Brilliant Cresyl Blue stain selects largest oocytes with highest mitochondrial activity, maturation-promoting factor activity and embryo developmental competence in prepubertal sheep

摘要

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 S 100A10 , 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.