OXIDATIVE STRESS AND REPRODUCTIVE FUNCTION: Oxidative stress and the long-term storage of horse spermatozoa

in Reproduction
Authors:
Fernando J PeñaLaboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain

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https://orcid.org/0000-0002-1311-2947
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Zamira GibbPriority Research Centre for Reproductive Science, University of Newcastle, New South Wales, Australia

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Correspondence should be addressed to F J Pena; Email: fjuanpvega@unex.es

This paper forms part of a special issue on Oxidative Stress and Reproductive Function. The guest editor for this section was Professor John Aitken, University of Newcastle, New South Wales, Australia

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In brief

The growing understanding of the mechanisms regulating redox homeostasis in the stallion spermatozoa, together with its interactions with energetic metabolism, is providing new clues applicable to the improvement of sperm conservation in horses. Based on this knowledge, new extenders, adapted to the biology of the stallion spermatozoa, are expected to be developed in the near future.

Abstract

The preservation of semen either by refrigeration or cryopreservation is a principal component of most animal breeding industries. Although this procedure has been successful in many species, in others, substantial limitations persist. In the last decade, mechanistic studies have shed light on the molecular changes behind the damage that spermatozoa experience during preservation. Most of this damage is oxidative, and thus in this review, we aim to provide an updated overview of recent discoveries about how stallion spermatozoa maintain redox homeostasis, and how the current procedures of sperm preservation disrupt redox regulation and cause sperm damage which affects viability, functionality, fertility and potentially the health of the offspring. We are optimistic that this review will promote new ideas for further research to improve sperm preservation technologies, promoting translational research with a wide scope for applicability not only in horses but also in other animal species and humans.

Abstract

In brief

The growing understanding of the mechanisms regulating redox homeostasis in the stallion spermatozoa, together with its interactions with energetic metabolism, is providing new clues applicable to the improvement of sperm conservation in horses. Based on this knowledge, new extenders, adapted to the biology of the stallion spermatozoa, are expected to be developed in the near future.

Abstract

The preservation of semen either by refrigeration or cryopreservation is a principal component of most animal breeding industries. Although this procedure has been successful in many species, in others, substantial limitations persist. In the last decade, mechanistic studies have shed light on the molecular changes behind the damage that spermatozoa experience during preservation. Most of this damage is oxidative, and thus in this review, we aim to provide an updated overview of recent discoveries about how stallion spermatozoa maintain redox homeostasis, and how the current procedures of sperm preservation disrupt redox regulation and cause sperm damage which affects viability, functionality, fertility and potentially the health of the offspring. We are optimistic that this review will promote new ideas for further research to improve sperm preservation technologies, promoting translational research with a wide scope for applicability not only in horses but also in other animal species and humans.

Introduction

Preservation of mammalian spermatozoa is a fundamental component of many animal breeding industries (Pena et al. 2011). This preservation is primarily achieved either through refrigeration (between 5°C and 18°C depending on the species) or via cryopreservation and storage under liquid nitrogen at −196°C. While in some species, cryopreservation has been successfully used since the second half of the last century, there are many species for whom this technology is still suboptimal, and for these animals, refrigerated (often referred to as ‘chilled’ or ‘cooled’) semen storage is used as an alternative. In food-producing species, fecundity is a trait that is actively selected for, and as such, an indirect selection for good semen quality (Butler et al. 2020, 2021) has simultaneously occurred. However, in other species, such as the horse, selection based on conformation and athletic performance predominates, and the result of this is that these animals have not been selected for fertility or sperm quality. Moreover, in food-producing species, stud males are often culled at a relatively young age (to maintain high fertility and to increase the rate of genetic gain), while in those species selected based on conformation and sports performance, sires may become increasing commercially valuable with advancing age as their progeny begin to demonstrate their ability to perform in the show ring or the sports arena. As such, these animals may experience increasing demand as sires at more advanced ages, the implication being that sperm quality and fertility also decrease with age (Sieme et al. 2008, Smith et al. 2013, Turner 2019).

