GENES THAT AFFECT SPERM EGG INTERACTION IN MAMMALS

影响哺乳动物精卵相互作用的基因

• 批准号: 6697061
• 负责人: STEPHEN H PILDER
• 金额: $ 20.82万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-01 至 2006-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6697061
• 关键词: alleles; artificial chromosomes; cell cell interaction; complementary DNA; egg /ovum; fertilization; gene mutation; genetic mapping; genetic markers; genetic polymorphism; genetic transcription; genetics; laboratory mouse; polymerase chain reaction; recombinant DNA; sperm; testis

Sperm-oolemma penetration (SOP) is fundamental to fertilization. However, in mammals, the molecular basis of this dynamic process is incompletely understood. The P.I. has mapped two tightly- linked mutations on mouse Chromosome (Chr) 17 in the t complex, Stop1p and Stop1d (Sperm-t complex-oolemma penetration 1 proximal and Sperm-t complex-oolemma penetration 1 distal, respectively) whose activity significantly decreases the ability of affected sperm from penetrating zona pellucida-free eggs (the "stop" phenotype). Because these genes offer a key to understanding the mechanisms that regulate the SOP process, the PI will determine if the "stop" phenotype derives from a defect in the binding and/or fusion phase(s) of SOP by testing sperm from mice expressing the "stop" phenotype in novel in vitro sperm-oolemma binding assays (Specific Aim I). Since Stop1d appears to be a major regulator of "stop" expression, and because Stop1d has been mapped only to a moderate resolution of approximately 3 centiMorgans (cM), the PI will map the Stop1d locus to high resolution (approximately 0.1-0.6 cM) by marker analysis of novel recombinant Chr 17 homologs from mice either expressing or not expressing the "stop" defect, and whose polymorphic breakpoints reside in the 3 cM Stop1d region (Specific Aim IIA). High resolution mapping will allow the PI to physically isolate both Stop1p and Stop1d loci in overlapping contiguous bacterial artificial chromosomes (BAC contigs) spanning each locus from proximal to distal flanking markers (Specific Aim IIB). In order to isolate candidate Stop1p and Stop1d genes, The PI will construct a testis-expression transcript map of each locus by differential display-PCR of mutant versus control testis mRNAs, and by cDNA selection of Stop1p and Stop1d BACs (Specific Aim IIIA). To determine which candidates are most likely to regulate "stop" activity, the PI will perform sequence and expression analysis of testis transcripts mapped to the Stop1p and Stop1d loci, testing for nucleic acid and presumed amino acid differences between allelic transcripts, and the tissue and temporal expression status of allelic transcripts (Specific Aim IIIB). The success of these experiments will contribute significantly to our understanding of a process, SOP, essential to fertilization, and will provide us with a model system for its further dissection.

精子膜穿透（SOP）是受精的基础。然而，在哺乳动物中，这一动态过程的分子基础尚不完全清楚。P.I.已经在小鼠染色体（Chr） 17的t复合体上绘制了两个紧密相连的突变，分别是Stop1p和Stop1d（精子-t复合体-膜渗透1近端和精子-t复合体-膜渗透1远端），其活性显著降低了受影响精子穿透无透明带卵子的能力（“停止”表型）。由于这些基因为理解SOP过程的调控机制提供了关键，因此PI将在新的体外精子-膜结合试验中检测表达“停止”表型的小鼠精子，从而确定“停止”表型是否源于SOP结合和/或融合阶段的缺陷（特定目的1）。由于Stop1d似乎是“停止”表达的主要调控因子，并且由于Stop1d仅被定位到大约3 cM的中等分辨率，PI将通过对表达或不表达“停止”缺陷的小鼠的新型重组Chr 17同源物进行标记分析，将Stop1d位点定位到高分辨率（大约0.1-0.6 cM），其多态性断点位于3 cM Stop1d区域（Specific Aim IIA）。高分辨率制图将允许PI物理分离重叠的连续细菌人工染色体（BAC contigs）中的Stop1p和Stop1d位点，这些染色体跨越从近端到远端侧翼标记的每个位点（Specific Aim IIB）。为了分离候选的Stop1p和Stop1d基因，PI将通过突变与对照睾丸mrna的差异显示pcr，以及Stop1p和Stop1d BACs的cDNA选择，构建每个位点的睾丸表达转录图谱（Specific Aim IIIA）。为了确定哪些候选基因最有可能调控“停止”活性，PI将对睾丸转录本Stop1p和Stop1d位点进行序列和表达分析，检测等位基因转录本之间的核酸和氨基酸差异，以及等位基因转录本的组织和时间表达状态（Specific Aim IIIB）。这些实验的成功将大大有助于我们对受精过程SOP的理解，并将为我们进一步的解剖提供一个模型系统。