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

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 ,, ,, ,, and , 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 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.

In vitro embryos production of oocytes selected based on follicular size and by brilliant cresyl blue staining

“…Furthermore, we observed that nuclear maturation (MII) did not differ between BCB+ and BCBoocytes from the mixed, large, and small diameter groups. These results (MII) do not support previous studies, where it was shown that BCB+ oocytes are more competent to complete MII (ALM et al, 2005;LIU et al, 2018;RODRIGUEZ-GONZALEZ et al, 2002). Although our results on maturation (MII) did not differ between the BCB+ and BCB-oocytes within the same follicular group, there were marked differences between the follicular groups.…”

“…Oocyte competence is progressively acquired towards the end of folliculogenesis, when many cellular and molecular changes occur (ADONA et al, 2016;LIU et al, 2018). Such changes can influence various components of maturation, such as the quality of the follicular environment, follicle size, morphology of the cumulus-oocyte complex, and maturation conditions (DE BEM et al, 2014;SOARES et al, 2018).…”

Global transcriptome analysis of porcine oocytes in correlation with follicle size

“…In another model, brilliant cresyl blue (BCB) stain was widely used to characterize oocytes based on their potential developmental capacity (Catalá et al, ; Salviano, Collares, Becker, Rodrigues, & Rodrigues, ). Recently, Liu et al () used single‐cell RNAseq technology to analyze the transcriptome profiles of porcine oocytes categorized by the BCB stain. A total of 155 genes related to oocyte meiosis, cell cycle, tight junction, and metabolic pathways were differentially expressed between BCB‐negative and BCB‐positive immature germinal vesicle (GV) stage oocytes.…”

Long noncoding RNA 2193 regulates meiosis through global epigenetic modification and cytoskeleton organization in pig oocytes

“…The sense sequence of the negative control (NC) was (5′‐3′) UUCUCCGAACGUGUCACGUTT and the antisense sequence was (5′‐3′) ACGUGACACGUUCGGAGAATT. Microinjection of siRNA or antibody into porcine oocytes or 1‐cell embryos was performed using the procedure as described previously (Huan, Xie, Liu, Kong, & Liu, 2015, X. M. Liu et al, 2018a). Briefly, oocytes or embryos in the manipulation medium supplemented with 7.5 μg/ml cytochalasin B and 0.3% bovine serum albumin (BSA) were microinjected with approximately 10 pl siRNA or antibody using sterile femtotips and the FemtoJet express microinjector (Eppendorf).…”