Expression and Distribution of Nucleic Acid-sensing TLRs in Porcine Sperm
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摘要:
目的 Toll样受体（Toll like receptors, TLRs）作为天然免疫系统中重要的模式识别受体，在抵御病原微生物感染方面发挥着重要作用。研究猪精子核酸识别TLR的表达和分布情况，为精子TLR信号通路相关功能和抗微生物特性的研究奠定基础。 方法 采用RT-PCR技术检测公猪精子TLR3、7、8和9 mRNA的表达情况，利用免疫荧光方法检测了TLR3蛋白在猪精子上的分布。 结果 RT-PCR结果显示，TLR3、7、8和9 mRNA在猪精子均有表达；免疫荧光试验结果显示TLR3主要分布在精子头部的后部和中段。 结论 猪精子上核酸识别TLRs的存在以及TLR3分布的区域化提示猪精子可能对病毒及其衍生物感染做出应答，为进一步研究精子TLR信号通路提供依据。 Abstract:Objective Expression and distribution of the toll-like receptor (TLR) in porcine sperm were analyzed to facilitate the study on the related signaling pathway and antimicrobial properties. Method The mRNA expressions of TLR3, 7, 8, and 9 in pig sperm were detected by RT-PCR, and the distribution of TLR3 by immunofluorescence. Result The mRNA of TLR3, 7, 8, and 9 were all expressed in the sperm, and TLR3 mainly found in the posterior head and midpiece. Conclusion The expression of nucleic acid-sensing TLRs and the localizations of TLR3 in the porcine sperm revealed in this study gave clues of how the sperm might respond to attacks by virus and derivatives. The information paved the way for further investigation on the sperm TLR signaling pathway and pathogen recognition function. -
Key words:
- Nucleic acid-sensing TLRs /
- porcine sperm /
- mRNA expression /
- immunofluorescence
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图 1 猪精子TLRs mRNA的表达
注：1：脾脏；2：肺脏；3：精子；M：Marker
Figure 1. mRNA expression of TLRs in porcine sperm
Note: 1: Spleen; 2: Lung; 3: Sperm; M: Marker
图 2 免疫荧光染色显示TLR3在猪精子的分布情况
注：A：特异性免疫荧光染色后，TLR3阳性表达处呈绿色荧光；B：荧光与明场叠加图；C：阴性对照；D：C与明场叠加图
Figure 2. Immunofluorescence of TLR3 in porcine sperm
Note: A: TLR3 stained by green fluorescence; B: image of green fluorescence on bright background; C: negative controls for TLR3; D: image of C on bright background.
图 3 TLR3在猪精子的分布
注：A、B：免疫荧光显示TLR3在猪精子头部的后部和中段表达；C：精子结构示意图；1：顶体区；2：赤道区；3：核后帽区；4：中片；5尾部
Figure 3. Localization of TLR3 in porcine sperm
Note: A-B: TLR3 expressed in midpiece and posterior head of sperm; C: schematic diagram of sperm structure; 1: acrosome; 2: equatorial segment; 3: post nuclear cap; 4: midpiece; 5: tail
图 4 猪精子不同位置TLR3荧光强度
注：1：猪精子头部的后部；2：精子中段
Figure 4. Fluorescence intensity of TLR3 at locations of porcine sperm
Note: 1: posterior head of sperm; 2: midpiece of sperm.
表 1 RT-PCR引物序列
Table 1. RT-PCR primers and conditions
基因名称
Gene基因序列号
Genbank Accession引物序列（5′→3′）
Primer Sequences(5′→3′)扩增长度
Size/bp溶解温度
Annealing/℃Pig TLR3 NM_001097444.1 F: GGCTCTGGAAACACGCAAACC
R: GAGACCATGATATTGAGGTGGAGTA428 62 Pig TLR7 NM_001097434.1 F: CACCAGAAGGCAGCAGATTAA
R: ACTTGCGGTTGACTGAGGTTAT423 58 Pig TLR8 NM_214187.1 F: GGCAAAAGAATGGAGATAGACCC
R: CAGCTCACTGAAAGCAGCGTC347 60 Pig TLR9 NM_213958.1 F: ACTGGAGCAGCCTGACCCT
R: AGCAGCGAGATGCCAAAAC466 62 Pig GAPDH NM_001206359.1 F: CATCACTGCCACCCAGAAGA
R: ATTGTCGTACCAGGAAATGAGC409 60 
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