts with 4HNE. Similarly, decreased VDAC and ATP1A4 abundance has been reported in the spermatozoa of males with asthenozoospermia [95] and in patients with unilateral varicocele (a pathology correlated with OS) [96], respectively.

The deleterious impact of ROS extends beyond the plasma membrane to also include those membranes surrounding subcellular organelles such as the mitochondria. In contrast to somatic cells, sperm mitochondria are localized in one specific region of the sperm cell—the mid-piece of the flagellum. Within this domain, the mitochondria are organized end to end to form long spirals

that wrap tightly around the axoneme [97,98]. The correct functioning of mitochondria is especially important for sperm cells in terms of supplying the energy needed for e fficient movement. The elevated production of mitochondrial ROS and the resultant propagation of sperm plasma membrane LPO can, in turn, initiate secondary LPO in mitochondrial membranes. Similar to the plasma membrane, the peroxidation of lipids residing in the mitochondrial membranes also changes the fluidity of these structures, thus resulting in the upregulation of proton and electron leakage through the inner membrane [99,100]. Such a situation leads to the loss of mitochondrial membrane potential (ΔΨ), compromises the e fficiency of mitochondrial ATP generation, and triggers a cycle of elevated electron leakage and the generation of additional mitochondrial ROS, which collectively exacerbates the impact of OS on sperm function [18,101,102].

### *4.2. Dysregualtion of Sperm Metabolic Enzymes*

In human sperm cells, the majority of the energy needed to support motility appears to originate from glycolysis, which takes place in the sperm tail. However, it has been argued that mitochondrial oxidative phosphorylation (OXPHOS) also plays a secondary role in the bioenergetic pathways associated with motility. In this context, a key substrate for OXPHOS are the endogenous lipids present in the sperm membrane [103], but other metabolic substrates and their associated pathways have also been implicated [18,103,104]. Indeed, depending on the prevailing environmental conditions that sperm encounter on route to the site of fertilization, these cells may be able to utilize di fferent metabolic pathways.

As previously mentioned, the cytotoxic aldehydes generated during LPO can form adducts with multiple elements of the sperm proteome. Curiously, however, not all sperm proteins appear to display equivalent susceptibility to lipid aldehyde carbonylation reactions [105]. By way of example, the application of a ffinity-based isolation techniques coupled with mass spectrometry has revealed that 4HNE preferentially adducts to a relatively small number of putative targets in human spermatozoa [22]. Notably, many of these 4HNE targets serve as metabolic enzymes responsible for the production of energy needed to support sperm motility (depicted in Figure 2), including several glycolytic enzymes [phosphofructokinase (PFKP); aldolase A, fructose-bisphosphate (ALDOA); phosphoglycerate kinase (PGK), pyruvate kinase (PKM); lactate dehydrogenase C chain (LDHC)], enzymes involved in the TCA cycle [malate dehydrogenase 2, NAD (mitochondrial) (MDH2)] [22], electron transport chain [succinate dehydrogenase complex, subunit A, flavoprotein (SDHA) [106]; ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1 (UQCRFS1)] [22] and beta-oxidation [fatty acid amide hydrolase (FAAH); acyl-CoA synthetase long-chain family member 1 (ACSL1); hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit beta (HADHB); acetyl-Coenzyme A acetyltransferase 1 (ACAT1)] [22]. Moreover, 4HNE has also been found to adduct with solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 31 (SLC25A31), a mitochondrial carrier protein involved in the exchange of cytoplasmic ADP with mitochondrial ATP [22]. Although the consequences of damage caused by the insertion of bulky 4HNE (C-9) carbonyl adducts has ye<sup>t</sup> to be investigated across all targeted sperm proteins, it is reasonable to suspect that these modifications could elicit protein mis-folding, poor substrate recognition, and/or degradation of the protein itself [107,108]. Indeed, recent work by Aitken et al. [106] has demonstrated that 4HNE adduction to SDHA can activate mitochondrial electron leakage and disrupt ΔΨ in human spermatozoa. These data raise the prospect that 4HNE may primarily perturb the bioenergetic pathways that sustain sperm motility and thus provide a mechanistic model to account for the dysregulation of sperm function commonly reported in cells burdened by excessive ROS production [68]. Notably, the self-perpetuating nature of this pathway may also account for the ability of 4HNE to drive human spermatozoa towards an intrinsic apoptotic pathway.

