G-CSFR Was Expressed on Undifferentiated Cytotrophoblast Cells of Term Fetal Membranes
The staining pattern obtained for G-CSFR and cytoker-atin in fetal membranes is shown in Figure 2. The tropho-blast cells in fetal membranes exhibited diffuse staining for G-CSFR, which, in this respect, was similar to the staining pattern observed in first- and second-trimester interstitial cytotrophoblast (Fig. 1g). The intensity of staining was similar to that observed on second-trimester interstitial tropho-blast. Adjacent sections treated with mouse IgG were negative (Fig. 2c). buy flovent inhaler
Expression of Placental G-CSFR mRNA and Protein Was Gestationally Regulated
Quantitation of levels of G-CSFR mRNA in placental tissue throughout gestation was achieved by RPA (Fig. 3). The expected protected RNA fragments obtained with hybridization of RNA with the 32P-labeled G-CSFR cRNA Fragments corresponding to other G-CSFR transcripts were not detected. Two GAPDH fragments of 120 bp and ~ 125 bp were detected, and variations of band intensity indicated differences in loading. The intensity of G-CSFR and GAPDH bands in placental RNA samples was estimated by densitometry, and levels of G-CSFR mRNA were expressed as a ratio of G-CSFR band intensity to the sum of the two GAPDH band intensities as shown in Figure 3b. Levels of G-CSFR mRNA were highest in third-trimester placental samples (Fig. 3d), with a 4fold increase in intensity with respect to levels in the first trimester (p = 0.0002). Levels of G-CSFR in second-trimester tissues were 4-fold lower than those of first-trimester tissues (p = 0.0045).
FIG. 3. Quantitative analysis of G-CSFR mRNA in placental tissue throughout gestation by RPA. a) RPA analysis of placental G-CSFR mRNA from placentas of different gestational ages (lanes labeled in weeks). A 497-bp fragment corresponding to the class I receptor, and two protected fragments of 120 bp and —12 5 bp corresponding to GAPDH, were detected in all the mRNA samples analyzed. b) Densitometric analyses of the gel shown in a. The intensities of bands corresponding to G-CSFR mRNA were expressed as a ratio of those corresponding to GAPDH. c) Schematic representation of the predicted placental G-CSFR mRNA fragments protected by the 595-bp 32P-labeled G-CSFR probe. The hatched box represents the 81-bp insertion sequence present in the mRNA corresponding to the class III receptor. The sizes of the predicted fragments representing both class III (431 and 64 bp) and class I (497 bp) receptors are indicated. d) Bar graphs representing the mean G-CSFR mRNA values obtained from densitometric analysis in the first, second, and third trimesters of pregnancy, ± SEM. The data presented are representative of three independent experiments.