Cryopreservation of mammalian spermatozoa exposes these cells to major insults including hyperosmotic shock at freezing and hypoosmotic shock at thawing (Sieme et al. 2008), cold shock (White 1993) and exposure to toxic cryoprotectants (Sieme et al. 2016). This may result in osmotic-induced necrotic cell death in a high percentage of spermatozoa after thawing (Abdelnour et al. 2022). Furthermore, other forms of cell death may be induced by sperm preservation procedures, and non-lethal damage that seriously impacts fertility also occurs. Oxidative stress is the major driver of this damage, and as such, the present review aims to provide an updated overview of the recent knowledge gathered on sperm regulation of redox homeostasis, along with the mechanisms involved in redox deregulation during sperm preservation. Therefore, this review will focus on the equid – a species in which oxidative stress is inexorably linked to sperm function by virtue of metabolic strategy – while making reference to other species where relevant, in the hope that we may promote new ideas for further improvement of current sperm preservation techniques.

Sperm metabolism and redox regulation are closely interconnected

The spermatozoon is a highly metabolic, redox-regulated cell which is subjected to dramatic changes during its lifespan; these dramatic changes require that spermatozoa adapt their metabolism to changing environments and that their redox regulation must be accurately coordinated to their metabolism. In this review, it is extremely important to consider that most technologies in use for sperm preservation expose spermatozoa to, at least, sub-physiological and potentially highly toxic environments. The basic aspects of sperm metabolism have been recently reviewed (Gibb & Aitken 2016, Pena et al. 2021, Amaral 2022), but in brief, energetic metabolism is the process in which ATP is generated from the oxidation of carbon-based molecules and the concurrent reduction of electron carriers to provide electrons for the mitochondrial generation of ATP via the electron transport chain. The energy released in these thermodynamically favourable processes is then harnessed to phosphorylate ADP to ATP (Quijano et al. 2016, Trostchansky et al. 2016). The tight regulation of redox reactions is a key component of metabolism; these reactions involve the transfer of electrons from reduced organic molecules to acceptors, NAD+ and NADP+, or to oxygen. Reactive oxygen species (ROS) such as superoxide (O2) and hydrogen peroxide (H2O2) are by-products of these reactions, as approximately 1–3% of O2 reduced in the mitochondria during oxidative phosphorylation (OXPHOS) forms O2 (Halliwell & Gutteridge 2003), which is rapidly dismutated to the membrane-permeable H2O2 via the action of superoxide dismutase. Principal reactions of O2 (in addition to dismutation to H2O2) are the reaction with FeS centres and the reaction with nitric oxide (•NO) leading to the generation of peroxynitrite (ONOO). While •NO is a regulatory molecule with important functions in the stallion spermatozoa (Ortega Ferrusola et al. 2009), ONOO is a stable molecule and a potent oxidant able to react with CO2 and electrophilic transition metal centres, yielding potent oxidants in a process which has recently been thoroughly reviewed (Pena et al. 2022). H2O2 can also react with metal centres and produce the highly toxic hydroxyl radical (•OH). To maintain ROS homeostasis, spermatozoa are provided with sophisticated antioxidant systems in both the seminal plasma and within the cells themselves (Ozkosem et al. 2016, O'Flaherty & Matsushita-Fournier 2017, Fernandez & O'Flaherty 2018, Fernandez et al. 2019, Gibb et al. 2021, Gibb et al. 2016, Lee et al. 2017, O'Flaherty 2018, 2020, O'Flaherty et al. 2020). Many essential aspects of sperm functionality are redox-regulated, with the reversible oxidation of thiols in cysteine residues of key proteins acting as an ‘on-off’ switch controlling many sperm functions. However, if dysregulation occurs, these residues may experience irreversible oxidation and oxidative stress leading to sperm malfunction and ultimately cell death (Pena et al. 2019, Aitken 2020, Pintus & Ros-Santaella 2021).

