Characterization of H/358 Antibody by Western Blotting
We tested the specificity of the Hp58 antibody by Western blotting. Induction of protein expression was monitored by SDS PAGE (Fig. 3A). The induced GST-Hp58 fusion protein migrated at a molecular mass of 35 kDa consistent with the predicted amino acid sequence of the coding region of the rat cDNA isolated and cloned from the RNA fingerprinting (see Fig. 1A). The GST protein alone migrated at the smaller expected mass of 27.5 kDa (Fig. 3A). Aliquots from those same protein samples were analyzed concurrently by immunoblotting (Fig. 3B). Immunoreactiv-ity to the induced GST-Hp58 fusion protein was strongly evident (lane 2), while in the absence of IPTG induction, Hp58 immunoreactivity was barely detectable (lane 3). There was slight nonspecific binding of the antibody to the GST protein alone (lanes 4 and 5) owing to the excessive amount of GST protein in these lanes (compare Fig. 3, A and B). Taken together, those results confirmed that the antibody reacted specifically with Hp58.
Localization of H/358 Protein in the Postimplantation Endometrium
We next investigated the distribution of Hp58 in the postimplantation endometrium between Days 6-9 of pregnancy. The distribution of Hp58 transcripts in the mouse conceptus and placenta had been previously reported . However, no information about the cell-specific expression or the distribution of Hp58 protein in the maternal cells of the early postimplantation uterus was known. At Day 6 of pregnancy, when morphological evidence for decidualiza-tion at the antimesometrial aspect of the endometrium was distinguishable, Hp58 was strongly expressed in the nuclei of the luminal (Fig. 4A) and glandular epithelium (not shown). Some stromal cell nuclei were also positive at Day 6 of pregnancy. Immunoreactive Hp58 localized in the nucleus in the stromal cells, while in decidual cells both the nuclei and the cytoplasm were immunopositive. There was no specific immunoreactivity when sections were treated with preimmune serum (Fig. 6, B and F). At Day 7 of pregnancy, the luminal (Fig. 4C) and glandular (not shown) epithelial cells expressed Hp58. In the stromal compartment, decidual cells were immunopositive in both the nucleus and cytoplasm (Fig. 4C). The luminal and glandular epithelia were immunopositive at Day 8 of pregnancy (Fig. 4D). The immunoreactivity shown in Figure 4D was characteristic of that observed in the glandular epithelial cells at Days 6-8 of pregnancy. Decidualization had expanded through the stromal compartment at Day 9 of pregnancy, and Hp58 immunoreactivity was evident in the decidual nuclei and cytoplasm (Fig. 4E).
FIG. 3. Specificity of Hp58 antibody. Glutathione-S-transferase and GST-Hp58 proteins were induced in XL-1 bacteria containing the plasmids pGex or pGex-Hp58. Protein extracts (10 ^l/lane) were size fractionated by SDS-PAGE and analyzed by Coomassie blue staining and Western im-munoblotting. A) Protein induction was monitored by Coomassie blue-stained SDS polyacrylamide gels. Lane 1, IPTG-induced GST-Hp58 fusion protein; lane 2, GST-Hp58 without induction; lane 3, IPTG-induced GST; lane 4, GST without induction; lane 5, prestained molecular size standards. Arrowhead shows the GST-Hp58 fusion protein. B) Western blot of protein extracts reacted with Hp58 antibody. Lane 1, prestained molecular size standards; lane 2, IPTG-induced GST-Hp58 fusion protein; lane 3, GST-Hp58 protein without induction; lane 4, IPTG-induced GST; lane 5, GST without induction.
FIG. 4. Cell-specific localization of H^58 in the early postimplantation endometrium. Immunocytochemical localization of H^58 from the endometrium of rats at Days 6-9 of pregnancy. Representative sections from implantation sites analyzed from three different rats are shown. A) Day 6 pregnant rat treated with H^58 antibody. Arrowheads indicate early decidual cells. B) Day 6 pregnant rat treated with preimmune serum. C) Day 7 pregnant rat treated with H^58 antibody. Arrowheads indicate immunoreactivity in the decidual cells. D) Day 8 pregnant rat treated with H^58 antibody showing immunoreactivity in the luminal and glandular epithelium. E) Day 9 pregnant rat treated with H^58 antibody showing nuclear and cytoplasmic immunoreactivity in the decidual cells. F) Day 9 pregnant rat treated with preimmune serum. Bar = 25 ^m. L, Luminal epithelium; G, glandular epithelium.