BASIS FOR MALE INFERTILITY: MOLECULAR MODELS

哺乳动物的精子鞭毛产生一种受调节的动力，使精子细胞能够通过女性生殖道中一系列复杂的环境，并穿透卵子。然而，除了形态学描述之外，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.已经绘制并分离了轴突动力蛋白重链Dnahc8，该链在野生型小鼠中以睾丸特异性方式高水平表达。Dnahc8在某些雄性不育重组小鼠睾丸中的图谱位置及其表达方式与这些重组小鼠精子以等位基因特异性方式显示的鞭毛组织和波形缺陷（分别称为“无鞭”和“曲状”）完全一致。因此，Dnahc8是“无鞭”和“curlicue”突变的等位基因依赖表达的强有力候选者。由于Dnahc8的表达似乎对精子尾部的生物发生和功能都有深远的影响，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。为了验证Dnahc8负责“无鞭”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.将通过免疫光镜和电子显微镜确定Dnahc8在突变和对照睾丸和附睾尾精子中的亚细胞位置，并通过双杂交分析分离Dnahc8潜在的生精结合伙伴（Specific Aim I）。在执行Specific Aim I的同时，P.I.将直接测试Dnahc8通过从小鼠基因组中靶向删除在精子尾部组装和功能中的假设作用（Specific Aim II）。为了评估Dnahc8对人类男性不育的潜在意义，P.I.将把Dnahc8的人类同源定位到人类基因组的一个亚染色体区域，并将确定该同源是否证明了与精子鞭毛缺陷相关的人类男性不育的多态性诊断（Specific Aim 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的能力。

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.