Overview of the redox regulation systems in the stallion spermatozoa

Early studies suggested that OXPHOS in the mitochondria was the main source of ATP in stallion spermatozoa (Ortega Ferrusola et al. 2010), a fact that was later confirmed by others (Aitken et al. 2012, Darr et al. 2016a, Darr et al. 2016b, Gibb et al. 2014), and as such, a paradoxical relation between stallion fertility and the production of ROS was revealed (Gibb et al. 2014). Today it is understood that this paradox is related to the production of ROS as by-products of active OXPHOS pathways by those spermatozoa with intense mitochondrial activity, and as these cells are more motile and are better able to undergo ROS-dependent changes required to fertilise the oocyte – such as capacitation (Aitken 2017, Medica et al. 2022) – the stallions with these more metabolically active, high ROS-producing spermatozoa are also more fertile (Gibb et al. 2014). This phenomenon can be easily observed using flow cytometry and double staining with JC-1 (to test mitochondrial membrane potential) and CellRox Deep Red™, a probe that predominantly identifies the presence of O2•−. Using this technique, it is apparent that live spermatozoa with high mitochondrial membrane potential also produce the most O2•−, which suggests that the interpretation of ROS production must be carefully done (Ortiz-Rodriguez et al. 2019a), as it is oxidative stress, and not ROS per se, that leads to deleterious downstream effects. These studies have culminated in a renewed interest in studying the redox regulatory mechanisms of stallion spermatozoa (Fig. 1).

Figure 1
Figure 1

Overview of mechanisms involved in the control of redox homeostasis in the stallion spermatozoa. The SCLTA11-xCT channel incorporates cystine (oxidised form of the amino acid cysteine) in exchange for intracellular glutamate. Once cystine is incorporated into the cytosol, it is reduced to cysteine and used to synthesise glutathione (GSH). GSH is a cofactor of many antioxidant enzymes, acting as electron donor; after donating electrons, GSH is oxidised to GSSG. GSH prevents lipid peroxidation, protein oxidation, DNA oxidation and ROS-induced forms of sperm dead such as ferroptosis and apoptosis. Oxidised glutathione is recycled by electrons donated by NADH and NADPH provided principally, but not exclusively, by the metabolisation of glucose through the pentose phosphate pathway (PPP). The first line of defence against oxidative stress is superoxide dismutase, other families of proteins playing major role in the control of redox homeostasis are the peroxiredoxins (PRX), glutathione peroxidases (GPX) and thioredoxins (TRX).

Citation: Reproduction 164, 6; 10.1530/REP-22-0264

A recent review paper outlining the endogenous antioxidant strategies employed by spermatozoa of a range of domestic animal species has been published. While the rich antioxidant status of seminal plasma is widely renowned (Gaitskell-Phillips et al. 2020), during long-term storage of stallion spermatozoa, the majority of the seminal plasma is removed via centrifugation, and as such, the antioxidant capacity of isolated spermatozoa is rather more restricted by virtue of their being almost completely devoid of cytoplasm. While a comprehensive characterisation of stallion sperm antioxidants is yet to be published, we know that mammalian sperm in general have been shown to contain numerous endogenous antioxidants, including glutathione (GSH) (Nikodemus et al. 2011), glutathione peroxidase 4 (GPX4) and 5 (Drevet 2006, Barranco et al. 2016), glutathione-S-transferase and aldehyde dehydrogenase (Gibb et al. 2016), superoxide dismutase (Peeker et al. 1997) and, more contentiously, catalase (Lapointe et al. 1998, Žaja et al. 2020).

