Prostaglandins (PGs) are lipid signalling molecules that play critical roles in gestation by promoting corpus luteus maintenance or luteolysis, and have been suggested to play other roles in early pregnancy, including embryo–maternal crosstalk. The signalling roles of PGs and other lipids are often mediated by peroxisome proliferator-activated receptors (PPARs), transcription factors that regulate the expression of other genes through PPAR-responsive elements. PPARG is a PPAR expressed by bovine pre-implantation embryos whose inhibition by morpholino intrauterine infusion has been reported to impair embryo development. As this approach causes PPARG depletion in both conceptus and uterus, it is unknown whether PPARG-mediated signalling in the embryo is required for embryo development. The objective of this study was to determine whether PPARG is required for blastocyst formation. For that aim, we have evaluated embryo development in PPARG knockout (KO) bovine embryos generated by CRISPR-Cas9 technology. Invitro matured oocytes were allocated in two groups: one was injected with mRNA encoding for Cas9 and sgRNA against PPARG to generate KO embryos (C+G, n=191) and the other was injected with mRNA alone (C, n=148), serving as a microinjection control generating only wild-type embryos. Following fertilization, embryos were allowed to develop to Day 8 blastocysts invitro. No differences were found in cleavage and blastocyst rates between both groups (cleavage 78.5±3.4 vs. 78.4±4.2; Day 7 blastocyst 17.3±3.9 vs. 10.8±2.9; Day 8 blastocyst 20.4±6.2 vs. 16.9±4.7; C+G vs. C; mean±s.e.m.; ANOVA P>0.05). Blastocysts of the C+G group were genotyped by clonal sequencing to determine which embryos in the C+G group were KO (i.e. harboured only frame-disrupting, KO alleles). Twenty-eight out of the 32 blastocysts analysed were edited (87.5%), of which 6 (18.8%) were KO. These results show that PPARG is not required for blastocyst formation, because KO embryos develop to that stage, but do not rule out a possible role in further developmental stages.
Identification and expression analysis of genes associated with bovine blastocyst formation
