Ezrin相关蛋白网络在人精子获能过程中的功能研究

发布时间：2018-06-25 14:07

本文选题：ezrin + 蛋白网络　；参考：《南京医科大学》2008年博士论文

【摘要】： 受精是一个新个体产生的标志,是指精子和卵子自发融合成为受精卵的过程,要完成这一过程必须具备一定的条件,精子获能就是其中之一。获能是指精子获得使卵细胞受精的能力,是精子在自然状态下完成受精过程的一个必需条件。哺乳动物的精子在受精前都要经历获能过程。在精子获能过程中,对于精子质膜的修饰改变是这一过程的一个关键事件。但是迄今为止,对于这一重要事件分子机制的研究结果还不多,远不能阐明这一过程。 Ezrin蛋白是ERM蛋白家族的成员之一,也是该家族中被研究最多的蛋白。在体细胞中的大量研究显示,ezrin是细胞皮质骨架蛋白与细胞质膜固有膜蛋白之间的衔接蛋白,它的主要作用是通过对骨架蛋白与膜蛋白的衔接作用,引起细胞膜表面结构的重构,从而介导包括细胞黏附、变形、微绒毛形成等一系列的细胞事件。不仅如此,活化的ezrin还能够和Rho解离抑制因子——RhoGDI一起促进细胞表层骨架蛋白的形成,从而使actin聚合形成F-actin。因此,在体细胞中就存在一个由ezrin、actin、RhoGDI、Rho蛋白以及质膜蛋白共同形成的蛋白网络体系。这个蛋白网络的主要作用就是对细胞膜的表面结构进行组装。本实验室在前期研究工作中发现,ezrin和RhoGDI1表达于人睾丸组织中,那么,会不会前述与ezrin相关的蛋白网络体系也能在精子中发挥功能呢?因为在精子获能过程中也存在膜重构的现象。我们推测,这个网络体系可能作用于精子获能过程中的膜重构。针对这一问题开展了以下研究:(1)用ERM家族磷酸化位点的抗体行不同获能状态精子蛋白的Western Blot实验,结果显示在精子获能过程中,ezrin第567位苏氨酸发生了磷酸化修饰,这说明ezrin在获能过程中被活化。推测活化后的ezrin可以与下游一系列蛋白通过直接或间接相互作用参与膜的重构,故运用一系列免疫共沉淀试验进行验证。(2)用免疫共沉淀方法比较获能前后ezrin与RhoGDI1相互作用情况,结果显示,两者的相互作用在获能后增加;运用二维电泳结合银染和磷酸化染色方法,结果显示,RhoGDI1自身还在获能过程中发生了磷酸化修饰,前述两个结果均可导致RhoGDI1自Rho蛋白解离下来。对后者用免疫共沉淀的方法进行验证,结果符合预想,RhoGDI1与RhoA的相互作用在获能后减弱。这一结果提示RhoA可参与下游一系列通路导致F-actin形成。(3)对于这一系列事件的最终结果用F-actin特异性染料Texas Red-Phalloidin染色进行分析,结果显示,获能后,精子的F-actin染色尤其是头部的染色明显增强。说明获能过程中actin聚合。(4)据体细胞中的研究,ezrin可以和F-actin相互作用,故运用Co-IP方法对ezrin和F-actin的相互作用进行分析,结果显示获能后ezrin与F-actin的相互作用增强。这与体细胞中的现象类似,因此我们推测ezrin还可通过其氨基端与膜相互作用。(5)筛选了一个精子膜蛋白cd44,同样运用免疫共沉淀的方法比较其与ezrin相互作用情况,结果显示,ezrin在获能后与cd44相互作用增加,且ezrin不仅可以和cd44的未修饰形式相互作用,还可与其糖基化修饰形式相互作用。(6)最后,分析ezrin和actin在获能中可能的功能,分别用ezrin和actin抗体对获能和顶体反应进行阻断,结果显示,ezrin和actin抗体能够阻断精子获能但对于顶体反应则没有影响。总之,本研究为人精子获能分子机制提出了一个新的假说,即在人精子获能过程中,ezrin、RhoGDI1、actin、RhoA及膜蛋白可以通过相互作用形成一个蛋白网络体系,这样的网络可以调节获能过程中精子质膜重组、精子膜蛋白的重新分布,从而促进精子与卵子的受精过程。
[Abstract]:Fertilization is a sign of a new individual, which refers to the spontaneous fusion of sperm and egg into a fertilized egg. In order to complete this process, a certain condition must be provided, and the capacitation of the sperm is one of them. The ability to acquire the sperm to fertilize the egg cells is a necessary condition for the sperm to complete the process of fertilization in its self state. Mammalian spermatozoa undergo the process of capacitation before fertilization. In the process of sperm capture, modification of the sperm plasma membrane is a key event in this process. But so far, the results of the molecular mechanism of this important event are not much, far from clarifying the process.
Ezrin protein is one of the members of the ERM protein family and is also the most studied protein in the family. A large number of studies in somatic cells show that ezrin is a cohesive protein between the cytoskeleton and the membrane propria of the cell membrane. Its main role is to induce the cell membrane surface through the cohesion of the skeleton egg white and membrane protein. The reconstruction of the surface structure mediates a series of cell events, including cell adhesion, deformation, and microvilli formation. Not only so, the activated ezrin can also promote the formation of the cytoskeleton protein with the Rho dissociation inhibitor, RhoGDI, so that actin is polymerized to form F-actin., so there is a ezrin, a in somatic cells. Ctin, RhoGDI, Rho protein and plasma membrane proteins co formed a protein network system. The main function of this protein network is to assemble the surface structure of the cell membrane.
