**1. Introduction**

The spermatozoa expelled from the seminiferous tubules at the end of spermatogenesis lack progressive motility and the ability to fertilize the oocyte. It is only through epididymal maturation and functional capacitation that spermatozoa undergo the necessary transformational changes needed to fertilize.

Capacitation is a highly orchestrated set of reactions that ultimately culminates in the acrosome exocytosis reaction and spermatozoa obtaining their fertilizing competency as they approach the oocyte in the oviduct [1]. Due to the lack of transcriptional and translational activity within mature spermatozoa, any changes that the cell undergoes must occur through post-translational modifications of pre-existing proteins. A key modulator of cell signaling and enzymatic function are the levels of reactive oxygen species (ROS) [2–8].

High sensitivity to membrane lipid peroxidation requires eutherian spermatozoa to maintain a fine balance between the production and regulation of ROS [9–12]. High concentrations of oxygen radicals and peroxidation by-products have been well documented as contributors to male infertility [9,13–17]. Ideally, when ROS is maintained at low concentrations, the ROS molecules are utilized by spermatozoa to modulate cellular functions such as the initiation of capacitation and hyperactivated motility [1,7,18–22]. Spermatozoa are, therefore, critically dependent on their network of regulatory and detoxifying enzymes to ensure that optimal concentrations of ROS are maintained.

Eutherian spermatozoa are largely devoid of the cytoplasmic reservoir of antioxidant enzymes seen in most somatic cells, and therefore have a large reliance on the detoxifying capacity of the fluids in their surroundings. Epididymal secretions and semen are amongs<sup>t</sup> the most antioxidant rich fluids within the body, equipped with specialized ROS scavenging molecules and enzymes that aid in bu ffering the oxidative stress levels of sperm cells [23–27]. It is also through these secretions that surface-anchored detoxifying enzymes can be imparted to the plasmalemma of spermatozoa as they progress through the male reproductive system.

The proteomic analysis of epididymal secretions, semen and surface bound spermatozoon proteins helps to decode the detoxifying landscape of spermatozoa and identify the regulatory systems at play [28,29]. At the center of many detoxifying systems is the tripeptide thiol glutathione. Glutathione provides a recyclable source of reducing power and facilitates many groups of antioxidant enzymes residing within and on the sperm surface, such as glutathione reductases, glutaredoxins, thioredoxins, perioxiredoxins and glutathione-s-transferases.

Glutathione-s-transferases (GSTs) are a large super-family of phase II detoxification enzymes that are ubiquitously found throughout the body, and well represented within the male reproductive environment [30]. GSTs of the Mu, Pi, Theta, Alpha, Zeta and Omega classes have all been identified as components of the seminal plasma or as sperm resident proteins, with their own functionally distinct roles [31–34]. Furthermore, several classes of GST have recently been shown to have functional multimodality, acting in both sperm–egg interactions and redox regulation of the plasmalemma [31]. Harboring enzymes that can facilitate more than one cellular process may be a valuable asset for cells such as spermatozoa that have evolved to be streamlined and devoid of most cytosolic resources.

The Omega class of GSTs have also been shown to have multifunctionality [30,35,36]. Equipped with a cysteine residue at their activity site, GSTOs are not only able to facilitate glutathione reductase reactions but have also been shown to have dehydroascorbate reductase capabilities [30,35,36]. Moreover, GSTO2, one of only two functionally active enzymes within the Omega class, has been credited as having the highest levels of dehydroascorbate reductase functionality within mammals [37]. With the epididymis and seminal plasma having some of the highest concentrations of ascorbic acid (AA) found within the body, maintaining optimal concentrations of AA within the sperm may prove vital to its fitness [26]. Therefore, the enzymatic versatility of facilitating reactions in both glutathione and AA centered pathways may make GSTO2 a highly valuable surface-bound enzyme.

In addition to its previous characterization as a constituent of the postacrosomal sheath and perforatorium of the perinuclear theca [34,38], we demonstrate that GSTO2 is also present on the sperm surface in mouse and boar spermatozoa. Through surface protein isolation, indirect immunofluorescence, fluorescence immunohistochemistry, functional inhibition studies and computer assisted sperm analysis, we characterize the surface localization of GSTO2 isoforms and demonstrate the functional role of GSTO2 in facilitating sperm capacitation through oxidative regulation.
