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. .

概括 Flagellar内转运（IFT）是纤毛形成的进化保守机制。 IFT/纤毛的缺陷 与纤毛相关疾病有关。尽管IFT在体细胞组织中的作用已广泛 研究了，对其在精子鞭毛形成中的作用知之甚少，这是专门的纤毛纤毛 附件结构。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，发现正常精子需要所有分析的IFT 编队/功能。在IFT基因中，IFT25和IFT27具有特别的兴趣。这两个IFT形成一个 异二聚体通过其独特的特征结构。尽管这两个基因不需要纤毛 在体细胞中的组装，两者对于精子形成和功能都是必不可少的。具体消除每个 雄性生殖细胞中的这些基因几乎导致无菌表型。这些小鼠的精子是 免疫机能，并具有混乱的辅助结构，尤其是纤维鞘。睾丸前 AKAP4，AKAP4的前体蛋白，A-激酶锚蛋白（AKAP）和显着成分 精子纤维鞘增加；相反，成熟的AKAP4显着降低 IFT25和IFT27 CKO小鼠。 IFT25与Dynactin 4（DCTN4）（Dynein相关蛋白）相关。在 除IFT25外，IFT27还与信号肽肽酶（如2A（SPPL2A））相关联，该肽的作用为 一种蛋白酶，存在于发育精子鞭毛中。成熟AKAP4的形成也受到影响 SPPL2A KO小鼠。基于这些观察结果，我们提出了以下中心假设：1 ）ift25和 IFT27专用于功能至关重要的配件结构组件的运动和放置 精子和2）IFT25/IFT27复合物使用特定域形成精子鞭毛的IFT复合粒子 组装。为了检验这些假设，我们提出以下具体目的：1。 睾丸中的IFT25/IFT27复合成分对于正常精子形态发生必不可少的，尤其是 辅助结构的形成； 2。研究IFT25 CKO小鼠的精子附件结构缺陷 动态并开发一个体内系统，以跟踪活精子中的IFT25复合物运输 鞭毛装配；和3。探索IFT25/27在精子信号传导中破坏的功能后果。我们 提出IFT25/IFT27异二聚体形成包含SPPL2A和DCTN4的运输复合物 通过男性生殖细胞中的特定域，用于正常的精子辅助结构组装；我们期望 IFT25 CKO小鼠中附件结构的缺陷将在特定的发育步骤中发生。动态 可以跟踪活雄性生殖细胞中IFT25复合物的运输过程。我们假设SPPL2A 参与处理Pro-Akap4以成熟到AKAP4中，从而导致成熟精子中的正常PKA信号传导。 拟议的研究将阐明两种IFT蛋白在功能形成中的机制 精子和 建立一个平台来研究其他IFT成分在男性和女性繁殖中的作用。 。