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. 2024 Nov 22;13(12):1437.
doi: 10.3390/antiox13121437.

Treatment with Pterostilbene Ameliorates the Antioxidant Status of Bovine Spermatozoa and Modulates Cell Death Pathways

Christos Chavas  1 , Vasiliki G Sapanidou  1 , Konstantinos Feidantsis  2 , Sophia N Lavrentiadou  1 , Despoina Mavrogianni  3 , Ioanna Zarogoulidou  1 , Dimitrios J Fletouris  4 , Maria P Tsantarliotou  1
Affiliations

Treatment with Pterostilbene Ameliorates the Antioxidant Status of Bovine Spermatozoa and Modulates Cell Death Pathways

Christos Chavas et al. Antioxidants (Basel). .

Abstract

Reactive Oxygen Species (ROS) play an important role in sperm physiology. They are required in processes such as capacitation and fertilization. However, the exposure of spermatozoa to ROS generated from internal or external sources may create a potentially detrimental redox imbalance. Antioxidant supplementation in semen is now a rather common approach to protect spermatozoa from oxidative stress (OS) during their handling and/or cryopreservation. Supplementation with pterostilbene, a potent antioxidant, protects spermatozoa from OS and ameliorates their post-thawing characteristics and viability. In the present study, we used freezing/thawing as a model of natural ROS overproduction and investigated the molecular mechanisms modulated by pterostilbene. Specifically, bovine frozen/thawed spermatozoa were incubated with 10 or 25 μM pterostilbene for 60 min. Results have shown that in a dose-independent manner, pterostilbene decreased lipid peroxidation and increased intracellular GSH levels. Moreover, pterostilbene ameliorated energy production, as ATP and AMP/ATP levels were restored, and increased autophagy levels through AMP-activated protein kinase (AMPK) activation, which finally resulted in the inhibition of apoptotic cell death in bovine spermatozoa when exposed to OS. This study sheds light on spermatozoa redox state, the crosstalk between apoptotic and autophagic pathways, and its role in determining the beneficial or detrimental effect of ROS in spermatozoa.

Keywords: antioxidant; apoptosis; autophagy; mitochondria; oxidative stress; pterostilbene.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
MDA (A), total antioxidant capacity (B), and intracellular GSH (C) levels in bovine spermatozoa under the effect of 10 μM (P10) or 25 μM (P25) pterostilbene treatments. Values constitute means ± S.D. Asterisks (*) denote statistically significant differences compared to control (p < 0.05, n = 6). No statistically significant differences were found between the P10 and P25 groups.
Figure 2
Figure 2
Bax (A), Bcl-2 (B), Bax/Bcl-2 (C), and cleaved caspase (D) levels in bovine spermatozoa in the presence of 10 μM (P10) or 25 μM (P25) pterostilbene. Values constitute means ± S.D. Spermatozoa extracts from control, P10, and P25 groups were immunoblotted for Bax, Bcl-2, and cleaved caspases. The levels of β-actin were determined to verify equal loading. Representative blots are shown (Figures S2 and S3). Asterisks (*) denote statistically significant differences compared to the control (p < 0.05, n = 6). No statistically significant differences were found between P10 and P25 groups.
Figure 3
Figure 3
AMP (A) and ATP (B) levels and AMP/ATP (C) ratios in bovine spermatozoa treated with 10 μM (P10) or 25 μM (P25) pterostilbene. Values constitute means ± S.D. Asterisks (*) denote statistically significant differences compared to the control (p < 0.05, n = 6). No statistically significant differences were found between P10 and P25 groups.
Figure 4
Figure 4
Phospho AMPK/AMPK ratio (A), ubiquitin conjugates (B), LC3 II/I ratio (C), and SQSTM1/p62 (D) levels in bovine spermatozoa exposed to 10 μM (P10) or 25 μM (P25) pterostilbene treatments. Values constitute means ± S.D. Spermatozoa extracts from control, P10, and P25 groups were immunoblotted for phospho AMPK, AMPK, ubiquitin conjugates, LC3, and SQSTM1/p62. The levels of β-actin were determined to verify equal loading. Representative blots are shown (Figures S4–S7). Asterisks (*) denote statistically significant differences compared to the control (p < 0.05, n = 6). No statistically significant differences were found between P10 and P25 groups.
Figure 5
Figure 5
Summarized model of pterostilbene’s effect on biochemical and physiological stress responses in bovine spermatozoa exposed to oxidative stress.
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