Expression profiling and function analysis identified new cumulus cells-expressed genes and miRNAs predictive of oocyte developmental potential

Although studies have identified cumulus cells (CCs)-expressed genes/miRNAs that regulate cumulus expansion and/or CC apoptosis and may be used as markers for selection of competent oocytes/embryos, CCs-expressed genes/miRNAs whose expression levels are directly correlated with oocyte developmental potential (DP) are urgently needed. To identify CCs-expressed DP-directly-related genes/miRNAs, we first established CC models from mouse cumulus-oocyte-complexes that showed significantly different DP after maturation in vitro or in vivo. By conducting mRNA/miRNA sequencing and functional analysis using such in vitro and in vivo CC models, we identified and validated new sets of CCs-expressed genes/miRNAs, whose expression levels were directly correlated with oocyte DP. Thus, we identified and validated Spp1, Fn1, Sdc1 and Ngf as DP-beneficial genes, Fos and Jun as DP-detrimental genes, and miR-7686-5p, miR-133a-3p, novel-miR-239, novel-miR-193 and miR-339-5p as DP-detrimental miRNAs. Furthermore, the close similarities in top KEGG pathways among 4 different experiments suggest that CCs-expressed genes and/or miRNAs regulate oocyte DP mostly indirectly through regulating cumulus expansion and/or CC apoptosis. The new genes/miRNAs identified can be used as markers for selection of competent oocytes/embryos, and the data will contribute to elucidating oocyte maturation mechanisms. Overall design: Mouse COCs were cultured for 18 h in good (GMS) or poor maturation system (PMS) before harvesting CCs for isolation of mRNAs and miRNAs (Fig. 1). The isolated mRNAs and miRNAs were subjected to RNA- and miRNA-seq to identify differentially expressed (DE) genes and miRNAs, respectively. To select genes, the DE genes were subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and the top enriched genes obtained were subjected to protein–protein interaction (PPI) analysis to obtain the top degree genes. Candidate genes were selected from the top degree genes. To select miRNAs, target genes were predicted from the DE miRNAs and were overlapped with the DE genes identified by RNA-seq to determine the potential genes. The potential genes were subjected to KEGG to obtain the top enriched genes, which were subjected to PPI to get the top degree genes. Candidate miRNAs were predicted from the top degree genes. The candidate genes and miRNAs were subjected to an in vivo COC validation before functional analysis. For functional analysis, COCs were transfected with siRNA or gene overexpression (GOE) vectors of the candidate genes, or with mimics (MM) or inhibitors (IN) of the candidate miRNAs, and the transfected COCs were cultured in GMS before assessment of DP to determine the DP-related (DPR) genes or miRNAs, respectively.