Multipolar zygotic divisions are characterized by parental genome segregation errors. Multipolar zygotic divisions are characterized by parental genome segregation errors

The first mitotic division causes both parental genomes present in the zygote to segregate into two biparental diploid daughter cells. This fundamental tenet was challenged by the observation that blastomeres with different genome ploidy and distinct parental genotypes can coexist within individual embryos. We hypothesized that whole parental genomes can segregate into distinct blastomere lines during the multipolar division of the zygote, a phenomenon referred to as “heterogoneic” cell division. Here, we provide evidence of genome-wide segregation errors in two human blastocysts and further pinpoint its origin in a bovine model by mapping the genomic landscape of 82 blastomeres from 25 embryos that underwent multipolar division at the zygote stage using genome-wide SNP arrays and sequencing. In most embryos, the coexistence of androgenetic and diploid or polyploid blastomeres with or without anuclear blastomeres, androgenetic and anuclear blastomeres, and androgenetic and gynogenetic blastomeres within the same embryo provided proof that multipolar zygotic division coincides with heterogoneic segregation of the parental genome. By mapping the segregational origin of the genomic content, we deduced distinct segregation mechanisms underlying heterogoneic cell division including segregation by a tripolar spindle, the pronuclear extrusion of a paternal genome and, the operation of an ectopic paternal or private parental spindles. Polyspermic embryos expel excessive paternal genomes resulting in an androgenetic or polyploid blastomere. Confirming the results in human blastocysts we found genome-wide segregation errors to persist in bovine blastocysts. Overall design: The genome-wide haplotype and copy number of a total of 146 bovine samples (82 blastomeres and two fragments isolated immediately upon time-lapse observation of multipolar division and 62 single cells derived from six dissociated blastocysts developing after multipolar division) belonging to 17 bovine pedigrees was determined to understand the origin and further development of mixoploid and/or chimeric embryos. The semen from three bulls was used for embryo production. Ovarian tissue samples from the donor cows (mothers), semen from the bull (father) or father of the bull (paternal grandparent) and blood of the mother of the bull (maternal grandparent) were used to extract bulk DNA (DNeasy Blood and Tissue kit, Qiagen, Germany). Ovaries were collected separately and retrieval of the oocytes was followed by routine in vitro embryo production. Single blastomeres and fragments were isolated immediately upon identification of a multipolar division by digestion of the zona pellucida with pronase and mechanical dissociation. Single cells derived from embryos that developed to the blastocyst stage following multipolar division were isolated by a zona pellucida digestion and subsequent trypsinization and mehchanical dissociation. All samples and sibling embryos were whole-genome amplified (WGA) using commercial multiple displacement amplification kit (Repli-G Single Cell kit, Qiagen, Germany). WGA products and bulk (grand)parental and sibling DNA were then SNP genotyped on BovineHD BeadChip arrays using Infinium HD whole-genome genotyping assay (Illumina Inc., USA). Subsequently, the SNP logR and SNP B-allele frequency (BAF) values, as well as the discrete SNP genotype calls were obtained for each single cell and applied in further single-cell analysis to obtain genome-wide haplarithm profiles using a modified version of the siCHILD algorithm (Zamani Esteki et al. 2015).