**Figure 2.** The influence of 4-hydroxynonenol (4HNE) on sperm function. The lipid aldehyde 4HNE has repeatedly been shown to form adducts with sperm flagellum proteins associated with the motility apparatus, signaling pathways and metabolism. In the context of the mitochondria, 4HNE adduction has been linked to adverse effects on enzymes of beta-oxidation, the tricarboxylic acid (TCA) cycle and the electron transport chain (ETC), thus attenuating the energy production. Abbreviations: CI, ETC Complex I; CII, ETC Complex II; CIII, ETC Complex III; CIV, ETC Complex IV; DA, dynein arms; FS, fibrous sheath; MT, microtubules; ODF, outer dense fibers.

Such a model takes on added significance in view of proteomic data emerging from studies of the spermatozoa of asthenozoospermic patients. Whilst not universal, a common theme to emerge from this work is that proteins involved in energy production are generally downregulated in the defective spermatozoa of asthenozoospermic individuals [18,105,109]. Indeed, the majority of proteins so affected in asthenozoospermia are linked to glycolysis, pyruvate metabolism, tricarboxylic acid cycle (TCA), OXPHOS, beta-oxidation or alternative metabolic pathways [18,109,110]. By way of example, the glycolytic enzyme, glucose-6-phosphate isomerase (GPI), has been shown to be under-represented in asthenozoospermic individuals, as has the sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDHS) [110–113]; the activity of which is also attenuated after treatment of spermatozoa with ROS [112]. Moreover, several mitochondrial enzymes involved in the downstream conversion of pyruvate to acetyl-CoA, TCA cycle, ETC and a plethora of ATP synthase subunits have also been shown to be dysregulated. Similar reductions in protein abundance also extend to several enzymes involved in beta-oxidation of fatty acids [18,95,105,109,110]. Alternative proteomic studies of asthenozoospermia have revealed downregulation of one of the subunits of NADH dehydrogenase (NDUFA13), a key constituent of complex I of the ETC [114]. The knockdown of this protein in a mouse spermatocyte cell line led to the loss of ΔΨ, increased ROS production and apoptosis [114]. These data draw interesting parallels with the study of asthenozoospermic individuals by Nowicka-Bauer et al. [18], which clearly showed the downregulation of sperm mitochondrial metabolic pathways and ΔΨ; defects that were accompanied by increased production of mitochondrial ROS in the spermatozoa of asthenozoospermic patients. These data reinforce the notion that reduced sperm motility is predominantly associated with dysfunction of sperm mitochondria leading to elevated levels of ROS and a reciprocal reduction in energy production. However, it remains to be established what factor(s) are responsible for the under-representation of metabolic enzymes in the spermatozoa of asthenozoospermic individuals and

whether this is in any way linked to localized ROS generation within the vicinity of the developing germ cells.

### *4.3. Defects in the Sperm Motility Apparatus and Signaling Pathways*

The flagellum, which is responsible for the propagation of sperm motility, is constructed around a cytoskeletal structure known as the axoneme. The axoneme, in turn, consists of tubulin microfilaments arranged in a highly conserved 9 + 2 scheme (whereby 9 doublets surround one central pair). To each doublet of microtubules are attached two dynein arms (outer and inner), which act as motor proteins capable of 'walking' along the adjacent microtubules and e ffecting the sliding of microtubules relative to each other (reviewed in [115]). This creates an undulatory wave that is propagated along the length of the flagellum to generate the rhythmic beating patterns responsible for propelling sperm forward [116]. Aside from the core elements described above, the sperm axoneme is also supported by a family of accessory proteins known as outer dense fibers (ODF1–ODF4), which uniquely among mammalian spermatozoa, are localized around the microtubules and provide additional elasticity and stability to flagellum movement [117]. Given the fundamental importance of motility in terms of delivering spermatozoa to the site of fertilization, it follows that any defects in the axonemal structure, or the ODF accessory proteins, can compromise fertility [118]. Accordingly, several studies on asthenozoospermia have linked this condition with an attendant change in the levels of structural proteins residing in the flagellum. Among the most frequently reported of these are the dynein motor proteins, ODFs and those belonging to the tektin family of microtubule-stabilizing proteins [18,109,117].

