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.

Keywords: assisted reproductive technologies; conceptus elongation; developmental biology; embryo quality; embryo transfer; lineage markers; post-hatching embryo culture; viability.

Figure 1

Comparative developmental timeline between mice, humans, and farm ungulates. Different cell lineages are indicated. Embryo development in humans and farm ungulates is delayed compared to mice. Implantation takes place after blastocyst hatching in mice and humans, while in farm ungulates it occurs following conceptus elongation. Mouse epiblast develops in a cup shape after the blastocyst stage, while in humans and farm ungulates it forms a flat embryonic disc. E, embryonic day; ICM, inner cell mass; EPI, epiblast; HYPO, hypoblast; TE, trophectoderm.

Figure 2

Expression of lineage-specific markers in ovine embryos at different developmental stages. Fluorescence images of embryos stained for SOX2 (epiblast); SOX17/GATA6/FOXA2 (hypoblast/endoderm); CDX2/GATA3 (trophectoderm); T (mesoderm). Nuclei were counterstained with DAPI (merge). (a,b) D9 Hatched blastocysts; (c,d): D11 Spherical embryos; (e) D13 Tubular embryo; (f) D13 ED without Rauber's layer; (g) gastrulating ED; (h) section of a gastrulating ED. Scale bars = 50 μm for (a,b,f,h); 100 μm for (c,d,g); 500 μm for panel (e).

Figure 3

Lineage-specific markers expressed at different developmental stages in farm ungulate embryos. ED, embryonic disc; ICM, inner cell mass; TE, trophectoderm.