MICROTUBULE/KERATIN INTERACTIONS DURING SPERMATOGENESIS

精细胞壁炉架既是生殖生物学家和细胞生物学家着迷的来源，也是被忽视的来源。近年来，我们实验室有了一些值得注意的进展，重新引起了人们对套垫在精子核形状和尾部形态发生中的分子组织和功能的关注。例如，从啮齿动物身上分离出完整的套索，发现套架的核环含有角蛋白9(K9)，套架和精子尾轴丝的微管蛋白亚型与Sak 57有关(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，因为它的基因只在两个部位表达：足垫表皮和睾丸。此外，人类K9螺旋杆的一个亚域的点突变会导致表皮松解性掌跖角化皮病，这是一种与家族性内部恶性肿瘤(食道、支气管、乳腺和卵巢)有关的皮肤病。这一建议中的工作假设是：1.K9基因敲除的雄性小鼠将是“无披肩”的。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。追求以下目标：目标1将测试K9对于组装套筒的核周环是必不可少的。目的2将完成Sak57基因的克隆，为进一步研究该角蛋白在减数分裂前期和精子发生中的作用奠定基础。目的3将测试临时K9基因的表达与外套架的发育相关，以及肌动蛋白在顶体后位置稳定外套架。目的4将测试套架和尾巴中的微管蛋白异构体异常是否可以解释azh/azh突变体(精子头部形状异常)的核形状和尾巴异常。目的5将测试粗线期精母细胞中SAK57皮质下骨架的破坏将导致基因表达的停止和减数分裂的停止。设计K9表达缺失的小鼠模型，对生殖生物学和癌症研究都很重要。

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.