Toll样受体7/8在公猪生殖系统中的表达及其配体对猪X/Y精子分选效果评价

doi:10.11843/j.issn.0366-6964.2023.04.013

Abstract

Abstract: This study aimed to analyze the expression of Toll-like receptor 7/8 (TLR7/8) in boar sperm and reproductive organs, and to explore whether boar X sperm and Y sperm could be separated by their ligand treatment. In this study, the mRNA expression levels of TLR7 and TLR8 genes in testis, caupt, corpus, caudu and sperm of 3 healthy adult boars were analyzed by qRT-PCR. The protein expression pattern of TLR7 and TLR8 in testis and epididymis of boar was investigated by immunohistochemistry. Their expression in sperm of different species (healthy adult mice, bulls and boars) was analyzed by cellular immunofluorescence. Finally, the effects of TLR7 and TLR8 on sperm motility and X/Y sperm separation were investigated by co-incubating their ligand R848 with boar sperm. The results showed that TLR7/8 mRNAs were expressed in boar testis, caupt, corpus, caudu and sperm. TLR7/8 proteins were mainly expressed in germ cells in testis and in columnar cell microvilli in epididymis. TLR7/8 proteins were only expressed in the tail of mouse and bull X sperm, but not in Y sperm. However, TLR7/8 proteins were expressed in both boar X and Y sperms with no significant difference in expression pattern. And TLR7 protein was mainly located in the acrosome of boar sperm head, while TLR8 protein was mainly located in the tail of boar sperm. Compared with the control group, the sperm motility of the treated group with TLR7/8 ligand R848 was reduced, but the sex ratio of the upper sperms was not significantly different (P>0.05). In conclusion, there are no significant difference in the expression of TLR7/8 between boar X sperm and Y sperm, but their expression patterns are different from those of mouse and bull sperm, so TLR7/8 may not be suitable for the separation of boar X sperm and Y sperm.

Key words: TLR7/8, R848, boar, sex control

CLC Number:

S828.3

WU Changhua, CHEN Weidong, YANG Wenzheng, MO Jianxin, HONG Linjun, ZHANG Xianwei, HUANG Sixiu. Expression of Toll-like Receptors 7/8 in Boar Reproductive System and Evaluation of Effect of their Ligands on Boar X/Y Sperm Sorting[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(4): 1490-1499.

