Roles of Dickkopf-1 and its receptor Kremen1 during embryonic implantation in mice  Jing Li, Ph.D., Wei-Min Liu, Ph.D., Yu-Jing Cao, M.S., Sha Peng, Ph.D., Ying Zhang, Ph.D., En-Kui Duan, Ph.D.  Fertility and Sterility  Volume 90, Issue 4, Pages 1470-1479 (October 2008) DOI: 10.1016/j.fertnstert.2007.09.003 Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Localization of Dkk1 (A–C, G, H) and Kremen1 (D–F, J, K) proteins in mouse cleavage-stage embryos. Green represents Dkk1 or Kremen1 staining with fluorescein isothiocyanate conjugate–conjugated secondary antibodies, and red indicates nuclear staining with propidium iodide. (A, D) Two-cell mouse embryo; (B, E) eight-cell mouse embryo; (C, F) compacted eight-cell embryo; (G, J) early blastocyst stage; (H, K) blastocyst hatched from the zona pellucida; and (I, L) negative control, with primary antibodies replaced by preimmune IgGs. Bar = 20 μm. Fertility and Sterility 2008 90, 1470-1479DOI: (10.1016/j.fertnstert.2007.09.003) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Dickkopf-1 (B) and Kremen1 (C) antisense ODNs inhibit blastocyst attachment and outgrowth on fibronectin. Hatched blastocysts were incubated in indicated concentrations of Dkk1 or Kremen1 As-ODNs. Blastocysts incubated in normal culture media (control) or in 10 μM Dkk1 or Kremen1 S-ODNs (cnl) served as controls. (A) Efficacy of Dkk1 and Kremen1 As-ODNs on the expression of Dkk1 or Kremen1 in blastocysts after 48 hours of culture. (Aa) Dickkopf-1 S-ODNs (10 μM); (Ab) Dkk1 As-ODNs (10 μM); (Ac) Kremen1 S-ODNs (10 μM); (Ad) Kremen1 As-ODNs (10 μM); and (Ae) negative control, with primary antibodies replaced by goat IgG. Bar = 20 μm. Each group contains ≥40 blastocysts, and results are shown as the mean ± SEM of three replicates. ∗∗P<.01, ∗∗∗P<.001. Fertility and Sterility 2008 90, 1470-1479DOI: (10.1016/j.fertnstert.2007.09.003) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Immunostaining of Dkk1 (A–I) and Kremen1 (J–R) proteins in pregnant, pseudopregnant, and implantation-delayed uteri and artificial decidua. (A, J) Day 1 of pregnancy; (B, K) day 3 of pregnancy; (C, L) day 4 of pregnancy; (D, M) day 5 of pregnancy; (E, N) day 7 of pregnancy; (F, O) day 5 of pseudopregnancy; (G, P) P-treated implantation-delayed uterus on day 7 of pregnancy; (H, Q) implanted embryos in E2-induced receptive uterus after 48 hours of treatment; (I, R) artificial decidua on day 8 of pseudopregnancy; and (S, T) negative control, with primary antibodies replaced by goat IgG. Cross-sections were used for all samples except day 5, for which longitudinal sections were used. de = deciduas; em = embryo; ge = glandular epithelium; le = luminal epithelium; s = stroma; tr = trophoblast. Bar = 100 μm. Fertility and Sterility 2008 90, 1470-1479DOI: (10.1016/j.fertnstert.2007.09.003) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Expression of Dkk1 and Kremen1 proteins in uterus of ovariectomized mice was regulated by ovarian steroid hormones E2 and P. Western blots showed time-dependent expressions of Dkk1 and Kremen1 after a single injection of E2 (A) or P (B). β-Actin was used as a loading control. Densitometric analysis also is shown. Results are presented as the mean ± SEM of three replicates (∗P<.05; ∗∗P<.01; ∗∗∗P<.001). (C) Immunostaining of Dkk1 and Kremen1 in uterus after treatment with E2 or P. ge = glandular epithelium; le = luminal epithelium; s = stroma. Bar = 100 μm. Fertility and Sterility 2008 90, 1470-1479DOI: (10.1016/j.fertnstert.2007.09.003) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 5 Effects of Dkk1 As-ODNs on mouse embryo implantation in vivo. Dickkopf-1 S- or As-ODNs (1 μM) was injected into one of each of the lateral uterine horns on the morning of day 3 of pregnancy, and the uteri were harvested on day 4 or day 7 of pregnancy. (A, B) Numbers of implanted embryos were severely inhibited by Dkk1 As-ODNs on day 7 of pregnancy (3.48 ± 0.4 vs. 1.70 ± 0.3, n = 12, ∗∗P<.01), and (C) Dkk1 As-ODNs actually decreased the expression of both Dkk1 messenger RNA and protein on day 4 of pregnancy. An amplification of β-actin gene transcripts was used to confirm RNA integrity and efficiency. An antibody against β-actin was used in the same Western blot as a loading control. Densitometric analysis of reverse-transcription polymerase chain reaction and Western blots for Dkk1 are shown. Results are presented as mean ± SEM of three triplicates (∗∗P<.01). Fertility and Sterility 2008 90, 1470-1479DOI: (10.1016/j.fertnstert.2007.09.003) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions