MICROTUBULE/KERATIN INTERACTIONS DURING SPERMATOGENESIS

精细胞manchette对生殖和细胞生物学家来说既是一个迷人的来源，也是一个被忽视的来源。近年来，我们实验室取得了一些值得注意的进展，重新引起了人们对精子核形成和尾部形态发生中manchette的分子组织和功能的关注。例如，从啮齿类动物中分离完整的manchette，发现manchette的核周环含有角蛋白9 (K9)， manchette和精子尾轴突的微管蛋白同型体与Sak 57（用于生精细胞/精子相关角蛋白，Mr 57 kDa）相关。K9和Sak 57将被证明在精子发生中具有深远的发育意义。K9是特别有趣的，因为它的基因表达只发生在两个部位：脚垫表皮和睾丸。此外，人类K9螺旋杆亚域的点突变可引起表皮松解性掌足底角化病，这是一种与家族性内部恶性肿瘤（食道、支气管、乳腺和卵巢）相关的皮肤病。本提案的工作假设是：1。K9基因敲除后，雄鼠将“少曼切特”。2. Sak 57基因敲除的雄性小鼠的精子发生不会超过减数分裂前期。目标1将检验K9对于装配核壳的核周环是必不可少的。Aim 2将完成Sak 57的cDNA克隆，这是进一步敲除研究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将测试时间K9基因表达与顶体发育相关，肌动蛋白在顶体后位置稳定顶体。Aim 4将检验在azh/azh突变体（精子头型异常）中，颏部和尾部异常的微管蛋白同工型是否解释了核形状和尾部异常。Aim 5将测试粗线精母细胞中Sak 57皮层下框架的破坏将导致基因表达停止和减数分裂停止。K9表达为零的工程小鼠模型对于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.