Differences in Cytoplasmic Maturation Between the BCB+ and Control Porcine Oocytes Do Not Justify Application of the BCB Test for a Standard IVM Protocol

Abstract

The Brilliant Cresyl Blue (BCB) test relies on G6PDH activity and a simple protocol for the selection of higher quality oocytes. Although the BCB+ oocytes of all the species that have been investigated are characterized by superior quality when compared to BCB- counterparts, application of the test for embryo production still remains an open issue. The aim of our study was to compare BCB+ and the control oocytes (not subjected to the BCB test) in terms of selected aspects of cytoplasmic maturation (mtDNA copy number, mitochondria distribution, relative transcript abundance of six marker genes). The results of our study revealed more relevant differences within the BCB+ and the control oocytes (before and after IVM) than between the two categories of oocytes. There was no difference in the transcript abundance of the BCB+ and the control oocytes in 5 out of 6 analyzed genes (BMP15, GDF9, ATP5A1, EEF1A, ZAR1) and in mtDNA content (pre-IVM 179609 vs. 176595 and post-IVM 187243 vs. 246984, respectively). With regard to mitochondria distribution in pre- and post-IVM oocytes, there was nonsignificant tendency for a more frequent occurrence of the expected patterns in the BCB+ group. The results of the present study do not support the application of BCB staining in a routine IVM protocol due to relatively high similarity in selected parameters characterizing cytoplasmic maturation of BCB+ and control oocytes. This high similarity may results from the limited amount of less competent BCB- oocytes (10%) still present among nonselected oocytes of proper morphology.

The making of an embryo in a basal metazoan: Proteomic analysis in the sea anemone Nematostella vectensis

“…Oxidative phosphorylation combines electron transport with cell respiration and ATP synthesis and is the main generator of cellular energy during oocyte maturation . In mammals, ATP5A1 was shown to have higher expression during oocyte maturation than after fertilization ; a similar pattern was seen in our study. Another highly expressed protein at the maturation stage was a transcription factor belonging to the cold‐shock proteins, which were implicated in coupling transcription translation during oogenesis .…”

Metabolic control of oocyte development: linking maternal nutrition and reproductive outcomes

“…Nonetheless, BCB staining has not been used to evaluate the G6PDH activity in human oocytes yet. Moreover, most recently, BCB staining of pig oocytes at the immature stage was found to compromise mitochondrial functions throughout oocyte maturation and, therefore, is not suitable for application in a standard in vitro maturation protocol [72, 73]. Therefore, BCB staining-based oocyte quality evaluation still remains an open issue.…”

Single-cell transcriptome sequencing reveals that cell division cycle 5-like protein is essential for porcine oocyte maturation

“…COCs were washed thoroughly in Hepes-buffered Tyrode medium containing 0.01% polyvinyl alcohol, and then subjected to the BCB test according to a previous protocol reported in pigs with some modifications (24). Briefly, COCs were incubated in Dulbecco's PBS (DPBS) supplemented with 13 M BCB (BCB medium) for 90 min at 39°C in a humidified 5% CO 2 incubator.…”

“…Oocytes discerned by BCB staining had different properties, such as abundance of maternal transcripts (9,(12)(13)(14)(15)(16), mitochondrial numbers and function (17)(18)(19), lipids content (19), and total cell number and cell survival rates of blastocysts (4,20). In pigs, the BCB test also seems to be capable of predicting the outcomes of oocyte maturation (21-23), although some caution arises recently (24,25). Oocytes with better developmental competence could also be used to improve the success rate of production of transgenic pigs with the somatic cell nuclear transfer technique (26,27).…”