Published 01/01/2014
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Hyperglycaemic Conditions Perturb Mouse Oocyte in Vitro Developmental Competence Via Beta-O-Linked Glycosylation of Heat Shock Protein 90

T.J. , R.D. , B.H. , G.R. , F.L. , S.M.

Keywords
Introduction
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.
Resume
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.
Method
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Results
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Conclusion
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
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