Meiotic spindles are critical to ensure chromosome segregation during gamete formation. Oocytes lack centrosomes and use alternative microtubule-nucleation mechanisms for spindle building. How these mechanisms are regulated is still unknown. Aurora kinase A (AURKA) is essential for mouse oocyte meiosis because in pro-metaphase I it triggers microtubule organizing-center fragmentation and its expression compensates for the loss of the two other Aurora kinases (AURKB/AURKC). Although knockout mouse models were useful for foundational studies, AURK spatial and temporal functions are not yet resolved. We provide high-resolution analyses of AURKA/AURKC requirements during meiotic spindle-building and identify the subcellular populations that carry out these functions: 1) AURKA is required in early spindle assembly and later for spindle stability, whereas 2) AURKC is required in late pro-metaphase, and 3) Targeted AURKA constructs expressed in triple AURK knockout oocytes reveal that spindle pole-localized AURKA is the most important population controlling spindle building and stability mechanisms.

• Keywords
• cell biology,
• Publication type
• Journal Article MeSH

The Aurora protein kinases are well-established regulators of spindle building and chromosome segregation in mitotic and meiotic cells. In mouse oocytes, there is significant Aurora kinase A (AURKA) compensatory abilities when the other Aurora kinase homologs are deleted. Whether the other homologs, AURKB or AURKC can compensate for loss of AURKA is not known. Using a conditional mouse oocyte knockout model, we demonstrate that this compensation is not reciprocal because female oocyte-specific knockout mice are sterile, and their oocytes fail to complete meiosis I. In determining AURKA-specific functions, we demonstrate that its first meiotic requirement is to activate Polo-like kinase 1 at acentriolar microtubule organizing centers (aMTOCs; meiotic spindle poles). This activation induces fragmentation of the aMTOCs, a step essential for building a bipolar spindle. We also show that AURKA is required for regulating localization of TACC3, another protein required for spindle building. We conclude that AURKA has multiple functions essential to completing MI that are distinct from AURKB and AURKC.

• MeSH
• Spindle Apparatus genetics   MeSH
• Aurora Kinase A genetics   MeSH
• Aurora Kinase B genetics   MeSH
• Aurora Kinase C genetics   MeSH
• Cell Nucleus Division genetics   MeSH
• Fetal Proteins genetics   MeSH
• Humans MeSH
• Meiosis genetics   MeSH
• Mice MeSH
• Oocytes growth & development   metabolism   MeSH
• Microtubule-Organizing Center metabolism   MeSH
• Polo-Like Kinase 1 MeSH
• Spindle Poles genetics   MeSH
• Protein Serine-Threonine Kinases genetics   MeSH
• Microtubule-Associated Proteins genetics   MeSH
• Cell Cycle Proteins genetics   MeSH
• Proto-Oncogene Proteins genetics   MeSH
• Chromosome Segregation genetics   MeSH
• Gene Expression Regulation, Developmental genetics   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Aurkb protein, mouse MeSH Browser
• Aurkc protein, mouse MeSH Browser
• Aurora Kinase A MeSH
• Aurora Kinase B MeSH
• Aurora Kinase C MeSH
• Fetal Proteins MeSH
• Protein Serine-Threonine Kinases MeSH
• Microtubule-Associated Proteins MeSH
• Cell Cycle Proteins MeSH
• Proto-Oncogene Proteins MeSH
• TACC3 protein, mouse MeSH Browser

Formation of the hatching mouse blastocyst marks the end of preimplantation development, whereby previous cell cleavages culminate in the formation of three distinct cell lineages (trophectoderm, primitive endoderm and epiblast). We report that dysregulated expression of Wwc2, a genetic paralog of Kibra/Wwc1 (a known activator of Hippo-signaling, a key pathway during preimplantation development), is specifically associated with cell autonomous deficits in embryo cell number and cell division abnormalities. Division phenotypes are also observed during mouse oocyte meiotic maturation, as Wwc2 dysregulation blocks progression to the stage of meiosis II metaphase (MII) arrest and is associated with spindle defects and failed Aurora-A kinase (AURKA) activation. Oocyte and embryo cell division defects, each occurring in the absence of centrosomes, are fully reversible by expression of recombinant HA-epitope tagged WWC2, restoring activated oocyte AURKA levels. Additionally, clonal embryonic dysregulation implicates Wwc2 in maintaining the pluripotent epiblast lineage. Thus, Wwc2 is a novel regulator of meiotic and early mitotic cell divisions, and mouse blastocyst cell fate.

