GPx6在大白猪附睾细胞中的表达及其与繁殖性能的相关性

doi:10.11843/j.issn.0366-6964.2021.09.011

摘要/Abstract

摘要： 本试验旨在研究谷胱甘肽过氧化物酶6（glutathione peroxidase 6，GPx6）在大白公猪附睾细胞中的表达和定位，并探究其与公猪繁殖性能的相关性。选取15月龄的公猪和母猪各3头，取睾丸、附睾、前列腺、尿道球腺、精囊腺、卵巢和输卵管，提取蛋白，通过蛋白免疫印迹和免疫组化（IHC）检测组织和细胞中GPx6蛋白的表达和定位；根据评定大白公猪受精能力的数学模型，按照公猪配种胎次≥20胎、3次配种公猪为同一头的标准，筛选并采集符合要求的20头公猪精液，同时，统计相对应的1 279头母猪的生产成绩，计算得到公猪繁殖性能指标（包括窝产总仔数、窝产活仔数、分娩率和繁殖力）。提取精子蛋白和精清蛋白，通过BCA和ELISA检测精子和精清中GPx6蛋白的含量。使用SPSS软件的独立样本t检验及单因素方差分析（one-way ANOVA），进行差异显著性分析，用双变量Pearson进行相关性分析，P<0.05表示差异或相关显著。结果表明，GPx6蛋白在附睾中高表达，IHC结果显示，GPx6蛋白在附睾的顶细胞、基底细胞、晕细胞、主细胞和精子中表达，在肌样细胞中不表达；精清蛋白中GPx6的含量是精子蛋白的7倍，精液中GPx6蛋白的含量与窝产活仔数、窝产总仔数呈负相关关系。结果提示，GPx6在附睾的顶细胞、基底细胞、晕细胞、主细胞和精子中表达，且其在精液中的含量会影响公猪的繁殖性能，这为GPx6对公猪受精能力影响的研究奠定了理论和试验基础。

关键词: 公猪, 附睾, 繁殖性能, 谷胱甘肽过氧化物酶6

Abstract: This study aimed to investigate the location and expression of glutathione peroxidase 6(GPx6) in epididymal cells of Large White boars as well to explore their correlation with reproductive performance of boars. Three female and male Large White pigs at 15 months old were selected, respectively. Testis, epididymis, prostate, urethral bulb gland, seminal vesicle, ovary and fallopian tube were collected for protein extraction. Expression and localization of GPx6 protein in different tissues and cells were detected by Western blot and immunohistochemistry (IHC). Boar fertilization ability was evaluated based on a mathematical mode, according to the standard of breeding parity ≥ 20 births and 3 times of breeding semen from the same boar, 20 Large White boars were selected along with their semen collected. Further, productive performance data of the 1 279 boar-paired sows were collected for evaluating the reproductive performance of those 20 breeding boars including total number born per litter (TNB), number born alive per litter (NBA), farrowing rate (FR) and fecundity. Protein from semen and sperm samples were extracted, BCA and ELISA were carried on to detect GPx6 content in sperm and seminal plasma. SPSS software was used for data statistics, while independent sample t-Test and one-way ANOVA were carried out for significant difference analysis, bivariate Pearson was used for correlation analysis, and P<0.05 indicated significant difference or correlation. The results indicated that GPx6 protein was highly expressed in epididymis, and IHC results showed GPx6 was expressed in apical cells, basal cells, halo cells, principal cells and spermatozoa from epididymis whereas was not expressed in myoid cells. The concentration of GPx6 protein in seminal plasma was as 7 times higher than that in sperm, meanwhile, the concentration of GPx6 protein in semen was negatively correlated with NBA and TNB. This study suggests that GPx6 is expressed in apical cells, basal cells, halo cells, principal cells and spermatozoa of epididymis, and its content in semen affects reproductive performance of boars, which provides a theoretical and experimental foundation for the research of function of GPx6 protein in boar fertility.

Key words: boar, epididymis, reproductive performance, glutathione peroxidase 6

中图分类号:

S813.83

陈云, 王凯, 周乐乐, 卫恒习, 李莉, 张守全. GPx6在大白猪附睾细胞中的表达及其与繁殖性能的相关性[J]. 畜牧兽医学报, 2021, 52(9): 2484-2490.

