Ovarian hyperstimulation (OH) is used in fertility treatments to stimulate superovulation and improve the chances of successful fertilisation. However, the increased levels of estrogen and progesterone prevent essential aspects of the plasma membrane transformation of uterine epithelial cells required for uterine receptivity. Changes in the actin cytoskeleton of uterine epithelial cells are a key component of uterine receptivity. Previous studies using a detergent permeabilisation technique to penetrate the plasma membrane demonstrated a loss of the actin terminal web but also resulted in limited visualisation of the subcellular details due to extraction artefacts. The influence of OH treatment on the actin cytoskeleton has not been studied. This study aims to develop a novel microscopy technique to study the actin cytoskeleton from the time of fertilisation and implantation in both normal and OH pregnancy.
A ‘single-process’ correlative light and electron microscopy (CLEM) protocol was developed which allows multi-platform imaging without compromising membrane integrity where a single ultrathin section is imaged in both the fluorescent and electron microscope. Correlative overlay data revealed that during normal pregnancy apical microvilli are progressively lost towards implantation and the thick, continuous terminal web is replaced by a thinner and irregular actin band. In contrast, the apical terminal web at the time of implantation in OH pregnancy was continuous beneath the apical plasma membrane and corresponded to an increase in length and number of microvilli.
These novel correlative microscopy results during normal pregnancy show that the actin terminal web of uterine epithelial cells is disrupted at the time of implantation, however in OH pregnancy the terminal web is maintained. This provides further evidence that the actin cytoskeleton remodelling is an essential component of implantation and that the maintenance of the actin terminal web of OH treated rats may contribute to the non-receptivity of the uterus in this model.