Figure 1.
Expression of OPN in the mouse uterus during early pregnancy.
(A) Real-time PCR was performed to quantify the Opn mRNA levels in the mouse uterus during early pregnancy (D1: day 1, D4: day 4, D5-NI: inter-implantation sites on day 5, D5-IS: implantation sites on day 5, D8-IS: implantation sites on day 8). (B) Immunohistochemistry was used to detect the OPN protein localizations in the mouse uterus during early pregnancy (D1-L: ligated uterine horn on day 1 of pregnancy, LE: luminal epithelium, GE: glandular epithelium, EM: embryo, AM: anti- mesometrium). (C) Opn mRNA level in the mouse uterus on day 8 of pregnancy and artificial decidualization. (D) OPN protein level in the mouse uterus on day 8 of pregnancy and artificial decidualization. (E) Opn mRNA level during the in vitro decidualization of mESC. (F) Dtprp mRNA level during the in vitro decidualization of mESC. *, p<0.05; error bars, S.E. All of the experiments were repeated three times.
Figure 2.
Ovarian steroids regulate OPN expression in vivo and in vitro.
(A) Real-time PCR of Opn mRNA expression in ovariectomized mouse uteruses after hormone injections (Oil: sesame oil, E2: estradiol-17β, P4: progesterone, E2+P4: estradiol-17β plus with progesterone). (B) A western blot was performed to confirm the hormone regulation of OPN expression in mouse uteri; GAPDH was used as an internal reference. (C) Real-time PCR of Opn mRNA expression in ovariectomized mice uterus after the injection of estrogen alone or estrogen plus with ICI182,780, an estrogen receptor α antagonist (E2+ICI, estradiol-17β plus with ICI182,780). (D) A western blot was performed to confirm the hormone regulation on OPN expression in mouse uteri; GAPDH was used as an internal reference. (E) Real-time PCR of Opn mRNA levels after the mESCs were treated with steroids hormones. (F) Western blot analysis for the steroids hormones regulation of OPN expression in mESCs; GAPDH was used as an internal reference. G. Real-time PCR of Opn mRNA expression after the mESCs were treated with progesterone and RU486. (H) Western blot analysis for the progesterone regulation of OPN expression in mESCs, GAPDH was used as an internal reference. *, p<0.05; error bars, S.E. All of the experiments were repeated three times.
Figure 3.
OPN protein level in uterine flushing fluids.
(A) Western blot analysis for OPN protein expression levels in uterine flushing fluids. (B) Ponceau red staining of the western blot membranes (C-M: conditioned medium for embryo culture). All of the experiments were repeated three times.
Figure 4.
(A) Unhatched mouse blastocysts were collected at 08:00 on day 4 of the pregnancy and cultured with BSA (control) or rOPN at the concentrations of 0.1 µg/mL, 1.0 µg/mL or 10.0 µg/mL. (B) The hatching rate of blastocysts in vitro cultured with rOPN at concentrations of 0.1 µg/mL, 1.0 µg/mL or 10.0 µg/mL. *, p<0.05; error bars, S.E. (C) Unhatched mouse blastocysts were collected at 08:00 on day 4 of the pregnancy and cultured with IgG (control) and anti-OPN antibody at concentrations of 0.01 µg/mL, 0.1 µg/mL or 1.00 µg/mL. (D) The hatching rate of blastocysts in vitro cultured with anti-OPN antibodies at concentrations of 0.01 µg/mL, 0.1 µg/mL or 1.00 µg/mL. All of the experiments were repeated three times.
Figure 5.
(A) The adhesion rate of blastocysts in FN pre-coated dishes with BSA (control), rOPN or anti-OPN antibodies. (B) The adhesion rate of blastocysts in FN pre-coated dishes with BSA (control), rOPN or RGD peptides. *, p<0.05; error bars, S.E. All of the experiments were repeated three times.
Figure 6.
OPN silencing limits trophoblast outgrowth and invasion in vitro.
(A) Real-time PCR of Opn mRNA levels in OPN-knockdown mESC by OPN siRNA transfection (NC: negative control siRNA, si-OPN: OPN siRNA). (B) A western blot confirmed the knockdown effect of OPN-targeted siRNA on OPN protein expression in mESCs or on secreted OPN proteins in the culture medium. (C) The adhesion rate of mouse blastocysts in control and OPN-knockdown mESCs. (D) OPN-targeted, siRNA-pretreated mESCs significantly decreased the trophoblast-spreading areas in the mESCs compared to the control group. The data for calculating the mean spreading area included at least ten embryos for each group, and all of the experiments were repeated at least three times. *, p<0.05; error bars, S.E. (E). Immunofluorescence was performed to detect the trophoblast outgrowth and invasion in OPN-targeted siRNA pretreated mESCs (Control, negative control siRNA pretreated mESC; si-OPN, OPN-targeted siRNA). Mouse embryos were marked by E-cadherin (Green signal), and nuclei were marked by DAPI (Blue). All of the experiments were repeated three times.
Figure 7.
OPN regulates the expression and enzymatic activity of MMP-9 in the trophoblast.
(A) Immunofluorescence was performed to detect the in situ OPN protein expression in OPN siRNA-pretreated or negative control siRNA-pretreated mESCs. (B) Immunofluorescence was performed to detect the trophoblast MMP-9 expression in OPN siRNA-pretreated or negative control siRNA-pretreated mESCs. Mouse embryos were marked by MMP-9 (Green signal), and nuclei were marked by DAPI (Blue). (C) Gelatin zymography was used to detect the enzymatic activity of MMP-9 and MMP-2 in the mESC-embryo culture medium, and MMP-9 enzymatic activity was inhibited by OPN silencing in the mESCs, when compared with negative controls. All of the experiments were repeated three times.
Figure 8.
Proposed schematic diagram for OPN roles in mouse embryo implantation and decidualization.
Mouse blastocysts entering the uterus and hatching from the zona pellucida are key steps for embryo activation and depend on the synchronization of embryo development and uterine receptivity. The ovarian estrogen (E2) surge induces OPN expression in the glandular epithelium, and OPN proteins are then secreted into uterine cavity to promote blastocyst hatching and adhesion to the luminal epithelium during implantation. After the blastocyst implants into uterine endometrium, uterine stromal cells undergo decidualization, which is characterized by extensive proliferation and differentiation. OPN is highly expressed in decidual cells and regulated by progesterone (P4). Hypothetically, OPN should be able to promote trophoblast cell invasion by regulating the expression and enzymatic activity of MMP-9. In conclusion, OPN expression during peri-implantation and decidualization may contribute to embryo activation and invasion in mice.