CHEN Yun, WANG Kai, ZHOU Lele, WEI Hengxi, LI Li, ZHANG Shouquan. Expression of GPx6 in Epididymal Cells of Large White Boar and Its Correlation with Reproductive Performance[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(9): 2484-2490.

参考文献 30

[1]	甄林青, 王立蕊, 付杰丽, 等.家畜精液质量相关生物标记的研究进展[J].畜牧兽医学报, 2016, 47(4):645-651.ZHEN L Q, WANG L R, FU J L, et al.The research progress in semen quality related biomarker in livestock[J].Acta Veterinaria et Zootechnica Sinica, 2016, 47(4):645-651.(in Chinese)
[2]	ZHU Z D, LI R N, FAN X T, et al.Resveratrol improves boar sperm quality via 5'AMP-activated protein kinase activation during cryopreservation[J].Oxid Med Cell Longev, 2019, 2019:5921503.
[3]	吕松洁, 付丽, 范文华, 等.线粒体靶向抗氧化剂Mitoquinone对湖羊冻精损伤的保护作用[J].畜牧兽医学报, 2019, 50(12):2554-2559.LÜ S J, FU L, FAN W H, et al.Protective effect of mitochondria-targeted antioxidant Mitoquinone on the damage of Hu sheep frozen sperm[J].Acta Veterinaria et Zootechnica Sinica, 2019, 50(12):2554-2559.(in Chinese)
[4]	ZHAO F F, WHITING S, LAMBOURNE S, et al.Melatonin alleviates heat stress-induced oxidative stress and apoptosis in human spermatozoa[J].Free Radic Biol Med, 2021, 164:410-416.
[5]	FRIESEN C R, NOBLE D W A, OLSSON M.The role of oxidative stress in postcopulatory selection[J].Philos Trans R Soc B Biol Sci, 2020, 375(1813):20200065.
[6]	CHEN Y, WANG K, ZHANG D L, et al.GPx6 is involved in the in vitro induced capacitation and acrosome reaction in porcine sperm[J].Theriogenology, 2020, 156:107-115.
[7]	ARTHUR J R.The glutathione peroxidases[J].Cell Mol Life Sci, 2000, 57(13-14):1825-1835.
[8]	DEAR T N, CAMPBELL K, RABBITTS T H.Molecular cloning of putative odorant-binding and odorant-metabolizing proteins[J].Biochemistry, 1991, 30(43):10376-10382.
[9]	SHEN J Y, ZHOU C L, ZHU S E, et al.Comparative transcriptome analysis reveals early pregnancy-specific genes expressed in peripheral blood of pregnant sows[J].PLoS One, 2014, 9(12):e114036.
[10]	张守全, 陈志林, 冯美莹, 等.一种评定大白公猪受精能力的数学模型及其建立方法:中国, 201510431755.1[P].2015-11-11.ZHANG S Q, CHEN Z L, FENG M Y, et al.Mathematic model for evaluating fertility of the Large White, and establishment method thereof:CN, 201510431755.1[P].2015-11-11.(in Chinese)
[11]	BELA K, HORVÁTH E, GALLÉ A, et al.Plant glutathione peroxidases:emerging role of the antioxidant enzymes in plant development and stress responses[J].J Plant Physiol, 2015, 176:192-201.
[12]	MORTAZAVI S M J, MORTAZAVI S A R, PAKNAHAD M.Arabidopsis plants exposed to gamma radiation in two successive generations show a different oxidative stress response[J].J Environ Radioact, 2017, 171:253-254.
[13]	PASSAIA G, QUEVAL G, BAI J, et al.The effects of redox controls mediated by glutathione peroxidases on root architecture in Arabidopsis thaliana[J].J Exp Bot, 2014, 65(5):1403-1413.
[14]	RODRIGUEZ MILLA M A, MAURER A, HUERE A R, et al.Glutathione peroxidase genes in Arabidopsis are ubiquitous and regulated by abiotic stresses through diverse signaling pathways[J].Plant J, 2003, 36(5):602-615.
[15]	TAN L L, XUE X G, DU J, et al.Probing the molecular toxic mechanism of lead (II) ions with glutathione peroxidase 6 from Arabidopsis thaliana[J].Spectrochim Acta A Mol Biomol Spectrosc, 2020, 226:117597.
