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

畜牧兽医学报 ›› 2023, Vol. 54 ›› Issue (4): 1490-1499.doi: 10.11843/j.issn.0366-6964.2023.04.013

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

吴昌华¹, 陈伟东¹, 杨文政¹, 莫健新², 洪林君¹, 张献伟^2*, 黄思秀^1*

1. 华南农业大学动物科学学院/国家生猪种业工程技术研究中心, 广州 510642;
2. 岭南现代农业科学与技术广东省实验室云浮分中心, 云浮 527400

收稿日期:2022-09-22 出版日期:2023-04-23 发布日期:2023-04-27
通讯作者: 张献伟,主要从事动物遗传育种与繁殖研究,E-mail:zxianw@163.com;黄思秀,主要从事动物遗传育种与繁殖研究,E-mail:sxhuang815@scau.edu.cn
作者简介:吴昌华(1996-),男,苗族,湖南凤凰人,硕士,主要从事动物遗传育种与繁殖研究,E-mail:1697311946@qq.com
基金资助:
广东省重点领域研发计划项目（2021B0707010007）；广东省乡村振兴战略专项“广东省畜禽地方品种保护与开发利用提升工程”（2018143）

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

WU Changhua¹, CHEN Weidong¹, YANG Wenzheng¹, MO Jianxin², HONG Linjun¹, ZHANG Xianwei^2*, HUANG Sixiu^1*

1. National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou 510642, China;
2. Yunfu Branch, Guangdong Laboratory for Lingnan Modern Agricultural Science and Technology, Yunfu 527400, China

Received:2022-09-22 Online:2023-04-23 Published:2023-04-27

摘要/Abstract

摘要： 旨在分析Toll样受体7/8（TLR7/8）在公猪精子及生殖器官中的表达情况，并探讨是否能通过其配体处理的方式分离猪X精子和Y精子。本研究采用qRT-PCR分析3头健康成年公猪睾丸、附睾头、附睾体、附睾尾以及精子中TLR7和TLR8基因的mRNA表达水平，利用免疫组化检测公猪睾丸和附睾中TLR7和TLR8蛋白的表达，并通过细胞免疫荧光分析其在不同物种（健康成年小鼠、公牛和公猪）精子中的表达情况，将其配体R848与猪精子共孵育，研究其对精子活力以及X/Y精子分离的影响。结果表明，TLR7和TLR8 mRNA在公猪睾丸、附睾头、附睾体、附睾尾组织以及精子中均表达；免疫组化结果显示，TLR7/8蛋白在睾丸中主要表达于生殖细胞，在附睾中主要表达于柱状细胞微绒毛中；细胞免疫荧光结果表明，TLR7和TLR8蛋白只表达于小鼠和牛X精子尾部，Y精子中不表达，但TLR7和TLR8蛋白在公猪X和Y精子中都表达且表达模式无显著差异，TLR7蛋白主要表达于猪精子头部顶体区域，TLR8蛋白主要表达于猪精子尾部；与对照组相比，用TLR7和TLR8配体R848孵育猪精子后，精子活力降低，但上层精子的性别比例无显著差异（P>0.05）。以上结果表明，TLR7和TLR8在猪X精子和Y精子间表达无显著差异，但其表达模式与小鼠和牛存在差异，因此TLR7/8可能不适用于猪X精子和Y精子的分离。

关键词: TLR7/8, R848, 公猪, 性别控制

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

中图分类号:

S828.3

吴昌华, 陈伟东, 杨文政, 莫健新, 洪林君, 张献伟, 黄思秀. 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[J]. 畜牧兽医学报, 2023, 54(4): 1490-1499.

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.

参考文献 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)

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

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

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