PPARG is dispensable for bovine embryo development up to tubular stages.

Following blastocyst hatching, ungulate embryos undergo a prolonged preimplantation period termed conceptus elongation. Conceptus elongation constitutes a highly susceptible period for embryonic loss and the embryonic requirements during this process are largely unknown, but multiple lipid compounds have been identified in the fluid nourishing the elongating conceptuses. Peroxisome proliferator-activated receptors (PPARs) mediate the signaling actions of prostaglandins and other lipids and, between them, PPARG has been pointed out to play a relevant role on conceptus elongation by a functional study that depleted PPARG in both uterus and conceptus. The objective of this study has been to determine if embryonic PPARG is required for bovine embryo development. To that aim, we have generated bovine PPARG KO embryos in vitro by two independent gene ablation strategies and assess their developmental ability. In vitro development to Day (D) 8 blastocyst was unaffected by PPARG ablation, as total, inner cell mass and trophectoderm cell numbers were similar between WT and KO D8 embryos. In vitro post-hatching development to D12 was also comparable between different genotypes, as embryo diameter, epiblast cell number, and embryonic disc formation and hypoblast migration rates were unaffected by the ablation. The development to tubular stages equivalent to E14 was assessed in vivo, following a heterologous embryo transfer experiment, observing that the development of extra-embryonic membranes and of the embryonic disc was not altered by PPARG ablation. In conclusion, PPARG ablation did not impaired bovine embryo development up to tubular stages.

HH5 Double-Carrier Embryos Fail to Progress through Early Conceptus Elongation.

Massive genotyping in cattle has uncovered several deleterious haplotypes that cause pre-term mortality. Holstein Haplotype 5 (HH5) is a deleterious haplotype present in the Holstein Friesian population that involves the ablation of the Transcription Factor B1 mitochondrial (TFB1M) gene. The developmental stage at which HH5 double-carrier (DC, homozygous) embryos or fetuses die remains unknown and this is a relevant information to estimate the economic losses associated to the inadvertent cross between carriers. To determine if HH5 DC survive to maternal recognition of pregnancy, embryonic day (E)14 embryos were flushed from superovulated carrier cows inseminated with a carrier bull. DC E14 conceptuses were recovered at Mendelian rates but they failed to achieve early elongation, as evidenced by a drastic (>26-fold) reduction in the proliferation of extraembryonic membranes compared with carrier or non-carrier embryos. To assess development at earlier stages, TFB1M knockout (KO) embryos -functionally equivalent to DC embryos- were generated by CRISPR technology and cultured to the blastocyst stage -Day (D)8- and to the early embryonic disc stage -D12-. No significant effect of TFB1M ablation was observed on the differentiation and proliferation of embryonic lineages and relative mtDNA content up to D12. In conclusion, HH5 DC embryos are able to develop to early embryonic disc stage but fail to undergo early conceptus elongation, required for pregnancy recognition.