"The Aurora Kinase proteins are a family of serine/threonine kinases that have been shown to play fundamental roles in controlling M phase progression in somatic cells. Aurora Kinase A protein is known to be vital for proper spindle assembly and therefore, chromosome segregation. Previous reports have shown that Aurora Kinase B is vital for proper completion of karyokinesis and cytokinesis in somatic cells. The role of Aurora Kinase C in somatic cells has been found to be less clear; however it appears to play an important role in spermatogenesis. Little is known about the role of these Aurora Kinase proteins mouse oocytes during oogenesis, and even less is known about them in embryos during early development. The objective of these studies was to characterize the presence, localization, and function of Aurora Kinase B and Aurora Kinase C protein and mRNA in mouse oocytes and early embryos. Oocytes and embryos were collected from hormone stimulated CF-1 mice and cultured for varying amounts of time. Cumulus denuded oocytes were either fixed for immunofluorescence microscopy studies, lysed for analysis of mRNA levels through the use of reverse transcription PCR (rtPCR) and quantitative rtPCR (q-rtPCR), lysed for protein analysis employing Western blotting, treated with Aurora Kinase protein inhibitor drugs, or microinjected with a siRNA pool targeting Aurora Kinase B. Samples were processed for immunofluorescence analysis using markers of spindle morphology (tubulins), Aurora Kinase B, Aurora Kinase C, and Aurora Kinase B activity (phospho Histone H3). Analysis of relative levels of Aurora Kinase B and Aurora Kinase C mRNA were assessed by rtPCR and q-rtPCR methods. Western blotting was performed on oocytes and early embryos to quantitate Aurora Kinase B and C protein levels. Aurora Kinase inhibitors, Hesperadin and ZM447439, were added to culture medium with mouse oocytes to determine the effects of the loss of Aurora Kinase activity. siRNAs were used to inhibit Aurora Kinase B mRNA in early embryos to ascertain the effect of functional loss of this transcript on embryo development. Marked differences were observed in the localization of Aurora Kinase B when unfertilized oocytes or pre-zygotic genome activation (ZGA) embryos were compared to post-ZGA samples. There was no evidence of Aurora Kinase B protein localized to the mitotic spindle or resultant midbody in oocytes and blastomeres of early embryos. Western blotting results supported this data. Embryos fixed post-ZGA demonstrated Aurora Kinase B localization at midbodies between dividing cells, as was found in mouse embryonic fibroblast control cells. Aurora Kinase C protein was not demonstrable in mouse oocytes, embryos, or control cells using immunocytochemistry or Western techniques. In contrast, Aurora Kinase B and Aurora Kinase C mRNAs were both found to be present in mouse oocytes and early embryos. q-rtPCR data further supported this finding for Aurora Kinase B and revealed that the mRNA level of this transcript is relatively constant until ZGA at which point a decrease relative to the earlier stages was observed. Transcript levels recovered post-ZGA and were comparable to the pre-ZGA levels. Functional inhibition of the Aurora Kinase family through the use of Hesperadin or ZM447439 demonstrated the importance of these proteins for proper microtubule and spindle organization, as these drugs disrupted both karyokinesis and cytokinesis in mouse oocytes and blastomeres of early embryos. Aurora Kinase B targeting siRNA also established a role for Aurora Kinase mRNA in embryos at the 2-cell stage based on the disruption of the cell cycle that was observed in treated embryos. Given earlier reports showing the vital role of the Aurora Kinase proteins in proliferating somatic cells, knowledge of the expression and localization of these proteins in oocytes and early embryos is vital for the understanding of cell cycle control during oogenesis and early embryogenesis. Our data indicate that Aurora Kinase B mRNA may also play a role in early embryogenesis, demonstrating a need for analysis of transcript as well as protein. Our results, as well as outcomes of future experiments suggested by our work, may provide significant insight into cell cycle regulation differences between somatic and embryonic cells. These differences may have a profound impact upon manipulated embryos including those reconstructed through somatic cell nuclear transfer. "
| Identifer | oai:union.ndltd.org:wpi.edu/oai:digitalcommons.wpi.edu:etd-theses-1850 |
| Date | 28 May 2010 |
| Creators | Lima, Christine A |
| Contributors | Eric W. Overström, Advisor, Joseph B. Duffy, Committee Member, Raymond L. Page, Committee Member |
| Publisher | Digital WPI |
| Source Sets | Worcester Polytechnic Institute |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Masters Theses (All Theses, All Years) |
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