The extent of decidualization of the oil-injected horn was visualized by staining for alkaline phosphatase activity, an early decidual cell marker. Figure 3 shows a representative deciduomata-induced (A) and control uterine horn (B) stained for alkaline phosphatase activity. In the deciduomata-in-duced horn, a crescent of anti-mesometrial stroma surrounding the luminal epithelium stained intensely for alkaline phosphatase activity. Alkaline phosphatase staining was also detected in the endothelial cells of the myometrium and the mesometrium, as well as in uterine glands and in the myometrium (Fig. ЗА). In the control horn, alkaline phosphatase activity was not detected in endometrial stroma. Alkaline phosphatase staining was detected only in the uterine glands, the myometrium, and the endothelial cells of the myometrium and the mesometrium (Fig. 3B). In addition, alkaline phosphatase staining was blocked in deciduomata-induced uterine sections incubated in substrate reaction solution containing levamisole (data not shown).
In situ hybridization studies were conducted on deciduomata-induced and control mouse uteri for IGF-I and IGFBP-4 gene expression, as shown in Figure 4. At 24 h after oil injection, a weak IGF-I mRNA signal was detected in the endometrial stromal cells surrounding the lumen (Fig. 4A), and an intense IGFBP-4 mRNA signal was detected in a horseshoe shape of antimesometrial stroma (Fig. 4B). At 48 h after oil injection, a strong IGF-I mRNA signal was detected in the stromal cells adjacent to the myometrium (Fig. 4C), while IGFBP-4 mRNA signal was intense in the primary and secondary decidual zones (Fig. 4D). At 72 h after oil injection, IGF-I mRNA signal was detected in the myometrium and in the stromal cells underlying the myometrium (Fig. 4E). An intense IGFBP-4 mRNA signal was also detected in stromal cells underlying the myometrium, as well as in stromal cells surrounding the uterine lumen (Fig. 4F). No IGF-I or IGFBP-4 mRNA signal was detected in the control horns at 48 h (Fig. 4, G and H), and similar results were obtained at 24 and 72 h (data not shown). buy levaquin online
FIG. 3. Confirmation of decidualization by enzyme histochemistry for alkaline phosphatase activity. A) An artificial decidual reaction was induced in pseudopregnant mice by injecting mineral oil into one uterine horn. Brightfield photomicrograph of a uterine cross section shows the extent of decidualization (dark stain) and the presence of mineral oil (o) in the uterine lumen at 24 h after injection. B) Brightfield photomicrograph of the contralateral, uninjected uterine horn at 24 h after injection. A and В, X25.
FIG. 4. Cellular localization of IGF-I and IGFBP-4 mRNAs in cross sections of decid-uomata-induced mouse uteri by in situ hybridization. А, С, E) Darkfield photomicrographs showing cells labeled with an antisense IGF-I cRNA probe at 24, 48, and 72 h after mineral oil injection, respectively. B, D, F) Darkfield photomicrographs showing cells labeled with an antisense IGFBP-4 cRNA probe at 24, 48, and 72 h after mineral oil injection, respectively.