Effects of rhChM-I on the IGF-I-induced migration of Rcho-1 trophoblast cells. (A) Expression of Prl3d1, MMP-9, and ChM-I in Rcho-1 trophoblast cells. Rcho-1 trophoblast cells were seeded at 2 × 105 cells/well in 6-multiwell plates and cultured in growth medium (NCTC-135 medium containing 20% FBS) until reaching confluence (day 0; undifferentiated). The culture medium was then replaced with differentiation medium (NCTC-135 medium containing 10% horse serum), and the cells were cultured for seven days (day 7, differentiated) to promote their differentiation to trophoblast giant cells. EPCs were isolated at 7.5 days p.c. and cultured for two days in DMEM containing 10% FBS and 15 mM HEPES. Total RNA was then isolated and reverse-transcribed, and the expression of Prl3d1 (28 cycles), MMP-9 (28 cycles), and ChM-I (35 cycles) was analyzed by RT-PCR. GAPDH (28 cycles) was used as an internal control. The gel images are representative of three independent experiments. (B) Boyden chamber migration assay of Rcho-1 trophoblast cells. Serum-starved Rcho-1 trophoblast cells (1 × 105 cells/200 μl) were preincubated with or without rhChM-I for 20 min, and then seeded onto fibronectin-coated cell culture inserts in NCTC-135 medium containing 0.5% FBS. Chemotactic migration of Rcho-1 cells was induced by the addition of IGF-I (100 ng/ml) to the lower chamber. Cells were then allowed to migrate for 6 h in the presence of various concentrations of rhChM-I. Control cells were treated with 0.1% BSA/PBS and allowed to migrate in the absence of IGF-I. The number of cells that had invaded the undersurface of the insert was counted in five representative high power fields (under × 200 magnification) per insert. The values shown are percentages of the number of migrated cells compared with the control cells (22 ± 2.3 cells/field) and are the means ± SD of a triplicate assay. The data are representative of three independent experiments. The statistical significances were determined by the Student's t-tests as compared to IGF-I alone (*P < 0.05, one-tailed).