ABSTRACTGENETIC AND MOLECULAR CONTROL OF OOCYTE FUNCTIONByAshley L. SeveranceA high-quality oocyte will successfully accomplish three critical tasks: segregate chromosomes, reprogram its own genome and that of the incoming sperm to an embryonic state, and support the metabolism and early development of the embryo. This dissertation focuses on two critical determinants of oocyte quality, maintenance of the meiotic spindle and mRNA regulation during meiotic maturation. Chapter 3 of this dissertation focuses on the role of the translational repressor, EIF4EBP1, at the meiotic spindles. I show that blocking EIF4EBP1 phosphorylation, which is normally permissive of translation, disrupts the first meiotic spindle, and that inhibiting Polo-like kinase 1 (PLK1) affects EIF4EBP1 phosphorylation and spindle formation at both meiotic divisions. This exciting result suggests PLK1 as an important regulatory focus for controlling EIF4EBP1 and mRNA translation locally at the spindle, to enable the supply of essential proteins during meiotic maturation. Chapter 4 of this dissertation builds on the theme of understanding maternal mRNA regulation by characterizing the dynamic changes in the pool of mRNAs during oocyte maturation and addresses how this varies with oocyte quality. By comparing maturation changes in the transcriptome between two inbred strains (C57BL/6J and DBA/2J) and F1 hybrids between the two strains (BDF1), I discover differences in maternal mRNA regulation associated with superior BDF1 oocyte characteristics (i.e., oocyte hybrid vigor). This includes differences in regulating mRNAs related tomitochondrial physiology and histone production. I also show that many of the differencesbetween the three genotypes arise during maturation and thus in the absence of transcription,indicating differences in regulating mRNA degradation. Overall, a combination of processesunderlies the differential regulation of maternal mRNA in BDF1 oocytes compared to parentalstrains. A small amount of transgressive gene expression is seen, but the most prominentmechanisms responsible for differential mRNA regulation in BDF1 oocytes include “blending”(F1 expression levels intermediate between parental expression levels) and additivedominance. Interestingly, up to 25% of the mRNAs differ significantly between genotypes at theMII stage. This includes many proposed markers of oocyte quality, for which mRNAs differentialexpression between strains exceeded what was reported for differences associated with oocytequality. Because all three genotypes are fertile, this suggests that single markers may not bereliable indicators of oocyte quality. Overall, these results clearly show that the oocyte utilizescomplex regulatory mechanisms at both the meiotic spindles and throughout the ooplasm. Thisdissertation opens the door for many potential future directions to probe further intounderstanding these oocyte-specific adaptations. In the future, this research along with otherstudies could be useful to develop therapies to improve fertility outcomes in both agriculturalspecies and in humans.
Read
