title: Extending prematuration with cAMP modulators enhances the cumulus contribution to oocyte antioxidant defence and oocyte quality via gap junctions.
abstract: OBJECTIVE
  Can bovine oocyte antioxidant defence and oocyte quality be improved by extending the duration of pre-in vitro maturation (IVM) with cyclic adenosine mono-phosphate (cAMP) modulators?
  
  
  CONCLUSIONS
  Lengthening the duration of cAMP-modulated pre-IVM elevates intra-oocyte reduced glutathione (GSH) content and reduces hydrogen peroxide (H2O2) via increased cumulus cell-oocyte gap-junctional communication (GJC), associated with an improvement in subsequent embryo development and quality.
  
  
  BACKGROUND
  Oocytes are susceptible to oxidative stress and the oocyte's most important antioxidant glutathione is supplied, at least in part, by cumulus cells. A temporary inhibition of spontaneous meiotic resumption in oocytes can be achieved by preventing a fall in cAMP, and cyclic AMP-modulated pre-IVM maintains cumulus-oocyte GJC and improves subsequent embryo development.
  
  
  METHODS
  This study consisted of a series of 10 experiments using bovine oocytes in vitro, each with multiple replicates. A range of pre-IVM durations were examined as the key study treatments which were compared with a control. The study was designed to examine if one of the oocyte's major antioxidant defences can be enhanced by pre-IVM with cAMP modulators, and to examine the contribution of cumulus-oocyte GJC on these processes.
  
  
  METHODS
  Immature bovine cumulus-oocyte complexes were treated in vitro without (control) or with the cAMP modulators; 100 µM forskolin (FSK) and 500 µM 3-isobutyl-1-methyxanthine (IBMX), for 0, 2, 4 or 6 h (pre-IVM phase) prior to IVM. Oocyte developmental competence was assessed by embryo development and quality post-IVM/IVF. Cumulus-oocyte GJC, intra-oocyte GSH and H2O2 were quantified at various time points during pre-IVM and IVM, in the presence and the absence of functional inhibitors: carbenoxolone (CBX) to block GJC and buthionine sulfoximide (BSO) to inhibit glutathione synthesis.
  
  
  RESULTS
  Pre-IVM with FSK + IBMX increased subsequent blastocyst formation rate and quality compared with standard IVM (P < 0.05), regardless of pre-IVM duration. The final blastocyst yields (proportion of blastocysts/immature oocyte) were 26.3% for the control, compared with 39.2, 35.2 and 34.2%, for the 2, 4 and 6 h pre-IVM FSK + IBMX treatments, respectively. In contrast to standard IVM (control), pre-IVM with cAMP modulators maintained open gap junctions between cumulus cells and oocytes for the duration (6 h) of pre-IVM examined, and persisted for a further 8 h in the IVM phase. Cyclic AMP-modulated pre-IVM increased intra-oocyte GSH levels at the completion of both pre-IVM and IVM, in a pre-IVM duration-dependent manner (P < 0.05), which was ablated when GJC was blocked using CBX (P < 0.05). By 4 h of pre-IVM treatment with cAMP modulators, oocyte H2O2 levels were reduced compared the control (P < 0.05), although this beneficial effect was lost when oocytes were co-treated with BSO. Inhibiting glutathione synthesis with BSO during pre-IVM ablated any positive benefits of cAMP-mediated pre-IVM on oocyte developmental competence (P < 0.01).
  
  
  CONCLUSIONS
  It is unclear if the improvement in oocyte antioxidant defence and developmental competence reported here is due to direct transfer of total and/or reduced glutathione from cumulus cells to the oocyte via gap junctions, or whether a GSH synthesis signal and/or amino acid substrates are supplied to the oocyte via gap junctions. Embryo transfer experiments are required to determine if the cAMP-mediated improvement in blastocyst rates leads to improved live birth rates.
  
