L. BCB-negative and BCB-positive oocytes. These included genes such as manipulation and maturation of cumulusCoocyte complexes constitute the main channel to produce assisted reproductive technology-ready oocytes in mammals. To augment the developmental competence of oocytes cultured CAY10603 and and and 19.0 and 19.1%; 0.01) (Fig. 1representative images of porcine COCs after BCB staining (the number and percentage of BCB-negative and BCB-positive COCs. representative images of chromatin configuration of GV oocyte nuclei. percentages of GV oocytes classified into different types of chromatin configuration from BCB-control, BCB-negative, and BCB-positive groups. **, 0.01. During maturation (IVM), limited cumulus expansion was observed for the BCB-negative group, as compared with the BCB-control and BCB-positive groups (Fig. 27.8 and 18.3%; 0.001) (Fig. 2, and 76.8%; 0.05) and BCB-positive (52.3 76.5%; 0.05) groups (Fig. 268.7%; 0.01) and blastocyst (59.7 39.2%; 0.01) than the BCB-negative group (Fig. 2, and and maturation of COCs classified by BCB staining. morphology of COCs (IVM for 24 h) and denuded oocytes (IVM for 44 h), as well as representative images of live (and representative images of parthenotes cleaved and developed to blastocysts. percentages of parthenotes cleaved and developed to blastocysts for BCB-control, BCB-negative, and BCB-positive groups. representative images of Hoechst 33342-stained blastocysts. average cell number per blastocyst. **, 0.01. ***, 0.001. Single oocyte RNA-seq To understand the molecular mechanism underlying BCB’s ability to discern oocytes with better quality and developmental competence, single cell transcriptome sequencing was performed on three BCB-negative and three BCB-positive cumulus-denuded GV oocytes, respectively. Standard Bioinformatics pipelines were employed to analyze generated short reads (Fig. S2). Mapping rates of short reads, CAY10603 and statistics on their distribution in the pig genome are summarized in Table S1. Of more than 57 million clean reads produced, 63.81% (on average) can be mapped onto the pig reference genome. We obtained a relatively large number of sequence variations, such as single nucleotide polymorphisms, insertions and deletions, possibly due to breed differences Rabbit polyclonal to Caspase 4 between pig samples we used and the reference genome (a Duroc sow). For each sequenced GV oocyte, global gene expression analyses helped to discover the top 10 most abundantly expressed genes, within which 7 genes were common to all 6 sampled oocytes, including (Table S2). Between BCB-positive and BCB-negative oocytes, a total of 155 genes (44 down-regulated and 111 up-regulated in BCB-positive oocytes) were differentially expressed (with more than 2-fold changes, 0.05) (Fig. 3(38, 39), (40, 41), (42), (43), (44, 45), (46), and (47). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis on differentially expressed genes (DEGs) identified a number of important pathways (Fig. 3, and 0.05). Open in a separate window Figure 3. Single-oocyte transcriptome sequencing. gene expression analysis to identify up- and down-regulated genes in BCB-positive oocytes as compared with BCB-negative oocytes. gene enrichment analysis on differentially expressed genes. KEGG pathways enriched from differentially expressed genes. Table 1 Functional pathways of differentially expressed genes between BCB-negative and BCB-positive immature GV oocytes valuesignaling pathway5 (4.9%)0.13733970.698442099JAK-STATsignaling pathway3 (2.94%)0.17823760.6984420910Wnt signaling pathway3 (2.94%)0.18364450.6984420911Metabolic pathways13 (12.75%)0.18457720.6984420912Regulation of actin cytoskeleton5 (4.9%)0.25268150.69844209 Open in a separate window MAPK, mitogen-activated protein kinase. JAK-STAT, Janus kinase-signal transducers and activators of transcription. Single cell transcriptome sequencing can help reveal cellular heterogeneity (34). Several lines of evidence provided by single oocyte RNA-seq also discovered oocyte heterogeneity in the current study. First, correlation analyses on RNA-Seq data could not separate oocytes clearly into two CAY10603 groups (BCB-negative and BCB-positive) (Fig. S5sample 15 in the same BCB-positive group (Fig. S5and 13 other DEGs were selected for RT-qPCR, profiled on pooled or single oocyte samples for the BCB-control, BCB-negative, and BCB-positive groups, respectively (Tables S4CS6). However, no significant expression differences were found for.