Using the oocyte-specific Socs3-knockout mouse model, we studied the effect of Socs3 gene in oogenesis and early embryonic development, employing techniques such as oocyte in vitro maturation, embryo in vitro culture, embryo micromanipulations, immunofluorescence and confocal microscope, as well as molecular biology techniques including single cell genotyping. Whilst the first results suggested a potentially important role for this specific gene, it however proved technically impossible to unequivocally determine its precise role. However, given literature precedents and observations from these preliminary studies, we were able to establish a link to the mTOR signalling pathway, which proved more retractable to our research (employing essentially similar techniques). mTOR-regulated cap-dependent translation of mRNAs with TOP motifs in their 5’UTR regions was previously described to play a role during oocyte meiosis. Using confocal microscopy techniques we were able to confirm that mTOR mediated inactivation of a specific protein translation inhibitor protein (4EBP1 - via a mechanism of targeted phosphorylation) regulates cap-dependent translation during 8- to 16-cell transition of preimplantation mouse embryo development. This is the developmental point at which constituent embryonic cells become spatially segregated for the first time, as some cells are on the outside of the embryo (and contribute to later extraembryonic tissues) while other cells are inside and completely surrounded by other cells (and are pluripotent and thus can contribute to the subsequent foetal tissues). When we cultured embryos in media with relevant chemical inhibitors (of mTOR and of translation initiation complexes), prior to this transition, we identified a decrease in the number of generated inner cells at 16-cell stage embryos, which persists until the early blastocyst stage, but then in a process of remarkable regulation such deficits were compensated for prior to implantation. Using single cell embryo microinjection techniques, we altered the expression levels of selected genes involved in cap-dependent translation and recapitulated the previously observed phenotype. We then studied the molecular mechanism in more details, using low input mass spectrometry followed by functional testing of selected candidate proteins, as well as live cell confocal microscopy. Building on these findings, we uncovered and described an additional role of the mTOR and p38 signalling pathways in translational regulation in the late blastocyst stage, playing role in specification of the primitive endoderm cell lineage. In addition, we adapted a polysomal fractionation protocol for low sample input and combined it with RNA sequencing. This allowed us to map actively translating RNAs at the genome-wide level in mouse oocytes during meiosis and compare the oocyte translatomes between young and aged females, shedding more light on the cap-dependent and –independent translational regulation at this developmental stage.
The results of this project were published in 6 peer reviewed publications. One manuscript, constituting the main theme of the project, is at the advanced stage of preparation and is expected to be submitted to a peer-reviewed journal by the end of 2021. Furthermore, the results were presented at three international conferences (1 oral and two poster presentations), one invited oral presentation at an international conference (after finishing the project), and two seminars at the local academic institutions. In addition, the results were disseminated at 4 public engagement activities (for secondary school teachers, students, and primary school children). All this was achieved with two maternity breaks and Covid-19 pandemics lockdown (with attendant closed child day care).
