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.
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