References 36

[1]	OAKBERG E F.Duration of spermatogenesis in the mouse and timing of stages of the cycle of the seminiferous epithelium[J].Am J Anat, 1956, 99(3):507-516.
[2]	LEBLOND C P, CLERMONT Y.Definition of the stages of the cycle of the seminiferous epithelium in the rat[J].Ann NY Acad Sci, 1952, 55(4):548-573.
[3]	CUI K H, MATTHEWS C D.X larger than Y[J].Nature, 1993, 366(6451):117-118.
[4]	JOHNSON L A, WELCH G R.Sex preselection:high-speed flow cytometric sorting of X and Y sperm for maximum efficiency[J]. Theriogenology, 1999, 52(8):1323-1341.
[5]	ERICSSON R J, LANGEVIN C N, NISHINO M.Isolation of fractions rich in human Y sperm[J].Nature, 1973, 246(5433):421-424.
[6]	BORO P, NAHA B C, MADKAR A, et al.Sexing of semen in bulls:a mini review[J].Int J Appl Res, 2016, 2(4):460-462.
[7]	丁凤玲, 上官爱哨, 周扬, 等.荷斯坦公牛X和Y精子核形态差异及其影响因素分析[J].畜牧兽医学报, 2022, 53(3):766-777.DING F L, SHANGGUAN A S, ZHOU Y, et al.Morphological differences of X and Y sperm nuclei and their influencing factors in Holstein bulls[J].Acta Veterinaria et Zootechnica Sinica, 2022, 53(3):766-777.(in Chinese)
[8]	SOH Y Q S, ALFÖLDI J, PYNTIKOVA T, et al.Sequencing the mouse Y chromosome reveals convergent gene acquisition and amplification on both sex chromosomes[J].Cell, 2014, 159(4):800-813.
[9]	CHEN X L, ZHU H B, WU C J, et al.Identification of differentially expressed proteins between bull X and Y spermatozoa[J].J Proteomics, 2012, 77:59-67.
[10]	SHEN D, ZHOU C H, CAO M Y, et al.Differential membrane protein profile in bovine X- and Y-sperm[J].J Proteome Res, 2021, 20(6):3031-3042.
[11]	SCOTT C, DE SOUZA F F, ARISTIZABAL V H V, et al.Proteomic profile of sex-sorted bull sperm evaluated by SWATH-MS analysis[J].Anim Reprod Sci, 2018, 198:121-128.
[12]	LAXMIVANDANA R, PATOLE C, SHARMA T R, et al.Differential proteins associated with plasma membrane in X- and/or Y-chromosome bearing spermatozoa in indicus cattle[J].Reprod Domest Anim, 2021, 56(6):928-935.
[13]	CHOWDHURY M M R, XU L G, KONG R, et al.In vitro production of sex preselected cattle embryos using a monoclonal antibody raised against bull sperm epitopes[J].Anim Reprod Sci, 2019, 205:156-164.
[14]	刘志鹏.TLR7/8激动剂通过调节MDSCs向M1型巨噬细胞分化增强奥沙利铂在结直肠癌中的化疗疗效[D].广州:南方医科大学, 2020.LIU Z P.TLR 7/8 agonist enhances the efficacy of oxaliplatin in colorectal cancer via directing the myeloid-derived suppressor cells to tumoricidal M1-macrophages[D].Guangzhou:Southern Medical University, 2020.(in Chinese)
[15]	UMEHARA T, TSUJITA N, SHIMADA M.Activation of Toll-like receptor 7/8 encoded by the X chromosome alters sperm motility and provides a novel simple technology for sexing sperm[J].PLoS Biol, 2019, 17(8):e3000398.
[16]	UMEHARA T, TSUJITA N, ZHU Z D, et al.A simple sperm-sexing method that activates TLR7/8 on X sperm for the efficient production of sexed mouse or cattle embryos[J].Nat Protoc, 2020, 15(8):2645-2667.
[17]	REN F, XI H M, REN Y J, et al.TLR7/8 signalling affects X-sperm motility via the GSK3α/β-hexokinase pathway for the efficient production of sexed dairy goat embryos[J].J Anim Sci Biotechnol, 2021, 12(1):89.
[18]	刘伟东.基于X精子TLR7/8特异性蛋白激活的牛精子分选技术[D].杨凌:西北农林科技大学, 2022.LIU W D.Bovine sperm sorting based on X sperm TLR7/8 specific protein activation[D].Yangling:Northwest A&F University, 2022.(in Chinese)
