Following hatching, bovine and ovine blastocysts elongate into tubular and then filamentous conceptuses that remain free-floating for several days before attaching to the uterine lining. Elongation is marked by trophectoderm proliferation and changes in trophectoderm shape. The ultimate goal of this work is to identify uterine- and conceptus-derived factors that control peri-attachment conceptus development in cattle. Fibroblast growth factors (FGF) encompass a large family of mitogens, morphogens, and angiogenic factors produced by various tissues, including the bovine/ovine endometrium and conceptus. FGF2 and FGF10 are of particular interest because uterine production of FGF2 and conceptus production of FGF10 intensify as elongation takes place in cattle and sheep. The objective of this work was to determine if FGF2 and FGF10 stimulate bovine trophectoderm migration during culture. Migration assays were conducted with CT1 cells, a trophectoderm line established from a bovine in vitro-produced blastocyst outgrowth. Cells were seeded on 8-μm pore Transwell inserts (Corning Inc., Corning, NY, USA; 50,000 cells/insert) and submerged in serum-free DMEM containing treatments (0, 0.5, 5, 50, and 500 ng mL-1 of recombinant bovine FGF2 or human FGF10). After 12 h, cells that migrated onto the lower surface were fixed, stained, and processed for counting using epifluorescence microscopy. Migrated cells were counted in 5 non-overlapping locations on each of 4 replicate Transwell inserts for each treatment. Experiments were repeated on at least 3 different occasions. Analysis of variance was completed. Differences in individual means were partitioned further by completing pair-wise comparisons. Supplementation with 5 or 50 ng mL-1 of FGF2 increased (P = 0.06 and P = 0.002, respectively) migration of CT1 cells when compared with controls (327 ± 17 or 485 ± 40 cells, respectively, v. 162 ± 16 cells). Supplementation with 500 ng mL-1 of FGF2 further increased (P < 0.02) migration when compared with controls and cells exposed to lower levels of FGF2 (548 ± 116 cells). FGF10 also stimulated CT1 migration. Supplementation with 0.5 ng mL-1 of FGF10 increased (P = 0.06) cell migration v. controls (254 ± 48 v. 184 ± 24 cells). Supplementation with 5 ng mL-1 further increased (P < 0.007) cell migration (373 ± 29 cells). Exposure to greater FGF10 concentrations did not further enhance cell migration. To summarize, both FGF2 and FGF10 promoted CT1 migration, suggestive of a potential function in regulating trophectoderm development, differentiation, and/or morphogenesis during peri-attachment conceptus development. FGF10 appeared to be more potent than FGF2 at mediating CT1 migration. The reason for this disparity has not been resolved but likely involves differences in ligand affinities to certain receptor subtypes.
This project was supported by NRI Competitive Grant No. 2008-35203-19106 from the USDA-CSREES.
Roles of intracellular fibroblast growth factors in neural development and functions
