O-313 Meiotic progression of human oocytes is characterized by mRNA splicing events of specific meiosis and spindle associated transcripts

Abstract

Abstract Study question Is there a specific splicing of transcripts associated with the progression through meiosis in human oocytes? Summary answer Single oocyte mRNA sequencing (scRNA-seq) reveals that several meiosis- and spindle-associated genes are specifically spliced during final meiotic maturation. What is known already Final meiotic maturation in human oocytes entails a fine regulation of the expression of maternal mRNAs, for both final oocyte growth and the initial phases of embryo development and is key to reproductive success. However, very little is known about the post-transcriptional mechanism regulating this process. Alternative splicing (AS) is the process of intron removal and exon-ligation and contributes to transcriptome complexity by generating different protein isoforms from the same gene. The analysis of oocytes’ mRNAs splicing patterns during meiotic maturation might give important information on the regulation of key transcripts responsible for the acquisition of oocyte competence. Study design, size, duration Thirty-four women undergoing oocytes donation cycles were enrolled in this study, and 40 oocytes, processed and analysed individually, were included. The following groups were compared: 10 immature germinal vesicles (GV), 10 GVs that failed to undergo GV breakdown (GVBD) after 30 hours of in vitro maturation (IVM) in G2plus media (FTM-GV), 10 matured oocytes after IVM (IVM-MII) and 10 in vivo matured MII oocytes (IVO-MII). Participants/materials, setting, methods Total RNA from each oocyte was extracted with PicoPure RNA Extraction kit, cDNA was generated and libraries from single oocytes were constructed using OvationSoLo RNA-Seq System and sequenced on a HiSeq 2500, with 2x150bp reads for alternative isoform analysis. Data were processed and analysed using the Bioconductor package DEXSeq, and exons with adjusted p-value<0.05 were considered significantly different. EGSEA package was used to perform pathway analysis. Main results and the role of chance We obtained high-quality data from each sample, with nearly 96% of the reads mapping to the human reference genome. We analysed specifically the genes that did not present differences in global mRNA expression, but presented differences in single exons, indicating a differential splicing pattern. When comparing GV and FTM-GV, only 3 differentially spliced genes (DSG) were found, indicating that the two types of cells present similar splicing patterns. Conversely, the comparison of GV vs IVM-MII and GV vs IVO-MII resulted in 550 genes and 846 genes with differentially spliced exons, respectively, with a 55% transcripts overlap between the two comparisons. Pathway enrichment analysis from the KEGG database performed on the common DSGs between GV and MIIs identified oocyte meiosis, progesterone-mediated oocyte maturation and cell cycle as containing most of the spliced genes. Genes with known involvement in chromosome segregation and spindle assembly such as CENPC and AURKA, and cell cycle regulators like CDK1 and BUB1were within the most relevant spliced transcripts in the MII group. Our findings demonstrate that transcripts important for the acquisition of oocyte meiotic competence are specifically processed when the final phases of maturation are triggered, specifically after GVBD. Limitations, reasons for caution The immature oocytes used in this study were obtained after hormonal stimulation and LH priming. We did not perform a functional analysis of the spliced exons, therefore no speculations on the differential protein functions can be performed. Wider implications of the findings The analysis of DSG between GV and MIIs identified specific spliced exons from genes mainly involved in different aspects of oocyte meiosis. Our findings highlight the key role of post-transcriptional events during the final phases of human meiosis. Trial registration number NA