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Publication Date
2025-06-23
Type
doctoral thesis
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Abstract
Centromeres are chomosomal loci essential for correct chromosome segregation during cell division. Defects in centromere function can lead to aneuploidy and cancer. This chromosomal locus is epigenetically defined by the presence of the histone variant Centromere Protein A (CENP-A), which replaces histone H3 in the octamer core. Previous studies in Drosophila melanogaster and mouse have highlighted the importance of the inheritance of paternal and maternal CENP-A in the embryo. In this thesis, Bos taurus was selected as a mammalian model system to study centromeres during early embryogenesis because of its similarity to humans in terms of the timings of cell division and activation of the zygotic genome. Initially, we report the expression of multiple CENP-A variants in Bos taurus from analyses of RNA sequencing databases. This finding poses the question of whether any of these CENP-A-like transcripts has evolved a specific function, perhaps in embryogenesis or in the germ line. A long term goal was to study the consequences of perturbing centromere function on the first mitosis and on embryonic development. To enable such experiments, we developed a number of tools, such as methods for CENP-A depletion using Trim-Away, Fluoresent In Situ Hybridisation (FISH) probes to label centromere DNA sequences and markers of S- and M-phases in the bovine cell cycle. Parental genome clustering at the pronuclear interface was previously described as a critical step to prevent chromosome missegregation and aneuploidy in human and bovine embryos. Yet, despite the critical function of centromeres in chromosome segregation, the position and spatial organisation of the centromeres at this time was unknown. In this thesis, using bovine in vitro fertillisation (IVF) studies, we identify 20 hours post insemination as the time point when parental pronuclei cluster and are in S-phase. We find that centromeres are unexpectedly positioned away from the pronuclear interface at this time. This localisation poses questions about how centromeres become re-arranged during chromatin condensation to allow faithful chromosome segregation during the first mitotic division. The agricultural sector suffers economic losses resulting from poor bovine IVF outcomes. The causes of such poor success rates in bovine assisted reproduction remain unknown. We propose that monitoring cell cycle progression at 20 hours post insemination could be used in future bovine IVF studies as a potential indicator of successful outcomes.
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Publisher
University of Galway
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Attribution-NonCommercial-NoDerivatives 4.0 International