Resveratrol Improves Boar Sperm Quality via 5'AMP-Activated Protein Kinase Activation during Cryopreservation

Abstract

Mammalian sperm is highly susceptible to the reactive oxygen species (ROS) stress caused by biochemical and physical modifications during the cryopreservation process. 5'AMP-activated protein kinase (AMPK) is involved in regulating both cell metabolism and cellular redox status. The aim of the present study was to investigate whether the resveratrol protects boar sperm against ROS stress via activation of AMPK during cryopreservation. Boar sperm was diluted with the freezing medium supplemented with resveratrol at different concentrations (0, 25, 50, 75, 100, and 125 μM). It was observed that the addition of 50 μM resveratrol significantly improved the postthaw sperm progressive motility, membrane integrity, acrosome integrity, mitochondrial activity, glutathione (GSH) level, activities of enzymatic antioxidants (glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase), and the phosphorylation of AMPK. Meanwhile, the lipid peroxidation, ROS levels, and apoptosis of postthaw sperm were reduced in the presence of 50 μM resveratrol. Furthermore, when fresh boar sperm was incubated with the medium in the presence of 50 μM resveratrol and 30 μM Compound C (an AMPK inhibitor), the effects of the resveratrol were partly counteracted by the Compound C. These observations suggest that the resveratrol protects boar sperm via promoting AMPK phosphorylation. In conclusion, the addition of resveratrol to the freezing extenders protects boar sperm against ROS damage via promoting AMPK phosphorylation for decreasing the ROS production and improving the antioxidative defense system of postthaw sperm. These findings provide novel insights into understanding the mechanisms of resveratrol on how to protect boar sperm quality contrary to the ROS production during cryopreservation.

Figure 1

Effects of resveratrol on sperm progressive motility, membrane integrity, and acrosome integrity during cooling, freezing, and thawing incubation processes. Cooled: fresh sperm cooled from room temperature to 5°C. Cooled-Res: fresh sperm cooled from room temperature to 5°C with 50 μM resveratrol. Equil.: cooled sperm equilibrated for 30 min at 5°C. Equil.-Res: cooled sperm equilibrated for 30 min at 5°C with 50 μM resveratrol. Freezing-Res: freezing extender added with (+) or without (-) 50 μM resveratrol. Thawing-Res: thawing solution supplemented with (+) or without (-) 50 μM resveratrol. Data are the mean ± SEM (n = 5 independent replicates). Columns with different uppercase letters differ significantly (p < 0.05).

Figure 2

Effects of different concentrations of resveratrol on postthaw sperm lipid peroxidation (a), mitochondrial membrane potential (b), ROS level (c), GSH level (d), GPx activity (f), SOD activity (g), and catalase activity (h). Photomicrographs of the postthaw sperm stained with ROS and GSH probes, respectively (e): the red arrow indicates sperm with a high level of intracellular ROS (high green fluorescence level), the yellow arrow indicates sperm with a low intracellular ROS level (low green fluorescence level), and white arrow indicates the distribution of GSH in sperm. Bars = 30 μm.

Figure 3

Effects of different concentrations of resveratrol on sperm oxidative DNA damage (b–h). Negative control (a). Photomicrographs of the postthaw sperm stained with 8-OHdG (i). Columns with different uppercase letters differ significantly (p < 0.05). Bars = 30 μm.

Figure 4

Location of AMPK in postthaw boar sperm was analyzed by immunofluorescence (a). Effects of different concentrations of resveratrol on postthaw boar sperm AMPK phosphorylation (b–d). (b) Western blotting image is showing the expression of the p-AMPK, AMPK, and α-tubulin of postthaw boar sperm. (c, d) Quantitative expression of the p-AMPK and AMPK over α-tubulin generated from western blotting (b). (e) Western blotting image is showing the expression of the p-AMPK, AMPK, and α-tubulin of sperm in the H₂O₂-induced oxidative stress model. (f, g) Quantitative expression of the p-AMPK and AMPK over α-tubulin generated from western blotting (e). Data are the mean ± SEM (n = 3 independent replicates). Columns with different uppercase letters differ significantly (p < 0.05).

Figure 5

Effects of resveratrol, AMPK activator (AICAR), and inhibitor (Compound C) on the sperm GSH level (a), catalase activity (b), ROS level (c), lipid peroxidation (d), mitochondrial membrane potential (e), membrane integrity (f), and acrosome integrity (g) in the H₂O₂-induced oxidative stress model in vitro. Data are the mean ± SEM (n = 3 independent replicates). Columns with different uppercase letters differ significantly (p < 0.05).

Figure 6

Photomicrographs of the postthaw sperm stained with the Annexin V-FITC/PI assay kit: live sperm (AN-/PI-; blue arrow), early apoptotic sperm (AN+/PI-; white arrow), late apoptotic sperm (AN+/PI+; yellow arrow), and nonviable necrotic sperm (AN-/PI+; black arrow) (a). Effects of different concentrations of resveratrol on postthaw sperm apoptosis (b–h). Data are the mean ± SEM (n = 3 independent replicates). Columns with different uppercase letters differ significantly (p < 0.05).

Figure 7

Western blotting image is showing the expression of apoptosis proteins in postthaw boar sperm (a). (b) Quantitative expression of the Parp-1, cleaved caspase-3, cleaved caspase-9, and p53 over α-tubulin generated from western blotting. Data are the mean ± SEM (n = 3 independent replicates). Columns with different uppercase letters differ significantly (p < 0.05).