Figure 1.
Whole mount in situ immunohistochemistry of gd7.5 B6 implantation sites co-stained with PE-CD31 (red) and FITC-CD45 (green).
Low power image (A) is a for orientation. The mesometrial side of the uterus is uppermost; the central black region (*) represents the embryonic crypt. More CD31 expression is present mesometrially than anti-mesometrially or in lateral (L) decidua. Images B, D, F are of the same field (represented by the boxed M) of the mesometrial uterus. Images C, E, G are of the same field (represented by the boxed AM) from the anti-mesometrial uterus. Image (B) illustrates the larger, web-like, less linked vessels of the decidua basalis. These are distinctly different to the narrower, more frequently linked anti-mesometrial vessels shown in (C). (B) but not (C) contains high numbers of CD31+ leukocytes. Image (D) shows the enrichment of leukocytes in decidua basalis while image (E) shows that leukocytes present anti-mesometrially are less frequent and more heterogeneous in shape and size. Red and green are merged in images (F) and (G). Image (F) shows that many cells with co-expression of CD31+ and CD45+ are present mesometrially while image (G) shows their absence anti-mesometrially. Magnification bar in (A) is 200 µm; in B-G 50 µm.
Figure 2.
Quantitative realtime PCR analyses of Dll1 in mesometrial and anti-mesometrial uterine samples and in flow sorted uNK cells.
Relative mRNA expression of Dll1 by the mesometrial side (M) of the virgin, gd4.5, gd5.5 and gd6.5 uterus of of the gd10.5 mesometrial lymphoid aggregate of pregnancy (MLAp) and decidua basalis (DB) is shown in (A). Relative mRNA expression of Dll1 by anti- mesometrial (AM) virgin uterus and by gd4.5, gd5.5 and gd6.5 anti-mesometrial decidua is shown in (B). Relative mRNA expression of Dll1 at gd10.5 by CD3-CD122+DBA- (DBA-) and CD3-CD122+DBA+ (DBA+) CD1 decidual cells normalized to Hprt1(C) is shown in (C). Data are means±SEM from all replicate analyses of two independent experiments. * P<0.05, ** P<0.01, *** P<0.001.
Figure 3.
Histological analysis of gd6.5 B6 decidua basalis for expression of DLL1.
Photomicrographs of gd6.5 B6 decidua basalis stained with DBA lectin-FITC (green), anti-DLL1-PE (red) and DAPI (blue) demonstrate in (A) DBA lectin-reactive small, agranular uNK cells and immature uNK cells with a few cytoplasmic granules. In (B), the same field is imaged showing cells reactive with DLL1. In the merged image (C), the co-expression of DBA lectin and DLL1 is shown (B and C; arrows mark representative cells). Additional cells that were DBA- and not identified expressed DLL1. The 6.5gd DBA+DLL1+uNK cells were found in the mesometrial decidua basalis (Meso DB) a region indicated as above the horizontal line in drawing (D). BV, entry of major blood vessel branches from the uterine artery; C conceptus, including ectoplacental cone. The area enclosed by dashed lines represents the residual uterine lumen. Bars: A, B and C are 40 µm.
Figure 4.
Histological analysis of DLL1 expression in mesometrial sections of gd10.5 B6 and CD1 implant sites.
Photomicrographs A-L and O show gd10.5 implant site cryosections co-stained with DBA lectin-FITC (green) to identify uNK cells and anti-DLL1-PE (red). Panel M(i) is stained with only DBA lectin-FITC (green). Panels A–M(i) are B6 implant sites and panel O is a CD1 implant site. Panel M(ii) provides a diagram of the regions images were collected from for the other panels and has been marked to show the banding pattern seen for DLL1 expression. This should be compared to low power image M(i) that shows DBA lectin-stained uNK cell distribution in each banded area. These are labeled region “1” for the MLAp, region 2 for decidua basalis distal to the placenta (pdDB) and region 3 for decidua basalis proximal to the placenta (ppDB). Region 4, the AM decidua, is at the bottom of the image. “C” represents the conceptus, including the placenta and the green ring around “C” is DBA lectin-stained yolk sac endothelium surrounding the fetus. The black space between the yolk sac and the region labeled “3” is the placenta which is uNK cell deficient. Expression of DLL1 by gd10.5 DBA+ cells was stratified within the mesometrial side of the implant sites in both strains and no significant differences were noted. In B6 MLAp (A–C; labeled “1” in M(i) and diagrammed in M(ii)), DLL1 was strongly expressed only infrequently (B) and not by the smaller, immature uNK cells that proliferate in this region (arrow heads in A, C). In decidua basalis of B6 that was distal to the placenta (D–F) and CD1 (O), DBA+ uNK cells brightly expressed DLL1 (arrows in D–F; O). DBA+DLL1+ uNK cells appeared to surrounded vessels (*). Additional perivascular DLL1 staining was present that was not associated with DBA+ cells. The decidual region proximal to the placenta was devoid of DLL1+ cells but abundantly populated by DBA+ uNK cells (G). Neither DLL1+ nor DBA+ uNK cells were present in the highly regressed anti-mesometrial decidua (A-Meso; J-L). DBA-stained yolk sac endothelium was present in this region (arrows in J, L). N is a photomicrograph of the placenta distal decidua basalis in a section from an archived paraffin-embedded gd10.5 B6 implant site double stained using DBA lectin-horseradish peroxidase and Periodic Acid Schiff’s reagent [25]. The latter stain reveals all granulated uNK cells and shows cells of the DBA-PAS+ subset (yellow circle). This image shows the typical strong association of uNK cells with arterioles and with microvessels, including intravascular positions and supports interpretations of the fluorescence images. In M(ii), BV indicates entry of major blood vessel branches from the uterine artery. Bars: A, B, C, J, K, L, O: 40 µm; D, E, F, G, H, I: 20 µm; M: 200 µm.