Maternal heparin-binding-EGF deficiency limits pregnancy success in mice

Abstract

An intimate discourse between the blastocyst and uterus is essential for successful implantation. However, the molecular basis of this interaction is not clearly understood. Exploiting genomic Hbegf mutant mice, we show here that maternal deficiency of heparin-binding EGF-like growth factor (HB-EGF) defers on-time implantation, leading to compromised pregnancy outcome. We also demonstrate that amphiregulin, but not epiregulin, partially compensates for the loss of HB-EGF during implantation. In search of the mechanism of this compensation, we found that reduced preimplantation estrogen secretion from ovarian HB-EGF deficiency is a cause of sustained expression of uterine amphiregulin before the initiation of implantation. To explore the significance specifically of uterine HB-EGF in implantation, we examined this event in mice with conditional deletion of uterine HB-EGF and found that this specific loss of HB-EGF in the uterus still defers on-time implantation without altering preimplantation ovarian estrogen secretion. The observation of normal induction of uterine amphiregulin surrounding the blastocyst at the time of attachment in these conditional mutant mice suggests a compensatory role of amphiregulin for uterine loss of HB-EGF, preventing complete failure of pregnancy. Our study provides genetic evidence that HB-EGF is critical for normal implantation. This finding has high clinical relevance, because HB-EGF signaling is known to be important for human implantation.

Fig. 1.

HB-EGF deficiency defers the window of implantation, leading to compromised pregnancy. (A) Litter sizes from Hbegf^−/− mice are smaller than those of WT mice (Student t test, *, P < 0.01). Numbers within bars indicate numbers of mothers examined. Data are presented as mean ± SEM. (B and C) Ovulation and fertilization are comparable between WT and null mice. Numbers within bars in B indicate the number of mice with eggs/total number of mice examined, and those in C are number of mice with two-cell embryos examined. (D) Preimplantation embryo development is normal in Hbegf^−/− mice. Numbers within bars indicate number of mice examined. (E) HB-EGF deficiency leads to deferral of implantation as examined on day 4 evening (2000 h and 2400 h) and day 5 morning (0800 h) by the blue dye method. Numbers within bars indicate numbers of mice with implantation sites (IS) per the total number of mice examined. (F) Representative photographs of WT uteri with IS and Hbegf^−/− uteri without blue bands on day 4 midnight (2400 h). (G) Representative photomicrograph of unimplanted morphologically normal blastocysts recovered from Hbegf^−/− females without blue reaction at 2400 h of day 4.

Fig. 2.

Amphiregulin compensates for the loss of HB-EGF during implantation. (A) In situ hybridization of Hbegf in the luminal epithelium surrounding the blastocyst before and during the onset of implantation in WT uteri. (B) Areg is expressed much earlier and at higher levels in the absence of HB-EGF. (C) Ereg is first detected in the luminal epithelium at the site of blastocyst attachment in WT mice, but its expression is barely detectable even at 2400 h of day 4 in Hbegf^−/− uteri. Arrows indicate location of blastocysts (×40). Le, luminal epithelium; ge, glandular epithelium; s, stroma; Bl, blastocysts; Myo, myometrium.

Fig. 3.

Reduced preimplantation ovarian estrogen secretion retains uterine Areg expression in Hbegf null uteri. (A and B) Serum progesterone (P₄) and estradiol-17β (E₂) levels are significantly lower in Hbegf^−/− mice than WT females on day 4 morning (0800 h). Data are means ± SEM (*, P < 0.05; Student t test). Numbers within bars indicate numbers of mice examined. (C) RU486 (400 μg/mouse at 2400 h on day 3) abolishes Areg expression in WT uteri, whereas ICI-182780 (ICI) (125 μg per mouse at 2400 h on day 3) retains Areg expression at 0800 h and 1800 h on day 4 (×40). (D) Whereas P₄ (2 mg per mouse at 0800 h on day 4) enhances Areg expression, E₂ (3 ng per mouse at 0800 h on day 4) largely neutralizes the retained Areg expression in Hbegf^−/− uteri at 1800 h on day 4 (×100). However, Areg is normally reinduced in luminal epithelial cells at the site of blastocyst apposition on day 4 midnight (2400 h) in null females treated with E₂ (3 ng per mouse at 0800 h on day 4). Le, luminal epithelium; ge, glandular epithelium; s, stroma; Myo, myometrium.

Fig. 4.

Amphiregulin compensates for the loss of HB-EGF in delayed implanting mice. (A) Implantation rates in delayed implanting WT or Hbegf^−/− mice after E₂ injection (25 ng per mouse). Mice were ovariectomized on the morning of day 4 of pregnancy and administered P₄ from days 5 through 7. IS were examined 24 after E₂ injection on day 7 by the blue dye method. Numbers within bars indicate the number of mice with IS per total number of mice examined. (B) Average number of IS in WT and Hbegf^−/− mice. Data are presented as mean ± SEM. Numbers within the bars indicate the number of mice examined. (C) In situ hybridization showing maintained Areg expression in the glandular (ge) and luminal (Le) epithelia in P₄-primed delayed uteri (P₄, 6 and 24 h). Moreover, Areg is induced in the luminal epithelium surrounding the blastocyst as early as 6 h after E₂ injection (×40). S, stroma; Myo, myometrium. (D and E) Implantation of normal day 4 WT blastocysts transferred into delayed WT and Hbegf^−/− recipients on day 7 of pseudopregnancy. Immediately after transfer, the recipient received an E₂ injection (3 or 25 ng per mouse, s.c.); implantation was examined 24 h later. Numbers within the bar in D indicate the number of recipients with IS per the total number of mice examined, and those in E are the number of IS per the total number of blastocysts transferred. *, P < 0.05; Student t test.

Fig. 5.

Conditional deletion of uterine HB-EGF confers implantation defects similar to systemic Hbegf deletion. (A) LacZ staining indicates HB-EGF deletion specifically in uteri but not in lung or heart in PR-Cre^+/−/Hbegf^lox/lox mice (×40). (B) RT-PCR analysis of Hbegf mRNA revealing almost total deletion of Hbegf gene in PR-Cre^+/−/Hbegf^lox/lox uteri, but not in the heart. Hbegf mRNA levels were abundant in PR-Cre^+/−/Hbegf^lox/lox ovaries on days 1 and 4 of pregnancy. (C and D) Comparable levels of serum progesterone (P₄) and estradiol-17β (E₂) in Hbegf^lox/lox and PR-Cre^+/−/Hbegf^lox/lox mice on day 4 morning (0800 h). Numbers within the bars indicate number of mice examined. Data are means ± SEM. (E) Implantation is severely compromised in PR-Cre^+/−/Hbegf^lox/lox mice examined at 2400 h on day 4. Numbers within the bars indicate number of mice with IS per total number of mice examined. (F) Litter sizes of mice are significantly smaller in PR-Cre^+/−/Hbegf^lox/lox mice (*, P < 0.05; Student t test). Numbers within the bars indicate number of mice with litters per the total number of plug-positive mice. (G) In situ hybridization of uterine Areg in Hbegf^lox/lox and PR-Cre^+/−/Hbegf^lox/lox mice (×40). Areg expressed surrounding the blastocyst (arrows) during the late phase contributes to replace uterine HB-EGF's function during implantation.