167 THE SUPPLEMENTATION OF GRANULOCYTE-MACROPHAGE COLONY STIMULATING FACTOR (GM-CSF) PROMOTES THE DEVELOPMENT OF NUCLEAR TRANSFERRED BOVINE EMBRYOS

Abstract

Granulocyte-macrophage colony stimulating factor (GM-CSF) is secreted by epithelial cells lining the female reproductive tract in mice and several other species. GM-CSF receptors are present in the fertilized oocyte and in all subsequent stages of development, and in blastocysts it is expressed in both inner cell mass and trophectoderm cells. Recent studies suggest that GM-CSF can act as a survival factor for the developing embryo. The purpose of this study was to examine the effect of GM-CSF, as a medium supplement, on the development of nuclear-transferred bovine embryos. Oocytes were enucleated after in vitro maturation in TCM-199 supplemented with 10% fetal bovine serum (FBS), 1 mg/mL FSH, and 1 mg/mL estradiol-17² for 20 h. Enucleated oocytes were fused with bovine ear skin fibroblast cells by a DC pulse of 25 V/150 mm for 20 ms in Zimmerman cell fusion medium. For activation, reconstructed embryos were exposed to 10 mM Ca-ionophore for 5 min, followed by 2 mM 6-dimethylaminopurine for 3 h. NT embryos were subsequently cultured in CR2 medium without or with 10 ng/mL recombinant porcine GM-CSF at 39.0°C in 5% O₂, 5% CO₂ and 90% N₂. After 7 days of culture, blastocyst formation was observed. The number of inner cell mass (ICM) and trophectoderm (TE) cells was examined by differential staining. Apoptotic cells in blastocysts were detected by a terminal deoxynucleotidyl transferase-mediated d-UTP nick-end labeling (TUNEL) assay. Data were analyzed by chi-square and Student's t-test. Addition of GM-CSF to the medium significantly (P < 0.05) increased the proportion of embryos developing to the blastocyst stage (37.6 ± 12.0 and 54.7 ± 13.9% for control and GM-CSF groups respectively). No differences in the total cell number and the ratio of ICM to total cells were detected between the control group (125.4 ± 35.7 and 38.5 ± 9.7%) and the GM-CSF group (123.8 ± 35.1 and 34.2 ± 13.1%). The mean proportion of apoptotic cells in blastocysts was not different between the control (5.4 ± 5.4%) and the GM-CSF (5.3 ± 3.9%) group. Our results showed the beneficial effect of GM-CSF on the development of NT bovine embryos. These results suggest that GM-CSF might be a useful molecule for increasing development of NT bovine embryos. Further studies are necessary to verify the mechanism of GM-CSF on the development of bovine NT embryos.