Regulation of membrane domains and mitochondrial dynamics during normal oocyte maturation and embryogenesis and in response to physiological stress

Oocytes acquire developmental competence during the final stages of folliculogenesis and during oocyte maturation to enable successful preimplantation embryo development. It is increasingly evident that the peri-conceptual environment surrounding the oocyte has a significant impact on the developmental competence and long-term health of offspring. Thus alterations to oocytes are emerging as significant contributors to the reduced conception and pregnancy rates that are commonly observed in conditions such as maternal diabetes or obesity. This thesis investigated a number of mechanisms by which alterations to oocytes, particularly lipids and oxidative stress, impinge upon the developmental program, namely the mitochondria and differentiation potential of embryonic cells. Firstly, the role of membrane functional domains in oocyte developmental competence was examined. Oocytes are cholesterol-laden cells and I sought to determine whether oocyte cholesterol stores are important for establishing membrane structure and functional lipid rafts during embryogenesis (via cholesterol depletion). I established that cholesterol depletion resulted in a significant reduction of cholesterol in the oocytes and disrupted oocyte raft distribution as verified by staining. I also showed that cholesterol depletion resulted in a failure to cleave in some embryos and delayed cleavage in others. Even when they developed to morphologically normal blastocysts, comprehensive analysis demonstrated that cholesterol depletion of oocytes led to embryos with reduced cell number, lower neutral lipid content and a disruption in raft distribution. These experiments underscored the importance of oocyte cholesterol stores for preimplantation development and membrane lipid raft formation and function during embryogenesis. To extend these findings, lipid raft and mitochondrial phenotypes were examined in oocytes and embryos of mice with high-fat diet (HFD)-induced obesity. I showed that oocytes and embryos from HFD mice exhibited disorganised lipid raft distribution and that mitochondrial activity was significantly reduced in the oocytes. Interestingly, BGP-15 treatment, an orally active compound that is currently in human clinical trials for type II diabetes, was able to restore oocyte mitochondrial activity, lipid raft distribution in the oocytes and to a lesser extent in the HFD embryos. In particular, there was a massive induction of mitochondrial DNA (mtDNA) copy number in oocytes following BGP-15 treatment to HFD mice. To understand the mechanism by which BGP-15 improves oocyte developmental competence, I focused on the mitochondria, essential organelles that are critical for embryo development and transgenerationally inherited. The kinetics of mtDNA replication and ability of BGP-15 to counteract stress was examined in multiple species and in vitro models of lipid-induced or oxidative stress. There was an accumulation of mtDNA levels during oocyte maturation from germinal vesicle (GV) to metaphase II (MII) oocytes in the mice and a significant mtDNA replication during embryo development in the mice, bovine and macaque. Supplementation of in vitro maturation (IVM) culture medium with high lipids suppressed mtDNA levels in the oocytes and embryos and this was partially prevented by BGP-15; an observation consistent in both mice and bovine embryos. Thus, supplementation of maturation medium with BGP-15 provides a means to increase mtDNA in both oocytes and embryos, thereby improving oocyte viability and developmental competence, particularly in the context of elevated lipids. Building on these results, BGP-15 was tested as a supplement to in vitro fertilisation (IVF) medium for its ability to counteract oxidative stress in cumulus-oocyte complexes (COCs) exposed to hydrogen peroxide (H2O2). Exposure of COCs to H2O2 significantly impaired cleavage rate following IVF. Embryo cell numbers, reactive oxygen species, mtDNA levels were altered but restored to control levels by BGP-15 treatment. Thus, oxidative stress in COCs recapitulates many of the defects seen with obesity and these are similarly normalised by BGP-15. Taken together, this study demonstrates that changes or alterations, as a result of environmental insult, before conception alter the phenotype of oocytes and that this is retained through to the course of later preimplantation development. I also demonstrated that BGP-15 is able to alleviate the effects of cellular stress via effects on mitochondria. These findings provide important benchmarks for future pre-clinical and clinical evaluations of preconception pharmaceuticals. This research is significant because increasing numbers of women seeking pregnancy have an altered follicular environment due to obesity, diabetes or poor diet; and my research has generated critically needed knowledge towards understanding how these lifestyle factors affect fertility and identified possible avenues for new therapies.Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 201