Abstract
The brilliant cresyl blue (BCB) test is used in both basic biological research and assisted reproduction to identify oocytes likely to be developmentally competent. However, the underlying molecular mechanism targeted by the BCB test is still unclear. To explore this question, we first confirmed that BCB-positive porcine oocytes had higher rates of meiotic maturation, better rates of cleavage and development into blastocysts, and lower death rates. Subsequent single-cell transcriptome sequencing on porcine germinal vesicle (GV)-stage oocytes identified 155 genes that were significantly differentially expressed between BCB-negative and BCB-positive oocytes. These included genes such as cdc5l, ldha, spata22, rgs2, paip1, wee1b, and hsp27, which are enriched in functionally important signaling pathways including cell cycle regulation, oocyte meiosis, spliceosome formation, and nucleotide excision repair. In BCB-positive GV oocytes that additionally had a lower frequency of DNA double-strand breaks, the CDC5L protein was significantly more abundant. cdc5l/CDC5L inhibition by short interference (si)RNA or antibody microinjection significantly impaired porcine oocyte meiotic maturation and subsequent parthenote development. Taken together, our single-oocyte sequencing data point to a potential new role for CDC5L in porcine oocyte meiosis and early embryo development, and supports further analysis of this protein in the context of the BCB test.
Highlights
The brilliant cresyl blue (BCB) test is used in both basic biological research and assisted reproduction to identify oocytes likely to be developmentally competent
We first tested to see if differences existed between two different controls, by comparing cumulus– oocyte complexes (COCs) incubated with the maturation medium and Dulbecco’s phosphatebuffered saline (DPBS) under the same culture conditions, respectively
We examined the chromatin configuration of oocytes at germinal vesicle (GV) stage, which were classified by two different methods, as non-surrounded nucleolus (NSN) and surrounded nucleolus (SN) [36], or as GV0 –GV4, respectively [37] (Fig. 1C)
Summary
The brilliant cresyl blue (BCB) test is used in both basic biological research and assisted reproduction to identify oocytes likely to be developmentally competent. The underlying molecular mechanism targeted by the BCB test is still unclear To explore this question, we first confirmed that BCBpositive porcine oocytes had higher rates of meiotic maturation, better rates of cleavage and development into blastocysts, and lower death rates. Subsequent single-cell transcriptome sequencing on porcine germinal vesicle (GV)-stage oocytes identified 155 genes that were significantly differentially expressed between BCB-negative and BCB-positive oocytes. These included genes such as cdc5l, ldha, spata, rgs, paip, wee1b, and hsp, which are enriched in functionally important signaling pathways including cell cycle regulation, oocyte meiosis, spliceosome formation, and nucleotide excision repair. Our single-oocyte sequencing data point to a potential new role for CDC5L in porcine oocyte meiosis and early embryo development, and supports further analysis of this protein in the context of the BCB test. To augment the developmental competence of oocytes cultured in vitro, a variety of techniques and methods have been pursued, such as the brilliant cresyl blue (BCB) test, zona imaging, optimization of culture system, and screening of biomarkers [3, 4]
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