Human Genetics Institute of New Jersey

My thesis project specifically involves the development of an Aurkc^-/- mouse model for studying human AURKC function, and using that model to characterize AURKC splice variants and sterility-associated mutants in oocytes.  I hypothesized that we could use Aurkc^-/- mice to study the human protein because they are 87% identical in their catalytic domain.  I proved this hypothesis by showing that both the human and mouse protein were both able to rescue the loss of function phenotype in the Aurkc^-/- oocytes (misalignment at metaphase, loss of substrate phosphorylation, metaphase I arrest).  Three splice variants of AURKC have been previously identified in testis tissue, but their presence in oocytes was unknown.  I collaborated with Dr. Nathan Treff at the Reproductive Associates of New Jersey where I performed qRT-PCR on single oocytes samples and found expression of all three variants in every single sample.  The next step was to analyze the functional relevance of these variants in my mouse oocyte model.  Through immunocytochemistry, confocal microscopy, and live imaging, I showed that the three variants do not function equally in female meiosis and have the best function when expressed simultaneously as is found in oocytes. 

            The final portion of my thesis research involves 3 mutations in the coding region of AURKC discovered in sterile men with 100% tetraploid sperm with multiple flagella.  I created Gfp-tagged cRNA constructs of the mutant AURKC and injected these into my Aurkc^-/- oocytes to characterize the phenotypes.  I found one mutant that does not localize or support meiotic progression, but when co-expressed with the wild type AURKC to simulate the heterozygous condition, actually causes cells to prematurely extrude a polar body.  Another mutant localizes only some of the time and is able to phosphorylate the substrate although to a lesser extent than the wild type.  These findings indicate that one mutant may have partial dominant negative function while the other might be only a partial loss of function which could both be extremely useful analytical tools in the laboratory for studying AURKC function in meiosis.