131 RNA-Seq TRANSCRIPTOME PROFILING OF INDIVIDUAL RHESUS MACAQUE OOCYTES AND PRE-IMPLANTATION EMBRYOS

Abstract

The objective of this study was to perform whole transcriptome sequencing (RNA-Seq) on individual rhesus macaque oocytes and intracytoplasmic sperm injection-derived embryos. Nine developmental stages were assayed (n = 2–3 per stage): germinal vesicle (GV), meiosis I (MI), and meiosis II (MII) oocytes and pronuclear (PN), 2-cell (2C), 4-cell (4C), 8-cell (8C), morula (MO), and blastocyst (BL) stage embryos. Rhesus females were superovulated with hrFSH and oocytes collected by follicular aspiration of ovaries recovered at necropsy 35 h after hCG treatment. Based on GV and polar body (PB) presence/absence, oocytes were classified as GV, MI, and MII. Embryos were produced by intracytoplasmic sperm injection of MII oocytes and cultured in HECM-9 media. Individual oocytes and embryos were collected at different developmental stages and frozen until use. The Clontech SMARTer Ultra Low Input RNA Kit for Illumina Sequencing was used for cDNA synthesis (oligo dT) and amplification. Sequencing libraries were created from fragmented cDNA with the Illumina TruSeq DNA kit. Single 100-bp reads were produced with a HiSEqn 2000 apparatus. Read alignment was done in CLC Genomics Workbench software to the RhesusBase genome annotation (53 788 genes). A gene was considered expressed if reads per kilobase of transcript model per million mapped reads (RPKM) was >0.4. Principal component analysis (PCA), hierarchical clustering, and differential gene expression analyses were performed with the DESEqn 2 package in R. On average, 22 311 310 reads were produced per sample with 63% aligning to the rhesus transcriptome. A total of 14 527 genes were detected in all replicates of at least one stage, with an average of 8855 genes per stage. The PCA and hierarchical clustering of all expressed genes discriminated between samples of different stages, except for 2C and PN, which were indistinguishable. All oocytes grouped to the same subcluster and close to PN-4C embryos, forming another subcluster. The 8C embryos constituted their own subcluster, which was closer to MO and BL than to other pre-implantation stages. Of genes expressed exclusively in embryo samples (n = 2823), 42% were expressed beginning at the 8C stage, 39% began expression at MO or BL, and 18% started expression between PN to 4C stage. The highest numbers of differentially expressed (DE) genes between consecutive stages were for MI-MII, MII-PN, 4C-8C, and 8C-MO, with an average of 5306 DE genes, whereas only 1060 were found in other comparisons. When gene expression was compared relative to the MII stage, 8C, MO, and BL had the highest numbers of DE genes (8965, 9635, and 9100, respectively, v. an average of 4713 for all other embryo stages). The high proportion of embryo-specific genes beginning expression at the 8C stage along with large numbers of DE genes observed between MII and 8C and the isolation of 8C samples to a unique expression cluster with PCA/hierarchical clustering indicates that major embryonic genome activation occurs at the 8C stage in the rhesus macaque. Overall, the dataset represents a comprehensive resource for analysis of polyadenylated transcript levels throughout early development in a nonhuman primate species.