MICROTUBULE/KERATIN INTERACTIONS DURING SPERMATOGENESIS

精子细胞的柄是生殖和细胞生物学家的一个既迷人又被忽视的来源。 近年来，在我们的实验室中有几个值得注意的发展，重新引起了人们对精子核成形和尾部形态发生中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乳突，发现乳突的核周环含有角蛋白9（K9），乳突和精子尾部轴丝的微管蛋白亚型与Sak 57（生精细胞/精子相关角蛋白，Mr 57 kDa）相关。 K9和Sak 57在精子发生中具有重要的发育意义。 K9是特别感兴趣的，因为它的基因表达只发生在两个位点：脚垫表皮和睾丸。 此外，人K9的螺旋杆的亚结构域中的点突变引起表皮增生性掌跖角化病，这是一种与家族性内部恶性肿瘤（食管、支气管、乳腺和卵巢）相关的皮肤病。在这个建议的工作假设是：1。K9基因敲除雄性小鼠将“无曼切特”。2. Sak 57基因敲除雄性小鼠的精子发生不会超过减数分裂前期。目标1将测试K9对于Manchette的核周环的组装是必不可少的。 目的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必要步骤。 目的3将测试的时间K9基因的表达与发展的肩袖和肌动蛋白稳定的肩袖在顶体后的位置。 目的4将测试异常微管蛋白亚型在肩和尾部帐户核成形和尾部异常azh/azh突变体（异常精子头部形状）。 目的5将测试粗线期精母细胞中Sak 57皮层下框架的破坏将导致基因表达的停止和减数分裂的停滞。 工程小鼠模型，其中K9的表达是无效的，是重要的生殖生物学和癌症研究。

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.