Heparin-binding EGF-like growth factor interacts with mouse blastocysts independently of ErbB1: a possible role for heparan sulfate proteoglycans andErbB4 in blastocyst implantation

Blastocyst implantation requires molecular and cellular interactions betwee n the uterine luminal epithelium and blastocyst trophectoderm. We have prev iously shown that heparin-binding EGF-like growth factor (HB-EGF) is induce d in the mouse luminal epithelium solely at the site of blastocyst appositi on at 16:00 hours on day 4 of pregnancy prior to the attachment reaction (2 2:00-23:00 hours), and that HB-EGF promotes blastocyst growth, zona-hatchin g and trophoblast outgrowth. To delineate which EGF receptors participate i n blastocyst activation, the toxicity of chimeric toxins composed of HB-EGF or TGF-alpha coupled to Pseudomonas exotoxin (PE) were used as measures of receptor expression. TGF-alpha or HB-EGF binds to EGF-receptor (ErbB1), wh ile HB-EGF, in addition, binds to ErbB4.The results indicate that ErbB1 is inefficient in mediating TGF-alpha-PE or HB-EGF-PE toxicity as follows: (i) TGF-alpha-PE was relatively inferior in killing blastocysts, 100-fold less than HB-EGF-PE, (ii) analysis of blastocysts isolated from cross-bred egfr (+/-) mice demonstrated that HB-EGF-PE, but not TGF-alpha-PE, killed egfr(- /-) blastocysts, and (iii) blastocysts that survived TGF-alpha-PE were neve rtheless killed by HB-EGF-PE, HB-EGF-PE toxicity was partially mediated by cell surface heparan sulfate proteoglycans (HSPG), since a peptide correspo nding to the heparin-binding domain of HB-EGF as well as heparitinase treat ment protected the blastocysts from the toxic effects of HB-EGF-PE by about 40%, ErbB4 is a candidate for being an HB-EGF-responsive receptor since RT -PCR analysis demonstrated that day 4 mouse blastocysts express two differe nt erbB4 isoforms and immunostaining with anti-ErbB4 antibodies confirmed t hat ErbB4 protein is expressed at the apical surface of the trophectoderm c ells. It is concluded that (i) HB-EGF interacts with the blastocyst cell su rface via high-affinity receptors other than ErbB1, (ii) the HB-EGF interac tion with high-affinity blastocysts receptors is regulated by heparan sulfa te, and (iii) ErbB4 is a candidate for being a high-affinity receptor for H B-EGF on the surface of implantation-competent blastocysts.