335 DEVELOPMENTAL ABILITY OF PIG OOCYTES TREATED WITH EGF DURING IN VITRO MATURATION

Abstract

Epidermal growth factor (EGF) is a major follicular factor affecting maturation of oocyte in many species. The insulin-like growth factor II (IGF2) gene is an imprinted gene in embryonic development that functions primarily as a regulator of cell growth and differentiation. Thus, this study examined the maturation and developmental ability of in vitro-fertilized (IVF) pig immature oocytes cultured in maturation medium supplemented with EGF. The blastocysts derived from these oocytes were further examined for the expression level of IGF2 as a cell survival activity. Pig immature oocytes were cultured in TCM-199 medium (with no supplement) with or without 10 ng/mL EGF for 42–14 h, and then matured oocytes were co-incubated with 5 × 105 sperm/mL in modified Tris-buffered medium containing 1 mM caffeine sodium benzonate and 0.1% bovine serum albumin (BSA) for 6 h for IVF. Subsequently, embryos were cultured in 50 μL of NCSU-13 containing 0.4% BSA for 7 days at 39°C in a humidified atmosphere of 5% CO2 in air. Total cell numbers in blastocysts were examined by fluorescence staining with Hoescht 33342, and the expression level of IGF2 was analyzed with a fluorescence-monitored quantitative real-time reverse transcriptase-polymerase chain reaction method. We found that pig oocytes matured with EGF showed significant improvement of their development ability (Table 1). Presence of EGF in TCM-199 medium significantly increased (P < 0.05) the rates of maturation, sperm penetration, male pronucleus (MPN) formation, cleavage, and blastocyst formation. Furthermore, blastocysts derived from oocytes cultured with EGF were 24-fold higher in the relative expression level of IGF2 than those without EGF. Therefore, these data suggest that pig oocytes matured in medium supplemented with EGF increases the developmental ability and cell viability during cell divisions following IVF. In conclusion, EGF may increase cytoplasmic as well as nuclear maturation of pig immature oocytes. Table 1. Improvement of developmental ability of pig oocytes matured with EGF This work was supported by the Research Project on the Production of Bio-organs, Ministry of Agriculture and Forestry, Republic of Korea.