Expression and activation of the membrane-cytoskeleton protein ezrin during the normal endometrial cycle  Orkun Tan, M.D., Turkan Ornek, M.D., Ahmed Fadiel, Ph.D., Kelley S. Carrick, M.D., Aydin Arici, M.D., Kevin Doody, M.D., Bruce R. Carr, M.D., Frederick Naftolin, M.D., Ph.D.  Fertility and Sterility  Volume 97, Issue 1, Pages 192-199.e2 (January 2012) DOI: 10.1016/j.fertnstert.2011.10.039 Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Representative micrographs of ir-T-ezrin (A–C) and ir-p-ezrin (D–F) from serial sections in early, mid-, and late proliferative phases of normal endometrium. (A) Immunoreactive total ezrin is present in early proliferative endometrium. (B) Immunoreactive total ezrin in the mid-proliferative phase is mainly concentrated at the luminal aspect of glandular cells. (C) In late proliferative phase, ir-T-ezrin staining around the newly forming secretory vacuoles is noted (yellow arrows). (D) Only minimal ir-p-ezrin was present in the lateral and basal aspects of the glandular cells in the early proliferative phase. In the mid- (E) and late (F) proliferative phases, ir-p-ezrin was present at the glandular apical surfaces. Secretory vacuoles in the late proliferative phase started to form and migrate toward the glandular lumen and stained with ir-p-ezrin (F; yellow arrow). Occasional glandular cells were stained with ir-T-ezrin (red arrows). Immunoreactive total ezrin and ir-p-ezrin were minimal in stromal cells. Original magnification, ×40 for A and D; ×20 for B and E; ×60 for C and F. Inset B and E: negative control slides. Original images taken with Zeiss Axio Observer wide-field microscopy under different magnifications. The representative images are original; therefore, the background colors and contrast are different among images under different magnifications. Fertility and Sterility 2012 97, 192-199.e2DOI: (10.1016/j.fertnstert.2011.10.039) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Representative micrographs of ir-T-ezrin (A–C) and ir-p-ezrin (D–F) in endometrial tissues during the early, mid-, and late secretory phases of normal endometrium. (A) In the early secretory phase the secretory vacuoles migrating toward the glandular apical surfaces are densely stained with ir-T-ezrin. (B, C) In the mid- and late secretory phases, glandular cells are heavily stained with ir-T-ezrin. (D) Immunoreactive phospho-ezrin is present around the secretory vacuoles and glandular apical surfaces, though weaker than that of ir-T-ezrin. (E, F) Immunoreactive phospho-ezrin staining was most intense in the glandular cells and their apical surfaces of the mid- and late secretory phases. Immunoreactive total ezrin and ir-p-ezrin in the stroma of secretory phase were weak. Original magnification, ×20 for A–F. Original images without editing of background color or contrast. Fertility and Sterility 2012 97, 192-199.e2DOI: (10.1016/j.fertnstert.2011.10.039) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Representative micrographs of ir-T-ezrin and ir-p-ezrin in glandular cells in normal human endometrial tissue from the mid-secretory phase. Using computational imaging techniques, some of these cells from the set square in A were further amplified. Original micrograph magnifications, ×80 in A; ×200 in B: ×2,000 in C: ×3,000 in D and E. Ezrin expression in the glandular cells localized on the apical pole of the cells (A). With digital magnification, heavy ezrin immunostaining is noted on the luminal epithelium and microvilli (B) and on pinopodes (C–E). + and ∗ indicate apical and basal aspect of the glandular cell, respectively. Stars in D and E indicate pinopodes. Arrowheads in D and E indicate microvilli. (F) Representative image of dense ir-p-ezrin staining on microvilli (yellow arrows) and luminal aspect of epithelial cells (red arrow). Fertility and Sterility 2012 97, 192-199.e2DOI: (10.1016/j.fertnstert.2011.10.039) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Representative micrographs of ezrin in mid-secretory phase. The secretory vacuoles (red arrows) in the mid-secretory phase were surrounded by progressively intensified ir-T-ezrin staining (A–D). When secretory vacuoles reach the luminal membrane, the cytoplasm surrounding the vacuoles was most heavily stained with ir-T-ezrin. Dense ir-T-ezrin of the microvillus is obvious (D, black arrow). A–D show the same section with different magnifications. Original magnification, ×60 for A, ×80 for B, ×100 for C; ×160 for D. Yellow arrow indicates magnified image. Original images without editing of background color or contrast. Fertility and Sterility 2012 97, 192-199.e2DOI: (10.1016/j.fertnstert.2011.10.039) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Distribution of endometrial stromal and glandular ezrin (A) and phospho-ezrin (B) immunostaining intensity (H-SCORE) during the early, mid-, and late proliferative and secretory phases of normal endometrium. Stroma = stromal cells; Gland = glandular cells. Bars represent mean ± SEM. The expression level of ezrin in the glandular cells was significantly higher than that of the stromal cells. ∗P<.05, significantly higher in glandular cells as compared with stromal cells for the same endometrial phase. ∗∗P<.05, significantly higher in glandular cells in the secretory phase as compared with that of the proliferative phase. EP = early proliferative; MP = mid-proliferative; LP = late proliferative; ES = early secretory; MS = mid-secretory; LS = late secretory. Fertility and Sterility 2012 97, 192-199.e2DOI: (10.1016/j.fertnstert.2011.10.039) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 2 Total ezrin (A; ezrin) and p-ezrin (B) protein expressions were analyzed by Western blotting in proliferative and secretory phases of normal endometrium. Endometrial tissues were not subcategorized as early, mid-, late proliferative or secretory phases. M = molecular marker; P = proliferative phase; S = secretory phase. Using molecular marker, the band size was confirmed to be for total ezrin and phospho-ezrin (80 kDa). These results were compared with β-actin, which was used as an internal control. (C) Both total ezrin and p-ezrin expressions were higher in the secretory phase as compared with that of the proliferative phase. Bars represent mean ± SEM. ∗P<.05, significant increase in the secretory phase compared with the proliferative phase. OD = optic densitometry. Fertility and Sterility 2012 97, 192-199.e2DOI: (10.1016/j.fertnstert.2011.10.039) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions