title: Dithiothreitol prevents age-associated decrease in oocyte/conceptus viability in vitro.
abstract: The present study was designed to ascertain whether the negative effects on reproductive potential of post-ovulatory ageing in vitro of oocytes can be prevented by antioxidant therapy. Mouse metaphase II (MII) oocytes were aged in vitro for 12 h prior to insemination in the presence of varying concentrations of L-ascorbic acid, 6-methoxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), L-cystine dihydrochloride, ethylenediaminetetraacetic acid (EDTA), beta-mercaptoethanol and DL-dithiothreitol (DTT). In-vitro ageing of oocytes was associated with lower fertilization rate, higher proportion of concepti exhibiting cellular fragmentation at 24 h post-insemination and lower percentage of concepti reaching the blastocyst stage. Ascorbic acid, Trolox and EDTA had no effect on cellular fragmentation or potential of oocytes for development. However, the probability of an oocyte reaching the blastocyst stage was decreased (P < or = or = 0.05) in oocytes incubated in the presence of L-cystine (50 and 500 microM) and beta-mercaptoethanol (5, 50 and 500 microM) when compared to control aged oocytes. Age-associated cellular fragmentation at 24 h post-insemination was partially prevented (P < or = 0.05) by incubating oocytes in the presence of beta-mercaptoethanol (500 microM). DTT (50 and 500 microM) increased (P < or = 0.05) fertilization rate and number of cells at 81 h post-insemination to levels similar to those exhibited by control oocytes. Furthermore, both age-associated fragmentation at 24 h post-insemination (P < or = 0.05) and decreased potential of oocytes for development to the blastocyst stage (P < or = 0.05) were prevented, at least in part, by culturing oocytes in the presence of DTT (50 microM). Although the mechanism by which DTT exerts its beneficial effects on aged oocytes remains to be elucidated, it may protect oocytes by preventing oxidation of free thiol groups and/or altering a redox-independent signalling pathway that mediates cellular fragmentation and death.
authors.affiliation: Department of Paediatrics, Obstetrics and Gynaecology, Faculty of Medicine, University of Valencia, Spain.
authors.forename: J J
authors.initials: JJ
authors.lastname: Tarín
authors.forename: J
authors.initials: J
authors.lastname: Ten
authors.forename: F J
authors.initials: FJ
authors.lastname: Vendrell
authors.forename: A
authors.initials: A
authors.lastname: Cano
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D016922:Cellular Senescence / Q000187:drug effects*; D003470:Culture Media; D004229:Dithiothreitol / Q000494:pharmacology*; D005314:Embryonic and Fetal Development / Q000187:drug effects*; D005260:Female; D005978:Glutathione / Q000378:metabolism; D066298:In Vitro Techniques; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D009865:Oocytes / Q000187:drug effects* / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects; D013439:Sulfhydryl Reagents / Q000494:pharmacology*
chemical_list: D000975:Antioxidants; D003470:Culture Media; D013439:Sulfhydryl Reagents; D005978:Glutathione; D004229:Dithiothreitol