154 THE EFFECT OF FIBROBLAST GROWTH FACTOR-4 ON THE DEVELOPMENT OF NUCLEAR TRANSFER BOVINE EMBRYOS

Abstract

Fibroblast growth factor-4 (FGF-4) has been shown to be preferentially produced in the inner cell mass (ICM) of mouse blastocysts, to stimulate the proliferation of mouse trophectoderm (TE) cells and to repress their transformation and differentiation into giant trophoblasts. Recent studies have shown that nuclear transfer (NT) bovine embryos have aberrant allocations of ICM and TE cells (Koo et al., 2002 Biol. Report. 67, 487–492) and aberrant expression of FGF-4 gene (Daniels et al., 2000 Biol. Reprod.63, 1034–1040). In this study, we examined whether recombinant human FGF-4 (rhFGF-4) stimulates development of nuclear transfer bovine embryos. As donor cells for NT, bovine ear skin fibroblast cells of passage 5 to 8 were used. Oocytes were enucleated after in vitro maturation in TCM 199 supplemented with 10% FBS, 1 μg mL⁻¹ FSH and 1 μg mL⁻¹ estradiol-17β for 20 h. Enucleated oocytes were fused with donor cells by a DC pulse of 25 V/150 μm for 10 μs in Zimmerman cell fusion medium. For activation, fused oocytes were exposed to 10 μM Ca-Ionophore for 5 min, followed by 2 mM 6-dimethylaminopurine for 3 h. NT embryos were subsequently cultured in CR2 medium (containing 0.5% BSA) without or with rhFGF-4 (1, 10 and 100 ng mL⁻¹) at 39.0°C in 5% O₂, 5% CO₂ and 90% N₂. After 7 days of culture, blastocyst formation was observed. Apoptotic cells in blastocysts were detected by a terminal deoxynucleotidyl transferase-mediated d-UTP nick end-labeling (TUNEL) assay and total cell number was examined by propidium iodide (PI) counterstaining. Data were analyzed by chi-square test and Student’s t-test. Supplementation of serum-free medium with rhFGF-4 increased the proportion of embryos developing to the blastocyst stage (18.4, 29.4 and 23.5% for 0, 1 and 10 ng mL⁻¹ FGF-4, respectively) and total cell number of blastocysts (66.3 ± 11.4, 75.9 ± 25.5 and 74.4 ± 22.4 for 0, 1 and 10 ng mL⁻¹ FGF-4). Particularly, 100 ng mL⁻¹ FGF-4 significantly (P < 0.05) increased the proportion of blastocysts (40.4%) and total cell number of blastocysts (86.7 ± 26.5) when compared with TCM 199 medium alone. FGF-4 also decreased the mean proportion of apoptotic cells in blastocysts (10.6 ± 7.8, 7.4 ± 5.3, 8.6 ± 5.3 and 7.5 ± 4.1% for 0, 1, 10 and 100 ng mL⁻¹ FGF-4). Our results suggest that FGF-4 may play a role in the early development of NT bovine embryos and might be a useful molecule for increasing development of NT bovine embryos in serum-free culture systems.