Effect of epidermal growth factor on preimplantation development and its receptor expression in porcine embryos

The present study aimed to determine the influence of exogenous epidermal growth factor (EGF) on in vitro preimplantation porcine embryo development and its mRNA expression for EGF receptor (EGFR). Oocytes were aspirated from abattoir ovaries, selected and cultured in defined, protein-free media for 44 hr before in vitro fertilization (IVF). Thirty-six hours after IVF, two-cell stage embryos were selected and treated or cultured until embryo treatment. In experiment 1, compact morulae were selected on day 4 after IVF and randomly allocated into 5 groups: NCSU 23 with PVA as group 1; NCSU 23 with PVA and 0.1 ng/ml, 1.0 ng/ml, 10.0 ng/ml EGF as group 2, 3, 4, respectively; NSCU 23 with 0.4% BSA as group 5. In experiment 2, treatment groups were the same as in experiment 1 except that 0.1% crystallized BSA was added to both washing media and all treatment groups instead of PVA. In experiments 3 and 4, two-cell stage embryos were treated and cultured in the same experimental design as experiments 1 and 2, respectively. RT-PCR was used to detect the mRNA expression of EGF receptor in compact morulae and blastocysts. The PCR products were subjected to direct DNA sequencing. There was no significant improvement in the development rate of embryos from compact morulae to blastocysts in the presence of various EGF concentrations (0.1, 1.0, 10.0 ng/ml) versus without EGF addition. They were all significantly lower than those embryos cultured in the continuous presence of 0.4% BSA. However, when a reduced concentration (0.1%) of crystallized BSA was added to all the treatment groups, a significantly lower rate of embryo development was observed in control media (NCSU23 with 0.1% crystallized BSA) compared with those developed in culture media with 0.4% BSA. With the addition of EGF at 10 ng/ml (with 0.1% BSA), embryo development rates were significantly improved over the control group (P < 0.05) and were as good as those rates in 0.4% BSA culture group. When embryos were selected and treated from the 2-cell stage, they did not develop to blastocyst stages after five more days' culture without any protein (BSA) or growth factor addition. When 0.1% BSA was included in the media, blastocyst formation rates were significantly improved by EGF addition at the concentration of both 1.0 or 10 ng/ml (P < 0.05) as compared to 0.0 or 0.1 ng/ml. EGFR mRNA was detected in both compact morulae and blastocyst stages of porcine embryos and confirmed by direct DNA sequencing. Our results indicate that IVM-IVF porcine embryo developmental rates could be improved by the addition of EGF in the culture media with the presence of a reduced amount of defined BSA (>97% albumin). However, EGF alone was not able to elicit any stimulatory effects on embryo development in the absence of protein supplementation. Further studies are needed to investigate the potential synergistic factors in embryo culture media to eventually define the porcine embryo culture media.