Toll样受体信号影响精子运动的机制

发布时间：2018-02-02 02:27

本文关键词： TLR信号精子运动线粒体功能信号通路　出处：《扬州大学》2014年硕士论文　论文类型：学位论文

【摘要】：生殖道感染是引起男性不育及女性不孕的最重要原因之一,而感染能启动固有免疫应答。Toll样受体(TLR)作为固有免疫系统的重要分子,在固有免疫系统抵抗外界干扰方面发挥着重要作用。近年的研究发现TLR表达于作为生殖细胞的精子中,而精子运动被认为是成功受孕的关键。为了探索TLR信号对精子生物学功能的影响,我们首先观察了不同TLR激动剂对精子运动的影响,发现TLR激动剂能够显著抑制精子运动。本课题拟深入研究TLR信号影响精子运动的具体信号通路,并在此基础上进一步探究TLR信号通过影响精子的何种生物学过程最终导致了精子运动减慢。研究内容分为3个部分： 1.不同种类的TLR激动剂对精子生物学性能的影响目的：观察不同病原组分的TLR激动剂对小鼠精子运动的影响,并初步探讨TLR激动剂抑制精子运动的可能原因。方法：采用逆转录PCR的方法分析不同TLR在精子中的表达。利用计算机辅助精子分析(CASA)的方法观察不同TLR激动剂与小鼠精子孵育6h后的精子运动情况以及TLR激动剂处理不同时间对正常人精子运动的影响。利用流式细胞术的方法观察TLR激动剂处理6h后的小鼠精子凋亡情况。结果：除了TLR3表达量极低之外,其它的TLR在精子中均有表达。在TLR激动剂处理小鼠精子6h后,除了TLR3所对应的激动剂poly(I：C)对精子运动没有影响,其他TLR激动剂均能不同程度地抑制精子运动。另外,TLR激动剂抑制精子运动具有时间依赖性,Zymosan与LPS均只能在处理6h后才有效抑制精子运动,而R848在作用精子2h后即有抑制效应。与对照组相比,TLR激动剂在6h之内不能有效地促进精子凋亡的发生。结论：TLR激动剂能够有效抑制精子运动并且该效应并不由TLR激动剂促进精子凋亡引起的。 2.TLR信号影响精子运动的通路研究目的：探索TLR信号影响精子运动的确切信号通路方法：利用MyD88敲除小鼠研究TLR激动剂对该种小鼠精子运动的影响。使用LPS处理小鼠精子2h,4h,6h,提取蛋白,通过Western-blotting观察小鼠精子IRAK, PI3K, GSK3α, AKT, GSK3β, ERK, p65等分子磷酸化水平的变化,同时使用IRAK, PI3K,MEK/ERK, NF-κB等分子的抑制剂处理小鼠精子半小时后再给予TLR激动剂刺激,CASA观察信号通路被抑制精子运动的改变。使用PI3K抑制剂处理小鼠精子,Western-Blotting观察AKT及GSK3a磷酸化水平的变化。结果：TLR激动剂对MyD88敲除小鼠的精子运动无影响。TLR激动剂处理小鼠精子后可观察到IRAK, PI3K, GSK3a等分子磷酸化水平随处理时间的延长而上升,而AKT,GSK3β, ERK, p65等分子的磷酸化水平无明显变化。IRAK或PI3K通路被抑制后TLR激动剂不再能够显著抑制精子运动,而MEK/ERK, NF-κB通路被抑制后TLR激动剂依然能显著抑制精子运动。PI3K抑制剂处理小鼠精子后,AKT的磷酸化水平没有显著改变而GSK3α的磷酸化水平受到了显著地抑制。结论：TLR信号以MyD88/IRAK4/PI3K及GSK3a依赖而非AKT依赖的信号通路途径抑制精子运动。 3.TLR信号影响精子的生物学过程研究目的：探讨TLR信号通过影响精子的何种生物学过程最终抑制了精子运动,并将深入分析TLR信号通路在影响该生物学过程中的作用。方法：利用荧光素酶反应的方法检测TLR激动剂处理6h后小鼠精子ATP水平的变化,进而使用基因敲除小鼠分析TLR激动剂引起的精子ATP水平的变化是否依赖MyD88及PI3K。采用F1F。ATPase ELISA检测试剂盒检测TLR激动剂处理6h后正常人精子F1F。ATPase的活性变化。利用JC-1线粒体膜电位检测试剂盒通过流式细胞术检测TLR激动剂处理不同时间对小鼠精子膜电位的影响,并利用Western-blotting分析相关信号通路分子在TLR激动剂处理后转移到线粒体的情况。结果：与对照组相比,TLR激动剂处理后显著降低了小鼠精子ATP水平,并且TLR激动剂不能引起MyD88-/-及pik3cd-/-小鼠精子ATP水平的变化。TLR激动剂处理6h后能够引起正常人精子F1F。ATPase活性的显著上升,并以时间依赖的方式降低小鼠精子线粒体膜电位。最后,TLR激动剂处理6h后能够引起PI3K及GSK3a等信号通路分子转移到小鼠精子线粒体中。结论：TLR信号通过影响线粒体膜电位的方式间接消耗ATP从而影响精子运动,该影响依赖于MyD88及PI3K通路。
[Abstract]:Toll - like receptor ( TLR ) , as an important molecule of the innate immune system , plays an important role in the innate immune system ' s resistance to external interference . In recent years , TLR expression is considered to be the key to successful pregnancy . The study was divided into 3 parts : 1 . Effects of different types of TLR agonists on sperm biological properties Objective : To observe the effect of TLR agonist on sperm motility in mice and to investigate the possible causes of the inhibition of sperm motility by TLR agonists . Methods : The expression of TLR in