Cytoskeletal filaments and associated proteins during restricted trophoblast invasion in bovine placentomes: light and transmission electron microscopy and RT-PCR

Abstract

Trophoblast cell migration is unusual in epitheliochorial placentae but occurs in placentomes of cows as "restricted" trophoblast invasion of binucleated trophoblast giant cells (TGC). Migration may be induced by integrin binding to the extracellular matrix initiating two pathways: (1) conformational changes of the actin cytoskeleton induced by an accumulation of its associated proteins and (2) integrin-dependent phosphorylation of various protein kinases. In cow placentomes, actin, its associated proteins (alpha-actinin, vinculin) and a key protein kinase of the signal transduction cascade (phosphorylated mitogen-activated protein kinase, pMAPK) were localized by immunogold-silver enhancement and immunoperoxidase staining at the light- and transmission electron-microscopical levels. Findings were confirmed by amplification of specific mRNA transcripts by reverse transcriptase/polymerase chain reaction. Actin and alpha-actinin were co-localized apically in mononuclear trophoblast cells, along the cytoplasmic membrane of TGC and apically in maternal crypt cells. The actin and alpha-actinin immunoreaction occurred as a band of electron-dense particles beneath the cytoplasmic membrane. Vinculin labelling was membrane-associated in TGC and in fetal and maternal endothelial cells. MAPK was observed as nuclear clusters in both kinds of trophoblast cells and was less dense in single uterine epithelial cells. Most MAPK immunoreactivity was detected in the nuclei of the trophoblast epithelium but was also sometimes membrane-associated in the cytoplasm. Thus, actin, alpha-actinin, MAPK and vinculin may be involved in the regulation of TGC migration. "Restricted" trophoblast invasion could serve as a model for invasive processes.

Integrin Activation in Bovine Placentomes and in Caruncular Epithelial Cells Isolated from Pregnant Cows

“…This in vitro observation correlated with observations in vivo where ACTN expression was localized to the apicolateral compartment of the caruncular epithelial cells. Similar localization of ACTN was previously reported for the bovine placentome via electron microscopy with the immunoperoxidase/diaminobenzidine staining [32]. Further, apical ACTN linking to b-integrin subunits as a response to SPP1 (osteopontin)-mediated trophoblast-endometrial signaling in the ovine and porcine endometrial epithelium and cultured epithelial and trophectodermal cells has been demonstrated [22,23].…”

Development of an in vitro Model for Bovine Placentation: A Comparison of the in vivo and in vitro Expression of Integrins and Components of Extracellular Matrix in Bovine Placental Cells

“…All these cytoskeletal components have been localized before to define cell types within placental tissues of the cow [Lang et al, 2004;Pfarrer, 2005].…”

Morphological Characterization of Basally Located Uninucleate Trophoblast Cells as Precursors of Bovine Binucleate Trophoblast Giant Cells

“…Based on the ultrastructural appearance and the fact that actin is a main component of the microvilli frame in other epithelial cells [Fath and Burgess, 1995;Coch and Leube, 2016], we interpret this electron-dense material in both cell types as a part of the actin cytoskeleton. This interpretation is reinforced by immunogold [Lang et al, 2004] and immunohistochemical staining [Nakano et al, 2005]. Actin [Lang et al, 2004] as well as α 6 -and β 1 -integrin subunits and laminin [Pfarrer et al, 2003] have been suggested to be involved in TGC migration.…”

“…This interpretation is reinforced by immunogold [Lang et al, 2004] and immunohistochemical staining [Nakano et al, 2005]. Actin [Lang et al, 2004] as well as α 6 -and β 1 -integrin subunits and laminin [Pfarrer et al, 2003] have been suggested to be involved in TGC migration. As α 6 β 1 -integrin, also called VLA-6 (very late antigen-6) [Hemler et al, 1988], can interact indirectly with actin intracellularly [Alberts et al, 2015] and with laminin extracellularly Alberts et al, 2015], the simultaneous staining of laminin and α 6 -and β 1 -integrin subunits along the cell membrane of TGCs lead to the interpretation that these proteins cooperate in TGC migration [Pfarrer et al, 2003].…”