Intraflagellar transport (IFT) and sperm formation

鞭毛内运输 (IFT) 和精子形成

基本信息

批准号：
10596173
负责人：
Zhibing Zhang
金额：
$ 43.69万
依托单位：
WAYNE STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-04-01 至 2027-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10596173
关键词：
A kinase anchoring protein Affect Binding Carrier Proteins Categories Chlamydomonas Cilia Co-Immunoprecipitations Complex Cyclic AMP-Dependent Protein Kinases Dedications Defect Development Disease Dynein ATPase Dyskinetic syndrome Female Flagella Genes Genetic Diseases Genotype Germ Cells Human Human Genetics Infertility Knock-in Mouse Knockout Mice Knowledge Laboratories Link Maintenance Male Infertility Mass Spectrum Analysis Mediating Monitor Morphogenesis Motor Movement Mus Mutant Strains Mice Mutation Peptide Hydrolases Phenotype Phosphorylation Polycystic Kidney Diseases Process Protein Kinase Protein Precursors Proteins Proteomics Reproduction Research Retinal Degeneration Role Signal Transduction Somatic Cell Sperm Tail Spermatocytes Spermatogenesis Spermiogenesis Sterility Structure System Testing Testis Time Tissues Transmission Electron Microscopy alpha helix cellular imaging ciliopathy cilium biogenesis cilium motility comparative conditional knockout dynactin in vivo interest male mutant mouse model particle protein protein interaction signal peptide peptidase sperm cell sperm function sperm protein trafficking

项目摘要

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. .

概括鞭毛内运输（IFT）是纤毛形成的进化保守机制。 IFT/纤毛缺陷与纤毛相关疾病有关。尽管 IFT 在体细胞组织中的作用已被广泛研究研究人员对其在精子鞭毛形成中的作用知之甚少，精子鞭毛是一种特殊的运动纤毛，具有附属结构。使用条件敲除（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，并发现所有分析的 IFT 都是正常精子所必需的形成/功能。在 Ift 基因中，Ift25 和 Ift27 特别受关注。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体细胞中的组装，两者对于精子的形成和功能都至关重要。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。这些小鼠的精子不能活动，附属结构混乱，尤其是纤维鞘。睾丸亲水平 AKAP4，AKAP4 的前体蛋白，A 激酶锚定蛋白 (AKAP) 的重要组成部分精子纤维鞘增多；相反，成熟的AKAP4在 Ift25 和 Ift27 cKO 小鼠。 IFT25 与动力蛋白相关蛋白 dynactin 4 (DCTN4) 结合。在除了 IFT25 之外，IFT27 还与信号肽肽酶 2a (SPPL2a) 相关，其功能如下：一种蛋白酶，存在于发育中的精子鞭毛中。成熟 AKAP4 的形成也受到影响 Sppl2a KO 小鼠。基于这些观察，我们提出以下中心假设：1 ) IFT25 和 IFT27 致力于移动和放置对功能至关重要的附件结构部件精子，以及 2) IFT25/IFT27 复合物使用特定结构域形成精子鞭毛的 IFT 复合物颗粒组装。为了检验这些假设，我们提出以下具体目标： 1. 表征睾丸中的 IFT25/IFT27 复杂成分对于正常精子形态发生至关重要，特别是附属结构的形成； 2. 研究Ift25 cKO小鼠精子辅助结构缺陷动态地开发一个体内系统来跟踪 IFT25 复合物在活精子生殖细胞中的运输鞭毛组装； 3. 探讨 IFT25/27 破坏精子信号传导的功能后果。我们提出 IFT25/IFT27 异二聚体形成含有 SPPL2a 和 DCTN4 的运输复合物通过雄性生殖细胞中的特定区域进行正常精子辅助结构的组装；我们期望 Ift25 cKO小鼠的附属结构缺陷会在特定的发育阶段发生。动态可以追踪 IFT25 复合物在活雄性生殖细胞中的运输过程。我们假设 SPPL2a 参与将 AKAP4 前体加工成 AKAP4，从而在成熟精子中产生正常的 PKA 信号传导。拟议的研究将阐明两种 IFT 蛋白在功能性蛋白质形成中的机制。精子和建立一个平台来研究其他 IFT 成分在男性和女性生殖中的作用。。