In vivo oxidative stress alters thiol redox status of peroxiredoxin 1 and 6 and impairs rat sperm quality

Abstract

Oxidative stress, the imbalance between the production of reactive oxygen species (ROS) and antioxidant activity is a major culprit of male infertility. Peroxiredoxins (PRDXs) are major antioxidant enzymes of mammalian spermatozoa and are thiol oxidized and inactivated by ROS in a dose-dependent manner. Their deficiency and/or inactivation have been associated with men infertility. The aim of this study was to elucidate the impact of oxidative stress, generated by the in vivo tert-butyl hydroperoxide (tert-BHP) treatment on rat epididymal spermatozoa during their maturation process. Adult Sprague-Dawley males were treated with 300 μmoles tert-BHP/kg or saline (control) per day intraperitoneal for 15 days. Lipid peroxidation (2-thibarbituric acid reactive substances assay), total amount and thiol oxidation of PRDXs along with the total amount of superoxide dismutase (SOD), motility and DNA oxidation (8-hydroxy-deoxyguanosine) were determined in epididymal spermatozoa. Total amount of PRDXs and catalase and thiol oxidation of PRDXs were determined in caput and cauda epididymis. While animals were not affected by treatment, their epididymal spermatozoa have decreased motility, increased levels of DNA oxidation and lipid peroxidation along with increased PRDXs (and not SOD) amounts. Moreover, sperm PRDXs were highly thiol oxidized. There was a differential regulation in the expression of PRDX1 and PRDX6 in the epididymis that suggests a segment-specific role for PRDXs. In conclusion, PRDXs are increased in epididymal spermatozoa in an attempt to fight against the oxidative stress generated by tert-BHP in the epididymis. These findings highlight the role of PRDXs in the protection of sperm function and DNA integrity during epididymal maturation.

Figure 1

Reduced sperm motility and oxidative stress markers in tert-BHP-treated compared to controls rats. (a) Sperm motility, (b) lipid peroxidation observed in units of nmoles of TBARS and (c) sperm DNA oxidation expressed as percentage of cells showing strong 8-OHdG labeling. Results are expressed as mean ± s.e.m. ^#and *mean lower or higher than controls (P < 0.05), respectively (Mann–Whitney test, n = 6). (d) Rat spermatozoa nuclei showing 8-OHdG labeling. Spermatozoa were incubated without (control) or with 2 mmol l⁻¹ hydrogen peroxide (H₂O₂) for 1 h at 20°C. Specificity of the antibody was demonstrated by incubating H₂O₂-treated samples with antibody previously incubated with 8-OHdG as described in material and methods.

Figure 2

Expression of PRDXs in rat spermatozoa. Representative blots of rat sperm proteins under reducing conditions (n = 4); 0.1 × 10⁶, 0.4 × 10⁶ or 0.1 × 10⁶ spermatozoa were loaded in each well. HeLa cells solubilized in electrophoresis sample buffer were used as positive control. To test the specificity, 0.4 µml⁻¹ of anti-PRDX1 antibody were incubated with 2 μml⁻¹ of its antigenic peptide in TBS-T supplemented with 3% BSA for 2 h at room temperature. The anti-mouse IgG antibody alone did not recognize any protein bands.

Figure 3

Increased total amount and relative intensity of PRDX1 and PRDX6 in spermatozoa from treated rats. On the left, representative immunoblots of sperm proteins under reducing conditions (sample buffer with 100 mmol l−1 DTT); 0.1 × 10⁶, 0.4 × 10⁶ or 0.1 × 10⁶ spermatozoa were loaded in each well for PRDX1, PRDX4 and PRDX6, respectively. The loading control was done by re-blotting each membrane with an anti-α-tubulin (α-Tub) antibody. HeLa cells solubilized in electrophoresis sample buffer were used as positive control. On the right, relative intensities of PRDX1 (23 and 54 kDa), PRDX4 (27 kDa) and PRDX6 (26 kDa). PRDXs band Intensities were normalized to that of α-tubulin (α-Tub). Lanes correspond to the same gel. *P < 0.05 (t-test, n = 6).

Figure 4

SOD1 is unchanged in spermatozoa from control or tert-BHP-treated rats. On the left, a representative immunoblotting of sperm proteins under reducing conditions; 0.2 × 10⁶ spermatozoa were loaded in each well for SOD1. Membrane was re-blotted with anti-α-Tubulin (loading control). On the right, relative intensity of SOD1 normalized to that of α-tubulin (α-Tub); n = 6.

Figure 5

Thiol oxidation levels of PRDX1 and PRDX6 are increased in spermatozoa from treated rats. On the left, representative immunoblots of sperm proteins under nonreducing conditions (sample buffer without DTT) showing PRDX1 (a) and PRDX6 (b) labeling. 0.1, 0.4 or 0.1 × 10⁶ spermatozoa were loaded in each well for PRDX1, PRDX4 and PRDX6, respectively. The loading control was done by re-blotting each membrane with an anti-α-tubulin (α-Tub) antibody. HeLa cells solubilized in electrophoresis sample buffer were used as positive control. On the right, relative intensities of thiol oxidized PRDX1 and PRDX6. PRDXs band intensities were normalized to that of α-tubulin (α-Tub). (c) Thiol oxidation ratio of PRDX1 and PRDX6. The thiol oxidation ratio was obtained by dividing the relative intensities of thiol-oxidized PRDXs (obtained from samples under nonreducing conditions) by those of reduced PRDX (total, obtained from samples under reducing conditions). Then, values were normalized to 1 corresponding to the mean thiol-oxidation ratio of that of controls. *P < 0.05 (t-test, n = 6).

Figure 6

PRDX1 and PRDX6 are differentially expressed in rat epididymis after tert-BHP treatment. (a) Representative immunoblot of caput and cauda epididymis proteins (10 µg protein/lane). Loading controls were done re-blotting each membrane with anti-α-tubulin (α-Tub). 0.1 × 10⁶ spermatozoa were loaded for PRDX1 and PRDX6 as positive controls. (b) Relative intensities of PRDX1 and PRDX6 normalized to that of α-Tubulin *and #mean higher or lower than controls (P < 0.05), respectively (t-test, n = 6).

Figure 7

Catalase expression is not changed in epididymis due to tert-BHP treatment. Representative immunoblot of caput and cauda epididymis proteins (10 μg protein per lane). Loading controls were done re-blotting the membrane with anti-α-tubulin (α-Tub), n = 3.