Figure 3: Analysis of uterine expression for H2AK119ub1 and H3K27me3 during early pregnancy.

(A) Western blot analysis. Total protein extracts of uterine tissues were collected during early pregnancy on D4, D5-IS, and D6-8 IS and IIS. Experimentally induced deciduoma and control horns were also analyzed on D7 pseudopregnancy. Ut, uterus; IS, implantation site; IIS, interimplantation site; Co, control; DM, deciduoma. (B) Quantitation was achieved by direct analysis of the bands from (A). Fold changes were normalized by H2A. *Values are statistically different (P < 0.05). (C) IHC localization of H2AK119ub1 and H3K27me3. Representative uterine tissue sections on D4, D5-IS, and D7-IS in the M- and AM-pole locations are shown. Sections are shown at 100X and 400X (as insets). Red staining indicates the localization of immunoreaction. le, luminal epithelium; ge, glandular epithelium, s, stroma; ds, decidualizing stroma; pdz, primary decidual zone; sdz, secondary decidual zone; dec, decidual cells; e, embryo; tgc, trophoblast giant cells; M, mesometrial pole; AM, antimesometrial pole. Arrow indicates polyploid cells. (D) IF analysis of co-localized expression of H2AK119ub1 or Cbx4 together with Ring1B or H3K27me3 on D7-IS, respectively. Red (H2AK119ub1 or Cbx4) and green (Ring1B or H3K27me3) staining indicate immuno-localization of proteins. DAPI was used as nuclear stain. Arrow indicates direction of embryo (em) location. Pictures are shown at 400X. (E) Western blot analysis of Ring1B, Cbx4, H2AK119ub1, H3K27me3, Actin, H2A for non-polyploid (NP) vs. polyploid (P) decidual cells during experimental decidualization on D7. Quantitation (fold change) of Ring1B, Cbx4, and H3K27me3 was achieved by direct analysis of the bands against Actin, while that of H2AK119ub1 was done against H2A. *Values are statistically different (P < 0.05). These experiments were repeated at least three times with similar results.