Immunohistochemical Localization of COX-2 in the Bovine Uterus
As shown in Figure 2, immunostaining for COX-2 was most intense in the trophectoderm layer of the conceptus, particularly in uninucleate trophoblastic cells (Fig. 2, F and G). On the other hand, underlying mesoderm was negative (Fig. 2, F-I) and binucleate cells were weakly stained (Fig. 2, G-I).
In maternal tissues at all stages of the cycle or pregnancy, the staining intensity followed this order: LE > SG > deep glands (DG) > S. The exceptions are d16P (Fig. 2E) in which all maternal compartments expressed COX-2 at a similar level and d30P (Fig. 2I) where COX-2 was darker in SG than in LE (not shown). COX-2 staining was also reduced at attachment sites in fused epithelial cells (Fig. 2, H and I), and a faint signal was sometimes apparent in the myometrium (Fig. 6B). The compactness of the stroma did not permit the separate analysis of the three contributors to stromal COX-2, which are fibroblasts, capillaries, and leukocytes. No difference was observed between caruncular and intercaruncular regions (not shown). In general, intracellular localization of COX-2 was cytoplasmic and also perinuclear, particularly in trophoblastic cells (Fig. 6A). Again, nonimmune serum showed no staining (Fig. 6G).
FIG. 4. Immunohistochemical localization of GM-CSF in cryosections of uteri from cyclic (d7 [A], d16 [B], d18 [C]) or pregnant cows (d7 [D], d16 [E], d18 [F], d21 [G], d24 [H], d30 [I]). Red color (AEC) indicates positive staining; sections were counterstained with hematoxylin. Solid arrows indicate leukocyte-like cells in the stroma; dotted arrows indicate trophoblastic binucleate cells. LE, fused epithelium (FE), S, conceptus (C), and SG are identified. Final magnification X200.
FIG. 5. Relative regulation of GM-CSF immunostaining in the uterine LE (A) and S (B). The intensity of GM-CSF immunostaining was evaluated using image analysis. Values are means ± SEM of three samples from days of the cycle or pregnancy. Control values (d7P) were adjusted to 100% and averaged 0.53 IOD unit per |xm2 in the LE (A) and 0.42 IOD unit per |xm2 in the S (B). Staining for GM-CSF is increased with time during pregnancy in LE (regression analysis, P < 0.01).
FIG. 6. Immunohistochemical localization of COX-2 (A and B) and GM-CSF (C-F) in cryosections of uteri. Primary antibodies against COXs and GM-CSF were respectively substituted with nonimmune serum (G) and negative IgG1 control (H). A) COX-2 perinuclear staining is more obvious in trophoblastic cells (d21P). B-D) Deeper portions of uteri are illustrated at d16P (B and C) and d30P (D). E and F) Heterogeneity of fusion between trophoblast and maternal epithelium through time: comparison between ”advanced” d21P (E) and ”late” d24P (F). Red color (AEC) indicates positive staining; sections are counterstained with hematoxylin. Solid arrows indicate perinuclear staining; dotted arrows indicate trophoblastic binu-cleate cells; open arrows indicate blood vessels. LE, fused epithelium (FE), S, conceptus (C), deep glands (DG), and myometrium (MY) are identified. Final magnifications X400 (A), X100 (B, C, D, G, and H) and X200 (E and F).