Human embryonic genome activation initiates at the one-cell stage

Abstract

In human embryos, the initiation of transcription (embryonic genome activation [EGA]) occurs by the eight-cell stage, but its exact timing and profile are unclear. To address this, we profiled gene expression at depth in human metaphase II oocytes and bipronuclear (2PN) one-cell embryos. High-resolution single-cell RNA sequencing revealed previously inaccessible oocyte-to-embryo gene expression changes. This confirmed transcript depletion following fertilization (maternal RNA degradation) but also uncovered low-magnitude upregulation of hundreds of spliced transcripts. Gene expression analysis predicted embryonic processes including cell-cycle progression and chromosome maintenance as well as transcriptional activators that included cancer-associated gene regulators. Transcription was disrupted in abnormal monopronuclear (1PN) and tripronuclear (3PN) one-cell embryos. These findings indicate that human embryonic transcription initiates at the one-cell stage, sooner than previously thought. The pattern of gene upregulation promises to illuminate processes involved at the onset of human development, with implications for epigenetic inheritance, stem-cell-derived embryos, and cancer.

Keywords: embryonic genome activation (EGA); fertilization; human one-cell embryo; single-cell RNA-seq; totipotency; transcriptome; zygote.

Graphical abstract

Figure 1

Human embryonic transcription initiates at the one-cell stage (A) Schematic of human one-cell embryo development at the times after fertilization (Capmany et al., 1996). Pb₁, first polar body; Pb₂, second polar body; PN, pronuclei; PNMB, pronuclear membrane breakdown. (B) Brightfield images of representative human metaphase II oocytes (mII) and bipronuclear one-cell embryos (emb). Arrowheads indicate pronuclei. Pb₁, first polar body; Pb₂, second polar body. Scale bar, 50 μm. (C) t-SNE analysis (without filtering) of single-cell RNA-seq data for human mII oocytes (mII, n = 12) and bipronuclear (2PN) one-cell embryos (emb, n = 12). Circles correspond to oocytes from an African American/Hispanic donor and triangles correspond to embryos from Asian donors, with all other oocytes and embryos being of Caucasian origin and filled triangles and circles corresponding to symbols of (D). (D) Heatmap showing changes in gene expression levels (FDR < 0.1, log₂FC > 0.58) in human mII oocytes (mII) (n = 12) and bipronuclear (2PN) one-cell embryos (emb; n = 12) of (C), indicating donors (top) and the Z score scale (−4 to 4). Each patient is represented by a symbol to indicate the provenance of oocytes and embryos. (E) Single-cell qPCR of upregulated DEG transcripts in individual human oocytes (mII; n ≥ 3 independent biological replicates) and 2PN one-cell embryos (emb; n ≥ 3 independent biological replicates) (FDR < 0.1, log₂FC > 0.5). Different oocytes and embryos were used to those of (D). Corresponding log₂FC values from RNA-seq are indicated beneath histograms. Primer pairs flanked exon junctions except in the cases of TIGD5 and NFKB1A (which have a single exon) and PIAS3. Values are ± SEM and normalized against mII oocytes. (F) Pie chart showing functional classes of upregulated DEGs (FDR < 0.1, cpm > 1.0). Ψ, pseudogene; ERV, endogenous retrovirus; lncRNA, long non-coding RNA; AS, antisense; miRNA, microRNA; si/so, sense-intronic/sense-overlapping; pt, processed transcript. (G) Pie chart showing processing classes (FDR < 0.34) of upregulated DEGs (p < 0.05; cpm > 1.0). Letters indicate Ingenuity codes. See also Figure S1 and Table S1.

Figure 2

Human one-cell upregulated gene characteristics and pathways (A) Raw scRNA-seq density plots (Sashimi plots) along exons and exon junctions. Arcs representing splice junctions connecting exons and display the number of reads split across the junction (junction depth) in mII oocytes (mII) and 2PN one-cell embryos (emb). Genomic coordinates (chrom) and gene annotation tracks are aligned beneath each respective plot. Solid black bars above plots indicate regions of potential alternative splicing. (B) qPCR for transcripts in individual human monopronuclear (1PN) and tripronuclear (3PN) one-cell embryos (3 ≥ n ≥ 6 biologically independent oocytes or embryos per target). Values for metaphase II oocytes (mII) and bipronuclear one-cell embryos (2PN) from Figure 1E are included for comparison. Values are ± SEM and normalized against mII oocytes (∼1.0). Unpaired t tests indicate p < 0.2. (C) Ingenuity pathway analysis (IPA) of gene networks upregulated (FDR < 0.1, log₂FC > 0) in 2PN one-cell embryos. (D–F) Upstream transcription regulators inferred by IPA of upregulated gene networks (FDR < 0.1, log₂FC > 0) in 2PN one-cell embryos for E2F4 (D), MYC (E), and MYCN (F). See also Figure S2 and Table S1.