Increased Oxidative Stress Markers in the Testis of Sickle MiceTo evaluate whether testosterone deficiency in Sickle mice involves increased oxidative stress at the testicular level, we measured the protein expression of several markers of oxidative stress in the testis of Sickle, Hemi, and WT mice by Western blot analysis:gp91phox (a catalytic subunit of reactive oxygen species-producing enzyme NADPH oxidase) [23]; 4-HNE (a major product of lipid peroxidation of polyunsaturated fatty acids, which covalently binds to cysteine, lysine, and histidine residues of proteins and is an indicator of protein peroxidation and a biomarker for oxidative damage) [25]; and GPx-1 (an antioxidant enzyme that reduces hydrogen peroxide to water and lipid peroxides to their corresponding alcohols) [24]. Protein expression of gp91phox was significantly (P<0.05) increased in the testis of Sickle compared to that of WT mice, but it did not differ from that of Hemi mice (Fig 6A). The amount of 4-HNE modified proteins was significantly (P<0.05) upregulated in testes of Sickle and Hemi compared to WT mice (Fig 6B). Protein expression of GPx-1 did not differ among WT, Hemi and Sickle mouse testes (P>0.05, Fig 6C). These results suggest increased oxidative stress in the testis of Sickle mice involving upregulated NADPH oxidase. The intermediately increased expression of 4-HNE coupled with basal expression of gp91phox in the testis of Hemi mice suggest lesser oxidative stress effects that do not involve NADPH oxidase activation.
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