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

Abstract

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.

Effect of DHA on the quality of In vitro produced bovine embryos

“…Even though the developmental rates are similar, DHA could increase the percentage of embryos expressing SOX2 and, consequently, the percentage of embryos that would lead to gestation. Failure of epiblast development has been described in vitro and in vivo, and several authors reported that between 23% and 65% of in vitro produced and transferred embryos no longer had an embryonic disc, as reviewed by Ramos-Ibeas et al [37]. In the present study, regardless of the freezing day, 27.4% to 43.3% of the embryos lacked SOX2 cells and therefore epiblast, after thawing and 48h of culture (Table 4).…”

Resetting H3K4me3, H3K27ac, H3K9me3 and H3K27me3 during the maternal-to-zygotic transition and blastocyst lineage specification in bovine embryos

“…In a similar vein, active enhancers in TE demonstrated very little enrichment for CDX2 motifs, despite notable expression in the blastocyst (Fig. 6k) and presence of CDX2 protein 58,60 . Thus, in terms of genome regulation, CDX2 may play a more prevalent role during later stages of bovine blastocyst development.…”

“…6b ). Moreover, OCT4/POU5F1 expression remains widespread in human and bovine blastocysts, but is already restricted to the ICM in mouse 58,60 ( Fig. 6b ).…”

“…Such differences highlight the need to establish additional animal models, such as bovine, to investigate the molecular basis for preimplantation development in humans. Although certain phenomena that are present in both ungulates and rodents (e.g., ncH3K4me3) are uniquely absent in humans 20 , cattle and human embryos demonstrate striking similarities in developmental timing, morphology, and regulators of both EGA and blastocyst formation 12,58,60,71 . For example, bovine embryos may be an appropriate model to study the function of genes not expressed in mouse, such as PLAC8, for which the function in TE remains unknown.…”

In vitro culture of ovine embryos up to early gastrulating stages

“…These differences were not observed at E14, presumably because those embryos lacking an ED had already degenerated. These observations suggest that failures in epiblast development may underlie the intrinsic high embryo mortality reported during this period of development in both humans and ungulates ( Diskin and Morris, 2008 ; Macklon et al, 2002 ; Perez-Gomez et al, 2021 ). N2B27 was the only medium supporting epiblast survival and ED formation in in vitro -cultured sheep embryos.…”