Post-hatching development of the porcine and bovine embryo—defining criteria for expected development in vivo and in vitro

Abstract

Particular attention has been paid to the pre-hatching period of embryonic development although blastocyst development is a poor indicator of embryo viability. Post-hatching embryonic development in vitro would allow for establishment of more accurate tools for evaluating developmental potential without the need for transfer to recipient animals. Such a system would require (1) definition of milestones of expected post-hatching embryonic development in vivo; and (2) development of adequate culture systems. We propose a stereomicroscopical staging system for post-hatching embryos defining the following stages: (1) Expanded hatched blastocyst stage where the embryo presents an inner cell mass (ICM) covered by trophoblast. (2) Pre-streak stage 1 where the embryonic disc is formed. (3) Pre-streak stage 2 where a crescent-shaped thickening of the caudal portion of the embryonic disk appears. (4) Primitive streak stage where the primitive streak has developed as an axis of cell ingression of cells for meso- and endoderm formation. (5) Neural groove stage where the neural groove is developing from the rostral pole of the embryo along with a proportional shortening of the primitive streak; and (6) Somite stage(s) where paraxial mesoderm gradually condensates to form somites. Post-hatching development of bovine embryos in vitro is compromised and although hatching occurs and elongation can be physically provoked by culture in agarose tunnels, the embryonic disk characterizing the pre-streak stage 1 is never established. Thus, particular focus should be placed on establishing culture conditions that support at least some of the above-mentioned critical phases of development that in vivo occur within the initial two (pig) to three (cattle) weeks.

Introduction

While there seems to be no scarcity in the literature describing in vitro production (IVP) of bovine embryos up to the stage of hatching from the zona pellucida, relatively little work has been done on characterizing the events leading into the post-hatching process of gastrulation in embryos of the domestic species. This period is of outmost interest for at least two major reasons.

First, it is clear, at least in cattle and pigs, that embryos derived by different technologies such as IVP or cloning by somatic cell nuclear transfer display molecular deviations despite appearing rather normal from the light microscopical point of view. Hence, blastocyst development is a poor indicator of embryo viability. Although blastocyst formation requires initial steps of cell differentiation, this process is far less complex and demanding than the subsequent phase of post-hatching embryonic development. The prospect of being able to support post-hatching embryonic development in vitro would potentially allow for establishment of tools for evaluating accurately the developmental potential of embryos without using recipient animals for transfer. Such an evaluation would require (1) definition of milestones of expected post-hatching embryonic development in vivo; and (2) development of suitable culture systems allowing for such development to take place in vitro. Currently, the in vitro development of bovine embryos beyond hatching allows for establishment of hypoblast and contemporary signs of shedding of the polar trophoblast, i.e. Rauber's layer. Moreover, elongation may be achieved upon physical constraints in agar tunnels [1], [2]. However, further development of the epiblast is compromised. In the pig, IVP of embryos is still hampered by the high incidence of polyspermy and low rates of pronuclear formation as well as suppressed subsequent development of the embryos, at least when compared to cattle [3], [4], [5]. Thus, in this species, development of embryos in vitro after hatching poses even more scientific challenges than in cattle.

Second, under in vivo conditions, the majority of embryonic and fetal mortality in both pigs and cattle occurs within the post-hatching period when the embryo accomplishes a complex development and a critical dialogue between the embryo and the dam decisive for continued pregnancy to be established [6], [7], [8], [9].

The first part of this review includes an up-to-date description of the morphology of post-hatching or pre-implantation development in pigs and cattle with special attention to the period around gastrulation. Molecular insights from the mouse and rabbit on especially the pre-gastrulation period are included. Moreover, a simple stereomicroscopical staging system for post-hatching embryos is presented. More knowledge in domestic species on these stages of development may provide clues to overcome the present obstacles, as discussed in the second part of this review, in contemporary post-hatching in vitro culture systems.