Hyperglycaemic conditions perturb mouse oocyte in vitro developmental competence via beta-O-linked glycosylation of heat shock protein 90.
OBJECTIVE
What is the effect of beta-O-linked glycosylation (O-GlcNAcylation) on specific proteins in the cumulus-oocyte complex (COC) under hyperglycaemic conditions?


CONCLUSIONS
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.


BACKGROUND
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.


METHODS
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.


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.


RESULTS
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.


CONCLUSIONS
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.


CONCLUSIONS
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.


BACKGROUND
This work was supported by a programme grant from the National Health and Medical Research Council, Australia, ID 453556. J.G.T. is a recipient of funding from and a consultant to Cook Medical Pty Ltd. The other authors have no conflicts of interest to declare.
The Robinson Institute, School of Paediatrics and Reproductive Health, The University of Adelaide, Ground Floor, Norwich Centre, 55 King William Road, North Adelaide, SA 5006, Australia.
L A
LA
Frank
M L
ML
Sutton-McDowall
H M
HM
Brown
D L
DL
Russell
R B
RB
Gilchrist
J G
JG
Thompson
D000818:Animals; D005260:Female; D006031:Glycosylation; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D006943:Hyperglycemia / Q000378:metabolism*; D059471:In Vitro Oocyte Maturation Techniques; D051379:Mice; D008808:Mice, Inbred CBA; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism*
D018841:HSP90 Heat-Shock Proteins; D017351:N-Acetylglucosaminyltransferases; C449880:O-GlcNAc transferase
HSP90; O-GlcNAc; hexosamine pathway; hyperglycaemia; oocyte developmental competence