Abstract
Resveratrol (3,5,4'-trihydroxystilbene, RSV) has been widely used in mammalian cells, but whether it can be used during freezing boar semen is still unknown. The effects of RSV treatment during boar semen freezing on its anti-freezing ability, apoptosis, and possible apoptotic pathways were observed in this study. Sperm motility, mitochondrial membrane potential (ΔΨm), adenosine triphosphate (ATP) content, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL)-positive apoptotic state, and messenger RNA (mRNA) expression levels of apoptotic genes involved in different apoptotic pathways after freezing with or without RSV treatment were tested. The results showed that: (1) Compared with fresh sperm, the motility, normal acrosome rate, and plasma membrane integrity rate of frozen boar sperm decreased significantly (P<0.05), and RSV did not significantly increase the sperm motility (0.44 vs. 0.40, P>0.05), but it did significantly improve the normal acrosome rate (57.65% vs. 47.00%, P<0.05) and plasma membrane integrity rate (46.67% vs. 38.85%, P<0.05). (2) After freezing, most boar sperm showed low mitochondrial ΔΨm. RSV treatment could increase the rate of high mitochondrial ΔΨm of boar sperm. (3) RSV treatment significantly decreased reactive oxygen species (ROS) levels (58.65% vs. 88.41%, P<0.05) and increased the ATP content (0.49 μmol/L vs. 0.25 μmol/L, P<0.05) of boar sperm during freezing. (4) The apoptotic rate of the freezing group (80.41%) with TUNEL detection increased significantly compared to the fresh group (9.70%, P<0.05), and RSV treatment greatly decreased the apoptotic rate (68.32%, P<0.05). (5) Real-time polymerase chain reaction (RT-PCR) showed that not only the genes from the death receptor-mediated apoptotic pathway (tumor necrosis factor-α (TNF-α), Fas ligand (FasL), and Caspase-8), but also the genes from the mitochondria-mediated apoptotic pathway (manganese superoxide dismutase (MnSOD), B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), and Caspase-9) were both significantly changed after freezing. RSV treatment during freezing greatly changed their expression levels. Although RSV treatment during boar semen freezing did not significantly increase motility after thawing, it still played an efficient antioxidant role, which could enhance the mitochondrial function and decrease the apoptotic level induced by both the death receptor- and mitochondria-mediated apoptotic pathways.
概要
目的:阐明白藜芦醇(RSV)在猪精液冷冻保存中的应用效果及作用机制。
创新点在冷冻过程中添加RSV,观察其对猪冻后精子抗 冻能力的影响,并阐明其细胞凋亡作用机理。
方法:在猪精液的冷冻和解冻过程中添加1 mmol/L RSV,解冻后检测精子活力、线粒体膜电位、腺 苷三磷酸(ATP)含量、凋亡水平和凋亡通路中 相关基因的表达情况。
结论:(1)与鲜精相比,冷冻精液的活力、顶体完整 性和质膜完整性均显著降低,冷冻前后RSV 处理 未能显著提高精子活力(0.44 vs. 0.40,P>0.05), 但能显著提高顶体完整性(57.65% vs. 47.00%, P<0.05)和质膜完整性(46.67% vs. 38.85%, P<0.05)。(2)解冻后精子线粒体膜电位显著下 降,RSV 的添加能提高精子膜电位水平。 (3)冷冻解冻过程中添加RSV 能显著降低精子 的活性氧(ROS)水平(58.65% vs. 88.41%, P<0.05),增加精子的ATP 含量(0.49 μmol/L vs. 0.25 μmol/L,P<0.05)。(4)TUNEL 凋亡检测 后,冷冻精子的凋亡率(80.41%)与鲜精组 (9.70%)相比显著增加(P<0.05),RSV 处理 能显著降低冻精的凋亡比例(68.32%,P<0.05)。 (5)实时荧光定量聚合酶链反应(qRT-PCR) 的结果显示,猪精液冷冻后,无论是死亡受体介 导凋亡途径中的相关基因(肿瘤坏死因子α (TNF-α)、TNF 受体超族配体(FasL)和半胱 氨酸的天冬氨酸蛋白水解酶8(Caspase-8)), 还是线粒体介导凋亡途径中的相关基因(锰超氧 化物歧化酶(MnSOD)、B 淋巴细胞瘤-2(Bcl-2)、 Bcl-2 相关X 蛋白质(Bax)和半胱氨酸的天冬氨 酸蛋白水解酶9(Caspase-9)),均产生明显变 化,RSV 添加亦能显著改变其表达水平。综上所 述,猪精液冷冻解冻过程中RSV 添加虽未能显著 提高精子活力,但仍表现为抗氧化保护效果,体 现在改善了线粒体功能,并通过改变死亡受体和 线粒体介导的凋亡途径中相关基因的表达水平, 降低了冻后精子的细胞凋亡。
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Acknowledgments
We thank Dr. Da-wei YAO (College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China) for editing of the manuscript.
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Wei-hua HE, Xiu-jun DUAN, and He-shuang DI performed the experimental research and data analysis, wrote and edited the manuscript. Xiao-hu ZHAI participated in the study design, data analysis, and writing and editing of the manuscript. All authors have read and approved the final manuscript and, therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data.
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Wei-hua HE, Xiao-hu ZHAI, Xiu-jun DUAN, and Heshuang DI declare that they have no conflict of interest. All institutional and national guidelines for the care and use of laboratory animals were followed.
Project supported by the Jiangsu Agri-animal Husbandry Vocational College Academy Research Project (No. NSF201509) and the Jiangsu University Brand Speciality Construction Work Founded Project (No. PPZY2015C230), China
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He, Wh., Zhai, Xh., Duan, Xj. et al. Effect of resveratrol treatment on apoptosis and apoptotic pathways during boar semen freezing. J. Zhejiang Univ. Sci. B 21, 485–494 (2020). https://doi.org/10.1631/jzus.B1900520
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DOI: https://doi.org/10.1631/jzus.B1900520