O-179 Fertilization topologies that occur seldom or not at all in nature give rise to distinct functional classes of mouse embryos

Abstract Study question Would new embryo properties emerge if fertilization was imposed on oocytes in regions, such as the animal pole, where it does not take place naturally? Summary answer Oocyte at vegetal pole or equator results 2-cell embryos with distinguishable transcriptomes and functional peculiarities. What is known already There bias used basic clinical research, because: 1) sperm–oocyte fusion hardly occurs surface above meiotic spindle – pole; 2) region opposite - poorly accessible to sperm due tiny perivitelline space. These regions are also avoided during intracytoplasmic injection (ICSI) for fear of damaging losing nucleus into 2nd polar body. Pole materials held non-essential mouse development, but this conclusion relies body, which an unreliable topological marker. design, size, duration Oocytes were rotated using a micromanipulator fitted Nomarski optics, landmark. Between 9:30 10:30 am single heads microinjected (treatments) vs. naturally prevalent equatorial (control). This way two topologies possibly classes zygotes created that otherwise seldom all represented natural fertilization, vitro insemination conventional ICSI. Participants/materials, setting, methods Metaphase II collected from 8-week-old B6C3F1 mice stimulated 10 I.U. eCG+hCG. Sperm batch cryopreserved CD1 semen deposited via ICSI cortex half-way between poles i.e. equatorially. Zygotes cultured KSOM(aa) analyzed (triplicate more) for: cleavage rates, stage (RNAseq), blastocyst germ layers (immunostaining trophectoderm, primitive endoderm, epiblast), postimplantation development. Main role chance Although full development supported irrespective site, clustered by revealed single-cell RNAseq 21, 21 13 two-cell whose fertilized equator, respectively. When examining sister blastomeres together, 462 genes shared transcriptome differently expressed sites, class contributing most difference (adj.p<0.05, Wilcoxon test). true when separately: interblastomere differences exceeded those (72% 14% genes). Ontology analysis Enrichr pointed endomembrane system acquaintance oocyte polarity studies (PMID 10545249; PMID 29746690). Follow-up blastocysts blastomere contribution each layer less balanced class, measured linear correlation cell numbers (e.g. R2=0.00 R2>0.23 epiblast). Summing up, difficult reconcile these data mainstream view harmful. Rather they support topology defines transcriptomes. Limitations, reasons caution study. Mouse uses mercury-loaded piezo-driven needles, human not. The higher consistency one same spermatozoa traded off against lower developmental rates. Single-cell resolution posed limit depth. Results need confirmation other strains. Wider implications findings similarity observed after compared consistent model first zygotic driven apposing pronuclei. Trial registration number applicable