75 DEVELOPMENT OF BOVINE SCNT EMBRYOS RECONSTRUCTED USING RECIPIENT OOCYTES AND EGFP-TRANSFECTED CELLS THAT WERE CULTURED WITH VITAMIN C OR E

Abstract

The objective of this study was to investigate the development of bovine embryos produced by somatic cell nuclear transfer (SCNT) after treatment of recipient oocytes and/or gene-transfected donor cells with vitamin E and/or vitamin C. In the first experiment, oocytes derived from a local slaughterhouse were cultured in vitro for 22 h in TCM-199 medium supplemented with 100 µg mL⁻¹ vitamin E, 100 µg mL⁻¹ vitamin C, a combination of vitamins E (100 µg mL⁻¹) and C (100 µg mL⁻¹), or no addition. After culture, a total of 1139 oocytes were randomly examined for their meiotic stage and glutathione (GSH) concentration, and 1171 oocytes were enucleated and reconstructed with fetal lung cells that had been transfected with a plasmid containing the enhanced green fluorescence protein (EGFP) and neomycin-resistant genes. As a control, the same cell line without gene transfection and recipient oocytes matured without vitamin treatment were used for SCNT. The SCNT was performed according to the established procedure in our laboratory (Murakami et al. 2005 Cloning and Stem Cells 7, 77–81). The total cells and DNA fragmented cells of SCNT blastocysts were examined on Day 7 after SCNT (Day 0). Data were subjected to arcsin transformation before analysis, and tested by a post-hoc, Fisher's protected least significant difference PLSD test using the Statview programme (SAS Institute, Inc., Cary, SC, USA). Differences at a probability P < 0.05 were considered significant. The levels of GSH synthesis in the matured ocytes were higher (P < 0.05) in vitamin supplementation groups (6.8–9.0 pmol/oocyte) than in the non-supplementation group (6.7 pmol/oocyte). As shown in Table 1, the vitamin E supplementation improved the blastocyst formation, total cell number, and DNA fragmentation of EGFP-embryos compared with those parameters for EGFP-embryos obtained from recipient oocytes without vitamin treatment. However, the numbers of total cells and DNA-fragmented cells of EGFP-embryos were significantly lower (P < 0.05) than those of control embryos obtained by the SCNT using somatic cells without transfection, irrespective of vitamin treatment. In the second experiment, SCNT embryos were produced using 312 recipient oocytes treated with 100 µg mL⁻¹ vitamin E and EGFP-transfected donor cells that had been cultured in medium supplemented with various concentrations (0, 50, or 100 µg mL⁻¹) of vitamin E. Although the supplementation of 100 µg mL⁻¹ vitamin E decreased the proportion (30.8%) of blastocyst formation as compared with non-vitamin (42.8%) and 50 µg mL⁻¹ vitamin E (46.0%), the proportion (2.0%) of DNA fragmented cells in the SCNT blastocysts was significantly lower (P < 0.05) than that of other groups (3.2–3.6%). In conclusion, supplementation of vitamin E during oocyte maturation and donor cell culture improved the development and quality of gene-transfected SCNT bovine embryos.

Table 1. Development of SCNT embryos using recipient oocytes treated with vitamin C and E