Aurora Kinase-A Regulates Oocyte Spindle Organization During Early Stages of Meiosis. (2009)

Aurora kinases (AURKs) are a family of serine/threonine protein kinases that play pivotal roles in regulation of mitosis and meiosis. Previous studies revealed that inhibition of mouse oocyte AURK activities using the AURK inhibitor ZM447439, which inhibits all AURK isoforms, resulted in meiotic spindle disorganization, aberrant chromatin condensation, and interference of histone-H3 phosphorylation at various serine residues. However, specific functions of each individual AURK isoform during mouse oocyte meiosis are unknown. To begin to delineate isoform-specific functions, Aurk gene expression during mouse oocyte maturation was determined using quantitative real-time PCR. Results indicate that mouse oocytes contain transcripts for all three Aurk isoforms: A, B and C, with AurkA being predominant. Therefore, to begin to determine functions of AurkA, germinal vesicle-intact (GV-intact) oocytes were placed in 40μM isobutymethylxanthine (IBMX) to inhibit meiosis, microinjected with AurkA siRNA, and incubated for 24h prior to removal of IBMX to allow processing prior to meiotic resumption. Quantitative real-time PCR and Western blot analysis verified that AurkA siRNA significantly reduced AurkA transcript and protein levels prior to re-initiation of meiosis, yielding a 75-fold and 60-fold reduction, respectively. Conversely, AurkB and AurkC expression were not influenced. Oocyte developmental comparisons and statistical analysis with unpaired Student's t-test demonstrated that AurkA knockdown had no impact on GVBD at 2hr (Control 90% versus Treatment 92%; p>0.05), but significantly inhibited progression to MII at 17h (60% n=335) compared to controls (79% n=263; p<0.05). Chromosomal spreading of oocytes that did progress to MII demonstrated a significant increase in abnormal chromosome segregation following AurkA siRNA compared to controls (46% versus 24%, respectively, p<0.05). To begin to determine causes for meiotic arrest prior to MII, immunocytochemical analysis was performed on MI oocytes following 7h of culture after removal from IBMX using β-tublin antibody, pericentrin antibody, and Hoescht to visualize microtubules, microtubule organizing centers (MTOCs), and chromatin, respectively. Confocal microscopic examination revealed that although microtubule polymerization occurred, organization of the meiotic spindle was perturbed, with improper localization of MTOCs and misaligned chromatin following AurkA siRNA compared to controls (68% versus 14%, respectively, p<0.05). A similar pattern of abnormal spindle morphology was observed in AurkA siRNA oocytes arrested prior to MII at 17h, suggesting that meiotic arrest took place around MI, prior to the anaphase transition. Further studies utilizing Western blot analysis demonstrated a significant decrease in histone-H3 phosphorylation at ser10 following AurkA siRNA compared to controls, while no reduction was observed at ser28. These data suggest that AURKA is an oocyte histone-H3 kinase and plays a critical role in regulating chromosome and spindle dynamics during early stages of meiosis prior to the anaphase transition. (platform)