Date of Award
Doctor of Philosophy
James D. Godkin
Bert Erickson, Henry Kattesh, Jeff MacCabe, John Wilkinson
Bovine endometrial glandular epithelial and stromal cells were isolated by enzymatic digestion from uteri of cows harvested on days 15 - 17 of the estrous cycle. Cell types were separated by slow speed centrifugation and grown a on collagen matrix. In a separate study, isolated gland cells were grown on millicell inserts coated with Matrigel and cultured in the presence of stromal cells. Response of isolated cells and co-cultured cells to bovine serum albumin (BSA), recombinant bovine trophoblast protein-1 (rbTP-1), recombinant bovine interferon (brIFN) and oxytocin were analyzed by measuring prostaglandin (FSα) (PGF) and E2 (PGE) and protein secretion. Regulation of gene expression of prostaglandin synthase (PGS) in each cell type was also evaluated.
Gland cells treated with rbTP-1 and brIFN produced less (p < 0.01) PGF (ng/μg DNA) than BSA treated cells. However, PGE was unaffected (p > 0.05) by rbTP-1 or brIFN. Oxytocin stimulated glandular cells to release significantly (p < 0.01) higher concentrations of prostaglandin F and E (p < 0.01). Recombinant bTP-1 and brIFN were ineffective in reducing (p > 0.05) oxytocin stimulated PGF secretion by gland cells. Isolated stromal cells released HGF secretion by gland cells. Isolated stromal cells released PGF in levels below the detection limits of the assay. Stromal cell secretion of PGE was unaffected by rbTP-1 and brIFN. Oxytocin reduced (P < 0.01) PGE released by stromal cells when compared to cells cultured in the absence of oxytocin. Proteins synthesized and released in the medium were analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and fluorography. Percent incorporation of 35S methionine into radiolabeled proteins by isolated stromal cells was significantly (p < 0.01) higher than gland cells regardless of treatment. Patterns of proteins produced by glandular cells were different when compared to stromal cells. Glandular cells produced twenty unique proteins which were absent on fluorograms obtained from stromal cells.
Gland cells co-cultured with stromal cells released significantly (p < 0.01) higher concentrations of PGF (ng/μg DNA) than gland cells cultured in the absence of stromal cells. A significant (p < 0.01) reduction in PGF release occurred when co-cultured cells were incubated in the presence of rbTP-1 and brIFN. Only brIFN reduced (p < 0.01) PGE release by co-cultured cells. Secretion of PGF and PGE by co-cultured cells was unaffected (p > 0.05) by oxytocin treatment. Percent incorporation of radiolabeled amino acids into proteins were not affected by any treatment. However, protein secretion was significantly reduced as a result of glandular-stromal interactions. Differences were present in the array of proteins produced by co-cultured cells and gland cells cultured alone. Cocultured cells produced an acidic protein (45 KDa, pI 5.5-6.8) which was absent on fluorograms from gland cells cultured alone.
Total RNA from glandular and stromal cells was isolated and subjected to Northern and slot blot analysis. Glandular cells contained a major PGS mRNA transcript of ≈ 2.8 Kb. Expression of PGS mRNA in stromal cells was evident but barely detectable. After correcting for total RNA loading by comparison with rat β-actin, quantitative slot blot analysis demonstrated no significant difference between treatments by either cell type.
These results indicate that the major effect of the trophoblast interferon is on PGF production by uterine glandular epithelial cells. Oxytocin stimulated prostaglandin production by gland cells and the IFN’s diminished this response in some not all cases. Patterns of proteins synthesized by isolated glandular epithelial and stromal cells were distinct. However, there was no effect of the IFN’s on protein synthesis by either cell type. These data do not support the concept that trophoblast interferon modulates PGF production through alteration of PGS expression.
Smith, Scott Edward, "Characterization of bovine uterine glandular epithelial and stromal cells. " PhD diss., University of Tennessee, 1993.