title: Brilliant Cresyl Blue stain selects largest oocytes with highest mitochondrial activity, maturation-promoting factor activity and embryo developmental competence in prepubertal sheep.
abstract: The aim of this study was to test the Brilliant Cresyl Blue (BCB) stain to select prepubertal sheep oocytes for in vitro blastocyst production. Oocyte diameter, mitochondrial activity, maturation-promoting factor (MPF) activity and mRNA relative expression (RE) of genes related to metabolism (ATPase Na(+)/K(+) transporting α 1 (ATP1A1) and cytochrome c oxidase subunit 1 (COX1)) and constitutive function of the cell (cytoplasmic polyadenylation-element-binding protein (CPEB) and S100A10) were assessed. Immature oocytes were exposed to different BCB concentrations (13, 26, 39 and 52  μM) and classified according to their cytoplasm colouration as grown BCB+ (blue cytoplasm) and growing BCB- (colourless cytoplasm). Staining oocytes with 13  μM BCB during 60  min allows selection of (BCB+) the largest (123.66  μm) and most competent oocytes to develop to the blastocyst stage (21%) with a higher number of cells (69.71 ± 6.19 s.e.m.) compared with non-stained BCB- oocytes (106.82  μm, 9% and 45.91 ± 3.35 s.e.m. respectively). Mitochondrial activity, assessed by MitoTracker Orange CMTMRos probe, was significantly higher in BCB+ than in BCB- oocytes after in vitro maturation (3369 and 1565  AU respectively). MPF activity was assessed by CDC2 kinase activity assay showing significantly higher activity at metaphase II stage in BCB+ than in BCB- oocytes (1.479 ± 0.09 and 1.184 ± 0.05 optical density respectively). The genes analysed in this work, ATP1A1, COX1, CPEB and S100A 10, did not show significant effect in mRNA RE between BCB selected oocytes. In conclusion, BCB stains larger and more competent oocytes to develop to the blastocyst stage with more active mitochondria and MPF activity and higher blastocyst cell number.
authors.affiliation: Departament de Ciència Animal i dels Aliments, Facultat de Veterinària, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain
authors.forename: Maria Gracia
authors.initials: MG
authors.lastname: Catalá
authors.forename: Dolors
authors.initials: D
authors.lastname: Izquierdo
authors.forename: Svetlana
authors.initials: S
authors.lastname: Uzbekova
authors.forename: Roser
authors.initials: R
authors.lastname: Morató
authors.forename: Montserrat
authors.initials: M
authors.lastname: Roura
authors.forename: Roser
authors.initials: R
authors.lastname: Romaguera
authors.forename: Pascal
authors.initials: P
authors.lastname: Papillier
authors.forename: Maria Teresa
authors.initials: MT
authors.lastname: Paramio
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000166:cytology / Q000187:drug effects; D002478:Cells, Cultured; D004396:Coloring Agents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004622:Embryo, Mammalian / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D047108:Embryonic Development / Q000187:drug effects / Q000502:physiology*; D005260:Female; D066298:In Vitro Techniques; D016200:Maturation-Promoting Factor / Q000187:drug effects / Q000502:physiology*; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology*; D023421:Models, Animal; D009865:Oocytes / Q000166:cytology* / Q000187:drug effects*; D010078:Oxazines / Q000494:pharmacology*; D012741:Sexual Maturation / Q000502:physiology; D012756:Sheep
chemical_list: D004396:Coloring Agents; D010078:Oxazines; C006797:Brilliant Cresyl Blue; D016200:Maturation-Promoting Factor