The protective effects of alpha-ketoacids against oxidative stress on rat spermatozoa in vitro

Abstract

The aim of this study was to determine the effects of antioxidants, including alpha-ketoacids (alpha-ketoglutarate and pyruvate), lactate and glutamate/malate combination, against oxidative stress on rat spermatozoa. Our results showed that H(2)O(2) (250 micromol L(-1))-induced damages, such as impaired motility, adenosine triphosphate (ATP) depletion, inhibition of sperm protein phosphorylation, reduced acrosome reaction and decreased viability, could be significantly prevented by incubation of the spermatozoa with alpha-ketoglutarate (4 mmol L(-1)) or pyruvate (4 mmol L(-1)). Without exogenous H(2)O(2) in the medium, the addition of pyruvate (4 mmol L(-1)) significantly increased the superoxide anion (O(2)(-).) level in sperm suspension (P < or = 0.01), whereas the addition of alpha-ketoglutarate (4 mmol L(-1)) and lactate (4 mmol L(-1)) significantly enhanced tyrosine-phosphorylated proteins with the size of 95 kDa (P < or = 0.04). At the same time, alpha-ketoglutarate, pyruvate, lactate, glutamate and malate supplemented in media can be used as important energy sources and supply ATP for sperm motility. In conclusion, the present results show that alpha-ketoacids could be effective antioxidants for protecting rat spermatozoa from H(2)O(2) attack and could be effective components to improve the antioxidant capacity of Biggers, Whitten and Whittingham media.

Figure 1

Standard curve measured by lucigenin for H₂O₂. Various concentrations of H₂O₂ were prepared in mBWW medium and then were measured using lucigenin (250 μmol L⁻¹).

Figure 2

Effects of antioxidants on reactive oxygen species (ROS) levels in sperm suspensions. (A): ROS levels were assayed using lucigenin. (B): ROS levels were assayed using MCLA. B, mBWW; P, pyruvate (4 mmol L⁻¹); K, α-ketoglutarate (4 mmol L⁻¹); L, lactate (4 mmol L⁻¹); GM, glutamate/malate (4 mmol L⁻¹); CON, control; H, H₂O₂ (250 μmol L⁻¹); SOD, superoxide dismutase (0.1 mg mL⁻¹); CAT, catalase (0.2 mg mL⁻¹). ^*P < 0.05, compared with control; ^#P < 0.001, compared with H sample; (n = 3).

Figure 3

Effects of antioxidants on sperm motility. (A): Sperm motility was examined at the end of a 1-h treatment using computer-assisted sperm analysis (CASA). (B): Sperm motility was examined at the end of a 5-h treatment using CASA. CON, control; H, H₂O₂ (250 μmol L⁻¹); P, pyruvate (4 mmol L⁻¹); K, α-ketoglutarate (4 mmol L⁻¹); L, lactate (4 mmol L⁻¹); GM, glutamate/malate (4 mmol L⁻¹). ^*P < 0.001, compared with control; ^#P < 0.01, compared with H sample; (n = 4).

Figure 4

Effects of antioxidants on adenosine triphosphate (ATP) levels. ATP levels were assayed at the end of a 1-h treatment. CON, control; H, H₂O₂ (250 μmol L⁻¹); P, pyruvate (4 mmol L⁻¹); K, α-ketoglutarate (4 mmol L⁻¹); L, lactate (4 mmol L⁻¹); GM, glutamate/malate (4 mmol L⁻¹). ^*P< 0.01, compared with control; ^#P < 0.001, compared with H sample; (n= 4).

Figure 5

Effects of antioxidants on tyrosine phosphorylation. (A): Western blot assay was performed at the end of a 5-h treatment. (B): Quantification of the 95-kDa bands indicated by arrow 1. (C): Quantification of the two 80-kDa bands indicated by arrow 2. (D): Quantification of the 55-kDa bands indicated by arrow 3. 0 h, control sample incubated for 0 h. CON, control; H, H₂O₂ (250 μmol L⁻¹); P, pyruvate (4 mmol L⁻¹); K, α-ketoglutarate (4 mmol L⁻¹); L, lactate (4 mmol L⁻¹); GM, glutamate/malate (4 mmol L⁻¹). ^*P < 0.05, compared with control; ^#P < 0.05, compared with H sample; (n = 4).

Figure 6

Effects of antioxidants on acrosome reaction. Acrosome reaction was assayed at the end of a 4-h treatment using CTC staining. CON, control; H, H₂O₂ (250 μmol L⁻¹); P, pyruvate (4 mmol L⁻¹); K, α-ketoglutarate (4 mmol L⁻¹); L, lactate (4 mmol L⁻¹); GM, glutamate/malate (4 mmol L⁻¹). ^*P < 0.05, compared with control; ^#P < 0.05, compared with H sample; (n = 3).

Figure 7

The effects of various concentrations of H₂O₂ on sperm viability. Rat spermatozoa were incubated with various concentrations of H₂O₂ for 4h and then viability was assayed using Transgreen/PI staining.

Figure 8

Effects of antioxidants on sperm viability. (A): Sperm viability was examined at the end of a 4-h treatment using Transgreen/PI staining. (B): Sperm viability was examined at the end of a 24-h treatment using same technique. CON, control; H, H₂O₂ (500 μmol L⁻¹); P, pyruvate (4 mmol L⁻¹); K, α-ketoglutarate (4 mmol L⁻¹); L, lactate (4 mmol L⁻¹); GM, glutamate/malate (4 mmol L⁻¹). ^*P < 0.01, compared with control value; ^#P < 0.01, compared with H sample. (n = 4).

Figure 9

The effects of various concentrations of H₂O₂ (μmol L⁻¹) on sperm protein tyrosine phosphorylation. Rat spermatozoa were incubated with various concentrations of H₂O₂ for 5h and then tyrosine phosphorylation was assayed using PY20 antibody. α-Tubulin was a 55-kDa protein and used as internal control.