The role of TEAD4 in trophectoderm commitment and development is not conserved in non-rodent mammals.

The first lineage differentiation in mammals gives rise to the inner cell mass and the trophectoderm (TE). In mice, TEAD4 is a master regulator of TE commitment, as it regulates the expression of other TE-specific genes and its ablation prevents blastocyst formation, but its role in other mammals remains unclear. Herein, we have observed that TEAD4 ablation in two phylogenetically distant species (bovine and rabbit) does not impede TE differentiation, blastocyst formation and the expression of TE markers, such as GATA3 and CDX2, although a reduced number of cells in the inner cell mass was observed in bovine TEAD4 knockout (KO) blastocysts. Transcriptional analysis in bovine blastocysts revealed no major transcriptional effect of the ablation, although the expression of hypoblast and Hippo signalling-related genes tended to be decreased in KO embryos. Experiments were conducted in the bovine model to determine whether TEAD4 was required for post-hatching development. TEAD4 KO spherical conceptuses showed normal development of the embryonic disc and TE, but hypoblast migration rate was reduced. At later stages of development (tubular conceptuses), no differences were observed between KO and wild-type conceptuses.

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 mediate the signaling actions of prostaglandins and other lipids, and, between them, PPARG has been pointed out to play a relevant role in 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 knock-out embryos in vitro using two independent gene ablation strategies and assessed their developmental ability. In vitro development to Day 8 blastocyst was unaffected by PPARG ablation, as total, inner cell mass, and trophectoderm cell numbers were similar between wild-type and knock-out D8 embryos. In vitro post-hatching development to D12 was also comparable between different genotypes, as embryo diameter, epiblast cell number, embryonic disk formation, and hypoblast migration rates were unaffected by the ablation. The development of 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 disk was not altered by PPARG ablation. In conclusion, PPARG ablation did not impaired bovine embryo development up to tubular stages.

mtDNA content in cumulus cells does not predict development to blastocyst or implantation.

STUDY QUESTION: Is relative mitochondrial DNA (mtDNA) content in cumulus cells (CCs) related to embryo developmental competence in humans and/or the bovine model? SUMMARY ANSWER: mtDNA content in CCs provides a poor predictive value of oocyte developmental potential, both in vitro and following embryo transfer. WHAT IS KNOWN ALREADY: CCs are closely connected to the oocyte through transzonal projections, serving essential metabolic functions during folliculogenesis. These oocyte-supporting cells are removed and discarded prior to ICSI, thereby providing interesting biological material on which to perform molecular analyses designed to identify markers that predict oocyte developmental competence. Previous studies have positively associated oocyte mtDNA content with developmental potential in animal models and women. However, it remains debatable whether mtDNA content in CCs could be used as a proxy to infer oocyte developmental potential. STUDY DESIGN SIZE DURATION: mtDNA content was analyzed in CCs obtained from 109 human oocytes unable to develop to blastocyst, able to develop to blastocyst but failing to establish pregnancy or able to develop to blastocyst and to establish pregnancy. mtDNA analysis was also performed on bovine cumulus samples collected from 120 oocytes unable to cleave, oocytes developing into cleaved embryos but arresting development prior to the blastocyst stage or oocytes developing to blastocysts. PARTICIPANTS/MATERIALS SETTING METHODS: Human CCs samples were obtained from women undergoing IVF. Only unfrozen oocytes and embryos not submitted to preimplantation genetic testing were included in the analysis. Bovine samples were obtained from slaughtered cattle and individually matured, fertilized and cultured in vitro. Relative mtDNA was assessed by quantitative PCR analysis. MAIN RESULTS AND THE ROLE OF CHANCE: mtDNA content in human and bovine CCs did not differ according to the developmental potential of their enclosed oocyte. Moreover, mtDNA content in bovine oocytes did not correlate with that of their corresponding CCs. LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: The lack of correlation found between mtDNA content in human CCs and oocytes was also assessed in bovine samples. Although bovine folliculogenesis, mono-ovulatory ovulation and early embryo development exhibit considerable similarities with that of humans, they may not be fully comparable. WIDER IMPLICATIONS OF THE FINDINGS: The use of molecular markers for oocyte developmental potential in CCs could be used to enhance success rates following single embryo transfer. However, our data indicate that mtDNA in CCs is not a good proxy for oocyte quality. STUDY FUNDING/COMPETING INTERESTS: This research was supported by the Industrial Doctorate Project IND2017/BIO-7748 funded by the Madrid Region Government. The authors declare no competing interests.

Lineage Differentiation Markers as a Proxy for Embryo Viability in Farm Ungulates.

Embryonic losses constitute a major burden for reproductive efficiency of farm animals. Pregnancy losses in ungulate species, which include cattle, pigs, sheep and goats, majorly occur during the second week of gestation, when the embryo experiences a series of cell differentiation, proliferation, and migration processes encompassed under the term conceptus elongation. Conceptus elongation takes place following blastocyst hatching and involves a massive proliferation of the extraembryonic membranes trophoblast and hypoblast, and the formation of flat embryonic disc derived from the epiblast, which ultimately gastrulates generating the three germ layers. This process occurs prior to implantation and it is exclusive from ungulates, as embryos from other mammalian species such as rodents or humans implant right after hatching. The critical differences in embryo development between ungulates and mice, the most studied mammalian model, have precluded the identification of the genes governing lineage differentiation in livestock species. Furthermore, conceptus elongation has not been recapitulated in vitro, hindering the study of these cellular events. Luckily, recent advances on transcriptomics, genome modification and post-hatching in vitro culture are shedding light into this largely unknown developmental window, uncovering possible molecular markers to determine embryo quality. In this review, we summarize the events occurring during ungulate pre-implantation development, highlighting recent findings which reveal that several dogmas in Developmental Biology established by knock-out murine models do not hold true for other mammals, including humans and farm animals. The developmental failures associated to in vitro produced embryos in farm animals are also discussed together with Developmental Biology tools to assess embryo quality, including molecular markers to assess proper lineage commitment and a post-hatching in vitro culture system able to directly determine developmental potential circumventing the need of experimental animals.