Translational control in mouse folliculogenesis and oocyte development

The production of healthy, euploid oocytes from a finite pool of primordial follicles is crucial to the reproductive success of the mammalian female. Central to the production of a healthy oocyte is correct expression of a number of proteins which govern processes such as cell cycle, chromosome segregation and cell differentiation. Control of the maternal mRNA pool during oocyte maturation is crucial for the correct temporal and spatial expression of protein necessary for these processes. Control of the mRNA pool is assisted through the action of sequence-specific RNA-binding proteins, including those of the Musashi (Msi) family, which has been widely described to be a family of translational repressor proteins in stem cells while studies in the mammalian germline have been limited. Utilising both mRNA and protein expression analysis, both Msi-1 and Msi-2 were found to be expressed throughout mouse folliculogenesis, with Msi-1 primarily expressed with the cytoplasm of the mouse oocyte and granulosa cells, while Msi-2 is expressed within the nucleus of these cell types. Transgenic overexpression mice for both Msi homologs were utilised to determine the downstream mRNA targets of both homologs in the mouse ovary. Through the utilisation of Native protein-RNA immunoprecipitation techniques, coupled with mRNA and protein expression techniques such as qPCR and immunoblotting performed on WT and tg ovaries, Msi-1 was found to act as a translational control protein capable of both translational activating the c-mos transcript and translationally repressing the ccng2, robo3 and msi-2 transcript in the ovaries of 5 week old mice. Functional analysis performed through use of a Ligation-mediated poly(A) tail length (LM-PAT) assay, revealed the differences in these two functions could result from the ability of Msi-1 and/or its interacting proteins to manipulate the length of the poly(A) tail of target transcripts, which governs mRNA translation efficiency. While Msi-2 was found to act as a translational repressor of cdkn1c in the mouse ovary, a novel function of Msi-2 as a transcriptional control and/or splicing factor of m-numb was uncovered by this study, with Msi-2 found to complex with the transcriptional repressor and splicing proteins SFPQ and Nono. Global transcriptome analysis of ovaries excised from WT, tgMsi-1 and tgMsi-2 mice revealed the RNA-binding proteins to primarily influence the expression of genes governing processes such as cell cycle, chromosome segregation, cellular differentiation, organism development and cell survival, with all of these processes being tightly controlled in order to produce a healthy oocyte and subsequent zygote. Therefore, the studies outlined in this thesis represent the first in depth expression and functional analysis of the RNA-binding proteins Msi-1 and Msi-2, with results highlighting the complex network of transcriptional and translational control that exists within the mouse ovary.