In a similar context, studies on sperm protein adducts arising from direct 4HNE challenge have demonstrated that this aldehyde can readily bind to tubulin (TUBB), several members of the dynein family (DNAH5, DNAH17, DNALI1), and the ODF1 and ODF2 proteins [22] (see Figure 2). Notably, these findings align well with the proteomic deficits identified in the spermatozoa of asthenozoospermic individuals [18,109,110]. As an extension of this work, however, it has also been shown that core elements of the sperm fibrous sheath (a unique cytoskeletal structure that surrounds the axoneme and outer dense fibers and regulates the flexibility and shape of the flagellar beat [119]) are highly sensitive to 4HNE adduction. In particular, the major fibrous sheath components of A-Kinase Anchoring Protein (AKAP4 and AKAP3) and Rhophilin Associated Tail Protein 1B (ROPN1B), which contribute to the structural organization of the fibrous sheath [119,120], have been identified as dominant 4HNE targets [22,99,101]. Indeed, AKAP4 has recently been validated as a conserved target for 4HNE adduction in primary cultures of post-meiotic male germ cells (round spermatids) and in mature mouse and human spermatozoa [121]. Through the application of an exogenous 4HNE treatment regimen, we further demonstrated that 4HNE modification resulted in a substantial reduction in the levels of AKAP4 detected in round spermatids and mature spermatozoa alike. Moreover, reduced AKAP4 levels were correlated with dysregulation of the capacitation-associated signaling framework assembled around the AKAP4 sca ffold.

These data accord with the demonstration that the spermatozoa of male mice lacking AKAP4 display reduced progressive movement and are infertile [122]. Similarly, reduced levels of AKAP4 have also been implicated as a biomarker of human asthenozoospermic males [95]. In addition to AKAP4, alternative cAMP-responsive elements such as protein kinase A (PKA), have also been validated as primary targets of 4HNE-mediated modification [22], whilst seminal OS has been correlated with altered levels of several proteins mapping to the MAPK/ERK pathways [123]. Such defects have been correlated with a commensurate decrease in global levels of tyrosine phosphorylation in human spermatozoa [22]; a form of post-translational modification that underpins sperm motility, and is particularly important in the induction of hyperactivation [124]. Aside from the targets mentioned above, 4HNE also has the potential to modify sperm phosphatase activity, and hence motility, via the targeting of serine/threonine-protein phosphatase 2B catalytic subunit gamma isoform (PPP3CC) [22]. Indeed, male mice lacking PPP3CC display an infertility phenotype linked to a reduction in sperm motility [125]. Thus, whilst metabolic-related proteins are clearly over represented among those

targeted for ROS and lipid aldehyde attack [22], numerous other proteins implicated in sperm motility also appear sensitive to OS.

Recent evidence suggests that mammalian spermatozoa may possess an unfolded protein response (UPR); a conserved cellular pathway that is activated upon accumulation of unfolded or misfolded proteins during stress conditions [126]. The activation of the UPR triggers the expression of chaperones to assist protein refolding (reviewed in [127]), whilst also blocking the synthesis of other proteins via the phosphorylation of eukaryotic translation initiation factor-2 (eIF2 α) [128]. Santiago et al. [126] have reported increased levels of proteins involved in the UPR (heat shock proteins; HSF1, HSP90, HSPD1, HSP27; and eIF2 α) and reduced motility in human spermatozoa exposed to H2O2. Despite this, it is possible that prolonged OS within the testes can compromise the UPR owing to 4HNE adduction of key elements of this pathway including the 60 kDa Heat Shock Protein, Mitochondrial (HSPD1) [22]. If this were to be the case, then the inactivation of UPR could contribute to the downregulation of some proteins associated with asthenozoospermia. In any case, such broad-spectrum e ffects make it extremely challenging to determine whether individual sperm proteins play a dominant role in the pathophysiological responses to OS or whether this phenomenon is instead attributed to dysregulation of multiple targets. This situation is further complicated when considering that 4HNE and other forms of ROS have the potential to damage not only sperm proteins, but also the DNA comprising the paternal and mitochondrial genomes.
