Trapping Cumulus Products for Contraceptive Targeting

捕获积云产品以实现避孕目标

• 批准号: 6711163
• 负责人: JOHN J EPPIG
• 金额: $ 31.35万
• 依托单位: JACKSON LABORATORY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-26 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6711163
• 关键词: cell growth regulation; complementary DNA; contraceptives; cooperative study; egg /ovum; genetic markers; genetically modified animals; granulosa cell; growth factor; histology; hormone regulation /control mechanism; laboratory mouse; paracrine; receptor; recombinant proteins

DESCRIPTION (provided by applicant): The premise of this application is that cumulus cells produce paracrine regulators crucial for normal oocyte development, and that disruption of their function will lead to the production of oocytes that cannot undergo fertilization or mbryonic development. This project will use signal sequence trap (SST) methodology to identify and characterize paracrine factors produced by cumulus cells that promote oocyte development. In addition, the SST will identify transmembrane receptors expressed by cumulus cells. These receptors may be necessary for cumulus cell functions essential for oocyte development or ovulation. Specific Aim 1 is to produce and screen a cumulus cell SST library in order to isolate cumulus cell-secreted factors and transmembrane receptors. Specific Aim 2 is to characterize and clone cumulus cell cDNAs that contain putative signal peptide sequences. Novel sequences will be further characterized by Northern blot and RNase protection analyses using multiple tissues and by in situ hybridization using ovaries at key developmental stages to establish specificity of cellular expression. cDNA inserts exhibiting cumulus cell-specific expression will be used to isolate full-length cDNAs from a cumulus cell cDNA library. Sequence analysis of the full-length cDNAs will be performed, and novel sequences will be used for further studies, including the isolation of genomic clones from 129/SvEv mouse libraries. Specific Aim 3 is to assess the function of putative cumulus cell-expressed ligands and receptors, and to define their participation in the oocyte-granulosa cell regulatory loop. Null alleles of genes that encode novel cumulus cell-specific proteins (identified in Specific Aim 2) will be produced through collaboration with the Center's Transgenic Knockout Core Facility. Heterozygous mice will be bred to homozygosity to assess the effects of the null mutations on fertility. In addition, ovarian histology will be evaluated with an emphasis on follicle development, cumulus cells, and oocytes. At the molecular level, the expression of potential downstream marker genes in oocytes will be investigated. The effects of the null mutations and cumulus cell-expressed recombinant proteins on oocyte and cumulus cell function in vitro, including the ability of oocyte to resume and complete meiosis, undergo fertilization, and complete preimplantation development, will be determined. Success in these studies will generate exciting new knowledge of the fundamental mechanisms that govern the development of both oocytes and the somatic components of follicles, as well as provide a plethora of new targets for fertility control.

描述（由申请人提供）：本应用的前提是，积云细胞会产生对正常卵母细胞发育至关重要的旁分泌调节剂，并且其功能的破坏将导致产生无法承受肥料或mbryonic发育的卵母细胞。该项目将使用信号序列陷阱（SST）方法来识别和表征由促进卵母细胞发育产生的旁分泌因子。此外，SST将鉴定由积云细胞表达的跨膜受体。这些受体对于卵母细胞发育或排卵必不可少的积云细胞功能可能是必需的。具体目的1是生产和筛选积云细胞SST文库，以分离积云的细胞分泌因子和跨膜受体。特定的目标2是表征和克隆积云细胞cDNA，其中包含推定的信号肽序列。新的序列将进一步以北印迹和RNase保护分析使用多个组织和原位杂交分析，并在关键发育阶段使用卵巢来建立细胞表达的特异性。表现出表现库卢斯细胞特异性表达的cDNA插入物将用于分离整个细胞cDNA文库的全长cDNA。将对全长cDNA进行序列分析，并将新序列用于进一步研究，包括从129/SVEV小鼠文库中分离基因组克隆。具体目标 3是为了评估假定的卵石细胞表达的配体和受体的功能，并定义了它们参与卵母细胞粒细胞调节环。编码新型库卢斯细胞特异性蛋白（在特定目标2中鉴定）的基因的无效基因等位基因将通过与该中心的转基因敲除核心设施合作产生。杂合小鼠将被繁殖为纯合性，以评估无效突变对生育能力的影响。此外，将评估卵巢组织学，重点是卵泡发育，卵黄细胞和卵母细胞。在分子水平上，将研究卵母细胞中势下游标记基因的表达。零突变和育细胞表达的重组蛋白对体外卵母细胞和积云细胞功能的影响，包括卵母细胞恢复和完全减数分裂，接受施肥和完全植入前发育的能力。这些研究的成功将产生令人兴奋的新知识，即控制卵泡和卵泡的体细胞成分的基本机制，并为生育能力控制提供了许多新的目标。