The role of reactive oxygen species and oxidative stress in post-ovulatory ageing and apoptosis of the mammalian oocyte

Following ovulation, the MII stage oocyte awaits fertilization in the oviduct, or, in the case of assistive reproductive technologies (ART), in in vitro culture medium. In the absence of fertilization, however, the oocyte experiences a time-dependent deterioration in quality referred to as post-ovulatory ageing. Post-ovulatory ageing is associated with a decline in fertilization rate, as well as the production of poor quality embryos, an increased risk for post-implantation errors and production of offspring with compromised health. Although the consequences associated with post-ovulatory ageing are well defined, the molecular mechanisms which orchestrate this decline in oocyte quality, or conversely, act to prevent post-ovulatory ageing in the event that timely fertilization has occurred, are not well understood. In this thesis we decipher a critical role for reactive oxygen species (ROS) in the initiation of post-ovulatory ageing and apoptosis of the mammalian oocyte. Using a mouse model, we have characterised a time–dependent accumulation of intracellular ROS following retrieval of the ovulated oocyte. This elevation in ROS was found to instigate a self-perpetuating cycle of lipid peroxidation, electrophilic aldehyde production and mitochondrial damage; resulting in the initiation of an intrinsic apoptotic cascade. The elevation in levels of ROS and electrophilic aldehydes within the oocyte were directly associated with a decreased capacity to participate in fertilization and support embryo development. Importantly, research within this thesis has demonstrated that timely fertilization of the oocyte is associated with an up-regulation of glutathione peroxidase activity, and accelerated DNA repair by the base excision repair (BER) pathway. These post-fertilization changes in oocyte biochemistry aide in circumventing the otherwise inevitable initiation of post-ovulatory ageing by preventing the accumulation of ROS and oxidative DNA damage. In identifying the critical role for ROS and electrophilic aldehydes in post-ovulatory ageing and apoptosis of the oocyte, it was possible to select antioxidant and aldehyde-reactive compounds to attenuate the onset of these processes. Specifically, melatonin was found to significantly improve fertilization rate, embryo formation rate and embryo quality in oocytes aged for 8 and 16 h in vitro, as well as delay the initiation of apoptosis. Similarly, penicillamine was found to prevent the decline in fertilization rate and embryo formation rate associated with elevated levels of electrophilic aldehydes. The adaptation of these supplementation techniques for use in a human ART setting would be advantageous in lengthening the optimal window of time in which oocytes must be inseminated post-retrieval, as well as increasing the viability of re-insemination techniques such as rescue-ICSI; potentially minimising the likelihood that further ovarian stimulation cycles would be necessary following a failure to fertilize by IVF. Collectively, these data provide a significant contribution to the field of knowledge surrounding degeneration and apoptosis of the mammalian oocyte, and provide novel methodologies for attenuating these events in an in vitro setting.