Establishment and characterization of immortalized bovine endometrial epithelial cells.

Bovine primary uterine endometrial epithelial cells (EECs) are not ideal for long-term studies, because primary EECs lose hormone responsiveness quickly, and/or they tend to have a short life span. The aims of this study were to establish immortalized bovine EECs and to characterize these cells following long-term cultures. Immortalized bovine EECs were established by transfecting retroviral vectors encoding human papillomavirus (HPV) E6 and E7, and human telomerase reverse transcriptase (hTERT) genes. Established bovine immortalized EECs (imEECs) showed the same morphology as primary EECs, and could be grown without any apparent changes for over 60 passages. In addition, imEECs have maintained the features as EECs, exhibiting oxytocin (OT) and interferon tau (IFNT) responsiveness. Therefore, these imEECs, even after numbers of passages, could be used as an in vitro model to investigate cellular and molecular mechanisms, by which the uterine epithelium responds to IFNT stimulation, the event required for the maternal recognition of pregnancy in the bovine species.

The accumulation of macrophages expressing myeloid-related protein 8 (MRP8) and MRP14 in the spleen of BALB/cA mice during infection with Plasmodium berghei.

Splenomegaly is one of the typical symptoms of malaria. However, the pathogenesis of splenic enlargement still remains unclear. Spleen is a major organ for clearance of malaria parasites, but excessive response to the parasites can lead to splenomegaly. Myeloid-related protein (MRP) 8 and MRP14 are expressed by myeloid cells and are regarded as marker proteins of an immature and inflammatory subtype of macrophage. Previous studies have demonstrated that accumulation of MRP8(+) and MRP14(+) macrophages is associated with the pathological changes associated with various inflammatory diseases. In order to elucidate whether MRP8(+) and MRP14(+) cells are also involved in splenomegaly during malaria, we investigated expression of MRP8 and MRP14 in the spleens of mice infected with Plasmodium berghei. The MRP8 and MRP14 levels in the serum were analyzed by western blot, which confirmed that these proteins were elevated during infection compared with uninfected controls. Enlargement of the spleen was prominent at 7days of infection, and histological analysis of the spleens demonstrated deposition of malaria pigments and accumulation of mononuclear cells. Immunohistochemical staining of the tissue revealed the accumulation of cells expressing MRP8 and MRP14. In addition, the locations of those cells overlapped with CD11b(+) cells in the red pulp. These results suggest that splenomegaly in malaria is partly due to the accumulation of MRP8(+) and MRP14(+) macrophages.

Expression of mesenchymal-related genes by the bovine trophectoderm following conceptus attachment to the endometrial epithelium.

In the course of experiments to identify and characterize the factors that function in bovine conceptuses during peri-attachment periods, various transcripts related to the epithelial-mesenchymal transition (EMT) were found. In this study, RNA was extracted from different sets of days 17, 20, and 22 (day 0=day of estrous) bovine conceptuses and subjected to real-time PCR analysis as well as Western blotting, from which abundances of N-cadherin (CDH2), vimentin, matrix metalloproteinase 2 (gelatinase A, 72 kDa gelatinase, 72 kDa type IV collagenase) (MMP2), and matrix metallopeptidase 9 (gelatinase B, 92 kDa gelatinase, 92 kDa type IV collagenase) (MMP9) mRNAs were determined on day 22, concurrent with (CDH1) mRNA and protein downregulation. Transcription factors in EMT processes were then analyzed and changes in snail homolog 2 (Drosophila) (SNAI), zinc finger E-box binding homeobox 1 (ZEB1), zinc finger E-box binding homeobox 2 (ZEB2), twist homolog 1 (Drosophila) (TWIST1), twist homolog 2 (Drosophila) (TWIST2), and Kruppel-like factor 8 (KLF8) transcripts were found in day 22 conceptuses, while confirming SNAI2 expression by Western blotting. Immunohistochemical analysis revealed that the day 22 trophectoderm expressed the mesenchymal markers N-cadherin and vimentin as well as the epithelial marker cytokeratin. In attempts to identify the molecular mechanisms by which the trophectoderm expressed EMT-related genes, growth factor receptors associated with EMT were analyzed. Upregulation of the growth factor receptor transcripts, fibroblast growth factor receptor 1 (FGFR1), platelet-derived growth factor receptor, alpha polypeptide (PDGFRA), platelet-derived growth factor receptor, beta polypeptide (PDGFRB), and transforming growth factor, beta receptor II (70/80 kDa) (TGFBR2) mRNAs, was found on day 22. The analysis was extended to determine the integrin (ITG) transcripts and found high levels of integrin, alpha 4 (antigen CD49D, alpha 4 subunit of VLA-4 receptor) (ITGA4), integrin, alpha 8 (ITGA8), integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61) (ITGB3), and integrin, beta 5 (ITGB5) mRNAs on day 22. These observations indicate that after the conceptus-endometrium attachment, EMT-related transcripts as well as the epithelial marker cytokeratin were present in the bovine trophectoderm and suggest that the implantation process for noninvasive trophoblasts requires not only extracellular matrix expression but also partial EMT.