Formation of fertile oocytes requires orderly segregation of chromosomes during meiosis, which depends on proper assembly of a bipolar spindle and its actions. The PI has identified a novel kinase IAK1, one of very few components identified so far in the female meiotic spindle. The proposed study is to test the hypothesis that IAK1 is an important player of the meiotic chromosome segregation machinery.
Specific Aim 1 is to localize the IAK1 protein and kinase activity in female germ cells throughout meiosis. In addition, IAK1 expression patterns and activity will be examined in mouse mutants defective in either checkpoint pathways or MPF/MAPK signal transduction pathways.
Specific Aim 2 is to disrupt the function of IAK1 by injection of antisense oligonucleotides, kinase-negative mutant form of IAK1, and anti-IAK1 antibodies into oocytes. Spindle formation and phosphorylation as well as chromosome segregation will be assessed in the treated oocytes.
Specific Aim 3 is to determine the functional domains of the IAK1 kinase by introducing wild-type and mutant forms of IAK1 into maturing oocytes. The mutated IAK1 proteins will be taqged with FLAG octapeptides or green fluorescent protein and localized in the injected oocytes by confocal microscopy. Simultaneously, the kinase activity will be examined in the oocyte extracts.
Specific Aim 4 is to identify the IAK1-interacting proteins in meiotic germ cells. Immunoprecipitation and Western blotting will examine interaction of IAK1 with known IAK1-interacting proteins in other systems, such as Cdc37 and p100. Novel IAK1-interacting proteins will be identified in yeast two-hybrid screening system using a mouse oocyte/egg cDNA library.
| Shuda, Kristy; Schindler, Karen; Ma, Jun et al. (2009) Aurora kinase B modulates chromosome alignment in mouse oocytes. Mol Reprod Dev 76:1094-105 |
| Fenderson, Bruce A; De Miguel, Maria P; Pyle, April D et al. (2006) Staining embryonic stem cells using monoclonal antibodies to stage-specific embryonic antigens. Methods Mol Biol 325:207-24 |
| Spruck, Charles H; de Miguel, Maria P; Smith, Adrian P L et al. (2003) Requirement of Cks2 for the first metaphase/anaphase transition of mammalian meiosis. Science 300:647-50 |
