SPERM CHROMATIN PROTEOMICS

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH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 中心，不一定是研究者的机构。 男性不育症是一个长期存在的谜的重大医学关注。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1：染色体非整倍性和DNA去凝聚或损伤与生殖失败相关。鉴定对精子染色质结构和包装重要的保守蛋白质可以揭示不育的普遍原因。在这里，我们结合联合收割机蛋白质组学，细胞学和功能分析在秀丽隐杆线虫，以确定精子发生染色质相关的蛋白质是重要的生育能力。我们的策略采用了多个步骤：从可比的减数分裂细胞类型中纯化染色质，即那些经历精子发生或卵子发生的细胞;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共纯化的1，099种蛋白质（目前最广泛的目录）减少到132种蛋白质用于功能分析。通过RNA干扰结合蛋白质定位研究降低基因功能，揭示了保守的精子发生特异性蛋白质对DNA压缩、染色体分离和生育力至关重要。意想不到的作用，精子发生中也检测到其他过程中涉及的因素。我们的策略是寻找C.线虫对哺乳动物的作用达到了其目的：在小鼠相应线虫蛋白基因敲除中，37%（7/19）的小鼠产生雄性不育。因此，我们的清单为确定男性不育的原因和男性避孕药的目标提供了重要的机会。

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 potential1: 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.