Origin and development of uniparental and polyploid blastomeres

Abstract

Whole-genome (WG) abnormalities, such as uniparental diploidy and triploidy, cause fetal death. Occasionally, they coexist with biparental diploid cells in live births. Understanding the origin and early development of WG abnormal blastomeres is crucial for explaining the formation of androgenotes, gynogenotes, triploidy, chimerism, and mixoploidy. By haplotyping 118 bovine blastomeres from the first cleavages, we identified that heterogoneic division occurs in both multipolar and bipolar cleaving zygotes. During heterogoneic division, parental genomes segregate into distinct blastomeres, resulting in the coexistence of uniparental and biparental diploid or polyploid cells. After culturing the totipotent blastomeres to three preimplantation stages and exploring transcriptomes of 446 cells, we discovered that stress responses contribute to developmental impairment in WG abnormal cells, resulting in either cell arrest or blastocyst formation. Their dominance in preimplantation embryos represents an overlooked cause of abnormal development. Haplotype-based screening could improve in vitro fertilization outcomes.

Keywords: Molecular biology; Omics.

Graphical abstract

Figure 1

Experimental setup (A) Diagram illustrating the experimental design. Bovine embryos were created and cultured in vitro. After the first zygotic division, blastomeres were dissected and cultured to three preimplantation time points. Blastomere outgrowths were subsequently collected and dissected into single cells. Each dissected cell underwent genome and transcriptome separation followed by sequencing. Genome data were used for haplotyping and copy-number profiling, whereas transcriptome data were used for gene expression analysis. Genome and transcriptome sequencing (G&T-seq): protocol for isolating poly(A) RNA and genomic DNA from single cells. Genotyping-by-sequencing (GBS): sequencing-based protocol for genome-wide haplotyping and copy-number profiling. (B) Example images of blastomere outgrowths collected at T1, T2, and T3, respectively.

Figure 2

Whole-genome segregation errors occur in multipolar and bipolar first cleavages (A) Numbers of bipolar and multipolar cleaving embryos used, categorized based on the completeness of embryo-derived blastomere outgrowth haplotyping: fully haplotyped (All checked), partially haplotyped (Partial), or not haplotyped (Unknown), with the y axis representing the fraction of counts. (B) The inferred chromosomal constitution of 118 blastomeres in culture, with the x axis representing the fraction of counts. (C) Three categories of genome segregation patterns identified during the first cleavage. In the oocyte being fertilized, the second meiotic division is depicted, along with the likely number of sperms fertilizing the embryo as deduced from the haplotypes. The arrows surrounding the pronuclei indicate replication occurred. During the first cleavage, spindles segregating the sister chromatids are shown. In the absence of a spindle, the genome is extruded. Numbers are given for patterns that occurred more than once. ∗ in category 1 indicates the one embryo with bipolar first cleavage and WG segregation error. ∗ in category 3 indicates the four embryos with multipolar first cleavage and without WG segregation error. For androgenetic blastomeres, different colors indicate genetic material from different sperm. (D) Chromosomal segregation patterns for embryos with bipolar or multipolar first cleavage and the inferred number of sperm that fertilized the egg, with the y axis representing the fraction of counts. Normal: zygote cleaved without WG segregation error. Heterogoneic: zygote cleaved with WG segregation error. Bipolar and multipolar refer to embryos undergoing either bipolar or multipolar first cleavage, respectively. See also Tables S1 and S2.

Figure 3

Blastomeres with whole-genome abnormalities can reach blastocyst stage despite impaired developmental potential (A) Distribution of 53 biparental diploid blastomere outgrowths and 48 blastomere outgrowths with WG abnormalities across different time points of outgrowth collection, with the x axis representing the fraction of counts. (B) Developmental stage comparison of biparental diploid blastomere outgrowths and blastomeres outgrowths with WG abnormalities in (A), with the x axis representing the fraction of counts (Fisher’s exact test). (C) Schematic representation of the six blastomere outgrowths with WG abnormalities that reached the blastocyst stage at T3, along with their sibling blastomere outgrowths and original embryos. (D) Images of the two cleaved anuclear blastomere outgrowths and schematic diagrams illustrating the embryos from which they originated. In (C) and (D), the same symbols as in Figure 2C are employed. Fertilization and the first cleavage are not depicted for embryos containing blastomeres with an unknown haplotype (due to sample loss or haplotype failure). See also Figure S1 and Tables S1, S2, and S3.

Figure 4

Whole-genome abnormalities do not alter the preimplantation developmental program but hinder transcriptomic development UMAP for single-cell transcriptome data with cells colored by (A) time of outgrowth collection, (B) genome constitution, (C) and molecular developmental stage indicated by marker genes expression (D) pseudotime value inferred from trajectory analysis. The inferred pseudotime trajectories are indicated by black curved lines on the UMAP. (E) The numbers of biparental diploid cells and cells with WG abnormalities used for transcriptome analysis for each time point, with the y axis representing the fraction of counts. (F) Pseudotime comparison of biparental diploid cells and cells with WG abnormalities at each outgrowth collection time point [Student’s t test; the numbers of biparental diploid cells and cells with WG abnormalities for each time point are shown in (E)]. Each dot represents one cell, with the x axis indicating its pseudotime value extracted from (D). Cells are colored according to their corresponding molecular developmental stage in (C) using the same color scheme. Vertical gray bars indicate the mean pseudotime value of each group. See also Figures S2–S4 and Table S2.

Figure 5

Distinct preimplantation cellular states are governed by specific key transcription factors (A) UMAP displaying cell clustering based on regulon activity, with cells colored according to their macular developmental stage as depicted in Figure 4C. (B) Heatmap of regulon activity. Each row represents one regulon, and each column represents one cell. The plot is generated using rescaled area under the curve (AUC) values, with cells ordered according to their molecular developmental stage and pseudotime values. (C) Key regulons selected for each stage. See also Figure S5 and Table S4.

Figure 6

Whole-genome abnormalities induce stress responses during embryonic genome activation (A) Numbers of DE genes identified in gynogenetic (GG), androgenetic (AG), polyploid (PL), and combined WG-aberrant cells compared to biparental diploid cells for each stage. The "/" symbol denotes comparisons not conducted due to insufficient cells. (B) Gene expression dot plot for DE genes during major EGA. (C) Overall alterations in biological processes reflected by deviations in gene expression. "/" if no DE genes observed. See also Figures S3 and S6 and Table S5.