• Keywords
• blastocyst cell number, cell division, cell lineage decision, cell-fate, oocyte maturation, preimplantation mouse embryo,
• Publication type
• Journal Article MeSH

Homologous chromosome segregation during meiosis I (MI) in mammalian oocytes is carried out by the acentrosomal MI spindles. Whereas studies in human oocytes identified Ran GTPase as a crucial regulator of the MI spindle function, experiments in mouse oocytes questioned the generality of this notion. Here, we use live-cell imaging with fluorescent probes and Förster resonance energy transfer (FRET) biosensors to monitor the changes in Ran and importin β signaling induced by perturbations of Ran in mouse oocytes while examining the MI spindle dynamics. We show that unlike RanT24N employed in previous studies, a RanT24N, T42A double mutant inhibits RanGEF without perturbing cargo binding to importin β and disrupts MI spindle function in chromosome segregation. Roles of Ran and importin β in the coalescence of microtubule organizing centers (MTOCs) and MI spindle assembly are further supported by the use of the chemical inhibitor importazole, whose effects are partially rescued by the GTP hydrolysis-resistant RanQ69L mutant. These results indicate that RanGTP is essential for MI spindle assembly and function both in humans and mice.

• Keywords
• RanGTP, importazole, importin β, meiosis I, oocyte,
• MeSH
• Spindle Apparatus physiology   MeSH
• beta Karyopherins genetics   metabolism   MeSH
• Nuclear Proteins genetics   metabolism   MeSH
• Meiosis physiology   MeSH
• Microtubules metabolism   MeSH
• Mutation MeSH
• Mice MeSH
• Oocytes cytology   metabolism   MeSH
• Cell Cycle Proteins genetics   metabolism   MeSH
• ran GTP-Binding Protein genetics   metabolism   MeSH
• Chromosome Segregation MeSH
• Guanine Nucleotide Exchange Factors genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• beta Karyopherins MeSH
• Nuclear Proteins MeSH
• Cell Cycle Proteins MeSH
• ran GTP-Binding Protein MeSH
• Rcc1 protein, mouse MeSH Browser
• Guanine Nucleotide Exchange Factors MeSH

BACKGROUND: SIRT1 histone deacetylase acts on many epigenetic and non-epigenetic targets. It is thought that SIRT1 is involved in oocyte maturation; therefore, the importance of the ooplasmic SIRT1 pool for the further fate of mature oocytes has been strongly suggested. We hypothesised that SIRT1 plays the role of a signalling molecule in mature oocytes through selected epigenetic and non-epigenetic regulation. RESULTS: We observed SIRT1 re-localisation in mature oocytes and its association with spindle microtubules. In mature oocytes, SIRT1 distribution shows a spindle-like pattern, and spindle-specific SIRT1 action decreases α-tubulin acetylation. Based on the observation of the histone code in immature and mature oocytes, we suggest that SIRT1 is mostly predestined for an epigenetic mode of action in the germinal vesicles (GVs) of immature oocytes. Accordingly, BML-278-driven trimethylation of lysine K9 in histone H3 in mature oocytes is considered to be a result of GV epigenetic transformation. CONCLUSIONS: Taken together, our observations point out the dual spatiotemporal SIRT1 action in oocytes, which can be readily switched from the epigenetic to non-epigenetic mode of action depending on the progress of meiosis.

• Keywords
• Epigenetics, Histone code, In vitro maturation, Oocyte, SIRT1, Sirtuin 1,
• Publication type
• Journal Article MeSH

Meiotic oocytes lack classic centrosomes and, therefore, bipolar spindle assembly depends on clustering of acentriolar microtubule-organizing centers (MTOCs) into two poles. However, the molecular mechanism regulating MTOC assembly into two poles is not fully understood. The kinase haspin (also known as GSG2) is required to regulate Aurora kinase C (AURKC) localization at chromosomes during meiosis I. Here, we show that inhibition of haspin perturbed MTOC clustering into two poles and the stability of the clustered MTOCs. Furthermore, we show that AURKC localizes to MTOCs in mouse oocytes. Inhibition of haspin perturbed the localization of AURKC at MTOCs, and overexpression of AURKC rescued the MTOC-clustering defects in haspin-inhibited oocytes. Taken together, our data uncover a role for haspin as a regulator of bipolar spindle assembly by regulating AURKC function at acentriolar MTOCs in oocytes.

