Heparin-binding epidermal growth factor (HB-EGF) may improve embryonic development and implantation by increasing vitronectin receptor (integrin alphanubeta3) expression in peri-implantation mouse embryos

Abstract

Purpose: This study investigated the effects of HB-EGF on expression of integrin alphanubeta3 and implantation of embryos.

Methods: Two-cell embryos were recovered and cultured with or without 10 ng/mL HB-EGF for 96h. Expression of integrin alphanubeta3 in cultured embryos was examined by real time-RT-PCR and immunofluorescence analysis; embryos were cultured with or without HB-EGF, then transferred into the uteri of pseudo-pregnant female mice in order to analyze their implantation rate.

Results: HB-EGF improved embryonic hatching and outgrowth during extended culture, and up-regulated expression of integrin alphanubeta3 in both the preimplantation embryo and outgrowing blastocyst. Also, integrin alphanubeta3 subunits were localized at the pericellular borders and cell-cell contact areas. The number of successful implantation sites of transferred HB-EGF-treated embryos in the uterus was increased when compared to number of implantation sites with non-treated controls.

Conclusions: HB-EGF may improve implantation by accelerating expression of integrin alphanubeta3 in peri-implantation mouse embryos.

Fig. 1.

Blastocyst outgrowth area. Two-cell stage embryos were cultured for 92 h in the presence or absence of HB-EGF or HB-EGF & anti-HB-EGF antiserum. After termination of culture, surface area was measured using image analysis system. Mean±SEM; ^*p < 0.01.

Fig. 2.

Effect of HB-EGF on expression of integrin ανβ3 gene in embryonic development. Green line: post-hCG 48 h 2-cell stage embryos were cultured with KSOM medium; Blue line: post-hCG 48 h 2-cell stage embryos were cultured with KSOM plus HB-EGF (10 ng/mL); Sky blue line: post-hCG 48 h 2-cell stage embryos were cultured with KSOM plus HB-EGF and anti-HB-EGF antiserum (10 ng/mL, 5 μg/μL); Red line: negative control (do not load embryos). Mean±SEM (n=3); ^*p < 0.05.

Fig. 3.

Immunofluorescence intensity and localization of integrin αν and β3 in mouse embryo development. 2-cell embryos (post-hCG 48 h) were cultured in KSOM (A, D), or were cultured in KSOM containing 10 ng/ml HB-EGF (B, E), and 10 ng/ml HB-EGF plus 1 μg/μL of anti-HB-EGF antiserum (C, F). Bright field: A, B, C, D, E, F. Integrin αν antibody stained: A′, B′, C′. Integrin β3 antibody stained: D′, E′, F′. Mean±SEM (n=10); ^*p < 0.05.

Fig. 4.

Comparison of implantation sites on the morning of gestation day 3 in mouse uterus for transferred HB-EGF treated or not treated embryos. The number of implantation sites in the treatment group was compared to the number in the control group (KSOM only culture). Uterus transfer with HB-EGF treated embryos is shown in left horn; a control is shown in right horn.