Unarguably the most abundant of these endogenous antioxidants is GSH, which acts to protect spermatozoa against oxidative stress-induced loss of motility and viability. Interestingly, in addition to containing GSH, stallion spermatozoa also contain the enzymatic machinery to synthesise GSH de novo (Ortega-Ferrusola et al. 2019). These enzymes are glutamate-cysteine ligase (GCLC), and glutathione synthetase (GSS), which have been evidenced using Western blotting, with image flow cytometry revealing that GSS is distributed in the midpiece, and GCLC is present in the post-acrosomal region, midpiece and tail (Ortega-Ferrusola et al. 2019). The ability of stallion spermatozoa to synthesise GSH was further evidenced through the use of the specific inhibitor of the GCLC, l-Buthionine sulfoximide (Ortega-Ferrusola et al. 2019). The availability of cysteine is the rate-limiting step in the synthesis of GSH, and while cysteine can be synthesised from methionine, the enzymes necessary for this pathway are not present in stallion spermatozoa (Ortiz-Rodriguez et al. 2019b). In these cells, cysteine is incorporated through the SLC7A11-xCT channel that exchanges intracellular glutamate for extracellular cystine (the oxidised form of cysteine) (Ortiz-Rodriguez et al. 2020, Ortiz-Rodriguez et al. 2021b). Once inside the cell, the cystine is reduced to cysteine and used for the synthesis of GSH. This tripeptide acts as an electron donor in numerous reactions of important antioxidant enzymatic systems in the spermatozoa in humans, including the peroxiredoxin, thioredoxin and glutathione peroxidase family of proteins (O'Flaherty 2014, 2018, Liu & O'Flaherty 2017, Fernandez & O'Flaherty 2018, Fernandez et al. 2019, Pena et al. 2019, Scarlata & O'Flaherty 2020). GSH by itself may act as an antioxidant due to its readily oxidisable thiol groups, and this oxidised GSH (GSSG) is then reduced back to GSH by NADPH, which is produced via the pentose phosphate pathway (Urner & Sakkas 1999, Williams & Ford 2004, Miraglia et al. 2010, Luna et al. 2016). The equine ejaculate also contains non-enzymatic antioxidants in the seminal plasma such as hercynine and ergothioneine (Sotgia et al. 2020, Tirpak et al. 2021).

The role of lipid aldehydes in manifesting the deleterious effects of lipid peroxidation has recently drawn attention to aldehyde dehydrogenases in the protection of spermatozoa against oxidative stress. The functional importance of aldehyde dehydrogenase 2 (ALDH2) in stallion spermatozoa has recently been characterised (Gibb et al. 2016). ALDH2 is the mitochondrial isoform of the aldehyde dehydrogenases and is responsible for removing the toxic electrophilic aldehyde adducts (such as 4-hydroxynonenal, 4HNE) that are a downstream product of lipid peroxidation arising from oxidative stress. The activity of ALDH2 has been closely correlated with progressive motility and negatively correlated with 4HNE adduction in stallion spermatozoa (Gibb et al. 2016), with inhibition of this enzyme causing a spontaneous increase in 4HNE levels in viable cells and a corresponding decrease in both total and progressive motility. These observations agree with those of Aitken et al. (2012), who revealed that 4HNE preferentially adducts to succinate dehydrogenase, an important electron transport chain (ETC) complex, causing disruption of the ETC and an increase in the production of ROS. Given the importance of mitochondrial ATP production in driving stallion sperm motility, it is no surprise that any disruption to the flow of electrons and the subsequent reduction in OXPHOS would result in a loss of motility and functionality in these cells. Furthermore, electrophilic aldehydes may lead to the alkylation of proteins associated with flagellar movement (Moazamian et al. 2015), which further exacerbates the loss of functionality that is associated with the downstream products of oxidative stress and lipid peroxidation in stallion spermatozoa.

Another important enzyme responsible for sperm aldehyde detoxification is glutathione-S-transferase which has garnered considerable attention in the context of both in vivo and in vitro sperm aldehyde detoxification in a number of species (Rao & Shaha 2000, Hemachand & Shaha 2003, Kumar et al. 2014, Hering et al. 2015, Kwon et al. 2015, Roshdy et al. 2015). While GST was broadly considered to carry the majority of the burden of sperm aldehyde detoxification when compared directly with ALDH, it was found to be of lesser importance for the maintenance of stallion sperm functionality in an in vitro setting (Gibb et al. 2016). Nonetheless, it is apparent that stallion spermatozoa contain a range of antioxidants which can intercept perpetrators of oxidative stress from the source (ROS) through to downstream products such as electrophilic aldehydes.

