DIFFERENTIAL EXPRESSION OF VASCULAR ENDOTHELIAL GROWTH-FACTOR AND ITS RECEPTOR MESSENGER-RNAS IN THE MOUSE UTERUS AROUND THE TIME OF IMPLANTATION

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 (approximate to 4.2 kb) transcript for VEGF mRNA was detected in uterine poly(A)(+) samples, except for the presence of two other minor (approximate to 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 (approximate to 6.5-7.0 kb) for Flk-1 mRNA and two transcripts (approximate to 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. The results suggest that vWF-positive stromal cells on day 4 and cells in the antimesometrial decidual bed on days 5-8 correlated with the expression of Flk-1 mRNA, as did the vWF- and lectin-positive cells in the mesometrial decidual bed. This implies that cells involved in angiogenesis at the mesometrial pole express the VEGF receptor mRNAs. In contrast, perhaps the endothelial cells of the existing blood vessels in the stromal bed on day 4 and those in the antimesometrial decidual bed on days 5-8 accumulated the receptor mRNAs, suggesting an involvement of VEGF in changes in vascular permeability. Flk-1 mRNA was also detected in embryonic tissues on day 8. Collectively, the results suggest that VEGF participates in increased vascular permeability and/or angiogenesis occurring in the uterine vascular bed during implantation. Further, the data suggest that VEGF is involved in trophoblast differentiation and invasion, as well as in decidualization and placentation.