  
  CONCLUSIONS
  IVM offers significant benefits to infertile and cancer patients and has the potential to significantly alter ART practice, if IVM efficiency in embryo production could be improved closer to that of conventional IVF (using ovarian hyperstimulation). Pre-IVM with cAMP modulators is a simple and reliable means to improve IVM outcomes.
  
  
  BACKGROUND
  This work was supported by grants and fellowships from the National Health and Medical Research Council of Australia (1007551, 627007, 1008137, 1023210) and by scholarships from the Chinese Scholarship Council (CSC) awarded to H.J.L. and the Japanese Society for the Promotion of Science Postdoctoral Fellowship for Research Abroad awarded to S.S. The Fluoview FV10i confocal microscope was purchased as part of the Sensing Technologies for Advanced Reproductive Research (STARR) facility, funded by the South Australian Premier's Science and Research Fund. We acknowledge partial support from the Australian Research Council Centre of Excellence for Nanoscale BioPhotonics (CE140100003). We declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
authors.affiliation: Robinson Research Institute & School of Medicine, The University of Adelaide, Adelaide SA 5005, Australia College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China.
authors.forename: H J
authors.initials: HJ
authors.lastname: Li
authors.affiliation: Robinson Research Institute & School of Medicine, The University of Adelaide, Adelaide SA 5005, Australia Australian Research Council Centre of Excellence for Nanoscale BioPhotonics, Adelaide, Australia.
authors.forename: M L
authors.initials: ML
authors.lastname: Sutton-McDowall
authors.affiliation: Robinson Research Institute & School of Medicine, The University of Adelaide, Adelaide SA 5005, Australia.
authors.forename: X
authors.initials: X
authors.lastname: Wang
authors.affiliation: Robinson Research Institute & School of Medicine, The University of Adelaide, Adelaide SA 5005, Australia Department of Biological Production, Institute of Agriculture, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Sugimura
authors.affiliation: Robinson Research Institute & School of Medicine, The University of Adelaide, Adelaide SA 5005, Australia Australian Research Council Centre of Excellence for Nanoscale BioPhotonics, Adelaide, Australia.
authors.forename: J G
authors.initials: JG
authors.lastname: Thompson
authors.affiliation: Robinson Research Institute & School of Medicine, The University of Adelaide, Adelaide SA 5005, Australia Discipline of Obstetrics & Gynaecology, School of Women's & Children's Health, University of New South Wales, Sydney 2013, Australia r.gilchrist@unsw.edu.au.
authors.forename: R B
authors.initials: RB
authors.lastname: Gilchrist
mesh_terms: D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000262:Adenylyl Cyclases / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D002229:Carbenoxolone / Q000494:pharmacology; D002417:Cattle; D005576:Colforsin / Q000494:pharmacology; D054885:Cumulus Cells / Q000187:drug effects / Q000502:physiology; D000242:Cyclic AMP / Q000819:agonists* / Q000378:metabolism; D004477:Ectogenesis / Q000187:drug effects*; D020536:Enzyme Activators / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005307:Fertilization in Vitro / Q000187:drug effects; D017629:Gap Junctions / Q000187:drug effects* / Q000378:metabolism; D005721:Glutamate-Cysteine Ligase / Q000037:antagonists & inhibitors / Q000378:metabolism; D005978:Glutathione / Q000819:agonists* / Q000037:antagonists & inhibitors / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000037:antagonists & inhibitors / Q000378:metabolism; D059471:In Vitro Oocyte Maturation Techniques; D009865:Oocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects*; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology
chemical_list: D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D010726:Phosphodiesterase Inhibitors; D005576:Colforsin; D019328:Buthionine Sulfoximine; D006861:Hydrogen Peroxide; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione; D002229:Carbenoxolone; D015056:1-Methyl-3-isobutylxanthine
keywords: cyclic AMP; gap-junctional communication; glutathione; oocyte in vitro maturation; oocyte quality