Reprogramming barriers in bovine cells nuclear transfer revealed by single‐cell RNA‐seq analysis

Abstract

Many progresses have recently been achieved in animal somatic cell nuclear transfer (SCNT). However, embryos derived from SCNT rarely result in live births. Single‐cell RNA sequencing (scRNA‐seq) can be used to investigate the development details of SCNT embryos. Here, bovine fibroblasts and three factors bovine iPSCs (3F biPSCs) were used as donors for bovine nuclear transfer, and the single blastomere transcriptome was analysed by scRNA‐seq. Compared to in vitro fertilization (IVF) embryos, SCNT embryos exhibited many defects. Abnormally expressed genes were found at each stage of embryos, which enriched in metabolism, and epigenetic modification. The DEGs of the adjacent stage in SCNT embryos did not follow the temporal expression pattern similar to that of IVF embryos. Particularly, SCNT 8‐cell stage embryos showed failures in some gene activation, including ZSCAN4 , and defects in protein association networks which cored as POLR2K, GRO1, and ANKRD1. Some important signalling pathways also showed incomplete activation at SCNT zygote to morula stage. Interestingly, 3F biPSCNT embryos exhibited more dysregulated genes than SCNT embryos at zygote and 2‐cell stage, including genes in KDM family. Pseudotime analysis of 3F biPSCNT embryos showed the different developmental fate from SCNT and IVF embryos. These findings suggested partial reprogrammed 3F biPS cells as donors for bovine nuclear transfer hindered the reprogramming of nuclear transfer embryos. Our studies revealed the abnormal gene expression and pathway activation of SCNT embryos, which could increase our understanding of the development of SCNT embryos and give hints to improve the efficiency of nuclear transfer.

Dissecting the molecular features of bovine-arrested eight-cell embryos using single-cell multi-omics sequencing

“…After use of stringent quality-control filters to ensure the construction of a high-quality database, we obtained 14 single-cell DNA and RNA libraries. We reanalyzed the single-cell RNA-seq data extracted from the PRJNA727165 public data set [33] of bovine in vitro fertilization embryo samples for uniform manifold approximation and projection (UMAP) analysis.…”

“…Consistent with previous studies [23,24], pluripotency-related genes and oocyte-marker genes were highly expressed in both blastocyst-developed and arrested embryos, while granulosa cell marker genes were not expressed (Figure 1E). We combined our eight-cell embryo data with all stages of preimplantation embryos from a recent study [33] and performed an integrated single-cell RNA analysis. The global expression pattern of all samples further confirmed that blastomeres from eight-cell embryos were collected correctly (Supplementary Figure S1D).…”

Bovine blastocyst like structures derived from stem cell cultures

“…We collected single-cell sequencing data from published literature for comparative analysis. Two Bovine IVF single-cell sequencing raw FASTQ data were downloaded from the GEO database, including 179 IVF cells 35 sequenced using Smart-seq2 and 98 IVF cells 36 sequenced using STRT-seq.…”

“…Data availability 8 cell and 16 cell from GSE99210 (Single-cell RNA sequencing reveals developmental heterogeneity of blastomeres during major genome activation in bovine embryos) 28 . Zygote, 2 cell, 8 cell, morula and blastocyst from PRJNA727165 (Reprogramming barriers in bovine cells nuclear transfer revealed by single cell RNA seq analysis) 27 . Raw unprocessed data of gastrulation embryos was obtained from Dr. Peter L. Pfeffer 29 .…”

“…To determine the transcriptional states of bovine blastoid cells, we performed single-cell RNA-sequencing (scRNA-seq) using the 10x Genomics Chromium platform and carried out integrated analysis with Smart-seq2 single-cell transcriptomes derived from zygote 27 , 2-cell 27 , 8-cell 28 , 16-cell 28 , morula 27 and day 7.5 blastocyst stage IVF bovine embryos 27 as well as in vivo bovine blastocysts (see data availability). Joint uniform manifold approximation and projection (UMAP) embedding revealed blastoid-derived cells clustered with blastocyst-derived cells ( Fig.…”