*Antioxidants* **2019**, *8*, 601

**Figure 1.** The surface reactivity of glutathione-s-transferase omega 2 (GSTO2) on non-permeabilized mouse and boar spermatozoa using indirect immunofluorescence. Non-permeabilized fresh boar and mouse spermatozoa were stained using a GSTO2 specific (Y-12) antibody (GSTO2), or a GSTO-specific fluorescent inhibitor (Inhibitor). Nuclei were labelled with DAPI. To confirm the specificity of the antibody, the anti-GSTO2 antibody was preincubated with its blocking agen<sup>t</sup> (blocking peptide, the peptide used to raise the antibody) according to the manufacturer's instructions and a secondary-only control was done (Secondary Only). The GSTO inhibitor is a membrane permeable cell tracker probe (Invitrogen Molecular Probe C7025). The bar represents 10 μm.

**Figure 2.** GSTO2 reactivity to biotin-isolated surface proteins of ejaculated and capacitated boar spermatozoa. Immunoblotting using an anti-GSTO2 (Y-12) antibody on biotin-isolated surface proteins from ejaculated boar sperm (Lane 1) and capacitated boar sperm (Lane 2). The antibody was pre-incubated with its corresponding blocking peptide to show specificity in both the ejaculated boar sperm (Lane 3) and capacitated boar sperm (Lane 4) samples.

Due to the spermatozoon's lack of transcriptional and translational capabilities after the round spermatid stage of spermiogenesis and the absence of GSTO2 membrane reactivity previously reported during spermatogenesis [34], we aimed to investigate if there was an external source of the enzyme that was secreted during epididymal transport. Fluorescence immunohistochemical staining of porcine and murine epididymal sections (Figure 3) revealed the presence of GSTO2 within the caput, corpus and caudal regions of the epididymis. The reactivity was concentrated at the luminal aspect of the epithelium, however, in the mouse caput, the GSTO2 reactivity was also seen in distinct regions, that spanned from the basal to the luminal aspect of the epithelium.

**Figure 3.** GSTO2 reactivity in histological sections of boar (**A**) and mouse (**B**) epididymis. In both species, caput, corpus and cauda epididymal sections show heightened GSTO2 reactivity at the luminal aspect of the epididymal epithelium with some fissures of reactivity towards the basal membrane. In mouse caput epididymal sections, some isolated pockets of reactivity can also be seen, spanning the length of the epididymal epithelium. Nuclear material was stained with DAPI. The bars in both (**A**) and (**B**) is representative of 20 μm.

### *3.2. The Functional Significance of GSTO2 During Capacitation*

Many surface proteins secreted by the epididymis have been shown to function in the regulation of capacitation. Therefore, through functional inhibition, using a specific inhibitor that binds to the active site of GSTO enzymes (Figure S1), we sought to determine if GSTO2 has a role modulating some facet of the capacitation process. Inhibitor specificity was confirmed in both mouse and boar whole sperm through fluorescence gel electrophoresis (Figure S2).

Inhibition of GSTO2's catalytic site during in vitro capacitation in mice resulted in a dampening of the hallmark increase in tyrosine phosphorylation that occurs at the late stages of capacitation (Figure 4) without impairing sperm viability (Figure S3). These findings were also observed in boar spermatozoa (Figure S4). Most likely as a consequence of the diminished tyrosine phosphorylation events, mouse spermatozoa also demonstrated a significant decrease in their ability to successfully undergo acrosome exocytosis when GSTO enzymes were functionally inhibited prior to in vitro capacitation (Figure 5).

**Figure 4.** The level of protein tyrosine phosphorylation during in vitro capacitation in mouse spermatozoa. (**A**) Total protein tyrosine phosphorylation patterns at 0, 45 and 90 min after in vitro capacitation in DMSO-treated and capacitated (Capacitated), DMSO-treated and not capacitated (Not Capacitated) and GSTO-inhibited and capacitated (GSTO Inhibited) mouse spermatozoa samples. (**B**) The intensity of the total protein tyrosine phosphorylation levels, shown in the Western blot at each time point for each treatment, were quantified using Image J and normalized to the respective intensity of alpha tubulin. Total tyrosine phosphorylation relative intensities were further normalized so that all time 0 values were 1. All measurements are the averages of three trials, all performed with different mice. Error bars represent standard error and \* signifies statistical significance of *p* = 0.008 determined by a one-way ANOVA.

