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

Abstract

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