二花脸公猪不同耐冻性精子的蛋白质组学分析

doi:10.3864/j.issn.0578-1752.2022.23.014

摘要/Abstract

摘要： 目的二花脸猪是我国优良的地方猪种之一，冷冻精液可提高二花脸公猪的利用率，并加强其种质资源保护，但目前生产的二花脸猪冷冻精液基本不能满足生产需要。研究通过分析二花脸公猪不同耐冻性精子的蛋白质组学，以促进二花脸公猪精子耐冻性蛋白质标志物的筛选，从遗传水平分析精子耐冻性的影响因素，为提高精子耐冻性提供参考依据。方法通过冻存14头二花脸种公猪的精液，再对冷冻精液的冻后质量进行分析，根据精子冻后活率和冻后活力的高低筛选出耐冻性好和差（good freezability ejaculates, GFE和poor freezability ejaculates, PFE）的公猪各3头，利用串联质谱标记(tandem mass tag, TMT)技术对GFE组和PFE组的精子蛋白进行定量蛋白质组学分析，鉴定出GFE组和PFE组精子中的差异蛋白(differentially abundant proteins, DAPs)。利用GO富集分析对差异蛋白的功能进行注释，利用 KEGG富集分析对差异蛋白的生物通路进行注释，通过STRING蛋白质互作数据库进行差异蛋白的互作网络分析并利用Cytoscape软件构建蛋白质-蛋白质相互作用(PPI)网络。结果研究发现GFE组和PFE组间共存在138个差异蛋白，PFE组与GFE组相比,有109个DAPs上调，29个DAPs下调。GO富集分析表明，共124个DAPs富集到47个GO term，主要富集到“细胞进程”“单有机体过程”“分子结合”“细胞”“细胞组分”等GO term。KEGG富集分析表明，DAPs主要富集到“肾素-血管紧张素系统”“补体和凝血级联”“血小板活化”等通路。从PPI网络可见53个节点和69 条连接边线，有8个具有强互作关系的蛋白ALB、ACRBP、ACR、ZAN、ZPBP2、HSPA5、FGB、FGG，这些蛋白质的功能主要富集于“单受精”“有性生殖”“精细胞发育”等与动物生殖生理以及精子发生密切相关的功能。进一步分析表明，GFE组和PFE组间的DAPs主要包括氧化还原相关蛋白（GSTM3、PRDX5、ALB、PDIA1、PDIA4等）、精子发生与功能相关蛋白（HSPA5、MFGE8、DNAH1、DNAH7、CUL3等）、能量相关蛋白（HK1、PGM1等）以及凋亡与炎症相关蛋白（THBS1、ELSPBP1、CP等）；PPI网络图分析可知3个DAPs（MME、ANPEP和PRCP）在肾素-血管紧张素系统（RAS）中相互作用。结论二花脸公猪不同耐冻性精子的蛋白质组分存在明显差异，可能对精子的冷冻性能产生影响，这些DAPs可作为精子耐冻性的候选蛋白标志物。

关键词: 二花脸公猪, 精子耐冻性, TMT, 差异蛋白

Abstract:

【Objective】 Erhualian pig is one of the excellent indigenous pig breeds in China. Frozen semen can improve the utilization rate of Erhualian boar and strengthen the protection of germplasm resources. However, the frozen semen of Erhualian pigs can not meet the production demand. This study analyzed the proteomics of Erhualian boar sperm with different freezing tolerance to promote the screening of protein markers of Erhualian boar sperm freezing tolerance, analyze the influencing factors of sperm freezing tolerance from the genetic level, and provide reference basis for improving sperm freezing tolerance. 【Method】 In this study, semen from14 Erhualian boars were frozen and their quality was analyzed. Boars with good and poor freezability ejaculates (GFE and PFE, n=3) were selected respectively, according to the motility and progressive motility of frozen-thawed spermatozoa. Using tandem mass tag (TMT) analyzed the sperm proteomics of GFE and PFE, and the differentially abundant proteins (DAPs) of spermatozoa of GFE and PFE were identified. The functions of DAPs were annotated by GO enrichment analysis, and the biological pathways of DAPs were annotated by KEGG enrichment analysis. The interaction network of DAPs was analyzed by STRING protein interaction database, and the protein-protein interaction (PPI) network was constructed by Cytoscape software..【Result】 The study found that there were 138 DAPs between GFE and PFE. Compared with GFE, 109 DAPs were up-regulated and 29 DAPs were down-regulated in PFE. GO enrichment analysis showed that a total of 124 DAPs were enriched to 47 GO terms, mainly enriched in “cellular process” “single-organism process” “binding” “cell” “cell part” and so on. KEGG enrichment analysis showed that DAPs were mainly enriched in “Renin-angiotensin system” “Complement and coagulation cascades” “Platelet activation” and so on. There were 53 nodes and 69 edges in the PPI network, among which 8 proteins showed high combine_score, including ALB, ACRBP, ACR, ZAN, ZPBP2, HSPA5, FGB and FGG. The functions of these proteins were mainly enriched in GO terms closely related to animal reproductive physiology and spermatogenesis, such as “single fertilization” “sexual reproduction” and “spermatid development”. Further analysis shows that DAPs in GFE and PFE mainly included redox related proteins (GSTM3, PRDX5, ALB, PDIA1, PDIA4), spermatogenesis and sperm function related proteins (HSPA5, MFGE8, DNAH1, DNAH7, CUL3), energy related proteins (HK1, PGM1), apoptosis and inflammation related proteins (THBS1, ELSPBP1, CP), and PPI network analysis shows that three DAPs (MME, ANPEP and PRCP) interact in renin-angiotensin system (RAS)..【Conclusion】 There are significant differences in sperm protein components of Erhualian boar with different freezing tolerance, which may affect the freezing performance of sperm. These DAPs can be used as candidate protein markers of sperm freezing tolerance.

Key words: Erhualian boar, sperm freezing tolerance, TMT, differentially abundant proteins

童世锋,任智彬,林斐,葛雨竹,陶景丽,刘杨. 二花脸公猪不同耐冻性精子的蛋白质组学分析[J]. 中国农业科学, 2022, 55(23): 4743-4752.

TONG ShiFeng,REN ZhiBin,LIN Fei,GE YuZhu,TAO JingLi,LIU Yang. Proteomic Analysis of Sperm with Different Freezing Tolerance in Erhualian Boar[J]. Scientia Agricultura Sinica, 2022, 55(23): 4743-4752.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2022.23.014