**Figure 5.** Sperm capacitation is reduced by the presence of the GSTO inhibitor when incubated in a capacitation medium. Sperm treatment was done through incubation for 25 min before in vitro capacitation was induced. The acrosome exocytosis reaction (a hallmark of capacitation) was induced using progesterone after 60 min of capacitation. Sperm samples were fixed and stained with PNA-647 and 4',6-diamidino-2-phenylindole (DAPI) and scored based on acrosome labelling. At least 200 sperm per treatment were assessed per trial and grouped into acrosome intact or acrosome reacting/reacted. Statistical significance was determined by multiple t-tests and is denoted by \*, *p* = 0.0004.

When GSTO inhibited spermatozoa were analyzed with computer-aided sperm analysis significant decreases in total and progressive motility were observed (Figure 6, Panel A and B). A significant decrease in the overall curvilinear velocity was also observed in capacitated samples (Figure 6, panel C). Additionally, while a trend of higher linearity, a measured ratio of straight line and curvilinear velocity, was observed in GSTO inhibited samples they were not found to be significant (Figure 6, Panel D). These findings sugges<sup>t</sup> that the spermatozoa treated with the GSTO inhibitor were not as successful in reaching the ideal state of hyperactive activity when compared to the controls.

**Figure 6.** Computer-aided sperm analysis (CASA) of GSTO-inhibited and DMSO treated (Control) mouse spermatozoa afterin vitro capacitation. Sperm motility parameters were analyzed on capacitated mouse spermatozoa that were either treated with DMSO (Control) or GSTO inhibitor prior to a 60-min incubation in capacitating medium at 37 degrees Celsius. The average total motility (**A**) was the combination of the progressive and non-progressive motility scores for each sample, whereas (**B**) shows solely the comparison of progressive motility. Both the total and progressive motility differences between the two treatment groups were statistically significant. The curvilinear velocity of GSTO inhibited sperm were also significantly decreased compared to controls when both treatment groups were capacitated (**C**). A higher linearity was also observed when spermatozoa were treated with the GSTO inhibitor (**D**), but the differences between the control and inhibited treatments were not found to be statistically significant. Error bars represent standard error. Statistical significance was determined using multiple t-tests and is denoted by \*.

Further investigations looked into the peroxidation of lipids within the plasma membrane of mouse spermatozoa after in vitro capacitation using the BODIPY 581/591 C11 probe that fluoresces red in a neutral state but is modified to a green fluorescence when lipids undergo peroxidation. The ratio of green fluorescence intensity over total fluorescence intensity was used as a measure lipid peroxidation and revealed a significant increase in lipid peroxidation when GSTO activity was inhibited (Figure 7). These findings were reinforced by a significant increase in the overall cellular reactive oxygen species levels of spermatozoa treated with the GSTO inhibitor compared to controls (Figure S5). Lastly, when in vitro fertilization was performed in both mouse and swine, there was a significant decrease in the sperm's ability to successfully penetrate the oocyte (Figure 8).

**Figure 7.** The assessment of sperm membrane lipid peroxidation after in vitro capacitation in mouse spermatozoa. (**A**) Lipid peroxidation levels were assessed based on the red and green fluorescence intensities of cells treated with the BODIPY 581/591 C11 probe. The relative intensity was calculated as the intensity of the green fluorescence over total fluorescence intensity. Spermatozoa were treated with the BODIPY 581/591 C11 probe and then incubated with either the GSTO Inhibitor or DMSO (Control) for 25 min before in vitro capacitation. After 60 min of in vitro capacitation, samples were fixed and imaged to determine the red and green fluorescence intensities of each cell. At least 200 cells were individually imaged in each treatment group for each trial, and a representative image from the control and GSTO inhibited groups is shown in (**B**). Three trials were performed, each with different mice, and error bars represent standard error. Statistical significance was determined using a t-test with Welch's correction and is denoted by \*.

**Figure 8.** The assessment of sperm penetration during mouse and swine in vitro fertilization (IVF) using a low sperm concentration. Successful sperm penetration was assessed in both mouse (**A**) and swine (**B**) IVF models after 5–6 h of co-incubation in the fertilization droplet. Spermatozoa were pre-treated with either DMSO (Control) or GSTO inhibitor for 25 min before being placed in capacitation-inducing medium. Mouse sperm were incubated with cumulus–oophorus complexes at a concentration of 1 × 10<sup>5</sup>/mL and boar sperm was incubated with in vitro matured swine oocytes at a concentration of 1 × 10<sup>4</sup>/mL. Oocytes were washed and culture for 8 h (mouse) or 16 h (swine). Oocytes were fixed and stained with DAPI to assess pronuclear formation and sperm penetration. Statistical significance was assessed through a t-test with Welch's correction. The data represent the adjusted average of three replicates and error bars represent standard error. Statistical significance is denoted by \*.
