Northern Blotting and ISH Analyses
Porcine uterine ER mRNA was detected by Northern blot and ISH analyses using [a-32P]UTP- and [35S]UTP-labeled antisense cRNA probes generated from 0ER8 cDNA template. Using the [a-32P]UTP-labeled antisense ER cRNA probe, a single major transcript of approximately 6.2 kilobases (kb) was detected by Northern blot analysis of total uterine RNA (Fig. 6). Darkfield photomicrographs depicting age-related changes in the presence and distribution of ER mRNA, detected by ISH in uterine tissues obtained from intact and OVX gilts between birth and PND 120, are presented in Figure 7. In addition, nuclear ER immunostaining and ER ISH signal patterns for epithelial and stromal compartments of the endometrium on PND 0 and 15, the interval associated with appearance of endometrial GE, are illustrated in detail in Figure 8.
Results of ISH for ER mRNA were complementary to immunohistochemical data for detection of nuclear ER protein in the developing porcine endometrium (Figs. 4 and 8). Consistently, ER mRNA signal intensity was not affected by OVX at birth. Signal above background, observed when labeled sense cRNA probe was substituted for antisense probe in ISH procedures (Fig. 7, Ctl), was not significant in tissue sections from PND 0. Thus, there was no evidence of ER gene expression in uterine tissues obtained at birth (Figs. 7 and 8B). On PND 15, with the appearance of endometrial glands, a strong ISH signal was observed in association with GE, while a moderate signal was observed in endometrial S (Figs. 7 and 8D). Signal above background was negligible in LE for tissues obtained on PND 15 (Fig. 8D). In tissues obtained on and after PND 15, ER mRNA signal intensity remained strong for GE, while signal for S and LE increased such that maximum signal was observed in all cell types by PND 90 to 120 (Fig. 7). buy asthma inhaler
FIG. 6. Identification of a single major 6.2-kb mRNA transcript (arrow) by Northern blot hybridization analysis of total RNA from neonatal porcine uterine tissue (PND 120 shown) using an [a-32P]UTP-labeled antisense cRNA probe generated by in vitro transcription from fcoRI-linear-ized 0ER8 template. Relative migration positions of 28S and 18S rRNA are indicated (arrowheads).
FIG. 7. Localization of ER mRNA in developing porcine endometrium by ISH. A [35S]UTP-labeled antisense cRNA probe, generated by in vitro transcription from fcoRI-linearized 0ER8 cDNA template (see text), was used to detect porcine uterine ER mRNA in situ. Darkfield photomicrographs illustrate distribution of ER mRNA in cross sections of uterine tissues ob-. tained at birth (PND = DO), and from intact gilts (i; left column) and gilts OVX at birth (o; right column) on PND 15, 30, 60, 90, and 120. The image presented for PND 0 (top left) shows a complete section of the uterine wall. LE, endometrial S, and the perimetrial surface (arrow) are indicated. Endometrial GE is absent on PND 0. Signal above background was not observed in uterine tissues from PND 0 or when sense cRNA probe was substituted for antisense cRNA probe (Ctl; tissue from PND 15 shown). Overall, no systematic effects of OVX at birth were observed. On PND 15 (D15i and D15o), signal indicative of ER mRNA was intense over GE and moderate for S, but not significant for LE. Intense positive GE signal persisted through PND 120 (D120i and D120o; bottom). Otherwise, signal intensity over S and LE increased to stable levels by PND 60-90. Bar = 150 |xm.
FIG. 8. ER expression in porcine endometrium at birth (PND 0; A and B) and on PND 15 (C and D). A and С depict immunostaining of uterine cross sections for nuclear ER protein. В and D show darkfield photomicrographs illustrating relative presence and distribution of ER mRNA detected by ISH (see text). On PND 0, neither nuclear immunostaining (A) nor signal (above background) indicative of the presence of ER mRNA (B) was detected in uterine LE or S. GE is absent on PND 0 but present on PND 15. Nuclear ER immunostaining remained effectively absent in LE on PND 15 (C). However, occasional LE cell nuclei were observed to be ER positive (C, between arrowheads). Signal indicative of the presence of ER mRNA in LE was also absent in tissue from PND 15 (D, between arrowheads). In contrast, strong, regular nuclear immunostaining was observed for GE cells (C), and a strong ISH signal, indicative of the presence of ER mRNA, was detected over GE cells (D) on PND 15. Bar = 50 pirn.