72 Parthenogenetic bovine embryos display reduced cell proliferation during post-hatching development

Parthenogenetic mammalian embryos are unable to survive to term due to defects in genomic imprinting, an epigenetic phenomenon that mediates the paternal-specific expression of a small subset of genes. Parthenogenetic embryos lack paternal genetic material, and their development has been well studied in mice, where the lack of paternally expressed imprinted gene products causes severe growth retardation and intrauterine death after implantation. In contrast to mice, ungulates exhibit an extended pre-implantation development characterised by a massive proliferation of extra-embryonic membranes and the formation of an embryonic disc, and it is unclear whether the lack of paternally expressed imprinted gene products could affect cell proliferation before implantation. To answer that question, we have analysed the development of specific lineages in parthenogenetic bovine embryos compared with in vitro fertilised counterparts. In vitro-matured oocytes were allocated into two groups: one was fertilised following conventional protocols (IVF group) and the other was activated by calcium ionophore and 6-DMAP treatment (PA group). Following fertilisation or activation, presumptive zygotes were cultured in vitro up to Day 7 (D7) in SOF medium. D7 blastocysts were either kept in culture in SOF until D8 or transferred to N2B27 medium to allow post-hatching embryo development until D12. At D8 or D12, embryos were fixed in 4% paraformaldehyde. Cleavage and blastocyst rates were similar for both groups (cleavage: 88.4 ± 4 vs 87.7 ± 1.6; blastocyst: 37.9 ± 2.3 vs 46.3 ± 3.2, for IVF vs PA, respectively; t-test P < 0.05). D8 blastocysts (45/group) were analysed by IHC to determine the number of trophectoderm (CDX2+) and inner cell mass (SOX2+ cells). D8 IVF embryos showed a significantly higher number of total and CDX2+ cells compared with PA (total: 111.9 ± 7.7 vs 90.4 ± 6.2; CDX2+: 80 ± 5.7 vs 61.1 ± 4.6, for IVF vs PA, respectively; Mann-Whitney test P < 0.05), whereas differences in SOX2+ cell number were not statistically significant (30.3 ± 2.8 vs 24.2 ± 2.9, Mann-Whitney test; P = 0.06). D12 embryos (52 IVF and 50 PA) were analysed by IHC to identify cells allocated to the trophectoderm (CDX2+), hypoblast (SOX17+), or epiblast (SOX2+). D12 embryo diameter was significantly larger in IVF conceptuses compared with PA (730 ± 6 vs 420 ± 3 µm, for IVF and PA, respectively; Mann-Whitney test P < 0.05) and, whereas the percentage of embryos showing epiblast survival did not differ significantly (32/52 vs 22/50, for IVF and PA, respectively; chi-squared P = 0,07), the number of SOX2+ cells was significantly reduced in PA embryos (41.5 ± 6.9 vs 19.4 ± 3.8, for IVF and PA, respectively; Mann-Whitney test; P < 0.05). In conclusion, these results evidence that bovine parthenogenetic embryos show reduced cellular proliferation before implantation.

This work was supported by projects 757886-ELONGAN from ERC and PID2020-117501RB-I00 from MCINN.