SPERM CHROMATIN PROTEOMICS

精子染色质蛋白质组学

• 批准号: 7957805
• 负责人: BARBARA J MEYER
• 金额: $ 0.33万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7957805
• 关键词: Aneuploidy; Biology; Caenorhabditis elegans; Cataloging; Catalogs; Chromatin; Chromatin Structure; Chromosome Segregation; Chromosomes; Clinical; Computer Retrieval of Information on Scientific Projects Database; Coupled; Cytology; DNA; F Factor; Failure; Fertility; Fertilization in Vitro; Funding; Fungal Genome; Goals; Grant; Infertility; Institution; Male Contraceptive Agents; Male Infertility; Male Sterility; Mammals; Medical; Meiosis; Mus; Nematoda; Oogenesis; Process; Proteins; Proteomics; RNA Interference; Research; Research Personnel; Resources; Role; Source; Spermatogenesis; Technology; United States National Institutes of Health; base; cell type; gene function; knockout gene; male; reproductive; sperm cell; sperm protein

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Male infertility is a long-standing enigma of significant medical concern. The integrity of sperm chromatin is a clinical indicator of male fertility and in vitro fertilization potential: chromosome aneuploidy and DNA decondensation or damage are correlated with reproductive failure. Identifying conserved proteins important for sperm chromatin structure and packaging can reveal universal causes of infertility. Here we combine proteomics, cytology and functional analysis in Caenorhabditis elegans to identify spermatogenic chromatin-associated proteins that are important for fertility. Our strategy employed multiple steps: purification of chromatin from comparable meiotic cell types, namely those undergoing spermatogenesis or oogenesis; proteomic analysis by multidimensional protein identification technology (MudPIT) of factors that co-purify with chromatin; prioritization of sperm proteins based on abundance; and subtraction of common proteins to eliminate general chromatin and meiotic factors. Our approach reduced 1,099 proteins co-purified with spermatogenic chromatin, currently the most extensive catalogue, to 132 proteins for functional analysis. Reduction of gene function through RNA interference coupled with protein localization studies revealed conserved spermatogenesis-specific proteins vital for DNA compaction, chromosome segregation, and fertility. Unexpected roles in spermatogenesis were also detected for factors involved in other processes. Our strategy to find fertility factors conserved from C. elegans to mammals achieved its goal: of mouse gene knockouts corresponding to nematode proteins, 37% (7/19) cause male sterility. Our list therefore provides significant opportunity to identify causes of male infertility and targets for male contraceptives.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 男性不育症是一个长期存在的谜，引起了医学界的重大关注。精子染色质的完整性是男性生育力和体外受精潜力的临床指标：染色体非整倍性和 DNA 解凝或损伤与生殖失败相关。识别对精子染色质结构和包装很重要的保守蛋白可以揭示不育的普遍原因。在这里，我们结合秀丽隐杆线虫的蛋白质组学、细胞学和功能分析来鉴定对生育能力重要的生精染色质相关蛋白。我们的策略采用了多个步骤：从可比较的减数分裂细胞类型（即正在进行精子发生或卵子发生的细胞类型）中纯化染色质；通过多维蛋白质鉴定技术 (MudPIT) 对与染色质共纯化的因子进行蛋白质组学分析；根据丰度对精子蛋白进行优先排序；以及减去常见蛋白质以消除一般染色质和减数分裂因子。我们的方法将与生精染色质（目前最广泛的目录）共纯化的 1,099 种蛋白质减少到 132 种用于功能分析的蛋白质。通过 RNA 干扰降低基因功能，结合蛋白质定位研究揭示了保守的精子发生特异性蛋白质，对 DNA 压缩、染色体分离和生育能力至关重要。还检测到参与其他过程的因素在精子发生中的意外作用。我们寻找从线虫到哺乳动物保守的生育因子的策略实现了其目标：与线虫蛋白相对应的小鼠基因敲除，37%（7/19）导致雄性不育。因此，我们的清单为确定男性不育的原因和男性避孕的目标提供了重要的机会。