221 RNA-Seq ANALYSIS OF INNER CELL MASS AND TROPHECTODERM CELLS OF EQUINE BLASTOCYSTS

The objective of this study was to characterise the transcriptome profiles of inner cell mass (ICM) and trophectoderm (TE) cells from horse blastocysts. Equine blastocysts were collected by uterine flushing 8 days after ovulation in mares superovulated with reFSH and reLH (Meyers-Brown et al. 2011 Anim. Reprod. Sci. 128, 52–59). The ICM were isolated by immunosurgery, whereas polar TE cells were obtained microsurgically. Individual ICM (n = 2) and TE (n = 3) samples were snap frozen in liquid nitrogen and stored at –80°C until processing. Total RNA was extracted from each individual sample using the Arcturus PicoPure RNA isolation kit including DNAse treatment. The RNA was then amplified using the SPIA-based Ovation RNASeq-II kit (NuGEN Inc., San Carlos, CA, USA). Sequencing libraries were prepared using the TruSeq DNA sample preparation kit (Illumina, San Diego, CA, USA). Libraries bearing unique indexes per sample were pooled and sequenced in a single lane of a HiSEqn 2000 apparatus (Illumina) by a single run of 100 bp. Data analysis was performed using CLC Genomics Workbench. Equus caballus genomic sequences and annotation (EquCab2.0) were obtained from the National Center for Biotechnology Information (NCBI). Sequence analysis was performed using CLC Genomics Workbench and differential expression by DeSeq analysis. A total of 196 669 501 reads were produced. After discarding duplicated and bad quality reads, an average of 22 508 594 reads per sample was used for analysis. Using the RNASeq algorithms, 77% of reads mapped to annotated transcripts. Among the 22 380 annotated genes, 11 677 and 11 919 we detected as expressed [reads per kilobase of exon model per million sequences (RPKM) > 0.3] in all ICM and TE samples, respectively. The correlation of RPKM values for all the genes analysed between any pair of ICM or TE samples was >0.97, indicating a high repeatability of the assay. Global analysis of the transcriptome by means of unsupervised clustering indicated that ICM and TE samples clustered to different groups. Genes known to be specific to ICM and TE were expressed primarily in their respective tissue, including KLF4, SOX2, POU5F1, NANOG, DNMT3B, LIN28A, FOXA2, SALL4, and HNF4A for ICM, and CDX2, KRT8, ATP12A, GRHL2, and GRHL1 for TE. In addition, genes related to primitive endoderm development were more highly expressed in ICM than in TE and included GATA4, GATA6, and PDGFRA. The DeSeq analysis between ICM and TE samples indicated that 1934 genes were differentially expressed (adjusted P 2). Biological functions overrepresented among genes that were overexpressed in ICM cells (n = 1374) included cell-cell adhesion, cell morphogenesis involved in differentiation, negative regulation of DNA binding, and cell proliferation. Among genes overexpressed in TE samples (n = 560), gene ontology analysis indicated that the most overrepresented biological processes were lipid localization, placenta development, and organic acid transport. In summary, this study provides a comprehensive analysis of genes expressed in ICM and TE of equine embryos, which is a fundamental resource to understanding early embryo development in this species.