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Socs3 gene in oocyte maturation and fertilisation - a novel link between inflammation and infertility

Periodic Reporting for period 1 - OOCSOCS (Socs3 gene in oocyte maturation and fertilisation - a novel link between inflammation and infertility)

Reporting period: 2017-06-01 to 2019-05-31

Approximately one in six couples worldwide experience infertility problems. One of the biggest risk factors is female age, affecting the oocyte and consequently the embryo quality, as well as the correct functions of placenta. As the average age of women at the first pregnancy increases, fertility issues are expected to become even more frequent, resulting in clinical consequences for assisted reproductive technology treatments. In order to effectively treat fertility problems, it is crucial to understand molecular mechanisms of oogenesis and early embryonic development. Due to clear ethical considerations associated with research on humans, the mouse is a suitable model for such studies as it shares mechanistic characteristics of oocyte and embryonic development, as well as age-associated fertility problems, with humans.
Reproductive aging and problematic female fertility are linked to increased inflammatory signalling. We initially focused on a previously identified candidate gene involved in the regulation of the inflammatory response, as well as placenta development, Suppressor of cytokine signalling 3 (Socs3). However, our research led us towards a much more fundamental role of the mTOR intra-cellular signalling pathway, also associated with inflammation and aging. Specifically, we studied the process of mTOR-regulated cap-dependent translation (a specialized and highly regulated form of cellular protein synthesis) and its effect on the oocyte maturation and the preimplantation embryonic development stages. We uncovered a novel fundamental effect of mTOR signalling on the spatial positioning of cells in the early embryo. Such spatial positioning is tightly associated with the segregation of individual and functional specialization of distinct cell lineages in the early embryo and mTOR signalling regulates the number of the progenitor cells that will give rise to the embryo itself (found encapsulated inside the developing embryonic mass), as oppose to outer-residing cells that ultimately form extraembryonic tissue (i.e. placenta). We also developed a highly optimised low input protocol for genome-wide profiling of actively translated mRNAs (molecular intermediates between genes and synthesis of the proteins they encode), applied it to maturing mouse oocytes and identified important differences in those from aged versus young females; with potential consequences for fertility. In addition, we studied the novel interplay of the mTOR and p38-mitogen-activated-kinase pathways in translation regulation at the late blastocyst stage (i.e. prior to uterine implantation), affecting the specification of the primitive endoderm extraembryonic cell lineage (needed to support the post-implantation developmental stages).

As a direct consequence of this award and the above summarised experiments progression, this project has also substantially contributed to the career progression of the postdoctoral researcher, Dr. Gahurova, towards becoming an independent group leader at the University of South Bohemia in Czech Republic.
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).
We uncovered a novel role of mTOR pathway in mouse preimplantation development. As mTOR signalling is one of the major pathways associated with aging, metabolism and nutrient sensing, our results contribute to the increased understanding and consequent treatment of reproductive aging, and maternal malnutrition, as well as the optimisation of the assisted reproduction technologies such as in vitro fertilisation (IVF) of oocytes.
Active mTOR-regulated cap-dependent translation in mitotic cells in mouse preimplantation embryo