https://www.chinaagrisci.com/CN/Y2022/V55/I23/4743

图/表 5

表1

图1

图2

图3

图4

参考文献 34

[1]	刘鑫, 李振, 邓世阳, 顾岳清, 黄媛, 刘杨, 李东锋, 卢元鹏, 韦伟, 陈杰, 张立凡. 二花脸猪种质特性的分子基础研究进展. 畜牧与兽医, 2016, 48(12): 109-113.
	LIU X, LI Z, DENG S Y, GU Y Q, HUANG Y, LIU Y, LI D F, LU Y P, WEI W, CHEN J, ZHANG L F. Research Progress on molecular basis of Erhualian pig germplasm characteristics. Animal Husbandry & Veterinary Medicine, 2016, 48(12): 109-113. (in Chinese)
[2]	吴梦, 刘雪芹, 刘子嘉, 肖普英, 丁玉春, 刘作华, 葛良鹏. 猪精液超低温冷冻保存研究进展. 中国畜牧杂志, 2019, 55(7): 35-40. doi:10.19556/j.0258-7033.2019-07-035. doi: 10.19556/j.0258-7033.2019-07-035
	WU M, LIU X Q, LIU Z J, XIAO P Y, DING Y C, LIU Z H, GE L P. Research progress on cryopreservation of boar semen. Chinese Journal of Animal Science, 2019, 55(7): 35-40. doi:10.19556/j.0258-7033.2019-07-035. (in Chinese) doi: 10.19556/j.0258-7033.2019-07-035
[3]	YESTE M. Sperm cryopreservation update: Cryodamage, markers, and factors affecting the sperm freezability in pigs. Theriogenology, 2016, 85(1): 47-64. doi:10.1016/j.theriogenology.2015.09.047. doi: 10.1016/j.theriogenology.2015.09.047 pmid: 26506124
[4]	WATSON P F. Recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their post-thawing function. Reproduction, Fertility, and Development, 1995, 7(4): 871-891. doi:10.1071/rd9950871. doi: 10.1071/rd9950871 pmid: 8711221
[5]	MAŃKOWSKA A, BRYM P, PAUKSZTO Ł, JASTRZĘBSKI J P, FRASER L. Gene polymorphisms in boar spermatozoa and their associations with post-thaw semen quality. International Journal of Molecular Sciences, 2020, 21(5): 1902. doi:10.3390/ijms21051902. doi: 10.3390/ijms21051902
[6]	FRASER L, BRYM P, PAREEK C S, MOGIELNICKA-BRZOZOWSKA M, PAUKSZTO Ł, JASTRZĘBSKI J P, WASILEWSKA-SAKOWSKA K, MAŃKOWSKA A, SOBIECH P, ŻUKOWSKI K. Transcriptome analysis of boar spermatozoa with different freezability using RNA- Seq. Theriogenology, 2020, 142: 400-413. doi:10.1016/j.theriogenology.2019.11.001. doi: 10.1016/j.theriogenology.2019.11.001
[7]	PEDROSA A C, ANDRADE TORRES M, VILELA ALKMIN D, PINZON J E P, KITAMURA MARTINS S M M, COELHO DA SILVEIRA J, FURUGEN CESAR DE ANDRADE A. Spermatozoa and seminal plasma small extracellular vesicles miRNAs as biomarkers of boar semen cryotolerance. Theriogenology, 2021, 174: 60-72. doi:10.1016/j.theriogenology.2021.07.022. doi: 10.1016/j.theriogenology.2021.07.022 pmid: 34419697
[8]	CASAS I, SANCHO S, BALLESTER J, BRIZ M, PINART E, BUSSALLEU E, YESTE M, FÀBREGA A, RODRÍGUEZ-GIL J E, BONET S. The HSP90AA1 sperm content and the prediction of the boar ejaculate freezability. Theriogenology, 2010, 74(6): 940-950. doi:10.1016/j.theriogenology.2010.04.021. doi: 10.1016/j.theriogenology.2010.04.021 pmid: 20580074
[9]	PRIETO-MARTÍNEZ N, VILAGRAN I, MORATÓ R, RIVERA DEL ÁLAMO M M, RODRÍGUEZ-GIL J E, BONET S, YESTE M. Relationship of aquaporins 3 (AQP3), 7 (AQP7), and 11 (AQP11) with boar sperm resilience to withstand freeze-thawing procedures. Andrology, 2017, 5(6): 1153-1164. doi:10.1111/andr.12410. doi: 10.1111/andr.12410
[10]	LLAVANERA M, DELGADO-BERMÚDEZ A, FERNANDEZ- FUERTES B, RECUERO S, MATEO Y, BONET S, BARRANCO I, YESTE M. GSTM3, but not IZUMO1, is a cryotolerance marker of boar sperm. Journal of Animal Science and Biotechnology, 2019, 10: 61. doi:10.1186/s40104-019-0370-5. doi: 10.1186/s40104-019-0370-5 pmid: 31391940
[11]	GUIMARÃES D B, BARROS T B, VAN TILBURG M F, MARTINS J A M, MOURA A A, MORENO F B, MONTEIRO-MOREIRA A C, MOREIRA R A, TONIOLLI R. Sperm membrane proteins associated with the boar semen cryopreservation. Animal Reproduction Science, 2017, 183: 27-38. doi:10.1016/j.anireprosci.2017.06.005. doi: S0378-4320(16)30761-8 pmid: 28662881