Storage of spermatozoa: exposure to non-physiological concentrations of solutes and toxic compounds

Most of the extenders currently in use for the short-term storage of equine semen are derived from the classical Kenney’s extender (Kenney et al. 1975) containing a combination of skim milk, glucose and antibiotics, these extenders contain glucose concentrations up to 300 mM, which is well above physiological concentrations of glucose in either the epididymis or the oviduct (Pena et al. 2021). On average, these extenders maintain acceptable sperm viability up to 48 h; however, spermatozoa have evolved to survive in the reproductive tract of the mare for up to a week (Troedsson et al. 1998), and therefore, it is reasonable to infer that these traditional extenders do not provide an optimal physiologically equivalent environment to allow the spermatozoa to survive for their full potential lifespan after ejaculation. Nowadays, it is widely accepted that an excessive intake of carbohydrates undermines human health, leading to the development of diseases like metabolic syndrome, diabetes, obesity, dementia and cancer, leading to the coining of the term ‘carbotoxicity’ (Kroemer et al. 2018). In the same way, recent research in sperm metabolism also suggests that most extenders in use contain excessive concentrations of glucose that are harmful to spermatozoa (Ortiz-Rodriguez et al. 2021a) and that the formulation of extenders has to be reconsidered (Darr et al. 2016b, Hernandez-Aviles et al. 2020, Hernandez-Aviles et al. 2021). Mechanisms involved in glucose toxicity in spermatozoa have been recently described (Pena et al. 2021, 2022), but in brief, intermediate metabolites formed during glycolysis (dihydroxyacetone phosphate, glyoxal and methylglyoxal) may cause sperm toxicity, particularly glyoxal and methylglyoxal. These metabolites are oxoaldehydes, which due to their carbonyl groups are strong electrophiles which can steal electrons from, and thus oxidise, proteins, lipids, and DNA. Moreover, these compounds contribute to the generation of advanced glycation end products, causing further sperm damage. Recent research shows that storage of stallion spermatozoa in commercial media containing high-glucose concentrations increases both glyoxal and methylglyoxal levels, leading to a loss of sperm functionality that can be avoided through the reduction of glucose (Ortiz-Rodriguez et al. 2021a). Many other mechanisms may also be involved in the damage induced by high glucose concentrations, including the direct formation of ROS by glucose, activation of MAP kinase, Ca2+-mediated mitochondrial fission (Nishikawa et al. 2000, Terrell et al. 2012), and activation of the polyol pathway, which may inhibit GSSG recycling by consuming NADPH, leading to compromised redox regulation through GSH depletion (Brownlee 2001). High glucose concentrations also trigger a metabolic pathway that activates diacylglycerol protein kinase C and NADPH-oxidase, leading to the overproduction of ROS and mitochondrial damage. Subsequently, this mitochondrial damage leads to the elevated production of O2•−, which may inhibit glyceraldehyde 3-phosphate dehydrogenase and divert metabolites upstream of the glycolysis pathway, resulting in increased flux of dihydroxyacetone phosphate to diacylglycerol and the further activation of protein kinase C (Nishikawa et al. 2000, Brownlee 2001). Furthermore, dihydroxyacetone phosphate is the precursor of the oxoaldehyde methylglyoxal (Ihnat et al. 2007, Ceriello & Testa 2009), and these high glucose concentrations inevitably predispose cells to apoptosis, ferroptosis, necroptosis and other types of cell death (LaRocca et al. 2016). Recent findings from our group have revealed that this latter route may be active in stallion spermatozoa, a high glucose concentration makes stallion spermatozoa more responsive to the induction of ferroptosis using the GPX4 inhibitor RSL3.

