Single-Cell RNA Sequencing Reveals Molecular Differences Underlying Meiotic Competence of Brilliant Cresyl Blue (BCB) Selected Alpaca Oocytes.

Abstract

Identifying oocytes with high meiotic competence remains a major challenge for improving in vitro embryo production in South American camelids. Although Brilliant Cresyl Blue (BCB) staining has been used to distinguish alpaca oocytes with different meiotic and developmental competence, the biological and molecular feature associated with their meiotic competence remain poorly understood. This study characterized the transcriptomic profiles of immature alpaca (Vicugna pacos) oocytes classified as BCB-positive (BCB+) or BCB-negative (BCB-) using single-cell RNA sequencing. BCB+ oocytes exhibited a significantly larger ooplasm diameter, a higher proportion of condensed chromatin configurations, and higher in vitro maturation rates than BCB- oocytes. Transcriptomic analysis identified 2978 differentially expressed genes and revealed a clear separation between groups (PC1 = 64.8%). Differentially expressed genes with higher transcript abundance in BCB+ oocytes were enriched in DNA metabolism, cell cycle, oocyte meiosis, and proteasome pathways, whereas those with higher transcript abundance in BCB- oocytes were enriched in gene expression regulation, RNA metabolism, chromatin remodeling, and intracellular signaling. Protein-protein interaction (PPI) network analysis identified distinct hub genes in each group, including CDK1, CCNB2, MAD2L1, and AURKB in BCB+ oocytes and EP300, CREBBP, SMARCA4, and ARID1A in BCB- oocytes. These results provide the first single-cell transcriptomic characterization of BCB-classified alpaca oocytes and establish the molecular basis underlying the biological differences identified by BCB staining.