BASIS FOR MALE INFERTILITY: MOLECULAR MODELS

男性不育的基础：分子模型

• 批准号: 6520930
• 负责人: STEPHEN H PILDER
• 金额: $ 18.01万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-07-15 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6520930
• 关键词: cilium /flagellum motility; dynein ATPase; epididymis; fertility; flagellum; gene deletion mutation; gene expression; gene targeting; genetic models; genetically modified animals; germ cells; human genetic material tag; human tissue; laboratory mouse; male; male reproductive system disorder; molecular pathology; phenotype; protein localization; protein protein interaction; protein structure function; sperm motility; spermatogenesis; testis; yeast two hybrid system

The mammalian sperm flagellum produces a regulated locomotive force that allows the sperm cell to proceed through a set of complex environments in the female genital tract and penetrate the egg investments. However, little beyond morphological description is known about the control of mammalian sperm flagellar formation, stability, or movement. The P.I. has mapped and isolated an axonemal dynein heavy chain, Dnahc8, that is expressed at high levels in a testis-specific fashion in wildtype mice. The map position of Dnahc8 and its mode of expression in the testes of certain male-sterile recombinant lines of mice is completely consistent with the flagellar organization and waveform defects, known as "whipless" and "curlicue", respectively, displayed in an allele-specific manner by sperm from these recombinant mice. Thus, Dnahc8 is a strong candidate for the allele-dependent expression of the "whipless" and "curlicue" mutations. Because Dnahc8 expression appears to have profound effects on both sperm tail biogenesis and function, Dnahc8 offers a key to unraveling the mechanisms underlying mammalian sperm tail assembly and/or motility. In order to test the hypothesis that Dnahc8 is responsible for expression of "whipless" and "curlicue", and to begin to clarify the cellular mechanisms underlying these phenotypes, the P.I. will determine the subcellular location of Dnahc8 in both mutant and control testis and cauda epididymal sperm through immuno-light and electron microscopy, and isolate potential spermiogenic binding partners of Dnahc8 through two-hybrid analysis (Specific Aim I). Concurrent with the performance of Specific Aim I, the P.I. will directly test the postulated role(s) of Dnahc8 in sperm tail assembly and function via its targeted deletion from the mouse genome (Specific Aim II). In order to assess the potential significance of Dnahc8 to human male infertility, the P.I. will localize the human ortholog of Dnahc8 to a sub-chromosomal region within the human genome, and will ascertain if this ortholog demonstrates polymorphisms diagnostic of human male infertility related to sperm flagellar defects (Specific Aim III). These experiments will reveal information crucial to our understanding of the molecular basis of mammalian sperm tail development and function in fertilization, while contributing to our ability to diagnose and treat human male infertility.

哺乳动物精子鞭毛产生受调节的运动力，使精子细胞能够穿过女性生殖道中的一系列复杂环境并穿透卵子投资。 然而，除了形态学描述之外，人们对哺乳动物精子鞭毛形成、稳定性或运动的控制知之甚少。 P.I.绘制并分离出轴丝动力蛋白重链 Dnahc8，它在野生型小鼠中以睾丸特异性方式高水平表达。 Dnahc8的图谱位置及其在某些雄性不育重组小鼠系睾丸中的表达模式与鞭毛组织和波形缺陷完全一致，分别被称为“鞭毛”和“花饰”，由来自这些重组小鼠的精子以等位基因特异性方式显示。 因此，Dnahc8 是“whipless”和“curlicue”突变的等位基因依赖性表达的有力候选者。由于 Dnahc8 表达似乎对精子尾部生物发生和功能都有深远影响，因此 Dnahc8 为揭示哺乳动物精子尾部组装和/或运动的机制提供了关键。 为了检验 Dnahc8 负责“whipless”和“curlicue”表达的假设，并开始阐明这些表型背后的细胞机制，P.I.将通过免疫光学和电子显微镜确定突变型和对照睾丸以及附睾尾部精子中 Dnahc8 的亚细胞位置，并通过双杂交分析分离 Dnahc8 的潜在生精结合伴侣（具体目标 I）。在执行特定目标 I 的同时，P.I.将通过从小鼠基因组中定向删除 Dnahc8，直接测试 Dnahc8 在精子尾部组装和功能中的假设作用（具体目标 II）。 为了评估 Dnahc8 对人类男性不育症的潜在意义，P.I.将把 Dnahc8 的人类直向同源物定位到人类基因组内的亚染色体区域，并将确定该直向同源物是否表现出与精子鞭毛缺陷相关的人类男性不育症的多态性诊断（具体目标 III）。 这些实验将揭示对我们理解哺乳动物精子尾部发育和受精功能的分子基础至关重要的信息，同时有助于我们诊断和治疗人类男性不育症的能力。