**4. Discussion**

The present study demonstrates, for the first time, the presence of GSTO2 on the surface of spermatozoa and its participation in the regulation of ROS levels during capacitation. Surface protein biotinylation, indirect immunofluorescence with anti-GSTO2 antibodies, and a GSTO-specific fluorescent inhibitor revealed the surface localization of two GSTO2 isoforms covering the plasmalemma of both mouse and boar spermatozoa. The absence of GSTO2 reactivity on the surface of eutherian spermatids at the time of spermiation, and its presence on epididymal spermatozoa, suggests that the origin of the surface GSTO2 isoforms differs from the isoforms characterized within the perinuclear theca region of the sperm head [34].

After spermatogenesis, the spermatozoa released from the testis are fully formed but not adequately primed to interact with the oocyte. Transport in the epididymis allows sperm cells to mature and gain surface enzymes and molecules required to function optimally. Sperm maturation is driven exclusively by external factors within the luminal microenvironment of the epididymis and occurs in the absence of transcriptional and translational activity by the sperm [28,29,42–46]. Therefore, many of the proteins, enzymes, chaperones and cytokines that collectively contribute to sperm protection and function are acquired through the release of secretory products from the principal cells into the epididymal lumen [28,29,45,47,48]. Our immunohistochemical analysis of both mouse and boar epididymal segments suggests that GSTO2 is likely a constituent of these secretions. Since GSTO2 is considered a cytosolic protein that is non-glycosylated, it most likely finds its way into the epididymal lumen through the apocrine secretory pathway.

The caput epididymis is of critical importance in promoting sperm maturation and has been shown to have greater apocrine secretory activity than the epididymal regions it precedes [49]. Thus, the predominance of GSTO2 within the apical poles of the principal cells in the caput of both the mouse and boar epididymis is supportive of its role in promoting sperm maturation and enhancing fertilization capacity. The presence of two isoforms of GSTO2 on the sperm surface indicates a difference in their functional roles and/or temporal association with the sperm surface. The absence of the lower molecular weight isoform from the surface of boar spermatozoa after in vitro capacitation suggests

that it may be removed as the membrane reorganizes in preparation for sperm–oocyte interactions. Without the ability to di fferentiate between the isoforms, their specific organizations on the sperm surface cannot be fully realized. Moreover, while this is the first report of GSTO2 on the surface of eutherian spermatozoa, we cannot exclude the possibility that our GSTO fluorescent inhibitor is also interacting with a GSTO1 isoform, since the inhibitor does not di fferentiate between members of the GST Omega family. However, neither we nor others have been successful in identifying GSTO1 in eutherian sperm.

The regulation of oxidative stress levels within capacitation is a true balancing act. Spermatozoa optimally function on the knife's edge of beneficial versus detrimental oxidative stress levels and, therefore, must be equipped with the necessary antioxidant enzymes and substrates to e ffectively regulate their environment. When protective and antioxidant rich environmental barriers such as the seminal plasma are diluted within the female reproductive tract, spermatozoa must rely solely on membrane-bound, cytosolic, and mitochondrial antioxidant enzymes to maintain an e ffective oxidative equilibrium.

At low concentrations, reactive oxygen species (ROS) have been shown to positively a ffect capacitation and the acrosome exocytosis reaction and play central roles within most of the transduction pathways associated with the sperm acquiring its fertilizing ability [2–8,18]. Members of many of the oxidative-reductive regulating superfamilies' have been characterized as sperm-resident proteins, such as thioredoxins, glutaredoxins, glutathione peroxidases (GPXs) glutathione-S-transferases (GSTs) and peroxiredoxins (PRDXs) [23,31,50–55]. Located internally or on the surface of the sperm plasma membrane, these enzymes function in diverse roles regulating transduction cascades, cell signaling, sperm–oocyte interactions and oxidative stress, facilitated by antioxidant molecules such as glutathione (GSH) and ascorbic acid (AA) [17,25,53,54,56].

Ascorbic acid (AA) or vitamin C is a potent single electron donor that acts as a scavenger of ROS in most organ systems [53,57]. AA is used in energetically favorable oxidation reactions to neutralize reactive and damaging compounds that contain an unpaired electron, such as hydroxide [53]. In both the germinal epithelium and the epididymis, AA levels are significantly higher than that of blood plasma, and a high concentration of AA within the seminal plasma has been shown to be positively correlated with sperm count, sperm motility and normal sperm morphology [26,27,53,58]. Previous reports have also shown that AA is required to protect sperm from endogenous ROS production at all stages of development and maturation in both the germinal layer and the epididymis [59,60]. Therefore, its replenishment within epididymal transport and capacitation processes may prove vital in maintaining the oxidative-reductive homeostasis of sperm as they prepare for fertilization. The recycling of dehydroascorbate (DHA) into its reduced ascorbate state can be accomplished through various mechanisms, including the direct reduction by glutathione and the enzymatic reduction by various thiol transferases and NADPH-dependent reductases [61]. One such enzyme that can facilitate this reaction is glutathione-s-transferase omega 2 (GSTO2). Past reports have found that GSTO2 has the highest dehydroascorbate reductase functionality within mammalian systems, with a higher a ffinity for DHA than glutathione itself [30,37,62]. Therefore, in sperm maturation and capacitation, two highly oxidative processes that utilize AA as a major source of reducing power, the presence of a high a ffinity DHA recycling enzyme may be critical in maintaining the required concentration of AA to e ffectively defend against damaging levels of oxidative stress.

The sperm plasma membrane is a rich lipid bilayer that is highly susceptible to lipid peroxidation by oxidative stress [10–12,63]. Damage to the membrane structure has previously been shown to have wide reaching functional implications in sperm, as membrane fluidity and surface proteins have large roles in both capacitation and sperm–oocyte interactions [9,13–17]. Our results show that the absence of GSTO2's catalytic activity negatively impacts the spermatozoon's ability to prevent lipid peroxidation within the plasmalemma during capacitation, resulting in the impairment or dysregulation of membrane-associated processes such as the acrosome exocytosis reaction, sperm motility and, ultimately, the sperm's ability to penetrate the oocyte. Therefore, while GSTO2 may not have a direct role within each of these processes, it is possible that its functional involvement in the regulation of the membranes oxidative state may have indirect implications on the sperm's overall fitness.

Limitations of this study were that our functional inhibitor was permeable and did not discriminate between di fferent GSTO enzymes and isoforms. However, to date, only GSTO2 has been shown to reside on or within spermatozoa. We recently reported that GSTO2 isoforms within the post-acrosomal and perforatorial regions of the perinuclear theca (PT) facilitate the post-fertilization nuclear decondensation and male nuclear transition [50]. For this reason, we did not investigate any events beyond sperm penetration where the PT isoforms would functionally participate. That these PT-resident GSTO2s could be involved in the capacitation process is unlikely, as they are firmly anchored to the PT and require harsh solubilization agents such as 1M NaOH to release them from the PT [34]. Due to this insolubility, their catalytic sites could be masked until solubilization in the oocyte cytoplasm during fertilization. Even if they were involved, their coverage compared to the surface GSTO2 is limited, and thus their functional contribution would be expected to be proportionally small.
