Akademik Veri Yönetim Sistemi
38th Annual Meeting A.E.T.E., Utrecht, Hollanda, 15 - 16 Eylül 2022, ss.122
Endogenous maternal progesterone levels and exogenous progesterone supplementation
have been positively linked with conceptus length and embryo survival in cattle.
However, the mechanism by which progesterone enhances conceptus development is
unknown. Progesterone may act directly on the embryo or indirectly, by promoting
changes in uterine fluid composition favoring conceptus growth. To discriminate
between both possibilities, we have analyzed the development of bovine embryos
lacking progesterone receptor (PGR) generated by CRISPR technology. To that aim, we
have microinjected in vitro matured oocytes with mRNA encoding for Cas9 alone
(control group, C, solely composed by WT embryos) or combined with sgRNA against PGR
(C+G group, partially composed by KO embryos). Following fertilization and culture up
to Day 7 (D7) blastocyst, subsequent development was analyzed in vitro and in vivo,
assessing lineage development by immunostaining for CDX2 (trophectoderm), SOX17
(hypoblast) and SOX2 (epiblast), and conducting genotyping by miSeq. In vitro
development from D7 to D12 was 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). In C+G group, 22/45 D12
embryos analyzed were KO (i.e., contained only KO alleles). Embryo diameter at D12
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), and the proportion of
embryos attaining complete hypoblast migration was similar in WT (23/32, 72 %), edited
non-KO (12/20, 60 %) and KO (19/22, 86 %) embryos (Chi-square p>0.05). No differences
were noted either 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). To assess in
vivo development, 40 blastocysts from C+G group were transferred to two synchronized
recipient ewes. Pregnancy was supported by exogenous progesterone (CIDR-Ovis) and
conceptuses were recovered 9 days after embryo transfer at a developmental stage
equivalent to day 14 (E14). All intact conceptuses recovered from recipient ewes were
edited by CRISPR (23/23) and 10/23 were KO. Conceptus growth was not affected by
PGR ablation (1.3±0.3 vs. 3.7±1.3 cm, mean±s.e.m. for edited non-KO and KO,
respectively, Two-Way ANOVA p>0.05) and all conceptuses showed hypoblast migration.
Embryonic disc was present in 7/10 (70 %) KO and 12/13 (92 %) edited non-KO
conceptuses and embryonic disc size was not affected by the ablation (333±72 vs.
234±43 μm, mean±s.e.m. for KO and edited non-KO, respectively, Two-Way ANOVA
p>0.05). In conclusion, the ablation of PGR does not impair embryo development up to
E14, suggesting that progesterone-mediated conceptus growth enhancement is
indirectly mediated by triggering changes in the uterus.