Live-Cell Imaging of Human Oocytes and Regulation of Cohesin Removal in Meiosis II

Abstract

The “maternal age effect” describes the striking increase in risk of miscarriage and chromosomally abnormal embryos and children from women older than 35. Studies in mice have shown that the protein complex Cohesin is reduced in an age-dependent manner. This protein complex ensures accurate segregation during both rounds of meiosis, by holding the chromosomes together and providing a counteracting force to spindle microtubules. Despite a wealth of knowledge generated from human oocytes, there are few live-cell studies, in part due to the paucity of material. This thesis uses human oocytes specifically donated for research to assess the effect of age on alignment of chromosomes at metaphase I and -II, which is a predictor of missegregation. Using high-resolution live-cell microscopy, it is clear that increased age is associated with chromosomes that are misaligned in metaphase I and -II. At metaphase II, eggs arrest until they are fertilised by sperm. The regulation of how chromosomes separate at this point is poorly understood. While the bulk of Cohesin is removed in anaphase I, a small amount is “protected” by Shugoshin 2 and remains between the centromeres to allow for faithful segregation in meiosis II. Currently, there is poor experimental work to support the hypotheses proposed to explain how the mechanisms that protect Cohesin in meiosis I are removed in meiosis II. One of these hypotheses is the requirement of spindle tension to separate the protector, Shugoshin 2, from Cohesin in meiosis II. Here, I show that spindle tension is not required for deprotection and that alternative models should be considered, such as one which suggests that higher-order regulation around meiosis II resumption is orchestrated by the Anaphase Promoting Complex and its coactivator Cdc20, APC/CCdc20, a protein complex that is active at anaphase onset.