Orphan Receptor Kinase ROR2 is Expressed in the Mouse Uterus

Expression of Ror2, Wnt3a, Wnt5a and Dkk1 transcripts in the pregnant mouse uterus. Complementary DNA from gd10 MLAp, decidua and placenta was amplified for WNT- related genes.

Gestation day 10 mouse uterus was stained for ROR2 using immunohistochemistry. Peroxidase-DAB detection localized ROR2 expression to uterine NK-like cells (arrows) in the MLAp (A) and decidua (B). Placental labyrinth cells expected but not confirmed to be trophoblast cells (arrowheads) lining the maternal circulation (C) were also positive. Size bar = 50 μm.

Fluorescent immuno-detection reveals uterine NK cells express ROR2 while splenic NK cells do not. No differences were observed between spleens collected from virgin and pregnant animals. Shown here are virgin splenocytes. Using immunohistochemistry, gd10 implantation sites were stained for ROR2 expression and uterine NK cells (using DBA lectin) and counterstained using DAPI. Co-localization of these markers revealed uterine NK cells express ROR2 (A; bar = 20 μm). To ask if splenic NK cells also expressed ROR2, immunohistochemistry (B; bar = 5 μm) and flow cytometry (C) was done using a peripheral NK cell marker, NK1.1, on splenocytes. Splenic NK1.1 + cells were negative for ROR2.

ROR2 expressing uterine NK cells are absent in Rag2^−/−Il2rg^−/− mice, but appear after engraftment of WT bone marrow. Rag2^−/−Il2rg^−/− mice, genetically deficient in T, B and NK cells, also lack ROR2 expressing uterine NK cells. Adult mice conditioned with chemotherapeutic agent, 5-Fluorouracil (5FU), receive bone marrow intravenously 48 h later. These mice are then set up for pregnancy and their uteri become populated with ROR2 expressing uterine NK cells. The absence of these cells in pregnancies of knock-out mice (not shown) is further support that uterine NK cells express ROR2. Immunohistochemistry shows gd12 decidua basalis. Size bar = 20 μm.

Time-course fluorescent immunohistochemistry staining of the uterus. Virgin, gd6 and 12 uteri were stained for ROR2 (red). DBA lectin staining (green) was used to localize uterine NK cells and DAPI (blue) nuclear counterstaining was performed. In the virgin stroma, ROR2 expressing cells are rare (arrow). ROR2 expression was observed on the basal side of uterine gland and endometrial epithelial cells. Gestation day 6 mesometrial stroma showed positive ROR2 staining in both stromal cells (arrow) and uterine NK cells (arrowhead). At gd6, compared to the mesometrial stroma, the anti-mesometrial stroma appeared weaker in ROR2 expression. Uterine glands at gd6 expressed ROR2 on both the luminal and basal side. In gd12 implantation sites, MLAp area stromal cells (arrow) and uterine NK cells (arrowhead) expressed ROR2. Gestation day 12 uterine NK cells in the decidua also expressed ROR2. The uterine wall was also ROR2 positive. Size bar = 50 μm.

Peroxidase ROR2 immunohistochemisty of virgin mouse uteri. Expression of ROR2 was localized to the basal side of endometrial and uterine gland epithelial cells throughout the estrous cycle. Size bar = 20 μm.