[12]	王欣悦, 石田培, 赵志达, 胡文萍, 尚明玉, 张莉. 基于绵羊胚胎骨骼肌蛋白质组学的PI3K-AKT信号通路分析. 中国农业科学, 2020, 53(14): 2956-2963. doi:10.3864/j.issn.0578-1752.2020.14.018. doi: 10.3864/j.issn.0578-1752.2020.14.018
	WANG X Y, SHI T P, ZHAO Z D, HU W P, SHANG M Y, ZHANG L. The analysis of PI3K-AKT signal pathway based on the proteomic results of sheep embryonic skeletal muscle. Scientia Agricultura Sinica, 2020, 53(14): 2956-2963. doi:10.3864/j.issn.0578-1752.2020.14.018. (in Chinese) doi: 10.3864/j.issn.0578-1752.2020.14.018
[13]	刘锴栋, 莫亿伟, 冯少娴, 吴婉仪, 黎海利, 钟军弟, 袁长春. 番荔枝花发育不同阶段的差异蛋白质组分析. 中国农业科学, 2018, 51(1): 149-159. doi:10.3864/j.issn.0578-1752.2018.01.014. doi: 10.3864/j.issn.0578-1752.2018.01.014
	LIU K D, MO Y W, FENG S X, WU W Y, LI H L, ZHONG J D, YUAN C C. Comparative proteomic analysis in different developmental stages of sugar-apple (Annona squamosa L.) flowers. Scientia Agricultura Sinica, 2018, 51(1): 149-159. doi:10.3864/j.issn.0578-1752.2018.01.014. (in Chinese) doi: 10.3864/j.issn.0578-1752.2018.01.014
[14]	REGO J P A, MARTINS J M, WOLF C A, VAN TILBURG M, MORENO F, MONTEIRO-MOREIRA A C, MOREIRA R A, SANTOS D O, MOURA A A. Proteomic analysis of seminal plasma and sperm cells and their associations with semen freezability in Guzerat bulls. Journal of Animal Science, 2016, 94(12): 5308-5320. doi:10.2527/jas.2016-0811. doi: 10.2527/jas.2016-0811 pmid: 28046165
[15]	VAZQUEZ J M, MARTINEZ E A, MARTINEZ P, GARCIA-ARTIGA C, ROCA J. Hypoosmotic swelling of boar spermatozoa compared to other methods for analysing the sperm membrane. Theriogenology, 1997, 47(4): 913-922. doi:10.1016/S0093-691X(97)00046-0. doi: 10.1016/S0093-691X(97)00046-0 pmid: 16728040
[16]	SHAN X, YU T, YAN X, WU J L, FAN Y N, GUAN X Y, FANG F G, LIN Y H, ZHANG Y H, LI Y S, LIU Y. Proteomic analysis of healthy and atretic porcine follicular granulosa cells. Journal of Proteomics, 2021, 232: 104027. doi:10.1016/j.jprot.2020.104027. doi: 10.1016/j.jprot.2020.104027
[17]	MANDAL R, BADYAKAR D, CHAKRABARTY J. Role of membrane lipid fatty acids in sperm cryopreservation. Advances in Andrology, 2014, 2014: 190542. doi:10.1155/2014/190542. doi: 10.1155/2014/190542
[18]	LLAVANERA M, DELGADO-BERMÚDEZ A, OLIVES S, MATEO-OTERO Y, RECUERO S, BONET S, FERNÁNDEZ- FUERTES B, YESTE M, BARRANCO I. Glutathione S-transferases play a crucial role in mitochondrial function, plasma membrane stability and oxidative regulation of mammalian sperm. Antioxidants (Basel, Switzerland), 2020, 9(2): 100. doi:10.3390/antiox9020100. doi: 10.3390/antiox9020100
[19]	GOMES F P, PARK R, VIANA A G, FERNANDEZ-COSTA C, TOPPER E, KAYA A, MEMILI E, YATES J R, MOURA A A. Protein signatures of seminal plasma from bulls with contrasting frozen- thawed sperm viability. Scientific Reports, 2020, 10: 14661. doi:10.1038/s41598-020-71015-9. doi: 10.1038/s41598-020-71015-9
[20]	NAGDAS S K, BUCHANAN T, RAYCHOUDHURY S. Identification of peroxiredoxin-5 in bovine cauda epididymal sperm. Molecular and Cellular Biochemistry, 2014, 387(1/2): 113-121. doi:10.1007/s11010- 013-1876-3. doi: 10.1007/s11010- 013-1876-3
[21]	WANG P, WANG Y F, WANG H, WANG C W, ZAN L S, HU J H, LI Q W, JIA Y H, MA G J. HSP 90 expression correlation with the freezing resistance of bull sperm. Zygote (Cambridge, England), 2014, 22(2): 239-245. doi:10.1017/S096719941300004X. doi: 10.1017/S096719941300004X
[22]	VINCE S, ŽAJA I Ž, SAMARDŽIJA M, BALIĆ I M, VILIĆ M, ĐURIČIĆ D, VALPOTIĆ H, MARKOVIĆ F, MILINKOVIĆ-TUR S. Age-related differences of semen quality, seminal plasma, and spermatozoa antioxidative and oxidative stress variables in bulls during cold and warm periods of the year. Animal, 2018, 12(3): 559-568. doi:10.1017/S1751731117001811. doi: 10.1017/S1751731117001811 pmid: 28735578
