In contrast to other species, localized maternal mRNAs are not believed to be prominent features of mammalian oocytes. Due to the lack of transcription in the fully-grown oocyte, critical oocyte processes including cell cycle progression, chromosome segregation, formation and maintenance of the meiotic metaphase spindles, maternal mRNA recruitment and degradation, fertilization and egg activation are all under post-transcriptional, translational, or post-translational control. Despite advances in understanding mechanisms regulating the translational control of cytoplasmic maternal mRNAs, it is unknown whether localized maternal mRNAs exist in mouse oocytes and what mechanisms are responsible for their control. Maternal mRNAs were isolated from metaphase II (MII) mouse oocytes, microsurgically-removed MII spindle-chromosome complexes, and enucleated MII oocytes and analyzed by cDNA microarray analysis. The analysis identified enrichment for maternal mRNAs encoding spindle and other proteins on the mouse oocyte metaphase II (MII) spindle. Maternal mRNAs involved in cellular compartments and processes related to the cytoskeleton, chromatin/nucleus, and cellular signaling were enriched on the MII spindle. Using immunofluorescence and confocal microscopy, MIS18A, a protein encoded by a spindle-localized maternal mRNA, was confirmed to be associated with the MII spindle along with components of the ribosome translational machinery. The key translational regulator, EIF4EBP1, was observed to undergo a dynamic and complex spatially regulated pattern of phosphorylation at sites that regulate its association with EIF4E and its ability to repress translation. These phosphorylation variants appeared at different positions along the spindle at different stages of meiosis. Overexpression of EIF4EBP1 mutants had a profound effect on the maintenance of MII arrest. Approximately 24% of oocytes expressing a phosphodeficient (Threonine 69 to Alanine) EIF4EBP1 mutant underwent spontaneous activation, suggesting EIF4EBP1 phosphorylation is important for translation of maternal mRNAs and maintenance of MII arrest. These results indicate that dynamic spatially restricted patterns of EIF4EBP1 phosphorylation may promote localized mRNA translation to support spindle formation, maintenance, function, and other nearby processes. Regulated EIF4EBP1 phosphorylation at the spindle may help coordinate spindle formation with progression through the cell cycle. The discovery that EIF4EBP1 may be part of an overall mechanism that integrates and couples cell cycle progression to mRNA translation and subsequent spindle formation and function may be relevant to understanding mechanisms leading to diminished oocyte quality, and potential means of avoiding such defects. The localization of maternal mRNAs at the spindle is evolutionarily conserved between mammals and other vertebrates and is also seen in mitotic cells, indicating that EIF4EBP1 control of localized mRNA translation is likely key to correct segregation of genetic material across cell types. / Molecular Biology and Genetics
| Identifer | oai:union.ndltd.org:TEMPLE/oai:scholarshare.temple.edu:20.500.12613/3489 |
| Date | January 2014 |
| Creators | Romasko, Edward Joseph |
| Contributors | Latham, Keith, Engel, Nora, Soprano, Dianne R., Litvin, Judith, Sapienza, Carmen, Habas, Raymond |
| Publisher | Temple University. Libraries |
| Source Sets | Temple University |
| Language | English |
| Detected Language | English |
| Type | Thesis/Dissertation, Text |
| Format | 124 pages |
| Rights | IN COPYRIGHT- This Rights Statement can be used for an Item that is in copyright. Using this statement implies that the organization making this Item available has determined that the Item is in copyright and either is the rights-holder, has obtained permission from the rights-holder(s) to make their Work(s) available, or makes the Item available under an exception or limitation to copyright (including Fair Use) that entitles it to make the Item available., http://rightsstatements.org/vocab/InC/1.0/ |
| Relation | http://dx.doi.org/10.34944/dspace/3471, Theses and Dissertations |
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