The long term goal of this project is to identify biological roles for mammalian glycans in development and cancer. Glycosyltransferase gene mutants of mice, embryonic stem (ES) cells and CHO cells will be used to investigate the basis of cell-type specific blocks to differentiation and transformation caused by alterations in specific glycans. Our recent studies show that 1) oocyte-specific knockout of complex and hybrid N-glycans compromises ovulation and oocyte developmental competence, but fertilization, blastogenesis and implantation proceed in the absence of these glycans; 2) the bisecting GlcNAc on complex N-glycans of non-hepatocyte glycoprotein(s) promotes hepatocarcinogenesis and liver regeneration after partial hepatectomy; and 3) deletion of mouse protein O-fucosyltransferase 1 generates a canonical Notch signaling phenotype identifying the Pofut1 gene as an essential component of the Notch signaling pathway. We now propose in Specific Aim 1 to investigate the molecular basis of the requirement for complex and hybrid N-glycans in oogenesis, and to identify roles of mucin O-glycans and O-fucose glycans in early development after oocyte-specific deletion of the core 1 beta3GalT-1 (C1galt1) or Pofut1 genes.
In Specific Aim 2 the hypothesis that a triantennary, GlcNAc-terminating complex N- glycan is required for spermatogenesis will be investigated by deletion of a novel GlcNAc-transferase gene that is expressed almost exclusively in testis and by eliminating complex and hybrid N-glycans from spermatocytes, spermatogonia or Sertoli cells. The Pofut1 gene will also be deleted in spermatocytes and spermatogonia or Sertoli cells to identify roles for Notch signaling in spermatogenesis.
In Specific Aim 3 the hypothesis that the Mgat3 gene may function as a tumor suppressor in mammary gland will be tested in the MMTV-Polyoma middle T (PyMT) mammary tumor model which metastasizes to lung and in cell signaling assays. In addition, roles for N- and 0-fucose glycans and Notch signaling will be investigated in mammary gland development and transformation by selective deletion of the Mgat1 and Pofut1 genes in mammary epithelia before puberty using MMTVCre, or during pregnancy using whey acidic protein (WAP) Cre recombinase. ? ?
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