Changes in endometrial ezrin and cytokeratin 18 expression during bovine implantation and in caruncular endometrial spheroids in vitro

Abstract

Introduction: The feto-maternal interface during bovine implantation was studied in vivo and using three-dimensional bovine endometrial (BCECph) and trophoblast spheroids (CCS), each with underlying fibroblasts.

Methods: The expression of ezrin and cytokeratin 18 (CK18) was analyzed via immunohistochemistry (IHC), RT-PCR and western blotting in bovine endometrium (GD 18-44) with in vivo (VIVO) and in vitro-produced embryos (VITRO). BCECph were stimulated with cotyledon-conditioned media (CCM) and analyzed by TEM/SEM and IHC. CCS were stained (IHC) for TGC markers, to test if spheroidal trophoblast cells had differentiated into TGC.

Results: At GD 20, caruncular epithelium (CE) and uterine glands (UG) showed a loss of cytosolic ezrin and CK18 followed by a complete loss of both proteins. At GD 35 both reappeared in CE and UG. The endometrial expression pattern did not differ between VIVO and VITRO. RT-PCR and western blotting confirmed the presence of ezrin and CK18. All spheroids had an outer polarized, cytokeratin and ezrin positive epithelium (CE or trophoblast) with apical microvilli. Stimulation of BCECph with CCM induced similar changes in ezrin expression as observed in endometrial tissue. However, no ultrastructural alterations were found by transmission electron microscopy. Absence of TGC-specific glycoproteins in CCS indicated that TGC differentiation was not induced by three-dimensional culture conditions.

Discussion: Ezrin and CK18 are downregulated during implantation in cattle. The expression changes represent a temporal depolarization, which could be important for an establishment of bovine pregnancy. Our in vitro experiments demonstrate that the trophoblast could contribute to this change in vivo.

Keywords: Bovine placenta; Cytokeratin 18; Ezrin; Implantation; Three-dimensional culture.