Another common practice in the equine industry is to adjust commercial insemination doses to a given number of ‘progressively motile spermatozoa’ while ignoring the presence of dead or non-motile cells; this practice may result in a high number of dead spermatozoa in the commercial doses, and these cells represent an important source of ROS via an action which is mediated by amino acid oxidases (Shannon & Curson 1972, Upreti et al. 1998, Houston et al. 2015).

l-Amino acid oxidases: interactions between dead or dying sperm and amino acids

Spermatozoa are generally stored in the presence of exogenous amino acids. While they are not absolutely dependent on an exogenous source of amino acids for survival (demonstrating prolonged survival during in vitro incubation over several days in a simple balanced salt solution containing no protein or defined amino acid sources at all (Gibb et al. 2015)), several studies have revealed that certain amino acids have prosurvival effects during cryostorage (Ugur et al. 2019, Ahmed et al. 2020), chilled storage (He & Woods 2003), and during storage or incubation at higher temperatures, possibly due to their antioxidant activities (Lahnsteiner 2009) or as substrates for mitochondrial energy production. The latter role has been suggested by the recent identification of proteins associated with amino acid metabolism in stallion spermatozoa (Swegen et al. 2015, Martín-Cano et al. 2020). Furthermore, many mammalian semen extenders and sperm storage media contain complex biological components, such as egg yolk and skim milk (or isolated proteins from these sources), which provide a vast array of amino acid substrates.

While these amino acids provide a suite of beneficial actions, in the presence of dead and dying spermatozoa, they can become substrates for the production of H2O2, which then goes on to trigger an oxidative cascade, bringing about the premature demise of the remaining viable spermatozoa in the storage vessel (Shannon & Curson 1972, Shannon & Curson 1982, Aitken et al. 2015). This phenomenon has been identified in the spermatozoa of several mammalian species, including the horse (Aitken et al. 2015), human (Houston et al. 2015), bull (Shannon & Curson 1972, Shannon & Curson 1982) and ram (Upreti et al. 1998), and is attributed to the exposure of l-amino acid oxidases through the damaged membranes of these dead and dying cells, with one report revealing that l-amino acid activity is highly correlated with the percentage of non-viable cells (Shannon & Curson 1982). These l-amino acid oxidases then interact with amino acids in solution, with a particular preference for l-aromatic amino acids such as l-phenylalanine, l-tryptophan and l-tyrosine (Aitken et al. 2015).

Leucocyte contamination: production of ROS by immune cells

The stallion ejaculate contains many contaminants, including erythrocytes, leucocytes and epithelial cells, in addition to a wide range of fungi and bacteria. Pioneering work late last century revealed that contaminating leucocytes were the predominant source of ROS in the human ejaculate and that by removing leucocytes from sperm suspensions, only a relatively low proportion of samples could be stimulated to produce ROS under the influence of phorbol 12-myristate 13-acetate (Whittington & Ford 1999). Despite this finding, the contribution of contaminating leucocytes to oxidative stress during stallion sperm storage has received very little attention, though it should be noted that by processing ejaculates using density gradient centrifugation (also known as single layer colloidal (SLC) centrifugation) prior to storage, the majority of these leucocytes are removed (Ricci et al. 2009, Khodamoradi et al. 2020), which, in combination with the removal of dead and dying spermatozoa (Loomis 2006) and the majority of microbial contaminants (Morrell et al. 2014a, Guimaraes et al. 2015, Varela et al. 2018), may contribute to the improved longevity and fertility of SLC processed, liquid-stored spermatozoa (Morrell et al. 2014b, Heutelbeck et al. 2015, Gibb et al. 2018).

