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Abstract Background During normal zygotic division, two haploid parental genomes replicate, unite and segregate into two biparental diploid blastomeres. Results Contrary to this fundamental biological tenet, we demonstrate here that parental genomes can segregate to distinct blastomeres during the zygotic division resulting in haploid or uniparental diploid and polyploid cells, a phenomenon coined heterogoneic division. By mapping the genomic landscape of 82 blastomeres from 25 bovine zygotes, we show that multipolar zygotic division is a tell-tale of whole-genome segregation errors. Based on the haplotypes and live-imaging of zygotic divisions, we demonstrate that various combinations of androgenetic, gynogenetic, diploid, and polyploid blastomeres arise via distinct parental genome segregation errors including the formation of additional paternal, private parental, or tripolar spindles, or by extrusion of paternal genomes. Hence, we provide evidence that private parental spindles, if failing to congress before anaphase, can lead to whole-genome segregation errors. In addition, anuclear blastomeres are common, indicating that cytokinesis can be uncoupled from karyokinesis. Dissociation of blastocyst-stage embryos further demonstrates that whole-genome segregation errors might lead to mixoploid or chimeric development in both human and cow. Yet, following multipolar zygotic division, fewer embryos reach the blastocyst stage and diploidization occurs frequently indicating that alternatively, blastomeres with genome-wide errors resulting from whole-genome segregation errors can be selected against or contribute to embryonic arrest. Conclusions Heterogoneic zygotic division provides an overarching paradigm for the development of mixoploid and chimeric individuals and moles and can be an important cause of embryonic and fetal arrest following natural conception or IVF.

In vitro production of embryos via Intra-Cytoplasmic-Sperm-Injection (ICSI) has become popular in the horse industry. Although improving, the blastocyst yield in clinical practice remains sub-optimal (∼20%: Stout. JEVS 2020; 89:103011). The high incidence of developmental arrest of ICSI embryos is suggested a result of mitotic errors during the first zygotic cell divisions. A better understanding of the cause of these errors could help develop strategies to prevent mitotic errors in equine in vitro produced (IVP) embryos. In somatic cells, the centrosomes, composed of two centrioles surrounded by pericentriolar material are the major microtubule-organizing centers and play essential roles in spindle assembly and chromosome segregation. Mammalian oocytes lack centrosomes and although two centrioles are re-introduced by the spermatozoon at fertilization, centrosomes make only a minor contribution to zygotic spindle assembly (Schneider et al., J.Cell. Biol. 2021; 220(11):e202010106). Although not essential for spindle assembly, the role of the centrosomes in ensuring fidelity of chromosome segregation during zygotic cell division is unclear. The aim of this study was to evaluate the position and number of centrosomes in horse ICSI zygotes during the first mitotic division. Cumulus-Oocyte-Complexes (COCs) were obtained from Warmblood mares by Ovum-Pick-Up, maintained in H-SOF at room temperature overnight and then matured in vitro , fertilized by ICSI and cultured as described previously (Ducheyne et al. Reprod. Fertil. Dev. 2019; 31(12):1830). After 24h, injected oocytes were fixedand immunostained for alpha-tubulin, acetylated tubulin, (peri-)centrosome material (NEDD1) and chromatin (DAPI). For each zygote, confocal microscopy was performed using a Leica SPE II microscope and a 0.17µm interval z stack generated. Of 326 COCs collected, 177 reached MII and were injected and cultured further. Of the 177, 136 zygotes were fixed and imaged, 13 were at the two pronucleus, 11 at prophase/prometaphase, 14 at metaphase, 11 at anaphase or telophase, 54 at the two cell stage and 33 were either degenerated or poorly stained. During the two pronucleus and prophase/prometaphase stages, centrosomes mainly localized at the interface between the two pronuclei. In 56% (n=14/25) of zygotes in pro-metaphase and metaphase, independent spindles formed around each of the two parental pronuclei. The majority of dual spindles were closely adjacent and synchronous. Moreover, in 64.2% (n=9/14) of the metaphase zygotes, the (peri-)centrosomes were either malpositioned (e.g. not at the poles of the spindle) or fragmented. Fragmented or multiple centrosomes are known to predispose to abnormal chromosome-microtubule attachment and aberrant chromosome segregation. We therefore hypothesize that malposition and fragmentation of the centrosomes contributes to mitotic errors and developmental arrest of equine ICSI embryos. Acknowledgements to CAPES, Brazil, for financial support (Code 88882.433332/2019-01 and 88887.467895/2019-00) [ABSTRACT FROM AUTHOR]