| Popis: |
In cattle, mechanistic studies of endometrial function rely on cell lines or primary culture of cells harvested postmortem. Understanding the endometrial physiology in dairy cows is essential, because approximately 50% of pregnancies are lost in the first 3 wk of gestation for unknown reasons. The objective was to validate an in vivo, minimally invasive, and estrous cycle stage–specific method to obtain endometrial luminal epithelial cells for culture. The uterine body of 26 cows was sampled using a cytology brush (cytobrush) 4 d after estrus. The viability of cells was measured by flow cytometry (80% live cells) and epithelial identity was determined by anti-vimentin and anti-cytokeratin immunofluorescence and quantitative PCR for KRT18 and VIM. A pool of cells from 15 animals was passaged 4 times in culture until confluent and then treated with 0, 0.1, 1, or 10 ng/mL of recombinant bovine interferon-tau (rbIFN-τ). The relative expression of transcripts related to IFN-τ signaling (IFNAR1), early (IRF2) and late (ISG15, OAS1) response to IFN-τ stimulus, and other IFN-τ–stimulated genes (CCL8, CXCL10, and FABP3) was measured by quantitative PCR. The relative expression of KRT18 transcripts was similar across passages; the relative expression of VIM increased at passage 2, and IFNAR1 transcripts decreased in cultured compared with that in fresh cells. The relative expression of ISG15, OAS1, CCL8, and FABP3 increased in response to rbIFN-τ. In conclusion, culture of endometrial luminal cells collected by cytobrush was feasible, generating a monolayer enriched in epithelial cells, and therefore constitutes a novel model by which to study endometrial luminal epithelial cell function, including responses to IFN-τ. |
