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).
Nonimmune serum, used as a negative control for COX-1 antiserum, showed no reactivity (Fig. 6G). The number of COX-1-positive cells in the S did not vary throughout the estrous cycle (regression analysis, P > 0.5), whereas it was doubled from d7P to d16P, decreasing (regression analysis, R2 = 0.25, slope = —4, P < 0.005) to cycle-level afterward (Fig. 3A). When d7, d16, and d18 were compared across status (pregnant vs. cyclic) by regression analysis, an effect of status was found (increased in pregnant cows, P < 0.05).
FIG. 1. Immunohistochemical localization of COX-1 in cryosections of uteri from cyclic (d7 [A] and [I], d16 [B], d18 [C]) or pregnant cows (d7 [D], d16 [E], d18 [F], d21 [G], d24 [H]). Red color (AEC) indicates positive staining; sections are counterstained with hematoxylin. Solid arrows indicate leukocyte-like cells in the stroma; dotted arrows indicate trophoblastic binu-cleate cells. LE, fused epithelium (FE), S, and conceptus (C) are identified. (I) Magnification of d7C in (A). Final magnifications X200 (A-H) and X400 (I).
FIG. 2. Immunohistochemical localization of COX-2 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 are counterstained with hematoxylin. Dotted arrows indicate trophoblastic binucleate cells. LE, fused epithelium (FE), S, concep-tus (C), and SG are identified. Final magnification X100.
FIG. 3. Relative regulation of COX-1 (A) and COX-2 (B-D) immunostaining in the uterine S (A and D), LE (C), and conceptus (B). All values are means ± SEM of three samples from days of the cycle or pregnancy. A) COX-1-positive cells in the sub-epithelial stroma were counted in each field, which covered 1.2 mm2. An effect of day was detected by regression analysis within pregnant cows from d16P to d30P (decrease, P < 0.005). When d7, d16, and d18 were compared across status by regression analysis, an effect of status (increased in pregnant vs. cyclic cows, P < 0.005) was found. (B-D) The intensity of COX-2 immunostaining was evaluated by image analysis. B) Control values (d18P) were adjusted to 100% and averaged 4.2 IOD units per |xm2 in the conceptus. Staining for COX-2 was reduced with time (regression analysis, P < 0.005). C and D) Control values (d7P) were adjusted to 100% and averaged 0.95 IOD unit per |xm2 in the LE (C) and 0.47 IOD unit per |xm2 in the S (D). Staining for COX-2 was increased with time during pregnancy in LE (regression analysis, P < 0.005).