AURORA KINASES REGULATE MOUSE OOCYTE CHROMATIN REMODELING AND HISTONE H3 PHOSPHORYLATION

Abstract

Defects within machinery of the oocyte are responsible for the majority of human aneuploidies. Therefore, examination of factors regulating oocyte chromatin dynamics is important to fully understand this complex process and circumvent resulting embryonic aneuploidy, infertility and congenital defects. Aurora kinases (AURK) are a family of serine/threonine protein kinases involved in regulation of chromosome condensation, spindle dynamics and cytokinesis; all critical components responsible for regulating cellular aneuploidy. However, little attention has been paid to AURK in regard to mammalian oocyte maturation. Real time quantitative rtPCR identified Aurk-A, B and C transcript in germinal vesicle intact (GVI), germinal vesicle breakdown (GVBD), metaphase I (MI) and metaphase II (MII) oocytes. To determine affects of AURK inhibition on oocyte maturation, GVI oocytes were collected from eCG stimulated CF1 mice and matured in HTF medium supplemented with 0.3% w/v BSA and varying doses of AURK inhibitor ZM447439 (ZM). Inhibition of AURK had no affect on oocyte GVBD at 2h or development to MI at 7h at any dose examined, compared to control treatments. However, concentrations of 2.5, 5, 10 and 20 μM ZM prevented all oocytes from extruding the first polar body and progressing to MII. Concentrations of 0.675 and 1.25 μM ZM allowed a portion of oocytes to extrude the first polar body (35% and 31%, respectively), which was significantly less than the percentage of MII oocytes obtained from control treatments (73%), P<0.01. Immunocytochemical (ICC) analysis of ZM treatment oocytes indicated cells were arrested at an MI-like state. This arrest was not due to absence of the meiotic spindle, as β-tubulin staining indicated microtubules polymerized around condensed chromatin. However, chromatin was scattered around the metaphase plate compared to vehicle treated controls. This pattern was observed for all oocytes obtained from treatments containing 2.5, 5, 10 and 20 μM ZM. To examine affects of AURK inhibition on oocyte chromosome condensation in greater detail, GVI oocytes were matured to MI in the presence of 10 μM ZM and chromosomal spreads analyzed. Inhibition of AURK resulted in oocytes with a significant reduction in normal chromosome condensation (0%) compared to control treatments (95%), as evidenced by the inability to resolve distinct bivalents. Additionally, to determine effects of AURK inhibition on homologue separation and segregation, in vitro matured MI and anaphase I (AI) oocytes were matured to MII in presence or absence of 10 μM ZM. Treatment of MI and AI oocytes with ZM resulted in significantly fewer cells progressing to MII compared to controls, P<0.05 (MI- 53% vs 80%, AI- 58% vs 73%). Arrested oocytes, as well as those that progressed to MII in the presence of ZM, displayed scattered chromatin throughout the spindle. To determine if aberrant chromatin remodeling following AURK inhibition could be the result of alternations in histone- H3 phosphorylation, Western blot analysis and ICC were performed utilizing phospho specific histone-H3 antibodies. Inhibition of AURK with 10 μM ZM resulted in absence of histone-H3 phosphorylation at ser10 and ser28. These data suggest a ZM-sensitive AURK is an oocyte histone-H3 kinase and is regulating chromatin remodeling. Further research will examine unique roles for specific AURK isoforms during oocyte maturation. (platform)