Relocalisation and activation of integrins induced rapidly by oestrogen via G-protein-coupled receptor 30 in mouse blastocysts

Abstract

Integrins are the dominant and final adhesion molecules in the attachment process between the blastocysts and endometrium. It is necessary for oestrogen to rapidly activate mouse blastocysts so that they attach to the endometrial epithelium. Our previous study suggested that oestrogen can rapidly induce an increase in intracellular calcium in mouse blastocysts via G-protein-coupled receptor 30 (GPR30). Thus, we deduced that integrins may be involved in GPR30 mediation of the fast effect of oestrogen on mouse blastocysts in implantation. To prove our hypothesis, we used immunofluorescence staining and in vitro coculture of mouse blastocysts and endometrial epithelial cell line (EECs), Ishikawa cells, in the present study. We found that αv and β1 integrin clustered in mouse blastocysts, and that β3 integrin was relocalised to the apical membrane of blastocyst cells when embryos were treated with 1 μM 17β-estradiol (E2), 1 μM E2 conjugated to bovine serum albumin (E2-BSA) and 1 μM G-1, a specific GPR30 agonist, for 30 min respectively, whereas pretreatment with 1 μM G15, a specific GPR30 antagonist, and 5 μM 1,2-Bis(2-aminophenoxy)ethane-N,N,N′′,N′′-tetraacetic acid tetrakis (acetoxymethyl ester)(BAPTA/AM), a cellular Ca2+ chelator, blocked the localisation of integrins induced by oestrogen via GPR30 in mouse blastocyst cells. E2, E2-BSA and G-1 increased the fibronectin (FN)-binding activity of integrins in blastocysts, whereas G15 and BAPTA/AM blocked the activation of integrins induced by oestrogen via GPR30 in mouse blastocysts. Inhibition of integrins by Arg-Gly-Asp peptide in blastocysts resulted in their failure to adhere to EECs in vitro, even if oestrogen or G-1 was provided. Together, the results indicate the fast effect of oestrogen via the GPR30 membrane receptor further induces relocalisation and activation of integrins in mouse blastocysts, which play important roles in the adhesion of blastocysts to EECs.