*2.3. Antibodies and Reagents*

The central antibody was a goa<sup>t</sup> polyclonal anti-GSTO2 antibody (Y-12, Santa Cruz Biotechnology, Dallas, TX, USA), used at a concentration of 0.2 μg/mL for Western blot analysis, and 6.67 μg/mL for fluorescence immunocytochemistry. For the measure of protein tyrosine phosphorylation, the clone 4G10 anti-tyrosine phosphorylation antibody (Millipore-Sigma, 05-321, St. Louis, MO, USA) was used at a concentration of 0.1 μg/mL and standardized using an anti-alpha tubulin antibody (Sigma T6074, Burlington, MA, USA). For immunohistochemistry a rabbit-polyclonal anti-GSTO2 (Sigma Prestige, HPA048141, Burlington, MA, USA) was used at a concentration of 6.67 μg/mL. For Western blot analysis, a rabbit anti-goat IgG-HRP (horseradish peroxidase) (0.4 μg/mL, Santa Cruz Biotechnology, Santa Cruz, CA, USA) secondary antibody was used, and, for indirect immunofluorescence studies, a donkey anti-goat IgG-CFL (colorized fluorochrome) 555 (Santa Cruz, 2 μg/mL), or donkey-anti-rabbit-IgG-CFL 488 was used (2 μg/mL, Abcam, Cambridge, MA, USA). For the assessment of the acrosome exocytosis reaction lectin PNA (*Arachis hypogaea)* conjugated to the colorized fluorocrome 647 (Invitrogen, L32460, Waltham, MA, USA) was used at a concentration of 15 μg/mL. For peroxidation analysis, a BODIPY 581/591 C11 probe (4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid) (Invitrogen Molecular Probes, D3861) was used at a final concentration of 5 μM. For evaluation of the total reactive oxygen species, the Cellular ROS Detection Assay kit (ab186029Abcam, Cambridge, MA, USA) was used following the manufacturer's manual. For all enzymatic inhibition studies, a membrane permeable cell tracker probe (Invitrogen Molecular Probes C7025) was used. The probe has been shown to bind covalently and irreversibly to the active site of GSTO isozymes [39] and was used at a concentration of 100 μM in all our experiments. Any additional reagents were purchased from Millipore-Sigma (Burlington, MA, USA).

### *2.4. Fluorescence Electrophoresis and Western Blotting Analysis*

All sperm samples were freshly extracted and subsequently incubated with our inhibitory probe for 25 min at 37 or 38 ◦C, depending on the species. The samples were washed twice and then solubilized in a non-reducing sample buffer (200 mM Tris pH 6.8, 4% SDS, 0.1% bromophenol blue, 40% glycerol, 5% β-mercaptoethanol). A BLUeye pre-stained protein ladder (GeneDirex) was loaded along with approximately 1–2 million cells per lane and resolved on 4% stacking and 12% separating polyacrylamide gels, as described by Laemmli [40]. The gel was run at 100 volts for 110 min before being placed in transfer buffer and imaged in a fluorescence biophotonic chamber. After transfer to a polyvinylidene fluoride (PVDF) membrane (Millipore) for 120 min in Tris-glycine transfer buffer on ice using a Hoefer Transfer apparatus (Hoefer Scientific Instruments), the membrane was also imaged in the same fluorescence chamber. The membrane was then blocked in a 10% skim milk and phosphate-buffered saline (PBS) solution with 0.05% Tween-20 (PBS-T) for 30 min to prevent non-specific binding. The membrane was incubated with primary antibody overnight at 4 ◦C with slight agitation. The next day, the membrane was washed in PBS-T six times, each for five minutes, before a two hour incubation with secondary antibody conjugated to horseradish peroxidase. The membrane was then washed extensively and subjected to an immunodetection reaction that was visualized using Clarity Western ECL Substrate (Bio Rad Laboratories, Hercules, CA, USA). The membrane was exposed to X-ray film for developing. For the evaluation of tyrosine phosphorylation (PY) during capacitation, relative intensities were calculate using Image J. Total PY was calculated for each sample and normalized using the intensity of tubulin in each sample.
