STUDIES ON BOVINE EARLY EMBRYONIC DEVELOPMENT AND PLURIPOTENCY

Abstract

From the time of fertilization in the oviduct, the mammalian embryo undergoes a sequence of developmental events that leads to implantation in the uterus. These events that include many fundamental aspects of embryonic development appear to be evolutionarily conserved, at least until the blastocyst stage, and are considered semi-autonomous as they can be recapitulated in vitro. Thus, the contributions of the oviductal and uterine microenvironment where development till the blastocyst takes place, have been largely overlooked. Embryo development post-hatching of blastocysts shows considerable differences between species. In cows and other ruminants, the trophoblast cells undergo a phase of rapid undifferentiated proliferation resulting in an elongated embryo that occupies almost the entire length of both uterine horns. Implantation occurs at the end of this proliferative phase with differentiation marked by attachment to the uterine epithelium and a subpopulation of trophoblasts becoming binucleate. Despite years of extensive observations on these apparent morphological events, the exact molecular mechanisms regulating trophoblast proliferation and differentiation leading to implantation in cattle remain uncharacterized. My studies on pre-hatching embryo development focused on characterizing the oviductal secretome from both ex vivo collected oviductal fluid and secretions from in vitro cultured oviductal epithelium. My studies on post-hatching embryo development focused on identifying pathways associated with trophoblasts and preimplantation development. By profiling the transcriptome and proteome of in vitro cultured bovine trophoblasts, we revealed conditions that exhibit similar molecular and functional characteristics as that of the embryonic trophectoderm and can serve as models to study trophoblast development. Using this model system, I demonstrated that trophoblast proliferation and differentiation into binucleate cells in cattle is at least in part mediated through a signaling initiated by the growth factor TGFβ1. TGFβ1 occupation of its receptors activates the RhoGTPase protein RhoA. RhoA through its downstream effector protein ROCK activates signaling cascades that negatively regulate trophoblast proliferation, simultaneously inducing both morphological and functional differentiation of trophoblasts and formation of a subpopulation of binucleate cells. My studies have set the stage for exploring additional mechanisms regulating pre-implantation development in cattle. Moreover, my data describe core signaling events associated with trophoblast proliferation and differentiation in cows.