[16]	WANG K, TANG S F, HOU X M.Molecular mechanism investigation on the interactions of copper (II) ions with glutathione peroxidase 6 from Arabidopsis thaliana[J].Spectrochim Acta A Mol Biomol Spectrosc, 2018, 203:428-433.
[17]	WANG Z, TANG S F, HOU X M.Glutathione peroxidase 6 from Arabidopsis thaliana as potential biomarker for plants exposure assessment to di-(2-ethylhexyl) phthalate[J].Spectrochim Acta A Mol Biomol Spectrosc, 2020, 229:117955.
[18]	TADROS S F, D'SOUZA M, ZHU X X, et al.Gene expression changes for antioxidants pathways in the mouse cochlea:relations to age-related hearing deficits[J].PLoS One, 2014, 9(2):e90279.
[19]	XU X C, HE L Y, ZHANG A M, et al.Toxoplasma gondii isolate with genotype Chinese 1 triggers trophoblast apoptosis through oxidative stress and mitochondrial dysfunction in mice[J].Exp Parasitol, 2015, 154:51-61.
[20]	BRIGELIUS-FLOHÉ R, MAIORINO M.Glutathione peroxidases[J].Biochim Biophys Acta, 2013, 1830(5):3289-3303.
[21]	张建新, 王茜, 荀文娟, 等.GPxs家族基因在山羊不同组织和睾丸不同发育时期的表达特性研究[J].畜牧兽医学报, 2011, 42(5):650-657.ZHANG J X, WANG Q, XUN W J, et al.The expression of glutathione peroxidases family genes in different tissues and testes at different stages of male goats[J].Acta Veterinaria et Zootechnica Sinica, 2011, 42(5):650-657.(in Chinese)
[22]	VLAGRAN I, CASTILLO-MARTÍN M, PRIETO-MARTÍNEZ N, et al.Triosephosphate isomerase (TPI) and epididymal secretory glutathione peroxidase (GPX5) are markers for boar sperm quality[J].Anim Reprod Sci, 2016, 165:22-30.
[23]	TATENO H, KRAPF D, HINO T, et al.Ca2+ ionophore A23187 can make mouse spermatozoa capable of fertilizing in vitro without activation of cAMP-dependent phosphorylation pathways[J].Proc Natl Acad Sci U S A, 2013, 110(46):18543-18548.
[24]	MUDGAL R, SRINIVASAN N, CHANDRA N.Resolving protein structure-function-binding site relationships from a binding site similarity network perspective[J].Proteins, 2017, 85(7):1319-1335.
[25]	WALENSKY L D, ROSKAMS A J, LEFKOWITZ R J, et al.Odorant receptors and desensitization proteins colocalize in mammalian sperm[J].Mol Med, 1995, 1(2):130-141.
[26]	OTTAVIANO G, ZUCCARELLO D, MENEGAZZO M, et al.Human olfactory sensitivity for bourgeonal and male infertility:a preliminary investigation[J].Eur Arch Otorhinolaryngol, 2013, 270(12):3079-3086.
[27]	SOUTO P L, CARMOUY L S T, SANTOS C, et al.Seasonal differences in seminal plasma proteins from two bovine breeds adapted to a subtropical climate[J].Trop Anim Health Prod, 2021, 53(1):61.
[28]	DACHEUX J L, DACHEUX F.New insights into epididymal function in relation to sperm maturation[J].Reproduction, 2014, 147(2):R27-R42.
[29]	SAMANTA L, PARIDA R, DIAS T R, et al.The enigmatic seminal plasma:a proteomics insight from ejaculation to fertilization[J].Reprod Biol Endocrinol, 2018, 16(1):41.
[30]	栾兆进, 张家新.哺乳动物附睾特异GPx5功能及其调节机制的研究进展[J].中国畜牧兽医, 2018, 45(9):2500-2506.LUAN Z J, ZHANG J X.Research progress on function and regulation mechanism of specific GPx5 of mammalian epididymis[J].China Animal Husbandry & Veterinary Medicine, 2018, 45(9):2500-2506.(in Chinese)