Abundance of COX-2 in both horns of control cows was similar (control X ipsi vs. control X contra, paired f-test, P > 0.1), whereas levels of COX-2 were higher after treatment in ipsi horns compared with contralateral (IFN X ipsi vs. IFN X contra, paired f-test, P < 0.05).
The signal for COX-2 was not significantly altered (treatment; treatment X horn, ANOVA, P > 0.1), even when ipsi and contralateral horns were compared, in the stroma (Fig. 8D), the glandular epithelium, the blood vessels, and the myometrium (not shown). Nonimmune serum showed no staining (Fig. 7I).
COX-1-Positive Cells Are Not Significantly Decreased after IFN-т Treatment
In experiment 2, the weak expression of COX-1 was not regulated in the LE or S in response to IFN-т treatment (Fig. 7, A and B). Isolated COX-1-positive cells in the S were counted (Fig. 8A) after treatment, and their number was comparable with the untreated control (treatment; treatment X horn, ANOVA, P > 0.1). Again, nonimmune serum showed no reactivity (Fig. 7I).
GM-CSF Immunostaining Is Increased in LE During Early Pregnancy
The intensity of the red staining (Fig. 4) in the LE and S was evaluated by image analysis, first by comparing between days of the estrous cycle or pregnancy. In the LE (Fig. 5A), no significant effect of day was observed during the cycle (regression analysis, P > 0.5). On the other hand, GM-CSF was significantly stimulated during pregnancy in the LE from d7P to d30P (R2 = 0.25, slope = 11, P < 0.01). As with COX-2, GM-CSF was expected to follow the pattern of IFN-т secretion.
Immunohistochemical Localization of GM-CSF in the Bovine Uterus
Immunostaining for GM-CSF was observed in the con-ceptus at the surface of the trophectoderm at d18P (Fig. 4F) and sometimes in d21P-d24P in regions where the attachment process was not advanced (Fig. 6F). In maternal tissues, GM-CSF staining was concentrated in the apical portions of the LE and glandular epithelium, as well as in the basement membrane (Fig. 4). The staining grades were similar to those observed with COX-2: LE > SG > DG > S.
COX-2 Immunostaining Is Increased in LE During Early Pregnancy
The intensity of the red staining (Fig. 2) in the concep-tus, LE, and S was evaluated by image analysis (Fig. 3, BD). COX-2 staining linearly decreased with time (Fig. 3B) in the conceptus between d18P and d30P (R2 = 0.3, slope = —4, P < 0.005). In the LE, on the other hand, no significant effect of day was observed (Fig. 3C) during the cycle (regression analysis, P > 0.5), even though COX-2 intensity appeared stronger at d16C, the day of luteolysis. However, COX-2 was significantly stimulated during pregnancy (Fig. 3C) in the LE from d7P to d30P (R2 = 0.20, slope = 10, P < 0.005).
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).
Immunohistochemical Localization of COX-1 in the Bovine Uterus
The expression of COX-1 in the conceptus (Fig. 1, F-H) and major compartments (epithelium, stroma, blood vessels, and myometrium) of the uterus was either very weak or nonexistent, with the exception of intensely stained, leukocyte-like cells that were concentrated in the S but absent from the LE or glandular epithelium (Fig. 1). A few similar COX-1-positive cells could also be found deeper in the myometrium, particularly around blood vessels (not shown). COX-1 staining was both nuclear and cytoplasmic (Fig. 1I).
For COX-1 studies, an average of 10 pictures per section were taken, and the number of positive isolated cells in the subepithelial stroma were counted. For COX-2 and GM-CSF studies, an average of 20 pictures per section were taken. Areas of interest, either the conceptus, LE, or subepithelial stroma (S), were separately cropped and then submitted to densitometry analysis.
IHC was performed twice on a total of 12 sample blocks, one from each uterine horn of six animals (three controls and three treated). Two sections, one from a control animal the other from an IFN-treated animal, were mounted on each precoated slide. Sections were air dried, incubated at 60°C for 30 min, and kept at 4°C until used. Immunostaining procedures were performed at room temperature unless otherwise noted. First, slides were deparaffinized in xylene and washed with ethanol. Endogenous peroxidase activity was then quenched with 3% H2O2 in methanol for 20 min.
Sections were air dried and fixed with 4% paraformaldehyde in PBS (pH 10) for 30 min. Immunostaining procedures were immediately performed at room temperature unless otherwise noted. First, endogenous peroxidase activity was quenched with 3% H2O2 in PBS for 15 min. Nonspecific binding sites were then blocked with 10% goat serum in PBS for 1 h.