Estrogen-regulated Expression and Distribution of Id-1 in the Mouse Uterus

Estrogen-regulated expression of Id gene family in the mouse uterus. (A) cDNA microarray analysis for change of Id gene expression in uteri of OVX mice exposed by E2 for 6 h and 12 h. (B) Validation for the estrogenic efficiency of E2-treated uterus by expression pattern of known early and late E2-responsive genes (HB-EGF and LF). (C) RT-PCR analysis for Id gene expression in uteri of OVX mice exposed by E2 for 2, 4, 6, and 12 h (D and E) Effects of ICI (D) and P4 (E) on Id-1 expression in uteri of OVX mice. (F) Laser capture microdissection (LCM) procedure. Capture of LE (a–c), M (d–f), and S (g–i) cells from OVX mice uteri exposed to E2. Capture sites of each uterine cell were indicated by arrowhead. (G) Distribution of Id-1 mRNA in specific uterine cell types obtained from OVX mice uteri exposed to E2. Adult OVX mice were given a single injection of oil (vehicle, dashed line), E2, E2 plus P4 or ICI or were given ICI before the injection of E2. Abbreviation, ND, not detected; E2, estrogen; P4, progesterone; ICI (ICI 182,780); E, luminal epithelium; M, Muscle; S, Stroma. All bars indicate as the average and standard deviation from three independent experiments. Relative expression levels were calculated with respect to vehicle control. *P < 0.05, **P < 0.01.

Temporal expression patterns of Id-1 during implantation period and preimplantation embryo development. (A) On the evening of pregnancy day 4 (2200–2400 h), implantation sites were visualized by increased vascular permeability at the site of blastocyst attachment using intravenous injection of Chicago Blue B solution. (B) RT-PCR for Id-1 expression between implantation (Imp) sites and inter-implantation (Inter) site. HB-EGF is used as positive control for implantation site. (C) Real-time PCR analysis was performed to compare Id-1 expression level between Imp and Inter site, quantitatively (*P < 0.05). Gel electrophoresis and melting-curve analyses were performed to confirm the correct PCR product size and absence of nonspecific bands. (D) Temporal expression patterns of Id-1 mRNA. GV, germinal vesicle oocyte; MII, metaphase II oocyte; PN, pronuclear stage embryo; 2C, 4C, 8C, MO and BL represent two-cell, four-cell, eight-cell, morula, and blastocyst stage embryos, respectively. The amount of pSPTet3 mRNA added was the same (2 × 10⁵ copies) for each stages, the ratio between Id-1 and pSPTet3 products is a relative measure of the amount of the Id-1 mRNA. This experiment was performed three times and the data, which are expressed relative to the amount present in the GV oocyte, are expressed as the average and standard deviation (*P < 0.05). (E) Immunostaining of Id-1 in mouse oocytes and preimplantation embryos. Embryos at various developmental stages were collected and immunostained with Id-1. Immunostaining was carried out three times, and approximately twenty oocytes or embryos were localized in each experiment. The signals were detected with a FITC-conjugated secondary antibody (left panel) and the nucleus was stained with 4,6-diamidino-2-phenylindole (DAPI, center panel). Bright field (BE) photograph was shown in right panel. Scale bars, 20 μm.