Previously, we have shown by in situ hybridization that the expression of IGFBP-4, a potential IGF-I inhibitor, correlates to the expression pattern described above for IGF-I mRNA throughout the periimplantation period. To determine whether uterine IGFBP-4 and IGF-I mRNA expression can be demonstrated in the absence of an embryo, in situ hybridization studies were performed on uteri from mice with one oviduct ligated to prevent fertilization. In this study, the contralateral horn, which was left intact to permit fertilization and embryo implantation, served as a control. IGFBP-4 mRNA was not detected in either uterine horn by in situ hybridization on gestational Day 2 (data not shown). On gestational Day 3, signals for both IGFBP-4 (Fig. 2, A and B) and IGF-I (data not shown) mRNA colocalized to the uterine stromal cells underlying the luminal epithelium in both the ligated and control horns. Similar results were obtained for IGFBP-4 mRNA on gestational Day 4 (Fig. 2, С and D). On gestational Day 5, however, no IGFBP-4 mRNA signal was detected in the ligated horn (Fig. 2F) even though IGFBP-4 signal was detected in the control horn (Fig. 2E). Similarly, on gestational Day 6, immediately after embryo implantation, an intense IGFBP-4 mRNA signal was detected in the control horn throughout the endometrial stroma surrounding the implanted embryo (Fig. 2G), whereas no IGFBP-4 mRNA signal was detected in the ligated uterus (Fig. 2H). buy asthma inhalers
To determine whether IGF-I and IGFBP-4 were expressed beyond gestational Day 4 in response to a specific signal from the embryo or to decidualization of the uterine stroma, an artificial decidual reaction was induced in pseudopregnant mice. On Day 4 of pseudopregnancy, mineral oil was injected into one uterine horn of female mice to stimulate a decidual reaction. The contralateral, uninjected uterine horn served as a control.
FIG. 2. Cellular localization of IGFBP-4 mRNA in longitudinal sections of ligated and control mouse uteri on Days 3 through 6 of gestation by in situ hybridization. B, D, F, H) Darkfield photomicrographs of ligated uterine horns on gestational Days 3, 4, 5, and 6, respectively, showing cells labeled with an antisense IGFBP-4 cRNA probe. А, С, E, C) Darkfield photomicrographs of control uterine horns on gestational Days 3, 4, 5, and 6, respectively, labeled with an antisense IGFBP-4 cRNA probe. White dots indicate autoradiographic grains, le, luminal epithelium; i, implantation site; d, decidua; s, stroma; m, myometrium. A-H, X40 (reproduced at 70%).