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Simpson, Eva (2025) Dissecting the ultrastructure of meiotic chromosomes and the role of the chromosome periphery using advanced imaging. PhD thesis, University of Nottingham.

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Abstract

Understanding the three-dimensional structure of chromosomes is essential for elucidating the mechanisms that govern their behaviour during cell division. However, detailed ultrastructural analysis of chromosomes in oocytes has been limited by technical challenges associated with imaging large, spherical cells. This thesis describes the development and application of a three-dimensional correlative light and electron microscopy (3D-CLEM) pipeline to overcome these challenges and enable high-resolution visualisation of chromosomal architecture during meiosis.

The 3D-CLEM workflow was first established and optimised using mitotic cells, allowing for precise correlation between fluorescence and electron microscopy images. This approach led to the generation of a high-resolution 'mitotic atlas', enabling the structural classification and quantitative comparison of mitotic chromosomes. Notably, this work contributed to the identification and characterisation of a novel chromatin state—plateaud compaction—recently reported in Cisneros-Soberanis & Simpson et al., (2024).

Following successful implementation in mitotic systems, the pipeline was applied to mouse oocytes to investigate the organisation and function of the chromosome periphery during meiosis. Immunolabelling and correlative imaging confirmed the presence of the proliferation marker Ki-67 at the periphery of meiotic chromosomes, establishing its role as a key organiser of this structural domain. Targeted disruption of the periphery revealed its critical importance for maintaining chromosome integrity, with its removal resulting in altered chromatin morphology and disorganisation within the meiotic spindle. This exciting discovery led to a second publication currently in pre-print and under review with Nature Comms (Simpson et al., 2024)

Finally, the pipeline was extended to the study of human oocytes, where it enabled the first three-dimensional reconstructions of meiotic chromosomes. These early datasets demonstrate the challenges associated with analysis of these cell types and defines key characteristics to make this work possible. Following development of the technique for handling human oocytes the feasibility of 3D-CLEM for human oocyte research was proven and the data provides a foundation for future investigations into chromosomal architecture in human meiosis.

In summary, this work establishes and validates a novel 3D-CLEM approach for high-resolution structural analysis of chromosomes in large mammalian oocytes. The pipeline has been successfully applied to both mouse and human cells, yielding new insights into the role of the chromosome periphery and enabling enhanced visualisation of chromosome structure during meiosis. This methodological advancement opens new avenues for investigating the ultrastructure of chromosomes and associated sub-nuclear compartments in rare or challenging cell types.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Booth, Daniel McAinsh, Andrew Bates, David Alberio, Ramiro
Keywords:	Chromosomes; Ultrastructural analysis; Light and electron microscopy pipeline; Mammalian oocytes; Meiosis
Subjects:	QS-QZ Preclinical sciences (NLM Classification) > QU Biochemistry
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Medicine
Item ID:	81697
Depositing User:	Simpson, Eva
Date Deposited:	10 Dec 2025 04:40
Last Modified:	10 Dec 2025 04:40
URI:	https://eprints.nottingham.ac.uk/id/eprint/81697

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