Ovary-Independent Estrogen Receptor Expression: RESULTS(2)

19 Jan
2013

RESULTS(2)Western Blotting

Results of Western blot analyses of uterine tissue protein from PND 0, 15, and 120 are shown in Figure 3. In tissue homogenate from PND 0, no specific signal of the expected size for ER protein (approximately 66 kDa) was detected. However, a specific positive ER signal of the expected size was observed as a faint band in tissue homogenate from PND 15, and a distinct specific signal of appropriate size was observed in tissue homogenate from PND 120. The ER-specific signal observed in tissue homogenates from PND 15 and 120 was not seen when irrelevant rat IgG was substituted for the H222 antibody or when buffer alone was substituted for primary antibody. In addition to the ER-specific signal, bands indicative of nonspecific binding of secondary reagents were also observed. In tissue homogenate from PND 0, these included major bands at approximately 52.4 kDa and 27.5 kDa. The nonspecific 52.4-kDa band was also observed in homogenates from PND 15 and 120.

Immunohistochemistry

Figure 4 shows representative photomicrographs depicting age-related changes in nuclear immunostaining patterns for ER protein between birth and PND 120 in endometrium from intact and OVX gilts. Extranuclear background staining was observed in tissue sections from all days and was most pronounced in tissue from PND 0. Patterns of change in nuclear immunostaining for ER protein were not affected by OVX at birth. cialis professional 20 mg
fIG3Ovary-independent estrogenFIG. 3. Identification of uterine ER by Western blot analysis. Total protein (50 p-g) from homogenates of uterine tissue obtained at birth (Day 0), PND 15 (Day 15), and PND 120 (Day 120) was applied to each lane. For each sample, the first (left) lane was incubated with primary rat antihuman ER H222 antibody and secondary antibody (H222+/2nd Ab+), while the second lane received an equivalent amount of irrelevant rat IgC instead of H222 (H222-/2nd Ab + ). Relative migration positions for molecular weight markers are indicated at 66, 45, and 31 kDa (left). The approximate migration position for ER protein is indicated by the arrow (right). No ER-specific signal was detected in tissue homogenate from PND 0. A faint, ER-specific signal (H222+/2nd Ab+) was detected at the expected position in tissue homogenate from PND 15, and a strong signal was seen in tissue homogenate from PND 120. This signal was not detected when irrelevant rat IgG was substituted for H222 (H222-/2nd Ab+) or when primary antibody was omitted (Day 120; (H222 = 0/2nd Ab+). Nonspecific binding of Abl and Ab2 was observed for all samples (bands below 66 kDa; see text for details).

fIG4Ovary-independent estrogenFIG. 4. Immunolocalization of nuclear ER protein in developing porcine endometrium. Photomicrographs depict effects of postnatal age, from birth (PND 0 = DO; top) to PND 120 (D120; bottom), on presence and distribution of nuclear ER protein in endometrium of intact gilts (i; left column) and gilts OVX at birth (o; right column). Nuclear immunostaining intensity, designated as absent, weak, moderate, or strong (see Fig. 5), was evaluated in endometrial S, GE, and LE. No effects of OVX were observed. Nonspecific, extranu-clear background staining was evident in all tissues. Nuclear staining indicative of ER protein was not observed in tissue from PND 0 (top left), or in control sections (Ctl; top right; D120 shown) in which irrelevant rat IgG was substituted for the primary H222 antibody. On PND 15 (D15i and D15o), specific nuclear ER staining was strong in GE and weak to moderate in S, but was absent in LE, with the exception of a few individual LE cells that appeared weakly ER positive (arrowhead). In GE, nuclear ER staining remained strong through PND 120. Consistent, strong nuclear ER staining was observed in endometrial S by PND 60. Regular nuclear ER staining was not observed in LE until PND 60 and later. Bar = 150 (im.

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