148EPIDERMAL GROWTH FACTOR INDUCES BCL-XL GENE EXPRESSION AND REDUCES APOPTOSIS IN PORCINE PARTHENOTES DEVELOPING IN VITRO

Abstract

Epidermal growth factor (EGF) induces well-documented mitogenic and differentiating effects on murine and bovine preimplantation embryos. However, the effects of EGF on apoptosis and apoptosis-related gene expression in porcine embryos developing in vitro have not been evaluated. The objective of this study was to determine the effects of exogenous EGF in the presence and absence of BSA on the preimplantation development of porcine diploid parthenotes. In addition, we measured cell number, apoptosis, and expression of apoptotic-related genes of the blastocysts that developed in these culture conditions. In vitro-matured oocytes were parthenogenetically activated by square electrical direct current pulses. After 3h of culture in North Carolina State University (NCSU) 23 medium with 0.4% BSA containing 7.5mgmL−1 cytochalasin B, embryos were washed and cultured in NCSU 23 medium with 0.4% BSA for 48h. The general linear models (GLM) procedure in the Statistical Analysis System was used to analyze developmental rates. A paired Student’s t-test was used to compare relative gene expression. Presumptive diploid 4-cell parthenote embryos were randomly cultured in the same medium containing 0 or 10ngmL−1 EGF in the presence and absence of 0.4% BSA. More 4-cell embryos developed into blastocysts at Day 7 when BSA was present at 0.1% (54.8%±4.9) and 0.4% (55.1%±3.1) than when BSA was absent (43.0%±3.5, P<0.05). The addition of 10ngmL−1 EGF into the medium did not significantly increase the developmental rate (40.2±2.2 v. 35.1±2.1), but EGF in the presence of 0.1 and 0.4% BSA significantly increased the cell numbers per blastocyst (51.3±2.8 v. 37.8±2.5 in 0.1% BSA; 51.8±2.0 v. 42.7±2.3 in 0.4% BSA, P<0.01 for both comparisons). Furthermore, EGF treatment in the absence of BSA did not inhibit apoptosis in the blastocysts (6.8%±0.9 v. 8.8%±1.0), while addition of EGF in the presence of 0.4% BSA significantly reduced the degree of apoptosis in the blastocysts (4.0%±0.9 v. 9.3%±1.0, P<0.01). To investigate whether EGF modulates mRNA expression of apoptosis-related genes, mRNA was prepared from single blastocysts and each preparation was subjected to RT-PCR for Bcl-xL and Bak transcripts. EGF enhanced the relative abundance of Bcl-xL expression in the presence of BSA (P<0.01). The relative abundance of Bak expression was not altered by EGF treatment in either the presence or the absence of BSA. These results suggest that EGF and BSA synergistically enhance Bcl-xL gene expression, which may result in a net increase in cell number in porcine presumptive diploid parthenotes developing in vitro.