Importance of oocyte to cumulus cell bi-directional signalling on oocyte and subsequent embryo and foetal development and viability.

Oocyte in vitro maturation (IVM) possesses significant scientific and clinical benefits such as the elimination of dangerous side-effects like ovarian hyperstimulation syndrome. Unfortunately, due to a lack of understanding of the intricate processes involved, IVM success rates are low and cannot rival that of current IVF protocols involving hormonal stimulation. Recent scientific advancement has unveiled the existence of the oocytecumulus cell (CC) bi-directional regulatory loop and its importance to the development and survival of both cell types during folliculogenesis. This thesis therefore aimed to investigate the significance of these communication axes during IVM on oocyte and CC functions such as cumulus expansion, metabolism and oxidative stress levels and oocyte developmental competence into foetal development. To target oocyte to CC signalling, growth differentiation factor 9 (GDF9), the primary identified oocyte paracrine factor in the mouse, and its SMAD2/3 signalling pathway were investigated. FSH/EGF were examined as modulators of CC to oocyte signalling, as these can only exert their effects on oocyte maturation through the CCs. Maturation of mouse cumulus-oocyte complexes (COCs) in the absence of FSH/EGF and/or in the presence of SMAD2/3 inhibition resulted in the ablation of cumulus expansion whereas the addition of exogenous GDF9 to intact COCs significantly increased cumulus expansion. To assess the importance of cumulus expansion independent of oocyte and cumulus communications to embryo development, azaserine, an inhibitor of hyaluronan synthesis was used. Azaserine did not successfully attenuated cumulus expansion in this system however subsequent blastocyst formation was severely diminished. Cumulus expansion was therefore not indicative of oocyte developmental competence. Meiotic maturation was only affected by FSH/EGF but both signalling pathways affected sperm penetration. While blastocyst formation was unaffected, disrupted oocyte or cumulus signalling significantly decreased blastocyst inner cell mass (ICM) numbers. Conversely, addition of exogenous GDF9 with FSH/EGF during IVM increased ICM numbers. Significantly, exogenous supplementation of GDF9 during IVM increased foetal survival while inhibition of SMAD2/3 signalling had the opposite effect. Implantation rates and foetal weights were unaffected in both treatments. The effect of GDF-9 and SMAD 2/3 signalling on metabolism of the COC was examined. The existence of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB) isoforms were discovered in COCs and along with other metabolic gene transcripts, were significantly altered in the absence of FSH/EGF and with exogenous GDF9 and FSH/EGF although SMAD2/3 inhibition had no effect. Glycolytic activity however, was decreased in the absence of FSH/EGF and with SMAD2/3 inhibition but increased with exogenous GDF9. TCA cycle activity was only affected by FSH/EGF. The absence of FSH/EGF, SMAD2/3 inhibition and azaserine during IVM all resulted in increased oxidative stress levels in the oocyte. The work in this thesis also demonstrated that oocyte and CC signalling are co-dependent on each other as apart from cumulus expansion and slower developmental rates, perturbations of both signalling pathways simultaneously did not have additive effects on oocyte developmental competence or on CC metabolic functions. This thesis has therefore provided significance to the field of oocyte IVM through the evidence that oocyte-CC bi-directional communication during IVM is essential to oocyte viability and foetal outcomes.Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 201