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 C /K C transporting a 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 mM) and classified according to their cytoplasm colouration as grown BCBC (blue cytoplasm) and growing BCBK (colourless cytoplasm). Staining oocytes with 13 mM BCB during 60 min allows selection of (BCBC) the largest (123.66 mm) and most competent oocytes to develop to the blastocyst stage (21%) with a higher number of cells (69.71G6.19S.E.M.) compared with non-stained BCBK oocytes (106.82 mm, 9% and 45.91G3.35 S.E.M. respectively). Mitochondrial activity, assessed by MitoTracker Orange CMTMRos probe, was significantly higher in BCBC than in BCBK 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 BCBC than in BCBK oocytes (1.479G0.09 and 1.184G0.05 optical density respectively). The genes analysed in this work, ATP1A1, COX1, CPEB and S100A10, 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.

Mitochondrial Activity as a Biomarker of Gamete and Embryo Health

“…This notion is supported by their detection of upregulated levels of expression for genes involved in ATP synthesis and electron transport in the BCB − state. An association between oocyte developmental competence and mitochondria was also suggested by the recent studies of Silva et al [ 116 ] and Catala et al [ 117 ] in the bovine and ovine systems, respectively. Silva et al found that the central distribution of mitochondria in MII bovine oocytes that is consistent with normal nuclear and cytoplasmic maturation and higher rates of blastocyst formation on day 7 was associated with a BCB + status in the fully grown oocyte at the outset of maturation in vitro.…”

Effect of L-carnitine supplementation on maturation and early embryo development of immature mouse oocytes selected by brilliant cresyle blue staining

“…Brilliant Cresyle Blue (BCB) is a vital dye which stains the cytoplasm blue (BCB+) or colorless (BCB−) depending on G6PDH function and is a convenient test for selection for fully grown oocytes [8]. It has been reported that the competence of maturation and development increased in BCB+ oocytes in pig [9], sheep [10], and bovine [11].…”

Buffalo Embryos Produced by Handmade Cloning from Oocytes Selected Using Brilliant Cresyl Blue Staining Have Better Developmental Competence and Quality and Are Closer to Embryos Produced byIn VitroFertilization in Terms of Their Epigenetic Status and Gene Expression Pattern

“…Thus, oocytes that have completed their growth phase show decreased G6PDH and exhibit cytoplasm with blue coloration (BCB + ), whereas growing oocytes, which are expected to have a high level of active G6PDH, remain colorless (BCB -) after staining. BCB staining has been successfully used for the selection of oocytes for production of IVF embryos in several mammalian species such as pig (Roca et al, 1998), goat (Rodriguez-Gonzalez et al, 2002), sheep (Catalá et al, 2011), bovine (Pujol et al, 2004), and buffalo (Manjunatha et al, 2007).…”