Global MicroRNA Expression Profiling of Buffalo (Bubalus bubalis) Embryos at Different Developmental Stages Produced by Somatic Cell Nuclear Transfer and In-Vitro Fertilization Using RNA Sequencing

Abstract

Despite the success of cloning technology in the production of offspring across several species, its application on a wide scale is severely limited by the very low offspring rate obtained with cloned embryos. The expression profile of microRNAs (miRNAs) in cloned embryos throughout embryonic development is reported to deviate from regular patterns. The present study is aimed at determining the dynamics of the global expression of miRNA profile in cloned and in-vitro fertilization (IVF) pre-implantation embryos at different developmental stages, i.e., the two-cell, eight-cell, and blastocyst stages, using next-generation sequencing. The results of this study suggest that there is a profound difference in global miRNA profile between cloned and IVF embryos. These differences are manifested throughout the course of embryonic development. The cloned embryos differ from their IVF counterparts in enriched Gene Ontology (GO) terms of biological process, molecular function, cellular component, and protein class categories in terms of the targets of differentially expressed miRNAs. The major pathways related to embryonic development, such as the Wnt signaling pathway, the apoptosis signaling pathway, the FGF signaling pathway, the p53 pathway, etc., were found to be affected in cloned relative to IVF embryos. Overall, these data reveal the distinct miRNA profile of cloned relative to IVF embryos, suggesting that the molecules or pathways affected may play an important role in cloned embryo development.

Keywords: In-vitro fertilization (IVF); RNA sequencing; bovine; gene ontology; miRNAs; somatic cell nuclear transfer (SCNT).

Figure 1

Number of known and novel miRNAs in the three replicates of (A) 2-cell stage cloned (2CC_1, 2CC_2, and 2CC_3) and 2-cell IVF embryos (2CI_1, 2CI_2, and 2CI_3); (B) 8-cell stage cloned (8-CC_1, 8CC_2, and 8CC_3) and IVF embryos (8CI_1, 8CI_2, and 8CI_3); and (C) cloned blastocyst (BL_1, BL_2, and BL_3) and IVF blastocyst (BLI_1, BLI_2, and BLI_3).

Figure 2

Reads of (A) 2-cell stage cloned and IVF embryos (2CC_1, 2CC_2, 2CC_3, 2CI_1, 2CI_2, and 2CI_3), (B) 8-cell stage cloned and IVF embryos (8CC_1, 8CC_2, 8CC_3, 8CI_1, 8CI_2, and 8CI_3), and (C) cloned and IVF blastocyst (BL1, BL2, BL3, BLI_1, BLI_2, and BLI_3) were mapped to different chromosomes of Bos Tauras (reference genome-UMI 3.1.1). The maximum number of reads was found to map on chromosome number 25.

Figure 3

Hierarchical clustering analysis of miRNAs expressed differentially in cloned and IVF embryos based upon RPKM value, reflecting similar expression patterns between replicates of the same origin. (A) For 2-cell stage embryos, cloned replicates were named 2CC_1, 2CC_2, and 2CC_3. Similarly, for IVF replicates, the names were 2CI_1, 2CI_2, and 2CI_3. (B) For 8-cell stage embryos, the cloned replicates were named 8CC_1, 8CC_2, and 8CC_3. Similarly, for IVF replicates, the names were 8CI_1, 8CI_2, and 8CI_3. (C) For the blastocyst stage, the cloned replicates were named BL_1, BL_2, and BL_3. Similarly, for IVF replicates, the names were BLI_1, BLI_2, and BLI_3.

Figure 4

Venn diagram showing the overall commonly and uniquely expressed miRNAs in cloned and IVF (A) 2-cell (2CC and 2CI), (B) 8-cell (8CC and 8CI), and (C) blastocyst-stage (BL and BLI) embryos.

Figure 5

(A) MA plot depicting the expression pattern of up- and down-regulated miRNAs in cloned relative to IVF embryos at the 2-cell stage. The Y-axis represents the log fold ratio (M) and the X-axis is the mean average of normalized counts. The red dots represent differentially expressed miRNAs having adjusted p-values above the threshold value, whereas the black dots represent differentially expressed miRNA having p-values below the threshold. (B) Volcano plot showing differentially expressed up- and down-regulated miRNAs in cloned relative to IVF embryos at the 2-cell stage. The red dots indicate miRNAs significantly differentially expressed, whereas the black dots indicate miRNAs the expression of which was non-significant in the two groups. The dots towards the left, right, and top denote down-regulated, up-regulated, and most significantly expressed miRNAs, respectively. (C,D) MA plot and volcano plot for 8-cell stage cloned embryos relative to their IVF counterparts. (E,F) MA plot and volcano plot for blastocyst stage cloned embryos relative to their IVF counterparts.

Figure 6

Bar graph depicting the number of differentially expressed miRNAs up- or down-regulated in cloned relative to IVF (A) 2-cell stage, (B) 8-cell stage, and (C) blastocyst stage embryos at FC ≥ 2 (p < 0.05).

Figure 7

Bar graph depicting the number of commonly expressed miRNAs up- or down-regulated in cloned relative to IVF (A) 2-cell stage, (B) 8-cell stage, and (C) blastocyst stage embryos at FC ≥ 2 (p < 0.05).

Figure 8

KEGG pathway analysis shows the Wnt signaling pathway and the target genes affected by under- and over-presented miRNAs in cloned relative to IVF embryos.

Figure 9

Validation of RNA-seq data by qPCR analysis of 6 miRNAs expressed differentially in cloned and IVF 2-cell, 8-cell, and blastocyst stage embryos. The pattern and magnitude of relative expression levels were found to be similar in RNA-seq and qPCR data. Bars with different superscripts differ significantly (* p < 0.05; ** p < 0.01; *** p < 0.0001). Values are mean ± SEM.