title: SIRT1 signalling protects mouse oocytes against oxidative stress and is deregulated during aging.
abstract: OBJECTIVE
  Is SIRT1 involved in the oxidative stress (OS) response in mouse oocytes?
  
  
  CONCLUSIONS
  SIRT1 plays a pivotal role in the adaptive response of mouse germinal vesicle (GV) oocytes to OS and promotes a signalling cascade leading to up-regulation of the MnSod gene.
  
  
  BACKGROUND
  OS is known to continuously threaten acquisition and maintenance of oocyte developmental potential during in vivo processes and in vitro manipulations. Previous studies in somatic cells have provided strong evidence for the role of SIRT1 as a sensor of the cell redox state and a protector against OS and aging.
  
  
  METHODS
  GV oocytes obtained from young (4-8 weeks) and reproductively old (48-52 weeks) CD1 mice were blocked in the prophase stage by 0.5 µM cilostamide. Groups of 30 oocytes were exposed to 25 µM H2O2 and processed following different times for the analysis of intracellular localization of SIRT1 and FOXO3A, and evaluation of Sirt1, miRNA-132, FoxO3a and MnSod gene expression. Another set of oocytes was cultured in the presence or absence of the SIRT1-specific inhibitor Ex527, and exposed to H2O2 in order to assess the involvement of SIRT1 in the activation of a FoxO3a-MnSod axis and ROS detoxification. In the last part of this study, GV oocytes were maturated in vitro in the presence of different Ex527 concentrations (0, 2.5, 5, 10, 20 µM) and assessed for maturation rates following 16 h. Effects of Ex527 on spindle morphology and ROS levels were also evaluated.
  
  
  METHODS
  SIRT1 and FOXO3A intracellular distribution in response to OS was investigated by immunocytochemistry. Real-time RT-PCR was employed to analyse Sirt1, miR-132, FoxO3a and MnSod gene expression. Reactive oxygen species (ROS) production was evaluated by in vivo measurement of carboxy-H2DCF diacetate labelling. Spindle and chromosomal distribution in in vitro matured oocytes were analysed by immunocytochemistry and DNA fluorescent labelling, respectively.
  
  
  RESULTS
  Specific changes in the intracellular localization of SIRT1 and up-regulation of Sirt1 gene were detected in mouse oocytes in response to OS. Moreover, increased intracellular ROS were observed when SIRT1 activity was inhibited by Ex527. In aged oocytes Sirt1 was expressed more than in young oocytes but SIRT1 protein was undetectable. Upon OS, significant changes in miR-132 micro-RNA, a validated Sirt1 modulator, were observed. A negative correlation between Sirt1 mRNA and miR-132 levels was observed when young oocytes exposed to OS were compared with young control oocytes, and when aged oocytes were compared with young control oocytes. FoxO3a and MnSod transcripts were increased upon OS with the same kinetics as Sirt1 transcripts, and up-regulation of MnSod gene was prevented by oocyte treatment with Ex527, indicating that SIRT1 acts upstream to the FoxO3a-MnSod axis. Finally, the results of the in vitro maturation assay suggested that SIRT1 might be involved in oocyte maturation by regulating the redox state and ensuring normal spindle assembly.
  
  
  CONCLUSIONS
  The main limitation of this study was the absence of direct quantification of SIRT1 enzymatic activity due to the lack of an appropriately sensitive method.
  
  
  CONCLUSIONS
  The present findings may provide a valuable background for studying the regulation of SIRT1 during oogenesis and its relevance as a sensor of oocyte redox state and energy status. The antioxidant response orchestrated by SIRT1 in oocytes seems to decrease with aging. This suggests that SIRT1 could be an excellent pharmacological target for improving oocyte quality and IVF outcome in aging or aging-like diseases.
  
  
  BACKGROUND
  The work was supported by the Ministero dell'Università e della Ricerca Scientifica (MIUR) to C.T., F.A., C.D., A.M.D. The authors declare no conflict of interest.
authors: Department of Life, Health and Environmental Sciences, University of L'Aquila, Via Vetoio, 67100 L'Aquila, Italy.
authors: Giovanna
authors: G
authors: Di Emidio
authors: Department of Life, Health and Environmental Sciences, University of L'Aquila, Via Vetoio, 67100 L'Aquila, Italy.
authors: Stefano
authors: S
authors: Falone
authors: Department of Life, Health and Environmental Sciences, University of L'Aquila, Via Vetoio, 67100 L'Aquila, Italy.
authors: Maurizio
authors: M
authors: Vitti
authors: Department of Life, Health and Environmental Sciences, University of L'Aquila, Via Vetoio, 67100 L'Aquila, Italy.
authors: Anna Maria
authors: AM
authors: D'Alessandro
authors: Servizio di PMA, Azienda Ospedaliera Cannizzaro, Catania, Italy.
authors: Marilena
authors: M
authors: Vento
authors: Dipartimento Gian Filippo Ingrassia, Sezione di Biologia, Genetica, Genomica Cellulare e Molecolare Giovanni Sichel, Università degli Studi di Catania, Catania, Italy.
authors: Cinzia
authors: C
authors: Di Pietro
authors: Department of Life, Health and Environmental Sciences, University of L'Aquila, Via Vetoio, 67100 L'Aquila, Italy.
authors: Fernanda
authors: F
authors: Amicarelli
authors: Department of Life, Health and Environmental Sciences, University of L'Aquila, Via Vetoio, 67100 L'Aquila, Italy Infertility Service, San Salvatore Hospital, Via Vetoio, 67100 L'Aquila, Italy carla.tatone@univaq.it.
authors: Carla
authors: C
authors: Tatone
mesh_terms: D000818:Animals; D002227:Carbazoles / Q000494:pharmacology; D005260:Female; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008815:Mice, Inbred Strains; D009865:Oocytes / Q000187:drug effects / Q000254:growth & development*; D018384:Oxidative Stress; D015398:Signal Transduction; D056564:Sirtuin 1 / Q000032:analysis / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D000099156:Superoxide Dismutase 2
chemical_list: D002227:Carbazoles; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors; D006861:Hydrogen Peroxide; D056564:Sirtuin 1; D013482:Superoxide Dismutase; D000099156:Superoxide Dismutase 2; C550547:6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide; C474033:FoxO3 protein, mouse; C470545:Sirt1 protein, mouse
keywords: SIRT1; miRNA; oocyte aging; oxidative stress; sirtuins