Effect of epidermal growth factor in preimplantation development of porcine cloned embryos

Abstract

In this study, we determined the expression of epidermal growth factor (EGF) and its receptor (EGFr) gene, and the effect of exogenous EGF supplementation on preimplantation development of porcine somatic cell nuclear transfer (SCNT) embryos. In vitro matured gilt oocytes were fertilized with frozen-thawed semen in vitro or reconstructed with fetal fibroblasts by SCNT. In Experiment 1, total RNA was isolated from oocytes, preimplantation SCNT, or in vitro fertilization (IVF) embryos. The expression of EGF and EGFr mRNA was determined using reverse transcription-polymerase chain reaction (RT-PCR). In SCNT and IVF embryos, the EGF mRNA was detected in oocytes, 2-cell, 4-cell, 8-cell, morulae, and blastocysts, while EGFr mRNA was detected in oocytes, 2-cell, morulae, and blastocysts. In Experiment 2, SCNT embryos at 1-cell stage were cultured in North Carolina State University (NCSU)-23 medium supplemented with different concentrations of EGF (0.1, 1, or 10 ng/ml). Supplementing with 10 ng/ml EGF improved cleavage rate (82.8% vs. 76.8%, P<0.05), but not the rate of blastocyst formation compared to the control. At all concentrations, EGF increased (P<0.05) the total cell number in blastocysts (range 50.5-53.7 vs. 43.9). In Experiment 3, EGF (10 ng/ml) was added to NCSU-23 medium at the morula stage. The EGF did not affect blastocyst formation, total cell number in blastocysts or the ratio of inner cell mass (ICM) to total cell number. In conclusion, we demonstrated that EGF and EGFr mRNA are expressed in porcine IVF and SCNT preimplantation embryos, and that EGF increased the quality of blastocysts by increasing total cell numbers in porcine SCNT embryos.