**3. Results**

### *3.1. The presence of GSTO2 on the Plasmalemma of Mature Mouse and Boar Spermatozoa*

Glutathione-S-Transferase Omega 2 was observed on the plasmalemma of non-permeabilized mouse and boar spermatozoa through the use of indirect immunofluorescence (Figure 1). This reactivity was inhibited when the antibody (Y-12) was pre-incubated with its corresponding peptide block, and was absent when samples were incubated in only the secondary antibody. Furthermore, when spermatozoa were incubated with a Glutathione-S-Transferase Omega (GSTO)-specific inhibitory binding molecule, the entirety of the sperm showed reactivity, suggesting enzymes of the GSTO family are present outside of the perinuclear theca, the sole localization of GSTO enzymes currently documented within eutherian spermatozoa [34].

To further investigate the surface localization of GSTO2, surface proteins of non-permeabilized fresh and capacitated boar spermatozoa were isolated through the use of the Pierce Cell Surface Biotinylation and Isolation Kit (A44390, ThermoFisher Scientific, Waltham, MA, USA), run on an SDS-PAGE gel and probed with an anti-GSTO2 antibody. The findings revealed that two GSTO2 isoforms were present on the surface of fresh boar spermatozoa (Figure 2, Lane 1) but that, following in vitro capacitation, only the higher isoform remained (Figure 2, Lane 2). This reactivity was inhibited when the antibody was pre-incubated with its corresponding peptide block (Figure 2, Lanes 3 and 4).