Cryopreservation: osmotic shock causing mitochondrial damage and triggering oxidative stress

It is well-known that cryopreservation causes significant damage to spermatozoa, with a variable percentage of cells entering the process succumbing to osmotic stress-induced necrosis, while a significant proportion of the surviving population experiences variable degrees of cellular dysfunction (Pena et al. 2011) (Fig. 2). These sublethal changes have been termed capacitation-like or apoptosis-like changes, but together they reflect the reduced lifespan of cryopreserved spermatozoa in the female genital tract following thawing and insemination. One major factor explaining this damage is oxidative stress, with redox dysregulation being largely recognised as a major factor contributing to cryodamage, and as such, numerous trials using antioxidants have been conducted with variable success (Yeste et al. 2015, Yeste 2016, Delgado-Bermudez et al. 2019, Yanez-Ortiz et al. 2021). The cryopreservation process causes oxidative stress to the spermatozoa; the osmotic stress at thawing causes mitochondrial swelling, leading to malfunction of the electron transport chain, and increased production and release of O2•−. Once the antioxidant defences of the spermatozoa are surpassed, the increased production of OH leads to the formation of lipid hydroperoxides (LOO) and the production of the aforementioned toxic aldehydes such as 4HNE, with an abundance of polyunsaturated fatty acids in sperm membranes favouring the formation of these compounds in a self-perpetuating reaction.

Figure 2
Figure 2

Overview of the oxidative damage induced by cryopreservation on the stallion spermatozoa. Osmotic shock, especially at thawing cause membrane rupture in a variable number of the spermatozoa. Mitochondria also experience osmotic stress, resulting in mitochondrial malfunction and leakage of O2•−. If the antioxidant defences are surpassed, oxidative stress causes DNA, protein and lipid peroxidation with formation of lipid aldehydes such as 4HNE, which self-perpetuates the oxidative insult in a closed loop. Lipid peroxidation initially causes an increase in membrane permeability and activation of ferroptosis and apoptosis that finally leads to sperm death. Oxidation of proteins causes sperm malfunction and reduced fertility, DNA oxidation may lead to DNA fragmentation and increased early embryo mortality or may cause problems in the offspring due to oxidative damage in specific transcripts and/or oxidative modification of the epigenome.

Citation: Reproduction 164, 6; 10.1530/REP-22-0264

Recent studies have revealed that cryopreservation causes the rapid exhaustion of endogenous sperm antioxidants (Martin Munoz et al. 2015, Munoz et al. 2016), and moreover, detailed proteomic studies have revealed that superoxide dismutase (SOD1), which is the first line of defence against oxidative stress in the cell, is depleted as a consequence of cryopreservation (Gaitskell-Phillips et al. 2021). Interestingly, another protein dramatically affected by cryopreservation is aldo-keto reductase family 1 member b (AKR1B1). Among the many functions of this protein are the reduction of lipid carbonyls (including 4HNE), GSH-conjugates of carbonyls, and the phospholipid aldehydes which are generated following the oxidation of the membrane lipids phosphatidylcholine and phosphatidylethanolamine (Girouard et al. 2009, Shen et al. 2011, Perez-Patino et al. 2018). These recent proteomic findings underpin the concept that oxidative stress is an underlying contributor to cryodamage and highlight the importance of ‘omics’ technologies in the study of the biology of mammalian spermatozoa. Moreover, the impact of the oxidative stress on the offspring must be considered, as embryos obtained using cryopreserved spermatozoa show alterations in their transcriptome which are attributable to oxidative stress (Ortiz-Rodriguez et al. 2019c).

The oxidation of cardiolipin, a lipid of the outer mitochondrial membrane, causes the release of cytochrome c, which mediates the release of pro-apoptotic factors from the mitochondria; this activation of the mitochondrial pathway of apoptosis has been frequently reported as a typical feature of cryoinjury, though other forms of cryodamage-induced cell death also occur. Recently researchers have paid considerable attention to ferroptosis (Bromfield et al. 2019, Ou et al. 2020, Zhao et al. 2020), an iron-dependent nonapoptotic cell death that can be instigated through the inactivation of GPX4 and/or the inhibition of the cystine/glutamate antiporter SLC7A11-xCT. The presence of the SLC7A11-xCT antiporter in stallion spermatozoa combined with the observation that classical ferroptosis inductors lead to sperm death (Ortiz-Rodriguez et al. 2019b, 2020) argues in favour of the existence of this type of ROS-mediated cell death in spermatozoa. However, it would appear that the mechanisms of cell death are multi-factorial, and as such, the assessment of multiple simultaneous mediators of cell death during sperm storage investigations is warranted.