[23]	SOARES MORETTI A I, MARTINS LAURINDO F R. Protein disulfide isomerases: Redox connections in and out of the endoplasmic Reticulum. Archives of Biochemistry and Biophysics, 2017, 617: 106-119. doi:10.1016/j.abb.2016.11.007. doi: 10.1016/j.abb.2016.11.007
[24]	YU J, LI M, JI C L, LI X X, LI H J, LIU G Q, WANG Y T, LIU G Y, WANG T, CHE X N, LEI C Z, DANG R H, ZHAO F W. Comparative proteomic analysis of seminal plasma proteins in relation to freezability of Dezhou donkey semen. Animal Reproduction Science, 2021, 231: 106794. doi:10.1016/j.anireprosci.2021.106794. doi: 10.1016/j.anireprosci.2021.106794
[25]	WANG W L, TU C F, TAN Y Q. Insight on multiple morphological abnormalities of sperm flagella in male infertility: What is new? Asian Journal of Andrology, 2020, 22(3): 236-245. doi:10.4103/aja.aja_53_19. doi: 10.4103/aja.aja_53_19
[26]	STIVAL C, DEL C PUGA MOLINA L, PAUDEL B, BUFFONE M G, VISCONTI P E, KRAPF D. Sperm capacitation and acrosome reaction in mammalian sperm. Advances in Anatomy, Embryology, and Cell Biology, 2016, 220: 93-106. doi:10.1007/978-3-319-30567-7_5. doi: 10.1007/978-3-319-30567-7_5
[27]	KHAN I M, CAO Z B, LIU H Y, KHAN A, RAHMAN S U, KHAN M Z, SATHANAWONGS A, ZHANG Y H. Impact of cryopreservation on spermatozoa freeze-thawed traits and relevance OMICS to assess sperm cryo-tolerance in farm animals. Frontiers in Veterinary Science, 2021, 8: 609180. doi:10.3389/fvets.2021.609180. doi: 10.3389/fvets.2021.609180
[28]	HUANG Z H, DANSHINA P V, MOHR K, QU W D, GOODSON S G, O’CONNELL T M, O’BRIEN D A. Sperm function, protein phosphorylation, and metabolism differ in mice lacking successive sperm-specific glycolytic enzymes. Biology of Reproduction, 2017, 97(4): 586-597. doi:10.1093/biolre/iox103. doi: 10.1093/biolre/iox103 pmid: 29025010
[29]	VILAGRAN I, CASTILLO J, BONET S, SANCHO S, YESTE M, ESTANYOL J M, OLIVA R. Acrosin-binding protein (ACRBP) and triosephosphate isomerase (TPI) are good markers to predict boar sperm freezing capacity. Theriogenology, 2013, 80(5): 443-450. doi:10.1016/j.theriogenology.2013.05.006. doi: 10.1016/j.theriogenology.2013.05.006 pmid: 23768753
[30]	ZHU W H, YANG M, SHANG J N, XU Y L, WANG Y L, TAO Q Q, ZHANG L, DING Y Y, CHEN Y G, ZHAO D D, WANG C L, CHU M X, YIN Z J, ZHANG X D. miR-222 inhibits apoptosis in porcine follicular granulosa cells by targeting the THBS₁ gene. Animal Science Journal, 2019, 90(6): 719-727. doi:10.1111/asj.13208. doi: 10.1111/asj.13208
[31]	D'AMOURS O, FRENETTE G, BORDELEAU L J, ALLARD N, LECLERC P, BLONDIN P, SULLIVAN R. Epididymosomes transfer epididymal sperm binding protein 1 (ELSPBP₁) to dead spermatozoa during epididymal transit in bovine. Biology of Reproduction, 2012, 87(4): 94, 1-11. doi:10.1095/biolreprod.112.100990. doi: 10.1095/biolreprod.112.100990
[32]	HELLMAN N E, GITLIN J D. Ceruloplasmin metabolism and function. Annual Review of Nutrition, 2002, 22: 439-458. doi:10.1146/annurev.nutr.22.012502.114457. doi: 10.1146/annurev.nutr.22.012502.114457 pmid: 12055353
[33]	LEUNG P S, SERNIA C. The renin-angiotensin system and male reproduction: new functions for old hormones. Journal of Molecular Endocrinology, 2003, 30(3): 263-270. doi:10.1677/jme.0.0300263. doi: 10.1677/jme.0.0300263 pmid: 12790798
[34]	GIANZO M, SUBIRÁN N. Regulation of male fertility by the renin-angiotensin system. International Journal of Molecular Sciences, 2020, 21(21): 7943. doi:10.3390/ijms21217943. doi: 10.3390/ijms21217943

步骤 Procedure	温度 Temperature (℃)	时间 Time (min)	速率 Rate (℃/min)
0	6	0	0
1	4	2	1
2	1	2	1.5
3	-140	4.7	30
4	-140	10	0