Insights into the three-dimensional ultrastructure of uterine epithelial cells during pregnancy

Abstract

To initialise implantation in pregnancy, the blastocyst must first attach to the luminal uterine epithelial cells (UECs), which undergo extensive morphological changes from the time of fertilisation to implantation collectively known as the Plasma Membrane Transformation (PMT). This thesis utilises 3-Dimensional Scanning Electron Microscopy (3DSEM) and Correlative Light and Electron Microscopy (CLEM) to investigate UECs during the PMT in the rat. 3DSEM revealed that following mating, neutrophils and macrophages are internalised within UECs, crossing the apical plasma membrane, representing the first evidence of cell-in-cell structures in the uterine epithelium. 3DSEM then quantified morphological changes in UECs during early pregnancy, revealing that at the time of implantation there is a significant reduction in cell volume, with a concurrent significant increase in the mean lipid droplet volume and the total lipid droplet volume. A novel CLEM protocol was utilised to investigate the actin cytoskeleton of UECs, confirming that the terminal web becomes disorganised at the time of implantation, with a thin, inconsistent actin fluorescence signal corresponding with remaining irregular membrane protrusions. CLEM was applied to investigate the actin terminal web in a rat model of Ovarian Hyperstimulated pregnancy (OH), which revealed the actin terminal web was also disrupted at the time of implantation. The apical actin cytoskeleton regulator Keratin 19 was then investigated. Keratin 19 was significantly greater in abundance at the time of implantation in OH pregnancy compared to normal pregnancy, while a 26 kDa fragment was significantly increased in abundance at the time of implantation in normal pregnancy. Experiments in ovariectomised rats showed that Keratin 19 expression is regulated by oestrogen. This thesis utilised 3DSEM and CLEM to derive novel insights into the changes that occur in UECs required for the development of uterine receptivity in early pregnancy.