Improved cryotolerance and developmental potential of in vitro and in vivo matured mouse oocytes by supplementing with a glutathione donor prior to vitrification.
Can supplementation of media with a glutathione (GSH) donor, glutathione ethyl ester (GEE), prior to vitrification protect the mouse oocyte from oxidative damage and critical changes in redox homeostasis, and thereby improve cryotolerance?



GEE supplementation supported redox regulation, rapid recovery of spindle and chromosome alignment after vitrification/warming and improved preimplantation development of mouse metaphase II (MII) oocytes.



Cryopreservation may affect mitochondrial functionality, induce oxidative stress, and thereby affect spindle integrity, chromosome segregation and the quality of mammalian oocytes. GEE is a membrane permeable GSH donor that promoted fertilization and early embryonic development of macaque and bovine oocytes after IVM.



Two experimental groups consisted of (i) denuded mouse germinal vesicle (GV) oocytes that were matured in vitro in the presence or absence of 1 mM GEE (IVM group 1) and (ii) in vivo ovulated (IVO) MII oocytes that were isolated from the ampullae and exposed to 1 mM GEE for 1 h prior to vitrification (IVO group 2). Recovery of oocytes from both groups was followed after CryoTop vitrification/warming for up to 2 h and parthenogenetic activation.



Reactive oxygen species (ROS), spindle morphology and chromosome alignment were analyzed by confocal laser scanning microscopy (CLSM) and polarization microscopy in control and GEE-supplemented MII oocytes. The relative overall intra-oocyte GSH content was assessed by analysis of monochlorobimane (MBC)-GSH adduct fluorescence in IVM MII oocytes. The GSH-dependent intra-mitochondrial redox potential (EmGSH) of IVM MII oocytes was determined after microinjection with specific mRNA at the GV stage to express a redox-sensitive probe within mitochondria (mito-Grx1-roGFP2). The absolute negative redox capacity (in millivolts) was determined by analysis of fluorescence of the oxidized versus the reduced form of sensor by CLSM and quantification according to Nernst equation. Proteome analysis was performed by quantitative 2D saturation gel electrophoresis (2D DIGE). Since microinjection and expression of redox sensor mRNA required removal of cumulus cells, and IVM of denuded mouse oocytes in group 1 induces zona hardening, the development to blastocysts was not assessed after IVF but instead after parthenogenetic activation of vitrified/warmed MII oocytes from both experimental groups.



IVM of denuded mouse oocytes in the presence of 1 mM GEE significantly increased intra-oocyte GSH content. ROS was not increased by CryoTop vitrification but was significantly lower in the IVM GEE group compared to IVM without GEE before vitrification and after recovery from vitrification/warming (P < 0.001). Vitrification alone significantly increased the GSH-dependent intra-mitochondrial redox capacity after warming (EmGSH, P < 0.001) in IVM oocytes, presumably by diffusion/uptake of cytoplasmic GSH into mitochondria. The presence of 1 mM GEE during IVM increased the redox capacity before vitrification and there was no further increase after vitrification/warming. None of the reproducibly detected 1492 spots of 2D DIGE separated proteins were significantly altered by vitrification or GEE supplementation. However, IVM of denuded oocytes significantly affected spindle integrity and chromosome alignment right after warming from vitrification (0 h) in group 1 and spindle integrity in group 2 (P < 0.05). GEE improved recovery in IVM group as numbers of oocytes with unaligned chromosomes and aberrant spindles was not significantly increased compared to unvitrified controls. The supplementation with GEE for 1 h before vitrification also supported more rapid recovery of spindle birefringence. GEE improved significantly development to the 2-cell stage for MII oocytes that were activated directly after vitrification/warming in both experimental groups, and also the blastocyst rate in the IVO GEE-supplemented group compared to the controls (P < 0.05).



None LIMITATIONS, REASONS FOR CAUTION: The studies were carried out in a mouse model, in IVM denuded rather than cumulus-enclosed oocytes, and in activated rather than IVF MII oocytes. Whether the increased GSH-dependent intra-mitochondrial redox capacity also improves male pronuclear formation needs to be studied further experimentally. The influence of GEE supplementation requires also further examination and optimization in human oocytes before it can be considered for clinical ART.



Although GEE supplementation did not alter the proteome at MII, the GSH donor may support cellular homeostasis and redox regulation and, thus, increase developmental competence. While human MII oocyte vitrification is an established procedure, GEE might be particularly beneficial for oocytes that suffer from oxidative stress and reduced redox capacity (e.g. aged oocytes) or possess low GSH due to a reduced supply of GSH from cumulus. It might also be of relevance for immature human oocytes that develop without cumulus to MII in vitro (e.g. in ICSI cycles) for ART.



The study has been supported by the German Research Foundation (DFG FOR 1041; EI 199/3-2). There are no conflict of interests.
Institute of Gene Technology/Microbiology, University of Bielefeld, 33501 Bielefeld, Germany.
Tom
T
Trapphoff
Institute of Gene Technology/Microbiology, University of Bielefeld, 33501 Bielefeld, Germany.
Martyna
M
Heiligentag
Institute of Gene Technology/Microbiology, University of Bielefeld, 33501 Bielefeld, Germany.
Jenny
J
Simon
Institute of Gene Technology/Microbiology, University of Bielefeld, 33501 Bielefeld, Germany.
Nora
N
Staubach
Dynamic Cell Imaging, Faculty of Biology, University of Bielefeld, 33501 Bielefeld, Germany.
Thorsten
T
Seidel
Laboratory for Functional Genome Analysis (LAFUGA), Gene Center, Ludwig-Maximilians-University Munich, 81377 Munich, Germany.
Kathrin
K
Otte
Laboratory for Functional Genome Analysis (LAFUGA), Gene Center, Ludwig-Maximilians-University Munich, 81377 Munich, Germany.
Thomas
T
Fröhlich
Laboratory for Functional Genome Analysis (LAFUGA), Gene Center, Ludwig-Maximilians-University Munich, 81377 Munich, Germany.
Georg J
GJ
Arnold
Institute of Gene Technology/Microbiology, University of Bielefeld, 33501 Bielefeld, Germany EiRi@uni-bielefeld.de.
Ursula
U
Eichenlaub-Ritter
D000818:Animals; D005260:Female; D005978:Glutathione / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D008677:Metaphase / Q000187:drug effects / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D009865:Oocytes / Q000187:drug effects*; D010084:Oxidation-Reduction / Q000187:drug effects; D018384:Oxidative Stress / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism; D058989:Vitrification / Q000187:drug effects
D017382:Reactive Oxygen Species; C042431:S-ethyl glutathione; D005978:Glutathione
adenosine 5’-triphosphate; chromosome alignment; developmental potential; glutathione; mitochondria; oocyte maturation; proteome; redox regulation; spindle; vitrification