Following hatching, bovine and ovine conceptuses undergo a phase of massive development and remodelling that causes elongation and filamentation. Proper trophoblast cell development and interaction with the uterus is critical for the establishment and maintenance of pregnancy. Various growth factors, including several fibroblast growth factors (FGFs), are produced by the uterus, and at least two of these, FGF2 and FGF10, are released into uterus lumen during early pregnancy. Microarray analysis found that gene products associated with migration and invasion were altered in bovine blastocysts exposed to FGF2 or 10. The objective of this work was to determine if FGF2 and FGF10 impact bovine and ovine trophoblast cell migration. The ability of FGF2 and FGF10 to influence migratory ability of trophoblast cells was examined by using an in vitro transwell migration assay. The bovine trophoblast line, CT1, was used in the first study. After serum starvation, CT1 cells were seeded on the top of each transwell membrane (50 000/transwell) in the presence of vehicle, 0.5, 5, or 50 ng mL–1 bovine recombinant FGF2 or human recombinant FGF10. After 12 h, the transwell was fixed and stained with Hoechst 33342 (0.5 μg mL–1). Migrated cells were counted on five non-overlapping areas of each filter using epifluorescence microscopy. Supplementation with 0.5 ng mL–1 FGF2 increased the number of migrated CT1 cells when compared with controls (268.3 ± 58.3 v. 167.3 ± 47.7; P < 0.01). Supplementation with 5 or 50 ng mL–1 FGF2 further increased the number of migrated CT1 cells (297.0 ± 51.4 and 429.4 ± 98.3, respectively; P < 0.001). Adding 0.5 ng mL–1 FGF10 did not affect CT1 migration but providing 5 or 50 ng mL–1 FGF10 increased CT1 migration (399.8 ± 29.7 and 392.7 ± 58.6 v. 194.2 ± 40.3 for controls; P < 0.005). A subsequent study utilised the ovine trophoblast line, oTR1 in the migration assay (30 000 cells/transwell; 8 h migration assay). Adding 0.5 ng mL–1 FGF2 or FGF10 did not affect oTR1 migration number but exposure to holdout 5 or 50 ng mL–1 FGF2 or FGF10 increased oTR1 migrated cell numbers v. controls (P < 0.05). In a subsequent study, p38 mitogen-activated protein kinase (MAPK), ERK1/2 and JNK signalling cascades utilised by FGF2 and FGF10 in oTR1 cells were investigated. Western blot analysis indicated that both FGF2 and FGF10 induced ERK1/2 and p38 MAPK phosphorylation status. Interestingly FGF10 activated JNK but not p38 MAPK. Taken together, FGF2 and FGF10 stimulate trophoblast cell migration. This response could be mediated by an ERK1/2- or p38 MAPK-dependent system.
This project was supported by NRICGP number 2008-35203-19106 from the USDA-NIFA.
APC binds intermediate filaments and is required for their reorganization during cell migration