SPAM 1 ALLELES, SPERM PHENOTYPE AND FUNCTION

SPAM 1 等位基因、精子表型和功能

• 批准号: 6334336
• 负责人: PATRICIA ANASTASIA DELEON
• 金额: $ 26.55万
• 依托单位: UNIVERSITY OF DELAWARE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-06-01 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6334336
• 关键词: CHO cells; alleles; autoradiography; cell adhesion molecules; complementary DNA; confocal scanning microscopy; electron microscopy; fertilization; flow cytometry; fluorescent in situ hybridization; fusion gene; gene expression; genetic models; genetically modified animals; genotype; glycosylphosphatidylinositols; laboratory mouse; light microscopy; membrane proteins; messenger RNA; model design /development; nucleic acid quantitation /detection; phenotype; protein structure function; sperm

DESCRIPTION: (Scanned from the applicant's abstract) The long-term objective of our studies are to determine a) if the expression of PH-20 or the Sperm Adhesion Molecule 1, SPAM1 (genetic nomenclature), reveals a mechanism for transmission ratio distortion (TRD), b) if the Spam1 gene product is retained in individual spermatids and the regulatory mechanism that controls its mRNA distribution, and c) how overexpression of SPAM1 affects sperm function. The proposal seeks to determine if the murine Spam1 gene product, a sperm membrane protein (Spam1), is shared among conjoined spermatids and whether the sperm of a heterozygote or hemizygote are phenotypically distinct with respect to this antigen. The study will make use of a Robertsonian translocation {Rb(6.16) or Rb} resource in which heterozygotes show a TRD and in which Spam1 expression is reduced, as well as hemizygotes for an overexpressed Spam1 transgene to be constructed. The effects of overexpression of Spam1 on the fertilizing competence of sperm will also be addressed by taking advantage of the hemizygotes to generate transgenic mice for functional studies. Thus the proposal addresses several important problems in reproductive biology: the biochemical equivalence of sperm in a heterozygote, a fundamental question in testicular biology; the compartmentalization of mRNA and its regulation by co-translational assembly and sequences in the 3' UTR, and the impact of overexpression of Spam1 on mammalian fertilization. The following hypotheses will thus be tested. 1) There is a bimodal distribution of the quantity of Spam1 mRNA in spermatids and protein in sperm from Rb(6.16)/+ mice, compared to a unimodal one in +/+ and Rb/Rb mice, with the lower RNA deposits in Rb-bearing cells. The latter can be rescued by transgenesis to eliminate their reduced fertilizing ability and TRD in Rb carriers; 2) Mice hemizygous for an overexpressed Spam1 transgene will reveal whether or not Spam1 mRNA and protein are equally shared among conjoined spermatids to produce biochemically and functionally equivalent or different sperm. Homozygotes will determine the effect of overexpression on the rates of investment penetration, zona binding, and fertilization, 3) Co-translational assembly, sequences mediating GPI-linkage of Spam 1, and sequences in the 3' UTR of the gene mediate its mRNA compartmentalization which leads to TRD. The work promises to impact current thinking about critical principles underlying haploid gene expression, elucidate a mechanism for meiotic drive, increase our understanding of the molecular mechanisms that underpin mammalian fertilization, and could identify a genetic basis for altered sperm-egg interactions involved in human fertility.

描述：（从申请人的摘要中进行扫描） 我们的研究是确定a）pH-20或精子的表达 粘附分子1，spam1（遗传命名法），揭示了一种机制 传输率失真（TRD），b）如果保留了垃圾邮件基因产物 在单个精子和控制其mRNA的调节机制中 分布，c）SPAM1的过表达如何影响精子功能。这 提案旨在确定鼠spam1基因产物是否是精子膜 蛋白质（spam1）在相结合的精子中共享 相对于此而言，杂合子或半合子在表型上是不同的 抗原。该研究将利用罗伯逊主义的易位{RB（6.16）或 rb}杂合子显示trd的资源，其中spam1表达为 减少了过表达的Spam1转基因的半合子 构造。 Spam1过表达对受精的影响 精子的能力也将通过利用 半合子产生转基因小鼠进行功能研究。因此 提案解决了生殖生物学的几个重要问题： 精子在杂合子中的生化等效性，这是一个基本问题 睾丸生物学； mRNA的分区及其调节 3'UTR中的共同组装和序列，以及 Spam1对哺乳动物施肥的过表达。以下假设 因此将进行测试。 1）数量的双峰分布 与RB（6.16）/+小鼠的精子中的精子中的垃圾邮件mRNA和蛋白质相比 +/ +和rb/rb小鼠中的单峰型，较低的RNA沉积在rb中 细胞。可以通过转基因挽救后者，以消除其减少 RB载体中的施肥能力和TRD； 2）小鼠半合子 过表达的Spam1转基因将揭示SPAM1 mRNA和蛋白质是否揭示 同样在相互结合的精子中共享生物化学和 功能等效或不同的精子。纯合子将确定 过表达对投资渗透率，Zona Binding的影响， 和受精，3）共同组装，序列介导 垃圾邮件1的GPI链接，基因3'UTR中的序列介导了其mRNA 导致TRD的隔室化。这项工作有望影响电流 思考单倍体基因表达的临界原理， 阐明减数分裂驱动的机制，增加我们对 支撑哺乳动物施肥的分子机制，可以鉴定 与人类生育能力有关的改变精子相互作用的遗传基础。