title: Investigating chromosome ageing in mouse dictyate oocytes
Creators: name: Moolnangdeaw, Apiwat
Publisher: name: The University of Edinburgh
Year: 2025
Subjects: subject: chromosomes
subject: mouse model
subject: age-dependent changes
subject: chromosome 18
subject: aneuploidy
subject: mature oocytes
subject: oocyte cohesion
subject: fluorescence in-situ hybridisation
subject: ageing oocytes

description: Ageing mammalian oocytes are known to be prone to transmit an abnormal set of chromosomes to the next generation, a condition called aneuploidy. One of the potential contributory factors is the loss of chromosome cohesion during the oocytes' prolonged meiotic arrest. Mature oocytes from aged mice have decreased amounts of cohesin, a structural protein that helps maintaining chromosome cohesion, associated with their chromosomes. However, these studies on the age-dependent loss of chromosome cohesion in oocytes were based on mature oocytes due to the challenging task of accessing pre-ovulatory oocytes. Therefore, it is currently unknown at which stage of oogenesis age-dependent loss of chromosome cohesion occurs. It is essential to understand when this age-dependent loss of oocyte cohesion happens to help us avoid, prevent or even treat the subsequent complications. Thus, the main objective of my thesis is to investigate when age-dependent chromosome cohesion loss occurs during oogenesis in mice. Before investigating mouse oocytes, I tested a fluorescence in-situ hybridisation (FISH) method to monitor sister chromatid separation in a simpler experimental model to ensure the method’s compatibility with the detection of chromosome cohesion loss. FISH was carried out on Tex19.1 mouse embryonic stem cells (mES cells) that were previously shown to have defects in chromosome cohesion at the mitotic stage. Here I show that FISH signals hybridised to specific regions on mES cell chromosomes were significantly further apart in Tex19.1-/- mES cells, indicating increased sister chromatid separation. More importantly, FISH on mES has proved to be a promising method to study chromosome cohesion loss in mammalian cells. To carry out a more in-depth study of chromosome cohesion in oocytes in fixed tissue, FISH was performed on Tex19.1 and Ubr2 mouse ovarian sections, two genes implicated in regulating cohesion in oocytes. However, there was no detectable increase in chromatid distances at the peri-telomere in Tex19.1-/- and Ubr2-/- oocytes in primordial follicles. It is possible that Tex19.1 and Ubr2 regulate cohesion at later stage of oogenesis. Notably, these experiments showed the capability of FISH to probe chromatid separation in meiotic cells in fixed tissue. To understand the effect of ageing on chromatid cohesion in mouse oocytes at different follicular stages, I then performed FISH on 3-month, 6-month, and 9-month mouse ovarian tissue sections. Dictyate oocytes, in primordial, primary, and secondary follicular stages, were analysed for chromatid separation at the peri-centromeric and peri-telomeric regions of two chromosomes. These ageing oocytes showed significant increases in inter-chromatid distances at chromosome 1 from 3-month and 6-month to 9-month oocytes in all three follicular stages and at both peri-centromere and peri-telomere. Surprisingly, inter-chromatid distance increases were also detected during follicle development from primordial to secondary. However, no significant inter-chromatid distance change was detected in chromosome 18 of oocytes in primordial follicles. Taken together, although ageing may have different effects on chromosomes, age-dependent effects on chromosome arm separation were detected in dictyate oocytes. In conclusion, these findings have provided new insights into our understanding of chromatid cohesion in female mouse germ cells and suggest that combining FISH with high-resolution 3D microscopy can allow the investigation of age-dependent changes in chromosome cohesion in dictyate oocytes. Ultimately, this research serves as a beneficial resource for the future study of ageing chromosome cohesion in pre-ovulatory oocytes in mammals, including humans.