Molecular basis of aneuploidy: Functions of the aurora kinases, Rad51c, and CREB binding protein in female meiosis
ABSTRACT Aneuploidy is a clinically important condition that can affect the health and fertility of an individual as well as his or her offspring. Many cases of aneuploidy arise as a result of mistakes during meiosis in females. This thesis used the mouse as a model to study the roles of the aurora kinases, Rad51c, and CREB Binding Protein (CBP) during female meiosis. Quantitative RT-PCR, immunocytochemistry, and microinjection of fluorescently-tagged aurora kinases, allowed us to determine the expression and localization of all three aurora kinases in mouse oocytes. AURKA localizes to microtubule organizing centers (MTOCs) and meiotic spindles at metaphase I (Met I) and metaphase II (Met II), suggesting it may play a role in organizing both meiotic spindles. Both AURKA and AURKB are found at the midbody during telophase I (TI) and formation of the first polar body, indicating that they may participate in the asymmetric cytokinesis that occurs. AURKB is concentrated on the kinetochores at Met I, but absent from the chromosomes at Met II, while AURKC, the meiotic homolog, is found on the chromosomes during Met I and Met II. Treatment of oocytes with the pan-aurora kinase inhibitor ZM447439 caused defects in meiotic progression and chromosome alignment at Met I and Met II. Microinjection of a fluorescently-tagged AURKB rescued the chromosome alignment defect seen at Met I. These studies suggest that although AURKB is the primary aurora kinase responsible for chromosome alignment at Met I, AURKC is responsible for chromosome alignment at Met II. Through the creation of Rad51c hypomorphic mice, we show that RAD51C appears to play an early role in prophase I (PI) that affects ovulation and alignment of chromosomes at Met I, as well as a later role in Holliday Junction (HJ) resolution. Both functions may contribute to aneuploidy. Finally, the creation of mice lacking CBP specifically in the oocyte, reveals a role for CBP in female fertility. CBP acetylates histone H2B in the transcriptionally active Germinal Vesicle (GV) intact oocyte and its activity may be required either directly or indirectly for spindle formation at Met II. Taken together, these studies reveal specific roles for the aurora kinases, Rad51c, and CBP during female meiosis. Perturbation of any of these molecules can cause mistakes in meiosis leading to aneuploidy.