Mapping the 3D architecture of meiotic chromosomes

Develop advanced correlative light electron microscopy (CLEM) pipelines and use them to define, for the first time, the 3D architecture of all human oocyte (female egg)
chromosomes, in oocytes undergoing both normal and aberrant division.

WHY THIS IS IMPORTANT: For largely unknown reasons ~half of human oocytes display chromosome segregation errors, a leading cause of aneuploidy. This directly contributes to the 13% of women with fertility problems, 25% of women that will suffer a miscarriage and 0.3% of live births that carry a constitutional aneuploidy. In most other systems defective chromosome structure is a known cause of aneuploidy, but in human oocytes this remains almost completely unexplored. This is because of three main obstacles: A) Restricted access to human oocytes; B) limited availability of established techniques/tools, particularly those for ultra-structural analyses; C) lack of reference for "normal" human oocyte division - i.e. it is difficult to fully understand features of aberrant division without fully characterising normal oocyte division first.

Our lab is one of the few in the UK that does have access to human oocytes. Therefore, this project provides a unique and exciting opportunity to perform ground-breaking research by addressing points B and C above.
AIMS:
AIM1 - Establish CLEM imaging pipelines. Mammalian oocyte chromosomes occupy <2% of the total oocyte volume. A classic challenge in analysing these small and randomly positioned organelles by electron microscopy (EM) has been finding them. Conventional EM approaches would require the capture and imaging of nearly 1000 consecutive sections - an impossible economic and equipment burden. We will address this long-standing problem by developing bespoke correlative imaging pipelines, allowing the Euclidian position of chromosomes to be recorded within the oocyte using a light microscope, prior to EM processing. Essentially this provides a more realistic target for 3D electron microscopy imaging and the pathway for successful CLEM. Additional EM methods to be explored will include cryo-CLEM, an equally powerful, but potentially more future-proof approach. In the first instance these techniques will be established using pig oocytes, an excellent substitute for those of human.

AIM2 - Define the 'meiotic chromosome atlas'. The methods established in aim1 will next be optimised for analysing human oocytes. Here we will generate nanometre accurate digital
reconstructions of each and every human oocyte chromosome. This will be performed during all stages of meiotic division allowing the retrieval of the basic structural parameters surrounding chromosome compaction, condensation and global morphology. This dataset will provide the first ever reference for chromosome ultra-structure during normal human oocyte division.

AIM3 - Investigate the geometry of aneuploid chromosomes. Finally, we will now be in a position to exploit the 'atlas' and compare differences in chromosome ultra-structure, between oocytes undergoing normal and aberrant division. A full geometric survey will be performed using computational analyses and modelling.

OUTCOMES: Studying the triggers of aneuploidy in human oocytes is widely regarded as one of the most important and rapidly emerging fields in research. This high-impact work will contribute to our understanding of the molecular mechanisms underpinning infertility, spontaneous abortion and birth defects. Furthermore, the advanced imaging tools and data-sets embedded within will become a pioneering resource, not only for those in closely-related fields, but also for the wider cell and developmental biology community.