GlcNAcylation regulates protein function in a manner similar to 50 phosphorylation, with the two modifications often targeting the same or adjacent sites on a 51 protein in what has been described as a yin-yang relationship (Comer and Hart 2000; Butkinaree, 52 Park, and Hart 2010; Wang, Gucek, and Hart 2008; Haltiwanger et al. O-GlcNAcylation is involved controlling essential cellular processes such as cell cycle 54 regulation (Slawson and Hart 2003; Slawson et al. 65 In contrast with glycosaminoglycan-type glycosylation, the O-GlcNAc modification consists of a 66 single beta-O-linked N-acetylglucosamine residue, and no chain is formed by addition of further 67 residues.
Study question: What is the effect of beta-O-linked glycosylation on 3 specific proteins in the cumulus-oocyte complex under hyperglycaemic conditions?4 Summary answer: Heat shock protein 90 was identified and confirmed as being O-5 GlcNAcylated in mouse COCs under hyperglycaemic conditions , 6 causing detrimental outcomes for embryo development. 12 Study design, size, duration: This study was designed to examine the effect of hyperglycaemic 13 conditions on O-GlcNAc levels in the mouse 14 COC, and furthermore to identify potential candidate proteins which are targets of this 15 modification, and their roles in oocyte maturation.
STUDY QUESTION What is the effect of beta-O-linked glycosylation (O-GlcNAcylation) on specific proteins in the cumulus-oocyte complex (COC) under hyperglycaemic conditions? SUMMARY ANSWER Heat shock protein 90 (HSP90) was identified and confirmed as being O-GlcNAcylated in mouse COCs under hyperglycaemic conditions (modelled using glucosamine), causing detrimental outcomes for embryo development. WHAT IS KNOWN ALREADY O-GlcNAcylation of proteins occurs as a result of increased activity of the hexosamine biosynthesis pathway, which provides substrates for cumulus matrix production during COC maturation, and also for O-GlcNAcylation. COCs matured under hyperglycaemic conditions have decreased developmental competence, mediated at least in part through the mechanism of increased O-GlcNAcylation. STUDY DESIGN, SIZE, DURATION This study was designed to examine the effect of hyperglycaemic conditions (using the hyperglycaemic mimetic, glucosamine) on O-GlcNAc levels in the mouse COC, and furthermore to identify potential candidate proteins which are targets of this modification, and their roles in oocyte maturation. PARTICIPANTS/MATERIALS, SETTING, METHODS COCs from 21-day-old superovulated CBA × C57BL6 F1 hybrid female mice were matured in vitro (IVM). Levels of O-GlcNAcylated proteins, HSP90 and O-GlcNAc transferase (OGT, the enzyme responsible for O-GlcNAcylation) in COCs were measured using western blot, and localization observed using immunocytochemistry. For glycosylated HSP90 levels, and to test OGT-HSP90 interaction, immunoprecipitation was performed prior to western blotting. Embryo development was assessed using in vitro fertilization and embryo culture post-maturation. MAIN RESULTS AND THE ROLE OF CHANCE Addition of the hyperglycaemic mimetic glucosamine to IVM medium for mouse COCs increased detectable O-GlcNAcylated protein levels (by western blot and immunocytochemistry), and this effect was reversed using an OGT inhibitor (P < 0.05). HSP90 was identified as a target of O-GlcNAcylation in the COC, and inhibition of HSP90 during IVM reversed glucosamine-induced decreases in oocyte developmental competence (P < 0.05). We also demonstrated the novel finding of an association between HSP90 and OGT in COCs, suggesting a possible client–chaperone relationship. LIMITATIONS, REASONS FOR CAUTION In vitro maturation of COCs was used so that treatment time could be limited to the 17 h of maturation prior to ovulation. Additionally, glucosamine, a hyperglycaemic mimetic, was used because it specifically activates the hexosamine pathway which provides the O-GlcNAc moieties. The results in this study should be confirmed using in vivo models of hyperglycaemia and different HSP90 inhibitors. WIDER IMPLICATIONS OF THE FINDINGS This study leads to a new understanding of how diabetes influences oocyte competence and provides insight into possible therapeutic interventions based on inhibiting HSP90 to improve oocyte quality.L.A. Frank, M.L. Sutton-McDowall, H.M. Brown, D.L. Russell, R.B. Gilchrist, and J.G. Thompso