[19]	张倩, 崔燕, 余四九, 等.免疫细胞在牦牛附睾和输精管的分布规律[J].畜牧兽医学报, 2022, 53(6):1925-1933.ZHANG Q, CUI Y, YU S J, et al.Distribution of immune cells in the epididymis and vas deferens of Yaks(Bos grunniens)[J].Acta Veterinaria et Zootechnica Sinica, 2022, 53(6):1925-1933.(in Chinese)
[20]	STEGER K.Transcriptional and translational regulation of gene expression in haploid spermatids[J].Anat Embryol, 1999, 199(6):471-487.
[21]	陈才.精子mRNA高通量测序及检测结果评估[D].扬州:扬州大学, 2014.CHEN C.Sperm mRNA high-throughput sequencing and detection result evaluation[D].Yangzhou:Yangzhou University, 2014.(in Chinese)
[22]	SENDLER E, JOHNSON G D, MAO S H, et al.Stability, delivery and functions of human sperm RNAs at fertilization[J].Nucl Acids Res, 2013, 41(7):4104-4117.
[23]	石子旸, 张安迪, 马嘉忆, 等.哺乳动物精子RNAs的研究进展[J].国际生殖健康/计划生育杂志, 2018, 37(6):486-491.SHI Z Y, ZHANG A D, MA J Y, et al.Research progress in mammalian sperm RNAs[J].Journal of International Reproductive Health/Family Planning, 2018, 37(6):486-491.(in Chinese)
[24]	MICHAELIS K A, NORGARD M A, ZHU X X, et al.The TLR7/8 agonist R848 remodels tumor and host responses to promote survival in pancreatic cancer[J].Nat Commun, 2019, 10(1):4682.
[25]	PETES C, ODOARDI N, GEE K.The toll for trafficking:toll-like receptor 7 delivery to the endosome[J].Front Immunol, 2017, 8:1075.
[26]	WANG M, YANG Y L, CANSEVER D, et al.Two populations of self-maintaining monocyte-independent macrophages exist in adult epididymis and testis[J].Proc Natl Acad Sci U S A, 2021, 118(1):e2013686117.
[27]	曹随忠, 李计尚, 张于, 等.TLRs基因在牦牛雄性生殖器官中表达情况的检测[J].中国兽医科学, 2014, 44(2):193-198.CAO S Z, LI J S, ZHANG Y, et al.Expression profile of TLRs gene in the reproductive organs of male Bos grunniens[J].Chinese Veterinary Science, 2014, 44(2):193-198.(in Chinese)
[28]	任玲玲.TLR3和TLR7/8对小鼠妊娠子宫免疫功能的影响机制研究[D].广州:暨南大学, 2011.REN L L.Effect of TLR3 and TLR7/8 on immune function in pregnant uterus of mice[D].Guangzhou:Jinan University, 2011.(in Chinese)
[29]	JASIN M, ZALAMEA P.Analysis of Escherichia coli beta-galactosidase expression in transgenic mice by flow cytometry of sperm[J]. Proc Natl Acad Sci U S A, 1992, 89(22):10681-10685.
[30]	BRAUN R E, BEHRINGER R R, PESCHON J J, et al.Genetically haploid spermatids are phenotypically diploid[J].Nature, 1989, 337(6205):373-376.
[31]	HENDRIKSEN P J M.Do X and Y spermatozoa differ in proteins?[J].Theriogenology, 1999, 52(8):1295-1307.
[32]	DU PLESSIS S S, AGARWAL A, MOHANTY G, et al.Oxidative phosphorylation versus glycolysis:what fuel do spermatozoa use?[J].Asian J Androl, 2015, 17(2):230-235.
[33]	HU X Y, CHEN J, WANG L, et al.Crosstalk among Jak-STAT, Toll-like receptor, and ITAM-dependent pathways in macrophage activation[J].J Leukoc Biol, 2007, 82(2):237-243.
[34]	EVERTS B, AMIEL E, HUANG S C C, et al.TLR-driven early glycolytic reprogramming via the kinases TBK1-IKKε supports the anabolic demands of dendritic cell activation[J].Nat Immunol, 2014, 15(4):323-332.
[35]	YE J, MILLS B N, QIN S S, et al.Toll-like receptor 7/8 agonist R848 alters the immune tumor microenvironment and enhances SBRT-induced antitumor efficacy in murine models of pancreatic cancer[J].J Immunother Cancer, 2022, 10(7):e004784.
[36]	李志强.猪性控精子的蛋白质组学分析[D].保定:河北农业大学, 2021.LI Z Q.Proteomics analysis of pig sex-sorted sperm[D].Baoding:Hebei Agricultural University, 2021.(in Chinese)

