Intraflagellar transport (IFT) and sperm formation

摘要 鞭毛内转运(IFT)是纤毛形成的一种进化保守机制。IFT/纤毛缺陷 与纤毛相关的疾病有关。尽管IFT在体细胞组织中的作用已被广泛研究 在研究中，人们对它在精子鞭毛形成中的作用知之甚少。精子鞭毛是一种专门的活动纤毛，具有 附属结构。使用条件基因敲除(CKO)策略，我们的实验室分析了男性生殖细胞- 特定的IFT突变小鼠，发现所有分析的IFT都是正常精子所必需的 队形/功能。在IFT基因中，Ift25和Ift27尤其令人感兴趣。这两个IFT形成一个 异二聚体通过其独特的特征结构。虽然这两个基因不是纤毛所必需的 在体细胞中组装，两者对精子的形成和功能都是必不可少的。具体消除每一项 雄性生殖细胞中的这些基因导致了几乎相同的不育表型。这些小鼠的精子 静止的，有杂乱无章的附属结构，尤其是纤维鞘。睾丸促肾上腺皮质激素原水平 AKAP4是AKAP4的前体蛋白，是A-激酶锚定蛋白(AKAP)的重要组成部分 与之相反，成熟的AKAP4则显著减少。 Ift25和Ift27均为CKO小鼠。IFT25与动力蛋白相关蛋白dynactin 4(DCTN4)相关。在……里面 除了IFT25，IFT27还与2a(SPPL2a)等信号肽酶结合，后者的功能是 一种蛋白水解酶，存在于发育中的精子鞭毛中。成熟的AKAP4的形成也受到影响 Sppl2a KO小鼠。基于这些观察，我们提出了以下中心假设：1 )IFT25和 IFT27专门用于移动和放置对功能至关重要的附件结构组件 精子，以及2)IFT25/IFT27复合体利用特定的结构域形成精子鞭毛的IFT复合体 正在组装。为了检验这些假设，我们提出了以下具体目标：1.为了刻画 睾丸中对正常精子形态发生至关重要的IFT25/IFT27复杂成分，特别是 附属器的形成；2.研究Ift25 CKO小鼠精子附属器结构缺陷 动态地开发一种体内系统来跟踪IFT25复合体在活生殖细胞中的精子运输 3.探索IFT25/27在精子信号转导过程中的功能后果。我们 提出IFT25/IFT27异源二聚体形成含SPPL2a和DCTN4的转运复合体 通过男性生殖细胞中的特定区域进行正常的精子附属结构组装；我们预计 Ift25 CKO小鼠的附属结构缺陷会在特定的发育阶段发生。动态感 可以追踪IFT25复合体在活的男性生殖细胞中的运输过程。我们假设SPPL2a 参与处理原-AKAP4成熟为AKAP4，导致成熟精子中正常的PKA信号。 拟议的研究将阐明这两种IFT蛋白在功能形成中的机制。 精子和 建立一个平台，研究IFT的其他组成部分在雄性和雌性生殖中的作用。 。

Summary Intraflagellar transport (IFT) is an evolutionarily conserved mechanism for cilia formation. Defects in IFT/cilia have been linked to cilia-related diseases. Although the roles of IFT in somatic tissues have been extensively studied, little is known about its role in sperm flagella formation, which are specialized motile cilia with accessory structures. Using conditional knockout (cKO) strategies, our laboratory analyzed male germ cell- specific IFT mutant mice and discovered that all the analyzed IFTs are required for normal sperm formation/function. Among the Ift genes, Ift25 and Ift27 hold particular interest. The two IFTs form a heterodimer through their unique characterized structures. Although these two genes are not required for cilia assembly in somatic cells, both are essential for sperm formation and function. Specific elimination of each of these genes in male germ cells resulted in almost identical sterile phenotypes. Sperm from these mice were immotile and had disorganized accessory structures, especially the fibrous sheath. Levels of testicular pro- AKAP4, the precursor protein of AKAP4, an A-kinase anchor protein (AKAP) and significant component of the sperm fibrous sheath, were increased; on the contrary, the mature AKAP4 was significantly reduced in both Ift25 and Ift27 cKO mice. IFT25 associates with dynactin 4 (DCTN4), a dynein-associated protein. In addition to IFT25, IFT27 also associates with signal peptide peptidases like 2a (SPPL2a), which functions as a protease and is present in developing sperm flagella. The formation of mature AKAP4 was also affected in the Sppl2a KO mice. Based on these observations, we propose the following central hypotheses: 1 ) IFT25 and IFT27 are dedicated to the movement and placement of accessory structure components critical for functional sperm, and 2) The IFT25/IFT27 complex use specific domains to form IFT complex particles for sperm flagella assembling. To test these hypotheses, we propose the following Specific Aims: 1. To characterize the IFT25/IFT27 complex components in the testis essential for normal sperm morphogenesis, particularly the formation of accessory structures; 2. To investigate sperm accessory structure defects of Ift25 cKO mice dynamically and develop an in vivo system to track the IFT25 complex trafficking in live germ cells for sperm flagella assembly; and 3. To explore functional consequences of IFT25/27 disruption in sperm signaling. We propose that the IFT25/IFT27 heterodimer forms a transporting complex containing SPPL2a and DCTN4 through specific domains in male germ cells for normal sperm accessory structure assembly; we expect defects in accessory structures in the Ift25 cKO mice will occur at specific developmental steps. The dynamic trafficking process of the IFT25 complex in live male germ cells can be tracked. We hypothesize that SPPL2a is involved in processing pro-AKAP4 to mature into AKAP4, resulting in normal PKA signaling in mature sperm. The proposed research will elucidate the mechanisms of the two IFT proteins in the formation of functional sperm and build a platform to study the roles of other IFT components in male and female reproduction. .