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Journal of Zhejiang University SCIENCE B 2020 Vol.21 No.6 P.485-494

10.1631/jzus.B1900520


Effect of resveratrol treatment on apoptosis and apoptotic pathways during boar semen freezing


Author(s):  Wei-Hua He, Xiao-Hu Zhai, Xiu-Jun Duan, He-Shuang Di

Affiliation(s):  Jiangsu Agri-animal Husbandry Vocational College, Taizhou 225300, China

Corresponding email(s):   zhaixiaohu010@163.com

Key Words:  Resveratrol (RSV), Boar semen freezing, Antioxidant, Mitochondrial function, Apoptotic pathway


Wei-Hua He, Xiao-Hu Zhai, Xiu-Jun Duan, He-Shuang Di. Effect of resveratrol treatment on apoptosis and apoptotic pathways during boar semen freezing[J]. Journal of Zhejiang University Science B, 2020, 21(6): 485-494.

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author="Wei-Hua He, Xiao-Hu Zhai, Xiu-Jun Duan, He-Shuang Di",
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volume="21",
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pages="485-494",
year="2020",
publisher="Zhejiang University Press & Springer",
doi="10.1631/jzus.B1900520"
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%T Effect of resveratrol treatment on apoptosis and apoptotic pathways during boar semen freezing
%A Wei-Hua He
%A Xiao-Hu Zhai
%A Xiu-Jun Duan
%A He-Shuang Di
%J Journal of Zhejiang University SCIENCE B
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%@ 1673-1581
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%I Zhejiang University Press & Springer
%DOI 10.1631/jzus.B1900520

TY - JOUR
T1 - Effect of resveratrol treatment on apoptosis and apoptotic pathways during boar semen freezing
A1 - Wei-Hua He
A1 - Xiao-Hu Zhai
A1 - Xiu-Jun Duan
A1 - He-Shuang Di
J0 - Journal of Zhejiang University Science B
VL - 21
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SP - 485
EP - 494
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Y1 - 2020
PB - Zhejiang University Press & Springer
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DOI - 10.1631/jzus.B1900520


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.

猪精液冷冻过程中白藜芦醇处理对其凋亡和凋亡途径的影响

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.

关键词:白藜芦醇(RSV);猪精液冷冻;抗氧化剂;线粒体功能;凋亡途径

Darkslateblue:Affiliate; Royal Blue:Author; Turquoise:Article

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