Expression of Toll-like Receptors 7/8 in Boar Reproductive System and Evaluation of Effect of their Ligands on Boar X/Y Sperm Sorting

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References 36

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	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.
[2]	SHANG Xiuguo, YU Weiwei, ZHAO Yunxiang, ZHU Xiaoping, CAO Tingting, GAO Guangxiong. Estimation of Genetic Parameters and Analysis of Season Effects and Age Effects for Semen Traits in Landrace Boars [J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(10): 2425-2432.
[3]	LI Yu-hua, FU Jie-li, LI Pei-fei, YANG Qiang-zhen, WANG Li-rui, XIE Wei-yi, LI Xin-hong. Effects of Staphylococcus aureus on Motility and Protein Phosphorylation Modification of Boar Sperm during Storage in vitro [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(9): 1665-1673.
[4]	ZHA An-dong, XING Gui-ling, GAO Xiao-wen, ZHANG Long, DI Sheng-wei, WANG Xi-biao, NIU Bu-yue. Association Analysis between IL6 Gene Intron 3 Polymorphism and Piglet Diarrhea, Growth and Boar Reproduction Traits [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(9): 1674-1682.
[5]	DING Hai-sheng，XU De-quan，LUO Yan，LIU Min. Histological Comparison and mRNA Expression Analysis of Porcine Seminal Protein on Accessory Glands of Duroc and Meishan Boars [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(8): 1714-1719.
[6]	SUI Fen-fen，HAN Xing-fa，CAO Xiao-han，DU Xiao-gang，MENG Feng-yan，CHU Ming-xing，ZENG Xian-yin. Effects of Active Immunization against GnRH on Skatole Metabolism in Male Pigs [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(6): 1140-1146.
[7]	REN Zi-li，ZHAO Yan-ling，WANG Jian-zhou，LI Yu-xin，SHANG Peng，CHAMBA Yangzom. Effect of Vitamin C on Tibetan Boar Semen Preserved at 4 ℃ and Levels of the Sperm Global Genome DNA Methylation [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(5): 1057-1061.
[8]	ZHANG Zhen，FAN Xiao-rui，XI Hua-ming，LIANG Ya-jun，HE Jun-ping. Effect of Heat Stress on the Expression of TGF-β3 and Claudin-11 Protein in Mature Boar Testis [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(10): 2136-2142.
[9]	WANG Xin，DAI Jian-jun，WU Cai-feng,ZHANG Shu-shan，WU Yun-long，ZHANG De-fu. Preliminary Studies on Apoptosis and Apoptotic Pathways of Frozen Boar Spermatozoa [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2015, 46(8): 1341-1347.
[10]	LIU Yang，HUANG He-lu，DONG Hai-tao，JIN Yi. Effect of LDL from Pigeon Egg Yolk on the Expression of HSP70 and Apoptosis-related Gene in Cold Shock and Frozen-thawed Boar Spermatozoa [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2015, 46(6): 940-948.
[11]	ZHAO Ke-wei，ZHAO Xue-yan，ZHANG Zhi-yan，HONG Yuan，FAN Yin，REN Jun，XING Yu-yun. Fine Mapping of QTL and Identification of MEA1 as a Positional Candidate Gene Affecting Boar Reproduction Traits on SSC7 [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(3): 363-374.
[12]	ZHAO Na, ZHEN Lin-qing, HU Qi-meng, WANG Liang-liang, LI Xin-hong. Cryopreservation Procedure Induces the Alterations of “Apoptosis-Like” of Boar Sperm [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2013, 44(11): 1766-1774.
[13]	LI Zhong-qiu, LIU Chun-long, MA Hong, FU Bo, WANG Liang, PENG Fu-gang, MA Jian-zhang, LIU Di. Influence of Cold Stress on Transcription of HSP90 in Northeast Wild Boar Fibroblasts [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2012, 43(12): 1978-1983.
[14]	LIU Li-hua;MIN Ling-jiang;SHEN Wei;PAN Qing-jie. Analysis of Genetic Structure in Wild Boar and Their Crossbred Populations [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2009, 40(2): 173-179.
[15]	YANG Xiuqin;GUAN Qingzhi;YU Hao;GUO Lijuan;LIU Hui;LIU Di;. Cloning, Sequence Analysis and Expression of Two Transcript Variants of Wild Boar CAPN10 Gene [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2009, 40(11): 0-1614.