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).
COX-2 Immunostaining Is Increased in LE of the Ipsi Horn after IFN-т Treatment
After IFN-т treatment, the staining for COX-2 (Fig. 7, C-F) in the LE was not significantly enhanced (Fig. 8B) when only the effect of treatment was examined (ANOVA, P > 0.05). However, the effect of the interaction between treatment and side of the uterine horn (Fig. 8C) was significant (treatment X horn, P < 0.05). The striking stimulation of COX-2 observed in the ipsi horn to the corpus luteum (Fig. 7, E vs. F) reached a 3.6 ± 0.5-fold increase over untreated control (control X ipsi vs. IFN X ipsi, paired f-test, P < 0.01). On the other hand, the expression of COX-2 in the LE of the contralateral (contra) horn (Fig. 7, C vs. D) was not significantly different (Fig. 8C) between treatment and control (control X contra vs. IFN X contra, paired f-test, P > 0.5).
FIG. 7. Immunohistochemical localization of COX-1 (A and B), COX-2 (contralateral horn, C and D; ipsi horn, E and F), and GM-CSF (G and H) in paraffin-embedded endometrium from control animals (A, C, E, and G) or cows treated with roIFN-т (B, D, F, and H). Primary antibodies against COXs and GM-CSF were respectively substituted with nonimmune rabbit serum (I) and negative mouse IgG1 (J). Brownish-red color (AEC) indicates positive staining, and sections are counterstained with hematoxylin. Solid arrows indicate leukocyte-like cells in the stroma; open arrows indicate blood vessels. Lumen (L), LE, and S are identified. Final magnification X200.
FIG. 8. Relative regulation of COX-1 (A), COX-2 (B-D), and GM-CSF (E and F) immunostaining in the LE (B, C, and E) and subepithelial stroma (A, D, and F) of uteri from cows in response to roIFN-т (IFN) treatment. Values are means ± SEM of two samples (ipsi and contralateral) from each of three control animals and three treated heifers. Mean values of untreated controls (ctrl) were adjusted to 100%. A) COX-1-positive cells in the subepithelial stroma were counted, and the ctrl value averaged 23 COX-1-positive cells per 0.28-mm2-field. B-D) By using image analysis, the intensity of COX-2 immunostaining was evaluated. B) The ctrl value averages 2.2 IOD units per |xm2 in the LE. C) The ctrl value averages 1.6 IOD units per |xm2 in the LE of the ipsi horn. aTreatment increased COX-2 staining in the ipsi horn (treatment X horn, ANOVA followed by paired t-test, P < 0.05). D) The ctrl value averages 0.19 IOD unit per |xm2 in the stroma. E and F) By using image analysis, the intensity of GM-CSF immunostaining was evaluated. E) The ctrl value averages 2.4 IOD units per |xm2 in the LE. bTreatment increased GM-CSF staining (ANOVA followed by paired t-test, P < 0.05). F) The ctrl value averages 0.47 IOD unit per l^m2 in the stroma.