Cysteine improves boar sperm quality via glutathione biosynthesis during the liquid storage

Abstract

Objective: Sperm is particularly susceptible to reactive oxygen species (ROS) stress. Glutathione (GSH) is an endogenous antioxidant that regulates sperm redox homeostasis. However, it is not clear whether boar sperm could utilize cysteine for synthesis GSH to protect sperm quality from ROS damage. Therefore, the present study was undertaken to elucidate the mechanism of how cysteine is involved in protecting boar sperm quality during liquid storage.

Methods: Sperm motility, membrane integrity, lipid peroxidation, 4-hydroxyIlonenal (4-HNE) modifications, mitochondrial membrane potential, as well as the levels of ROS, GSH, and, ATP were evaluated. Moreover, the enzymes (GCLC: glutamate cysteine ligase; GSS: glutathione synthetase) that are involved in glutathione synthesis from cysteine precursor were detected by western blotting.

Results: Compared to the control, addition of 1.25 mM cysteine to the liquid storage significantly increased boar sperm progressive motility, straight-line velocity, curvilinear velocity, beat-cross frequency, membrane integrity, mitochondrial membrane potential, ATP level, acrosome integrity, activities of superoxide dismutase and catalase, and GSH level, while reducing the ROS level, lipid peroxidation and 4-HNE modifications. It was also observed that the GCLC and GSS were expressed in boar sperm. Interestingly, when we used menadione to induce sperm with ROS stress, the menadione associated damages were observed to be reduced by the cysteine supplementation. Moreover, compared to the cysteine treatment, the γ-glutamylcysteine synthetase (γ-GCS) activity, GSH level, mitochondrial membrane potential, ATP level, membrane integrity and progressive motility in boar sperm were decreased by supplementing with an inhibitor of GSH synthesis, buthionine sulfoximine.

Conclusion: These data suggest that boar sperm could biosynthesize the GSH from cysteine in vitro. Therefore, during storage, addition of cysteine improves boar sperm quality via enhancing the GSH synthesis to resist ROS stress.

Keywords: Boar Sperm; Cysteine; Glutathione Synthesis; Reactive Oxygen Species Stress.

Figure 1

Dynamic changes in the sperm membrane integrity by addition of different concentrations of cysteine to the diluted medium after 13 days’ preservation. Values are specified as mean±standard deviation (SD). ^a,b Columns with different lowercase letters differ significantly (p<0.05).

Figure 2

Effects of cysteine on sperm GSH level (A), activities of SOD (B) and catalase (C), ROS level (E) and lipid peroxidation (F) after 13 days’ preservation. Values are specified as mean±standard deviation (SD). GSH, glutathione; SOD, superoxide dismutase; ROS, reactive oxygen species; Cys, cysteine. * Denotes significant differences compared with the control (p<0.05).

Figure 3

(A–B) Western blotting analysis of the effect of cysteine on 4-HNE modifications in boar sperm after 5-days’ preservation. Western blotting image is showing the expression of 4-HNE modifications in boar sperm (A). (B) Quantitative expression of the 4-HNE over α-tubulin generated from western blotting (A). Immunofluorescent localization of 4-HNE in boar sperm (C). Data are the mean±standard deviation (SD). 4-HNE, 4-hydroxyIlonenal. ^a–c Columns with different lowercase letters differ significantly (p<0.05). Bars = 30 μm.

Figure 4

Effects of cysteine on sperm mitochondrial membrane potential (A), ATP level (B) and acrosome integrity (C) after 13 days’ preservation. Values are specified as mean± standard deviation (SD). Cys, cysteine. * Denotes significant differences compared with the control (p<0.05).

Figure 5

Western blotting detection of (A) the GCLC and (B) GSS enzymes in boar sperm. GCLC, glutamate cysteine ligase; GSS, glutathione synthetase; S1–4, sample1–4.

Figure 6

Effect of cysteine on the GSH level and the ROS damage induced by menadione in vitro condition. ROS level (A), GSH level (B), γ-GCS activity (C), mitochondrial membrane potential (D), ATP level (E) and membrane integrity (F). Data are the mean±standard deviation (SD). Cys, cysteine; Men, menadione; BSO, buthionine sulfoximine; γ-GCS, γ-glutamylcysteine synthetase. ^a–c Columns with different lowercase letters differ significantly (p<0.05).

Figure 7

Effect of cysteine on sperm progressive motility in the menadione-induced ROS stress model. Data are the mean±standard deviation (SD). ^a–c Columns with different lowercase letters differ significantly (p<0.05).

Figure 8

Mechanisms of cysteine involved in regulating boar sperm quality. Boar sperm could utilize the cysteine to generate GSH for reducing the ROS stress, thus improving sperm quality. BSO, buthionine sulfoximine.