MICROTUBULE/KERATIN INTERACTIONS DURING SPERMATOGENESIS

精子发生过程中微管/角蛋白的相互作用

• 批准号: 6521142
• 负责人: ABRAHAM L KIERSZENBAUM
• 金额: $ 23.76万
• 依托单位: CITY COLLEGE OF NEW YORK
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6521142
• 关键词: acrosome; actins; confocal scanning microscopy; developmental genetics; expression cloning; gene expression; gene targeting; genetically modified animals; keratin; laboratory mouse; laboratory rat; male; mass spectrometry; meiosis; microtubules; molecular assembly /self assembly; mutant; phenotype; protein isoforms; protein protein interaction; protein structure function; sperm; spermatogenesis; tubulin

The spermatid manchette is both a source of fascination and neglect for reproductive and cell biologists. In recent years, there have been severable notable developments in our laboratory that have rekindled attention to the molecular organization and function of the manchette in sperm nuclear shaping and tail morphogenesis. Examples include the isolation of intact manchettes from rodents, the findings that the manchette's perinuclear ring contains keratin 9 (K9) and that tubulin isoforms of the manchette and sperm tail axoneme are associated to Sak 57 (for spermatogenic cell/sperm-associated keratin, Mr 57 kDa). K9 and Sak 57 will prove to be of profound developmental significance in spermatogenesis. K9 is of particular interest because its gene expression occurs in two sites only: the footpad epidermis and testis. In addition, point mutations in a subdomain of the helical rod of human K9 cause epidermolytic palmoplantar keratoderma, a skin disease linked to familial internal malignancies (esophagus, bronchi, breast and ovary). The working hypotheses in this proposal are: 1. K9 gene knockout male mouse will be "manchette-less". 2. Spermatogenesis in a Sak 57 knockout male mouse will not advance beyond meiotic prophase. The following aims are pursued: Aim 1 will test that K9 is essential for the assembly of the perinuclear ring of the manchette. Aim 2 will complete the cDNA cloning of Sak 57, an essential step for further knockout studies to determine the role of this keratin in meiotic prophase and spermiogenesis. Aim 3 will test that the temporal K9 gene expression correlates with the development of the manchette and that actin stabilizes the manchette at a postacrosomal location. Aim 4 will test that abnormal tubulin isoforms in the manchette and tail account for nuclear shaping and tail abnormalities in the azh/azh mutant (for abnormal spermatozoon headshape). Aim 5 will test that the disruption of the Sak 57 subcortical framework in pachytene spermatocytes will result in the cessation of gene expression and arrest of meiosis. Engineering mouse models in which the expression of K9 is null, is important for both reproductive biology and cancer research.

精子细胞 manchette 既令生殖生物学家和细胞生物学家着迷，又被忽视。 近年来，我们实验室取得了一些显着的进展，重新引起了人们对精子核成形和尾部形态发生中 manchette 的分子组织和功能的关注。 例子包括从啮齿类动物中分离出完整的 manchette，发现 manchette 的核周环含有角蛋白 9 (K9)，并且 manchette 的微管蛋白亚型和精子尾轴丝与 Sak 57 相关（对于生精细胞/精子相关角蛋白，Mr. 57 kDa）。 K9 和 Sak 57 将被证明在精子发生中具有深远的发育意义。 K9 特别令人感兴趣，因为它的基因表达仅发生在两个部位：足垫表皮和睾丸。 此外，人类 K9 螺旋杆子结构域的点突变会导致表皮松解性掌跖角化病，这是一种与家族性内部恶性肿瘤（食道、支气管、乳房和卵巢）相关的皮肤病。该提案的工作假设是： 1. K9基因敲除的雄性小鼠将“无manchette”。 2. Sak 57 敲除雄性小鼠的精子发生不会进展到减数分裂前期之后。追求以下目标： 目标 1 将测试 K9 对于 manchette 核周环的组装至关重要。 目标 2 将完成 Sak 57 的 cDNA 克隆，这是进一步敲除研究以确定该角蛋白在减数分裂前期和精子发生中的作用的重要步骤。 目标 3 将测试颞 K9 基因表达与 manchette 的发育相关，以及肌动蛋白将 manchette 稳定在顶体后位置。 目标 4 将测试 manchette 和尾部中异常的微管蛋白亚型是否解释了 azh/azh 突变体（精子头部形状异常）中的核形状和尾部异常。 目标 5 将测试粗线期精母细胞中 Sak 57 皮质下框架的破坏是否会导致基因表达停止和减数分裂停滞。 K9 表达无效的工程小鼠模型对于生殖生物学和癌症研究都很重要。