Functional Roles of Eph A‐Ephrin A1 System in Endometrial Luminal Epithelial Cells During Early Pregnancy

Abstract

Eph and ephrin regulate diverse biological events such as proliferation, adhesion, migration, and angiogenesis through cell-to-cell interactions. However, little is known of their functional role and mechanisms of action in uterine endometrial cells. In the present study, we demonstrated the effects of the Eph and ephrin on interactions between blastocysts and endometrial luminal epithelial (pLE) cells in the pig that is regarded as an excellent biomedical animal model for research on the peri-implantation period of pregnancy. Results of this study indicated that among eight members of the Eph A family, expression of Eph A1, A2, A4, and A7 was strongly detected in endometrial epithelial cells during early pregnancy. Of these, for identification of signal transduction pathways induced by ephrin A1, a major ligand for Eph A, cell proliferation assays, and immunofluorescence and cell cycle regulation were analyzed following treatment of pLE cells with ephrin A1. Ephrin A1 stimulated proliferation of pLE cells as evidenced by abundant PCNA expression and an increase in the G2/M phase. Western blot analysis showed that ephrin A1 activated PI3K and MAPK signaling proteins in a time-dependent manner. Moreover, phosphorylation of AKT, ERK1/2, P38, and JNK proteins were suppressed by their inhibitors wortmannin, U0126, SB203580, and SP600125, respectively. Also, phosphor-AKT was reduced by ERK1/2 and P38 inhibitors. Ephrin A1-induced proliferation and migration of pLE cells was also blocked by those inhibitors. Collectively, these results suggest that ephrin A1 enhances interactions between porcine blastocysts and endometrial luminal epithelial cells by activating PI3K and MAPK signal transduction pathways. J. Cell. Physiol. 232: 1527-1538, 2017. © 2016 Wiley Periodicals, Inc.

Investigation of Eph‐ephrin A1 in the regulation of embryo implantation in sows

“…The expressions of Eph A1, A2 and A4 protein in the endometrial stroma underlying the luminal epithelium were the highest on pregnancy Day 18 (Fu, Fu, & Wang, ). In situ hybridization analyses revealed a cell‐specific localization of ephrin A1 mRNAs in the uterine luminal and the glandular epithelia on the pregnant Day 9, 10, 12, 13, 14, 20 and 30) (Lim et al, ). Muc1 proteins (another implantation related gene) were observed in luminal epithelium and stroma by the immunostaining, and the stained Muc1 proteins in the between attachment sites were stronger than that were at the attachment sites on Days 13 and 18 in pigs (Ren, Guan, Fu, & Wang, ).…”

“…We demonstrated that ephrin A1 gene could be silenced by shRNAs, and the reduced expressions of ephrin A1 led to the reduction of the capacity of cell migrations and adhesions. In pigs, ephrin A1 enhances interactions between porcine blastocysts and endometrial luminal epithelial cells by activating PI3K and MAPK signal transduction pathways (Lim et al, ). In mice, Haruko Fujii et al () have shown that Eph–ephrin interaction could induce repulsive forces to control cell position and movement (Fujii et al, ).…”

“…During the implantation period, several adhesion-related genes such as vascular endothelial growth factor (VEGF) (Tayade, Black, Fang, & Croy, 2006), obesity gene (known as Leptin or LEP) (Wang et al, 2014), leptin receptor (LEPR) (Fu et al, 2016) and Episialin (MUC1 or mucin 1) have previously been reported to be expressed in human endometrium and embryo. Among these adhesion-related genes, Erythropoietin-producing hepatocellular receptor-ligand (Eph-ephrin) system plays important roles in a variety of developmental processes including migration, adhesion, differentiation and proliferation of cells, tissue morphogenesis, vasculogenesis and neuronal plasticity through cell-to-cell communication (Kania & Klein, 2016;Lim, Bae, Bazer, & Song, 2016). The Eph-ephrin system is implicated in cell behaviour and morphology (Yamazaki et al, 2009) and restricts the spatial extent of ERK1/2 activation during the penultimate division of the pigment cell precursors (Haupaix et al, 2014).…”

Differential gene expression of Eph‐ephrin A1 and LEPR‐LEP with high or low number of embryos in pigs during implantation

“…These results suggested that low Eph‐ephrin A1 expression had advantageous effects on embryo number. Therefore, the results might be attributed to the function of Eph‐ephrin system, which can induce repulsive forces in cell migration and adhesion during embryo implantation in various mammals (Fujii, Fujiwara, Horie, Sato, & Konishi, ; Lim et al., ). LEP protein was also found in the uterus of Yorkshire sows during the pre‐attachment, post‐attachment period (Smolinska, Kaminski, Siawrys, & Przala, ).…”

“…The available data have identified some genes that regulate embryo attachment in pigs, and these genes include the following: peroxisome proliferator-activated receptor γ (PPARγ) (Wang, Kong, Hu, Fu, & Wang, 2011), Muc1 , erythropoietinproducing hepatocellular receptor-ligand (Eph-ephrin) system (Fu, Fu, Ren, Chen, & Wang, 2012), obesity gene (or known as leptin or LEP) (Wang, Fu, & Wang, 2014) and leptin receptor (LEPR) (Fu, Li, Li, Fang, & Ren, 2016). Among these genes, Eph-ephrin A1 is an important member of the Eph-ephrin system, which plays an important role in a variety of developmental processes including cell migration, adhesion, vasculogenesis and neuronal plasticity through cell-tocell communication (Kania & Klein, 2016;Lim, Bae, Bazer, & Song, 2016). Eph-ephrin A1 mRNA expression and protein expression are increased in the endometrium and embryo during implantation in humans (Frisen, Holmberg, & Barbacid, 1999;Fujiwara et al, 2002;Red-Horse et al, 2005), mice (Fujii et al, 2006) and embryo attachment in pigs (Fu, Fu, Ren, et al, 2012).…”