Conclusions and future directions

Spermatozoa are redox-regulated cells which depend upon a delicate balance between oxidants and antioxidants for proper function. In horse spermatozoa, a dependence upon OXPHOS means that these cells produce large amounts of ROS, which predispose them to redox dysregulation and oxidative stress. Furthermore, current practices of sperm preservation also trigger oxidative stress (Burnaugh et al. 2010, Mislei et al. 2020) and thus exacerbate the reduction in post-storage longevity. To overcome this, it is important that we continue to develop more physiologically appropriate sperm storage media based on our developing knowledge of stallion sperm metabolism (Gibb et al. 2018), and importantly, how this metabolism influences redox status and sperm longevity in vitro (Pena et al. 2022). In addition to the development of optimised media, it is important to maximise the quality of the ejaculate prior to storage through the use of minimal contamination semen collection techniques and colloidal centrifugation at processing to remove dead spermatozoa, bacteria and other contaminants (Balao da Silva et al. 2014, Gonzalez-Fernandez et al. 2012). Of final note, it will be through the use of high-throughput techniques, like proteomics, metabolomics, multiparametric flow cytometry and their corresponding bioinformatic analyses, that we will find molecular markers to identify ejaculates with high or low fertilising capacity (Aitken et al. 2020, Gaitskell-Phillips et al. 2022), which will be imperative for improving breeding efficiency into the future.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review.

Funding

The authors received financial support for their investigations from the Ministerio de Ciencia-European Fund for Regional Development (EFRD), Madrid, Spain, grants PID2021-122351OB-I00 and Junta de Extremadura-EFRD (IB 20008 and GR 21060). Figures were created with BIORENDER.

Author contribution statement

FP outlined the paper, created the figures, and wrote part of the paper, ZG wrote part of the paper and revised the figures, FP and ZG performed the literature review and approved the final draft for submission.

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    Figure 1

    Overview of mechanisms involved in the control of redox homeostasis in the stallion spermatozoa. The SCLTA11-xCT channel incorporates cystine (oxidised form of the amino acid cysteine) in exchange for intracellular glutamate. Once cystine is incorporated into the cytosol, it is reduced to cysteine and used to synthesise glutathione (GSH). GSH is a cofactor of many antioxidant enzymes, acting as electron donor; after donating electrons, GSH is oxidised to GSSG. GSH prevents lipid peroxidation, protein oxidation, DNA oxidation and ROS-induced forms of sperm dead such as ferroptosis and apoptosis. Oxidised glutathione is recycled by electrons donated by NADH and NADPH provided principally, but not exclusively, by the metabolisation of glucose through the pentose phosphate pathway (PPP). The first line of defence against oxidative stress is superoxide dismutase, other families of proteins playing major role in the control of redox homeostasis are the peroxiredoxins (PRX), glutathione peroxidases (GPX) and thioredoxins (TRX).

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    Figure 2

    Overview of the oxidative damage induced by cryopreservation on the stallion spermatozoa. Osmotic shock, especially at thawing cause membrane rupture in a variable number of the spermatozoa. Mitochondria also experience osmotic stress, resulting in mitochondrial malfunction and leakage of O2•−. If the antioxidant defences are surpassed, oxidative stress causes DNA, protein and lipid peroxidation with formation of lipid aldehydes such as 4HNE, which self-perpetuates the oxidative insult in a closed loop. Lipid peroxidation initially causes an increase in membrane permeability and activation of ferroptosis and apoptosis that finally leads to sperm death. Oxidation of proteins causes sperm malfunction and reduced fertility, DNA oxidation may lead to DNA fragmentation and increased early embryo mortality or may cause problems in the offspring due to oxidative damage in specific transcripts and/or oxidative modification of the epigenome.

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