Akademik Veri Yönetim Sistemi
19th International Congress on Animal Reproduction, Bologna, İtalya, 26 - 30 Haziran 2022, ss.177
BACKGROUND-AIM
TEAD4 plays an essential role on trophectoderm (TE) differentiation
in mice, where its ablation impedes blastocyst formation. However, in a
previous study we found that bovine embryos lacking TEAD4 (TEAD4 KO) develop to
blastocyst. The aim of this study was to determine if subsequent
preimplantation development was affected by the ablation.
METHODS
In vitro matured bovine oocytes were injected with mRNA encoding
for Cas9 and sgRNA against TEAD4 (C+G group). Following fertilization and
embryo culture to D7, blastocysts were transferred to a Day 6.5 recipient ewe.
Nine days after ET, conceptuses were recovered at a stage developmentally
equivalent to bovine E14. Recovered conceptuses were measured, fixed and subjected
to immunocytochemistry (ICC) to determine lineage development using antibodies
for CDX2 (TE), SOX17 (hypoblast) and SOX2 (epiblast). Following ICC embryos
from C+G group were genotyped by clonal sequencing to determine
which harbored only frame-disrupting alleles on TEAD4 sequence (i.e. KO
alleles).
RESULTS
32 Day 7 bovine blastocyst developed in vitro following microinjection
were transferred to both sides of the uterus (16+16) of a recipient ewe. Nine
days after ET 14 conceptuses (7+7) were recovered by uterine flushing. All conceptuses
were edited, 3 harbored at least one in-frame (non-KO) allele and 11/14 were
KO. The development of extraembryonic membranes (TE+hypoblast) was not affected
by the ablation, as conceptus length was comparable between KO and non-KO
conceptuses (10.9±3 vs. 9.8±2.3 mm, mean±s.e.m. for non-KO vs. KO, respectively,
t-test p>0.05) and all conceptuses but one KO (the smallest embryo) showed
complete hypoblast migration along the inner surface of the TE. A SOX2+ embryonic
disc (ED) was detected in all conceptuses but one KO (the same smallest
embryo), and ED diameter was similar in both groups (260±82 vs. 229±33 μm, mean±s.e.m.
for non-KO and KO, respectively, t-test p>0.05).
CONCLUSIONS
In contrast with the
crucial role observed in murine embryos, bovine embryos do not require TEAD4
for trophectoderm specification and maintenance up to tubular stages.