No abstract

Previous studies from our laboratory established that large M(r) mucin glycoproteins are major apically disposed components of mouse uterine epithelial cells in vitro. The present studies demonstrate that Muc-1 represents one of the apically disposed mucin glycoproteins of mouse uterine epithelia, and that Muc-1 protein and messenger RNA (mRNA) expression are regulated in the periimplantation mouse uterus by ovarian steroids. Muc-1 expression is exclusive to the epithelial cells of the uterus under all conditions examined. Muc-1 expression is high in the proestrous and estrous stages and decreases during diestrous. Both Muc-1 protein and mRNA decline to barely detectable levels by day 4 of pregnancy, i.e. before the time of blastocyst attachment. In contrast, Muc-1 expression in the cervix and vagina is maintained during this same period. Delayed implantation was established in pregnant mice by ovariectomy and maintained by the administration of exogenous progesterone (P). Initiation of implantation was triggered by coinjection of P-maintained mice with a nidatory dose of 17 beta-estradiol (E2). Muc-1 levels in the uterine epithelia of P-maintained mice declined to low levels similar to those observed on day 4 of normal pregnancy. Coinjection of E2 did not alter Muc-1 expression, suggesting that down-regulation of Muc-1 is a P-dominated event. This was confirmed in ovariectomized nonpregnant mice, which displayed stimulation of Muc-1 expression after 6 h of E2 injection. E2-Stimulated Muc-1 expression was inhibited by the pure antiestrogen, ICI 164,384. Although P alone had no effect on Muc-1 expression, it antagonized the action of E2. Injection of pregnant mice with the antiprogestin, RU486, a known implantation inhibitor, on day 3 of pregnancy restored high level expression of Muc-1 mRNA on day 4, indicating that down-regulation of Muc-1 is P receptor mediated. Collectively, these data indicate that Muc-1 expression in mouse uterine epithelium is strongly influenced by ovarian steroids. It is suggested that the loss of Muc-1 contributes to generation of a receptive uterine state.

Vascular endothelial growth factor (VEGF) is a mitogen for endothelial cells and an inducer of angiogenesis. VEGF is also known as a vascular permeability factor because it can stimulate vascular permeability. In the rodent, increased uterine vascular permeability occurs at the sites of blastocysts with the onset of the attachment reaction. This is followed by stromal decidualization and angiogenesis. We examined the temporal and spatial expression of VEGF and its receptors, Flk-1 and Flt-1, in the mouse uterus during the peri-implantation period (days 1-8) using Northern and in situ hybridization to assess the involvement of VEGF in the process of implantation. Primarily, a major (approximately 4.2 kb) transcript for VEGF mRNA was detected in uterine poly(A)+ samples, except for the presence of two other minor (approximately 3.7 and 2.5 kb) transcripts in decidual samples. The steady-state levels of these transcripts did not vary much during the peri-implantation period, except for an increase in day-8 decidual samples. Results of in situ hybridization experiments demonstrated accumulation of VEGF mRNA in the luminal epithelium on days 1 and 2. In contrast, stromal cells exhibited a modest level of signals on day 3. On day 4, luminal epithelial cells and those in the subepithelial stromal bed accumulated VEGF mRNA. On days 5-7, a clear cell type-specific accumulation of this mRNA was noted. On day 5 after the initial attachment reaction, luminal epithelial and stromal cells immediately surrounding the blastocyst exhibited accumulation of VEGF mRNA. On days 6-8, the accumulation occurred in cells in the decidual bed at both the mesometrial and antimesometrial poles. The embryo, especially the trophoblast giant cells, also accumulated VEGF mRNA on day 8. The expression of the VEGF receptors, Flk-1 and Flt-1, was also examined. A single transcript (approximately 6.5-7.0 kb) for Flk-1 mRNA and two transcripts (approximately 6.5 and 7.5 kb) for that of Flt-1 were detected in poly(A)+ uterine RNA samples. In situ hybridization studies showed accumulation of Flk-1 mRNA in a subset of cells in the stromal bed on day 4, but not in any uterine cell types on day 1. On days 5-8, cells in both the mesometrial and antimesometrial decidual beds exhibited accumulation of Flk-1 and Flt-1 mRNAs. Lectin binding (Dolichos biflorus agglutinin) was used to identify newly sprouting endothelial cells (angiogenesis), while an antibody to the von Willebrand factor (vWF) was employed to identify endothelial cells in general.(ABSTRACT TRUNCATED AT 400 WORDS)

A synchrony between the activated state of the blastocyst and differentiation of the uterus to the receptive state is essential to the process of implantation. This process is directed by progesterone (P4) and estrogen. The mechanism by which P4 differentiates the uterus, enabling estrogen to initiate implantation, is unknown but likely to involve localized induction of growth and differentiation factors. We have cloned the murine amphiregulin (AR) gene, a newly discovered member of the epidermal growth factor family, and demonstrate that its expression is implantation-specific and P4-regulated in the mouse uterus. A transient surge in AR mRNA levels occurred throughout the uterine epithelium on day 4 of pregnancy. With the onset of blastocyst attachment late on day 4, AR mRNA accumulated in the luminal epithelium exclusively at the sites of blastocysts. Thus, AR expression correlated first with rising P4 levels and then with the attachment reaction. The rapid induction of AR mRNA in the ovariectomized uterus only by P4 and abrogation of this induction by RU-486 (a P4 receptor antagonist) suggest that this uterine gene is regulated by P4. AR appeared to exhibit preferential phosphorylation of epidermal growth factor receptor in the uterus over that in the blastocyst. This is a first report of a P4-regulated uterine epithelial cell growth factor that is associated with epithelial cell differentiation during implantation. The association of AR in implantation is further documented by its down-regulation in the day 4 pregnant uterus in which uterine receptivity and implantation were disrupted by estrogen or RU-486 treatment on day 3. These results further indicate that the expression of the AR gene could serve as a molecular marker for the receptive state of the uterus for implantation.

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