Akademik Veri Yönetim Sistemi
19th International Congress on Animal Reproduction, Bologna, İtalya, 26 - 30 Haziran 2022, ss.177
BACKGROUND-AIM
Progesterone has been positively associated with conceptus length and
embryo survival in cattle. However, it is unclear whether this positive effect
is exerted through a direct effect upon the embryo or indirectly, by promoting changes
in uterine fluid composition. While progesterone supplementation to
conventional embryo culture up to Day 8 (D8) is not required for embryo
development, post-hatching embryo culture (D8 to D12) requires the addition of
multi-compound supplements containing progesterone. The objective of this study
was to determine the developmental effects of progesterone receptor (PGR)
ablation.
METHODS
In vitro matured bovine oocytes were injected with mRNA encoding for Cas9
alone (control group, C) or combined with sgRNA against PGR (C+G group).
Following fertilization and culture, blastocysts were cultured in a
post-hatching system up to D12. At D12, embryos were fixed and epiblast and
hypoblast development were analyzed by
immunocytochemistry for SOX2 and SOX17, respectively. Following
individualized image analysis, samples were recovered and genotyped by miSeq to
determine which
embryos in C+G group were KO.
RESULTS
PGR ablation did not impair blastocyst formation, as similar developmental
rates were obtained in C or C+G groups (33.4±2.6 vs. 26.7±4.3 %, mean±s.e.m.
for C and C+G,
respectively, t-test p>0.05). In vitro development from D7 to D12 was
also similar between groups (85.4±5.7 vs. 81.3±6.3, mean±s.e.m. for C and C+G,
respectively, t-test p>0.05). 22 out of 45 D12 embryos analyzed in C+G group
were KO. D12 embryo diameter was not affected by embryo genotype (772±74 vs.
648±64 vs. 731±44 μm, mean±s.e.m. for WT, edited non-KO and KO, respectively,
ANOVA p>0.05). The proportion of embryos showing complete hypoblast
migration was similar in WT (23/32, 72 %), edited non-KO (12/20, 60 %) and KO
(19/22, 86 %) embryos (Chisquare p<0.05). Epiblast survival (SOX2+ cells)
rates were also similar in WT (26/32, 81 %), edited non-KO (9/20, 45 %, 60 %)
and KO (12/20, 60 %) and no differences were noted on embryonic disc (ED)
formation rate (20/32 63 % vs. 6/20 30 % vs. 10/22 45 % for WT, edited non-KO
and KO, respectively Chi-square p>0.05).
CONCLUSIONS
Direct progesterone signaling to the embryo through PGR is not required for
hypoblast migration and ED formation.