Osteopontin is up-regulated in human decidual stromal cells Michael von Wolff, M.D., Michael K Bohlmann, M.D., Christiane Fiedler, Stefanie Ursel, Thomas Strowitzki, M.D.  Fertility and Sterility  Volume 81, Pages 741-748 (March 2004) DOI: 10.1016/j.fertnstert.2003.08.027

FIGURE 1 Immunostaining of osteopontin in human endometrium of the luteal phase and in decidua of the sixth to ninth week of pregnancy in representative tissue samples of fertile subjects. Immunohistochemistry showed maximal staining of osteopontin in glandular epithelial cells of secretory phase endometrium (A). Staining was not consistently strong in all glands. Some glands showed strong staining (A, black arrow), whereas others showed only weak staining (A, white arrow). Staining of stromal cells was weak, apart from some stromal cells with moderate staining intensity in the late secretory phase. Decidual tissue revealed moderate to strong staining in decidual glands (B, white arrow). In contrast to endometrial stromal cells, staining of decidual stromal cells was moderate to strong in all tissue samples (B, C, black arrows). Substituting for the primary anti-osteopontin antibody with nonimmune rabbit serum resulted in weak background staining in decidual stromal and epithelial cells (D). (Magnification: A, B, D, ×250; C, ×650). von Wolff. Osteopontin and decidua.Fertil Steril 2004. Fertility and Sterility 2004 81, 741-748DOI: (10.1016/j.fertnstert.2003.08.027)

FIGURE 2 Stromal cells were isolated by filtration and cultured in polystyrol culture plates for 4 to 5 days. Cells were stimulated with 17β-E2 (E2, 10−8 M = 2.7 ng/mL), P (Prog, 10−6 M = 310 ng/mL), IL-1β, IL-6, or LIF (10 ng/mL) or with culture supernatants of decidual natural killer cells (NK cells) for 6 and 24 hours (n = 5). Osteopontin mRNA concentrations were analyzed by RNase protection assay. The relative optical density values were calculated by normalizing the mRNA to the relative optical density of L32 and GAPDH. Concentrations of osteopontin mRNA are shown relative to unstimulated controls (relative optical density = 1.0). Natural killer cells were isolated from induced abortions and were stimulated with IL-15 and cultured for 2 days in culture plates, coated with decidual stromal cells. Culture supernatants were used for stimulation of stromal cells at a concentration of 10%. Osteopontin mRNA expression was analyzed after 6 hours of stimulation by RNase protection assay, and osteopontin protein concentrations in culture supernatants were analyzed after 24 hours of stimulation by ELISA (data not shown). von Wolff. Osteopontin and decidua.Fertil Steril 2004. Fertility and Sterility 2004 81, 741-748DOI: (10.1016/j.fertnstert.2003.08.027)

FIGURE 3 Osteopontin mRNA expression in endometrial stromal cells at different stages of decidualization in vitro. Stromal cells were obtained from four patients and were isolated and decidualized in four-cell cultures (C1–C4) by stimulation with 17β-E2 (0.5 ng/mL) and P (50 ng/mL) for 0, 7, 14, and 21 days. Decidualization was confirmed by concentration of PRL and IGFBP-1 in culture supernatants. mRNA analysis was performed by RNase protection assay. A representative mRNA expression profile is shown under each diagram; the corresponding mRNA expression of L32 and GAPDH is shown at the bottom. Cell culture experiments and RNase protection assays were performed in duplicates. von Wolff. Osteopontin and decidua.Fertil Steril 2004. Fertility and Sterility 2004 81, 741-748DOI: (10.1016/j.fertnstert.2003.08.027)

FIGURE 4 Osteopontin protein concentration in supernatants of endometrial stromal cells. Endometrial tissue was taken from hysterectomy specimens, and stromal cells were isolated by enzymatic tissue digestion, filtration, and use of antibody-coated magnetic beads. Cells were decidualized in four cultures (C1–C4) by stimulation with 17β-E2 (0.5 ng/mL) and P (50 ng/mL) for 0, 7, 14, and 21 days. Osteopontin in culture supernatants was analyzed by ELISA. Prolactin concentrations in culture supernatants were analyzed after 0, 7, 14, and 21 days to confirm decidualization of stromal cells. Cell culture experiments and ELISA were performed in duplicates. von Wolff. Osteopontin and decidua.Fertil Steril 2004. Fertility and Sterility 2004 81, 741-748DOI: (10.1016/j.fertnstert.2003.08.027)