sperm was analyzed by reverse transcription polymerase chain reaction ( RT - PCR ) . The effects of TLR agonist and TLR agonist on sperm motility were observed by computer - assisted sperm analysis ( CASA ) . Results : In addition to TLR3 expression , other TLR agonists were expressed in the sperm . After 6 h of TLR agonist treatment , the agonist poly ( I : C ) corresponding to TLR3 had no effect on sperm motility , and other TLR agonists inhibited sperm motility to a varying degree . In addition , TLR agonists inhibited sperm motility after 6 hours of treatment . The TLR agonist did not effectively promote sperm apoptosis within 6 hours compared with the control group . Conclusion : TLR agonists can effectively inhibit sperm motility and the effect is not caused by TLR agonists in promoting sperm apoptosis . 2 . TLR signaling pathway for sperm motility Objective : To explore the exact signal pathway of TLR signaling affecting sperm motility Methods : The effects of TLR agonist on sperm motility in mice were studied by using MyD88 knockout mice . After 2 h , 4 h , 6 h of LPS - treated mice , the changes of phosphorylation level were observed by Western - blotting . Results : TLR agonist has no effect on the sperm motility of MyD88 knockout mice . TLR agonist can be observed after treatment of sperm in mice . Conclusion : TLR signaling can inhibit sperm motility in the pathway of MyD88 / RK4 / PIK and GSK3 - dependent signaling pathway . 3 . Study on the biological process of TLR signaling affecting sperm Objective : To investigate the effect of TLR signaling on sperm motility , and to deeply analyze the role of TLR signaling pathway in the biological process . Methods : The changes of ATP level in human sperm after 6 hours of TLR agonist treatment were detected by luciferase reaction , and the changes of ATP level of sperm induced by TLR agonist were analyzed by using F1F . ATPase ELISA kit . The effect of TLR agonist on sperm membrane potential was detected by flow cytometry and Western - blotting was used to analyze the effect of TLR agonist on sperm membrane potential of mice . Results : Compared with the control group , TLR agonist treatment significantly reduced the level of ATP in the sperm of mouse sperm , and TLR agonist could not induce the change of ATP level of MyD88 - / - and pik3cd - / - mice . Conclusion : TLR signaling indirectly consumes ATP by affecting the mitochondrial membrane potential , which affects sperm motility , which is dependent on the MyD88 and PIK pathways .

【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R321

【共引文献】