RNA-Seq Profiling of Intact and Enucleated Oocyte SCNT Embryos Reveals the Role of Pig Oocyte Nucleus in Somatic Reprogramming

Abstract

The specific molecular mechanisms involved in somatic reprogramming remain unidentified. Removal of the oocyte genome is one of the primary causes of developmental failure in cloned embryos, whereas intact oocyte shows stronger reprogramming capability than enucleated oocyte. To identify the reason for the low efficiency of cloning and elucidate the mechanisms involved in somatic reprogramming by the oocyte nucleus, we injected pig cumulus cells into 539 intact MII oocytes and 461 enucleated MII oocytes. Following activation, 260 polyploidy embryos developed to the blastocyst stage whereas only 93 traditionally cloned embryos (48.2% vs. 20.2%, P < 0.01) reached blastocyst stage. Blastocysts generated from intact oocytes also had more cells than those generated from enucleated oocytes (60.70 vs. 46.65, P < 0.01). To identify the genes that contribute to this phenomenon, two early embryos in 2-cell and 4-cell stages were collected for single-cell RNA sequencing. The two kinds of embryos were found to have dramatically different transcriptome profiles. Intact oocyte nuclear transfer embryos showed 1,738 transcripts that were up-regulated relative to enucleated cloned embryos at the 2-cell stage and 728 transcripts that were down-regulated (|log2Ratio| ≥ 5). They showed 2,941 transcripts that were up-regulated during the 4-cell stage and 1,682 that were down-regulated (|log2Ratio| ≥ 5). The most significantly enriched gene ontology categories were those involved in the regulation of binding, catalytic activity, and molecular transducer activity. Other genes that were notably up-regulated and expressed in intact oocyte nuclear transfer embryos were metabolic process. This study provides a comprehensive profile of the differences in gene expression between intact oocyte nuclear transfer embryos and traditional nuclear transfer embryos. This work thus paves the way for further research on the mechanisms underlying somatic reprogramming by oocytes.

Fig 1

Schema of construction of traditional SCNT embryos(a), intact MII oocyte SCNT embryos(b) and chromosome exchange polyploid embryos(c).

Fig 2. Graphical representations of the developmental capacity of traditional SCNT embryos and intact oocyte SCNT embryos.

(a) Cleavage rate and blastocyst rate of enucleated oocyte SCNT and intact oocyte SCNT. (b) Number of cells in blastocysts generated from parthenogenesis, enucleated oocyte SCNT, and intact oocyte SCNT. (c) This cleavage rate and blastocyst rate in parthenogernesis are affected by the mechanical trauma associated with parthenogenesis. (d) The number of cells in blastocysts generated from intact oocyte parthenogenesis and mechanical trauma oocyte parthenogenesis. (e) The cleavage rate and blastocyst formation rate of intact oocyte SCNT after genome exchange.

Fig 3. Karyotyping of cumulus cells, enucleated oocyte SCNT embryos and intact oocyte SCNT embryos.

Fig 4. The shape of blastocysts generated from parthenogenesis, enucleated oocyte SCNT and intact oocyte SCNT.

Fig 5

Scatter plot of DEGs in (a) A vs. C; (b) B vs. D; (c) A vs. B; (d) C vs. D and GO functional classification of DEGs in (e) A vs. C and (f) B vs. D. Note: For A vs. C, A is the control group. Red points indicate genes up-regulated in C relative to A, green points represent genes down-regulated in C relative to A, and blue points represent genes that showed no differences or fold change below 2.

Fig 6. Cluster image of DEG levels of four samples.

Each column represents an experimental sample, and each row represents a gene. Differences in expression are shown in different colors. Red indicates up-regulation and green represents down-regulation.

Fig 7

Top 20 statistics of pathway enrichment for (a) A vs. C and (b) B vs. D.

Fig 8. Cluster image of DEG levels of transcription factor activity genes expressed in the nucleus.

Each column represents one sample, and each row represents one gene. Red indicates up-regulation and green indicates down-regulation.

Fig 9. Expression of selected genes in real-time quantitative RT-PCR.