Flutamide alters β-catenin expression and distribution, and its interactions with E-cadherin in the porcine corpus luteum of mid- and late pregnancy

Abstract

This study examined whether flutamideinduced androgen deficiency during mid- and late pregnancy in pigs affected luteal expression of adherens junction protein, β-catenin, and its interactions with Ecadherin. Flutamide (50 mg/kg body weight) was administered into pregnant gilts between days 43-49 (GD50F), 83-89 (GD90F) or 101-107 (GD108F) of gestation. Corpora lutea (CLs) were obtained on day 50, 90 or 108 of pregnancy (n=8-11 per each group). Total β-catenin and E-cadherin expression was examined at mRNA (real-time PCR) and protein (Western blot) level. Moreover, subcellular β-catenin fractions were extracted and immunoblotted. Immunohistochemistry was used for β-catenin localization. To determine whether flutamide disturbs β-catenin/E-cadherin mutual interactions, coimmunoprecipitation using anti-β-catenin antibody was performed. Furthermore, phosphorylation of Ecadherin was assessed. Flutamide exposure led to decreased β-catenin mRNA expression in all examined groups (p<0.001 or p<0.01), but protein level was lower only in the GD90F and GD108F groups (p<0.05). Ecadherin mRNA (p<0.05 or p<0.01) and protein (p<0.05) levels were up-regulated in all flutamidetreated groups when compared to controls. β-catenin was predominantly found in membranes of luteal cells with no significant changes after antiandrogen treatment. βcatenin/E-cadherin complexes were more abundant in the GD90F (p<0.05) and GD108F (p<0.01) groups than in controls due to enhanced E-cadherin phosphorylation at serine 838/840 in those animals (p<0.05). Overall, although androgen deficiency affected β-catenin expression in the CL of pregnancy in pigs, a compensatory mechanism by enhanced interactions with E-cadherin is possible. Thus, androgen signaling via androgen receptors appears to be crucial in the regulation of luteal cells cross-talk.