• Keywords
• Aurora kinase, Haspin, MTOC, Oocyte, Spindle,
• MeSH
• Spindle Apparatus metabolism   MeSH
• Aurora Kinase C metabolism   MeSH
• Intracellular Signaling Peptides and Proteins antagonists & inhibitors   metabolism   MeSH
• Metaphase MeSH
• Mice MeSH
• Oocytes metabolism   MeSH
• Microtubule-Organizing Center metabolism   MeSH
• Protein Serine-Threonine Kinases antagonists & inhibitors   metabolism   MeSH
• Protein Transport MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Aurora Kinase C MeSH
• Haspin protein, mouse MeSH Browser
• Intracellular Signaling Peptides and Proteins MeSH
• Protein Serine-Threonine Kinases MeSH

Polo-like kinase 1 (PLK1) orchestrates multiple events of cell division. Although PLK1 function has been intensively studied in centriole-containing and rapidly cycling somatic cells, much less is known about its function in the meiotic divisions of mammalian oocytes, which arrest for a long period of time in prophase before meiotic resumption and lack centrioles for spindle assembly. Here, using specific small molecule inhibition combined with live mouse oocyte imaging, we comprehensively characterize meiotic PLK1's functions. We show that PLK1 becomes activated at meiotic resumption on microtubule organizing centers (MTOCs) and later at kinetochores. PLK1 is required for efficient meiotic resumption by promoting nuclear envelope breakdown. PLK1 is also needed to recruit centrosomal proteins to acentriolar MTOCs to promote normal spindle formation, as well as for stable kinetochore-microtubule attachment. Consequently, PLK1 inhibition leads to metaphase I arrest with misaligned chromosomes activating the spindle assembly checkpoint (SAC). Unlike in mitosis, the metaphase I arrest is not bypassed by the inactivation of the SAC. We show that PLK1 is required for the full activation of the anaphase promoting complex/cyclosome (APC/C) by promoting the degradation of the APC/C inhibitor EMI1 and is therefore essential for entry into anaphase I. Moreover, our data suggest that PLK1 is required for proper chromosome segregation and the maintenance of chromosome condensation during the meiosis I-II transition, independently of the APC/C. Thus, our results define the meiotic roles of PLK1 in oocytes and reveal interesting differential requirements of PLK1 between mitosis and oocyte meiosis in mammals.

• MeSH
• Anaphase-Promoting Complex-Cyclosome metabolism   MeSH
• Nuclear Envelope metabolism   MeSH
• Kinetochores metabolism   MeSH
• Microscopy, Confocal MeSH
• Meiosis physiology   MeSH
• Mice MeSH
• Oocytes growth & development   MeSH
• Microtubule-Organizing Center metabolism   MeSH
• Image Processing, Computer-Assisted MeSH
• Polo-Like Kinase 1 MeSH
• Protein Serine-Threonine Kinases metabolism   MeSH
• Cell Cycle Proteins metabolism   MeSH
• Proto-Oncogene Proteins metabolism   MeSH
• Chromosome Segregation physiology   MeSH
• Blotting, Western MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Anaphase-Promoting Complex-Cyclosome MeSH
• Protein Serine-Threonine Kinases MeSH
• Cell Cycle Proteins MeSH
• Proto-Oncogene Proteins MeSH

Regulation of mRNA translation by cytoplasmic polyadenylation is known to be important for oocyte maturation and further development. This process is generally controlled by phosphorylation of cytoplasmic polyadenylation element binding protein 1 (CPEB1). The aim of this study is to determine the role of Aurora kinase A in CPEB1 phosphorylation and the consequent CPEB1-dependent polyadenylation of maternal mRNAs during mammalian oocyte meiosis. For this purpose, we specifically inhibited Aurora kinase A with MLN8237 during meiotic maturation of porcine oocytes. Using poly(A)-test PCR method, we monitored the effect of Aurora kinase A inhibition on poly(A)-tail extension of long and short cyclin B1 encoding mRNAs as markers of CPEB1-dependent cytoplasmic polyadenylation. Our results show that inhibition of Aurora kinase A activity impairs neither cyclin B1 mRNA polyadenylation nor its translation and that Aurora kinase A is unlikely to be involved in CPEB1 activating phosphorylation.

• MeSH
• Aurora Kinase A metabolism   MeSH
• Cyclin B1 genetics   MeSH
• mRNA Cleavage and Polyadenylation Factors chemistry   metabolism   MeSH
• Phosphorylation MeSH
• Meiosis * MeSH
• RNA, Messenger metabolism   MeSH
• Oocytes enzymology   metabolism   MeSH
• Polyadenylation MeSH
• Sus scrofa metabolism   MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Aurora Kinase A MeSH
• Cyclin B1 MeSH
• mRNA Cleavage and Polyadenylation Factors MeSH
• RNA, Messenger MeSH