In our previous study, we found that Ezrin and RhoGDI1 are expressed in human testicular tissue. Then, will the ezrin related protein network system also function in sperm, because membrane remodeling exists in the process of sperm capacitation. We speculate that this network system may play a role in the process of sperm capacitation. The following studies have been carried out in the following aspects: (1) the Western Blot experiment with the antibodies of the ERM family phosphorylation sites in different capacitated sperm proteins shows that the ezrin 567th threonine has phosphorylated modification during the process of sperm capacitation, which indicates that ezrin is activated during the capacitation process. The activated ezri is conjectured. N can participate in the reconstruction of membrane through direct or indirect interaction with a series of downstream proteins, so a series of immunoprecipitation tests are used to verify. (2) the interaction of Ezrin and RhoGDI1 before and after the immune coprecipitation is compared. The results show that the interaction between the two proteins is increased after the acquisition of energy; two dimensional electrophoresis combined with silver staining. And phosphorylation staining, the results showed that RhoGDI1 itself was also phosphorylated during the acquisition process. The previous two results could lead to the dissociation of RhoGDI1 from the Rho protein. The latter was verified by the immunoprecipitation method. The results agreed that the interaction between RhoGDI1 and RhoA weakened after the acquisition of energy. This result suggests RhoA. A series of downstream pathways may be involved in the formation of F-actin. (3) the final results of this series of events were analyzed with F-actin specific dye Texas Red-Phalloidin staining. The results showed that after the acquisition of energy, the staining of the sperm F-actin staining, especially on the head, was obviously enhanced. Ezrin can interact with F-actin, so Co-IP method is used to analyze the interaction between Ezrin and F-actin. The results show that the interaction between Ezrin and F-actin is enhanced after the acquisition of energy. This is similar to the phenomenon in somatic cells. Therefore, we speculate that ezrin can also interact with the membrane phase through its amino terminal. (5) a sperm membrane protein CD44 is screened. The interaction with ezrin was compared with the method of immunoprecipitation. The results showed that the interaction between Ezrin and CD44 increased after the acquisition of energy, and ezrin could not only interact with the unmodified forms of CD44, but also interact with the form of glycosylated modification. (6) finally, the possible functions of Ezrin and actin in the capacitation were analyzed with ezri, respectively. N and actin antibodies blocked the capacitation and acrosome reaction. The results showed that Ezrin and actin antibodies blocked sperm capacitation but had no effect on the acrosome reaction.
In conclusion, a new hypothesis is proposed for the molecular mechanism of human sperm capacitation, that ezrin, RhoGDI1, actin, RhoA and membrane proteins can form a protein network through interaction in human sperm capacitation, which can regulate the recombination of spermatozoa in the process of capacitation, the redistribution of the sperm membrane protein, and thus promote the redistribution of the sperm membrane protein. The fertilization process of spermatozoon and egg.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R321

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