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: 6351422
• 负责人: STEPHEN H PILDER
• 金额: $ 24.21万
• 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 2005-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6351422
• 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-卵膜穿透(SOP)是受精的基础。然而，在哺乳动物中，这种动态过程的分子基础还不完全清楚。P.I.在小鼠染色体(Chr)17上的t复合体Stop1p和Stop1d上定位了两个紧密连锁的突变(分别为精子-t复合体-卵膜穿透1近端和精子-t复合体-卵膜穿透1远端)，它们的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)。由于这些基因为了解SOP过程的调控机制提供了关键，PI将通过在新型体外精子-卵膜结合试验(特定目标I)中测试表达“STOP”表型的小鼠精子来确定“STOP”表型是否源于SOP结合和/或融合阶段(S)的缺陷。由于Stop1d似乎是“Stop”表达的主要调控因子，而且Stop1d仅被定位到大约3厘米的中等分辨率，PI将通过对表达或不表达“Stop”缺陷的小鼠的新型重组Chr 17同源物的标记分析，将Stop1d基因定位到高分辨率(约0.1-0.6 cM)，并且其多态断裂点位于3 cM的Stop1d区域(特定目标IIA)。高分辨率作图将使PI能够在物理上分离重叠的连续细菌人工染色体(BAC重叠群)中的Stop1p和Stop1d基因座，这些基因座横跨每个基因座从近端到远端的侧翼标记(特定目标IIB)。为了分离候选基因Stop1p和Stop1d，PI将通过突变和对照睾丸mRNAs的差异显示-聚合酶链式反应，以及通过对Stop1p和Stop1d BACs的cDNA选择(特异性目标IIIA)，构建每个基因座的睾丸表达转录图谱。为了确定哪些候选基因最有可能调节“停止”活性，PI将对定位于Stop1p和Stop1d基因座的睾丸转录产物进行序列和表达分析，测试等位基因转录产物之间的核酸和推测的氨基酸差异，以及等位基因转录产物的组织和时间表达状态(特定目标IIIB)。这些实验的成功将有助于我们理解对受精至关重要的SOP过程，并将为我们进一步剖析它提供一个模型系统。