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Biology of Reproduction 61, 1324-1330 (1999)
© 1999 Society for the Study of Reproduction, Inc.

Articles

Development and Characterization of Immortalized Ovine Endometrial Cell Lines1

Greg A. Johnsona, Robert C. Burghardta,b, Gary R. Newtonc, Fuller W. Bazera,b, and Thomas E. Spencer2,a

a Center for Animal Biotechnology and Genomics, Albert B. Alkek Institute of Biosciences and Technology, Texas A&M University System Health Science Center, and Department of Animal Science, Texas A&M University, College Station, Texas 77843 b Department of Veterinary Anatomy and Public Health, College of Veterinary Medicine, Texas A&M University, College Station, Texas 77843 c Cooperative Agricultural Research Center, Prairie View A&M University, Prairie View, Texas 77446

The objective of this study was to generate immortalized endometrial epithelial and stromal cell lines from the ovine uterus. Luminal (LE) and glandular epithelial (GE) cells and stromal (ST) cells were enzymatically isolated from the uterus of a Day 5 cyclic ewe (estrus on Day 0), and primary cultures were immortalized by transduction with a retroviral vector (LXSN-16E6E7) packaged by the amphotropic fibroblast line PA-317. Cells having integrated the vector were selected by resistance to the neomycin analogue G418 (0.6–0.8 mg/ml). Surviving cells were maintained in complete culture medium containing G418 (0.1 mg/ml) and subcultured for more than 40 passages. Phase-contrast microscopy revealed that LE and GE cells exhibited a cobblestone morphology whereas immortalized ST cells were spindle shaped. The epithelial origin of LE and GE was confirmed by positive cytokeratin immunostaining, and ST cells were vimentin positive. All cell lines were negative for smooth muscle {alpha}-actin staining. Western blot analyses of cell extracts revealed the presence of signal transducers and activators of transcription (STAT) proteins 1, 2, and 3. In the LE cells, interferon tau (IFN{tau}) induced nuclear translocation of STAT proteins 1 and 2 and up-regulated several IFN-inducible genes, including STATs 1, 2, and 3 and ubiquitin cross-reactive protein (UCRP/ISG17). In the LE cell line, IFN regulatory factor one was transiently up-regulated and then down-regulated by IFN{tau}. Immunostaining revealed the presence of nuclear estrogen receptor and progesterone receptor in all cell lines. These ovine endometrial cell lines provide useful in vitro model systems for the study of hormone and cytokine action, signal transduction pathways, cell-cell interactions, and gene expression in specific cell types of the ovine endometrium.

1 This work was supported in part by NIH Grant HD32534 to F.W.B.

2 Correspondence: Thomas E. Spencer, Center for Animal Biotechnology and Genomics, 444 Kleberg Center, Texas A&M University, College Station, TX 77843–2471. FAX: 409 862 2662; tspencer{at}ansc.tamu.edu




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