Intraflagellar transport (IFT) and sperm formation

鞭毛内运输 (IFT) 和精子形成

• 批准号: 10445709
• 负责人: Zhibing Zhang
• 金额: $ 40.53万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 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/10445709
• 关键词: A kinase anchoring protein; Affect; Binding; Carrier Proteins; Chlamydomonas; Cilia; Co-Immunoprecipitations; Complex; Cyclic AMP-Dependent Protein Kinases; 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; base; 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

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. .

总结 鞭毛内转运（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。虽然IFT在体细胞组织中的作用已经被广泛研究， 研究，很少有人知道它在精子鞭毛形成的作用，这是专门的运动纤毛， 附属结构使用条件性敲除（cKO）策略，我们的实验室分析了男性生殖细胞- 特异性IFT突变小鼠，并发现所有分析的IFT都是正常精子所需的 形成/功能。在Ift基因中，Ift25和Ift27特别令人感兴趣。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，两者都是精子形成和功能所必需的。具体消除每一个 雄性生殖细胞中的这些基因导致几乎相同的不育表型。这些小鼠的精子 不动，有紊乱的附属结构，特别是纤维鞘。睾丸激素水平 AKAP 4，AKAP 4的前体蛋白，A-激酶锚蛋白（AKAP）和细胞凋亡的重要组分。 精子纤维鞘，增加;相反，成熟的AKAP 4显着减少， Ift 25和Ift 27 cKO小鼠。IFFT 25与动力蛋白相关蛋白dynactin 4（DCTN4）结合。在 除了IFFT 25之外，IFFT 27还与信号肽肽酶如2a（SPPL 2a）相关，其功能为 一种蛋白酶，存在于发育中的精子鞭毛中。成熟AKAP 4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 ）IFT 25和 IFT 27专用于功能关键的附件结构部件的移动和放置 精子，和2）IFT 25/IFT 27复合物使用特定结构域形成精子鞭毛IFT复合物颗粒 组装为了验证这些假设，我们提出了以下具体目标：1。表征 在睾丸中的IFFT 25/IFFT 27复合物组分对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Ift 25 cKO小鼠精子附属结构缺陷 动态地开发一个体内系统来跟踪精子活生殖细胞中的IFT 25复合物运输 鞭毛组装;和3.探索精子信号中IFFT 25/27破坏的功能后果。我们 我提出，IFFT 25/IFFT 27异二聚体形成含有SPPL 2a和DCTN4的转运复合物 通过男性生殖细胞中的特定结构域进行正常精子附属结构组装;我们预期 Ift 25 cKO小鼠中附属结构的缺陷将在特定的发育阶段发生。动态 可以追踪活的雄性生殖细胞中的IFT 25复合物的运输过程。我们假设SPPL 2a 参与加工前AKAP 4成熟为AKAP 4，导致成熟精子中正常的PKA信号传导。 这项研究将阐明这两种IFT蛋白在功能性细胞因子形成中的作用机制。 精子和 建立一个平台，研究其他IFT组件在男性和女性生殖中的作用。 .