Scientia Agricultura Sinica ›› 2022, Vol. 55 ›› Issue (23): 4743-4752.doi: 10.3864/j.issn.0578-1752.2022.23.014

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles     Next Articles

Proteomic Analysis of Sperm with Different Freezing Tolerance in Erhualian Boar

TONG ShiFeng1(),REN ZhiBin1,LIN Fei2,GE YuZhu1,TAO JingLi1,LIU Yang1,*()   

  1. 1Department of Animal Genetics and Breeding, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095
    2Jiangsu Changshu Animal Husbandry Industry and Commerce Co., Ltd., Changshu 215500, Jiangsu
  • Received:2021-09-18 Accepted:2021-12-28 Online:2022-12-01 Published:2022-12-06
  • Contact: Yang LIU E-mail:sftong9518@163.com;yangliu@njau.edu.cn

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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

Table 1

Program freezing program for 0.5 mL straws of program freezers (Ice Cube)"

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

Fig. 1

Semen quality evaluation A: Percent differences in the levels of pre-frozen motility (P-F MOT), frozen-thawed motility (F-T MOT). B: Pre-frozen progressive motility(P-F PMOT), frozen-thawed progressive motility (F-T PMOT). C: Plasma membrane integrity (PMI). D: Mitochondrial membrane potential (MMP). Data are presented as Mean ± SEM (n=3), *** represents P<0.001 and ** represents P<0.01"

Fig. 2

Sperm protein identification and analysis A: Total protein and peptide identification results; B: PCA analysis results of GFE and PFE (Orange dots represent GFE, blue dots represent PFE); C, D: Differentially abundant proteins identification results (C: Red bar represents up-regulated proteins, blue bar represents down-regulated proteins; D: Red dots represent up-regulated proteins, blue dots represent undifferentiated proteins, and orange dots represent down-regulated proteins)"

Fig. 3

Functional annotation results of DAPs A: Top 20 of GO enrichment (Y axis: GO term, X axis: protein ratio, color change represents Q value). B: Top 20 of KEGG enrichment (Y axis: pathway, X axis: rich factor, the size of the circle represents protein number, color change represents Q value)"

Fig. 4

Protein-protein interaction (PPI) network A: PPI network of string analysis of all DAPs (The blue circles represent down-regulated proteins, the red circles represent up-regulated proteins, the size of the circle represents degree, and the color of the line represents combine_score); B, C, D: Overlapping subnets in PPI network (The blue circles represent down-regulated proteins, the red circles represent up-regulated proteins)"

[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
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