341 APOPTOSIS IN SOMATIC CELL CLONED AND EGFP TRANSGENIC BOVINE EMBRYOS

Abstract

The overall success rate achieved by cloning techniques to date is low, mainly due to deficiencies in nuclear reprogramming, gene expression and DNA fragmentation, which result in early and late embryonic losses. This study was carried out to compare the incidences of DNA fragmentation during development of IVF, parthenote (PT), nuclear transfer (NT) and transgenic-cloned (TG) embryos. Terminal deoxynucleotidyl transferase (TdT) nick-end labelling (TUNEL) with propidium iodide counterstaining was used for determination of DNA fragmentation and total cell number. Donor cells were fetal fibroblasts with or without transfection with EGFP, and cultured in DMEM + 15% FCS until confluent, for up to 5 days. At 19 h post maturation (hpm), enucleated oocytes were reconstructed with donor cells and activated at 24 hpm with the combination of ionomycin (5 μM, 5 min) and cycloheximide (10 μg/mL, 5 h) after electric fusion by a single DC pulse (1.6 KV/cm, 60 μs) delivered by a BTX 200. Parthenotes were produced by the same activation protocol at 24 hpm. The eggs and control IVF embryos were cultured in CR1aa at 39°C in a humidified atmosphere of 5% CO₂ in air. Differences among groups were analyzed using one-way ANOVA after arc-sine transformation of proportional data. Embryos at the 8-cell stage in all treatments, IVF, PT, NT and TG, showed DNA fragmentation. The apoptotic cell index (total number of apoptotic nuclei/total number of nuclei) of Day 7 blastocysts was significantly (P < 0.05) higher in TG and NT embryos (17/91, 18.6 ± 4.0% and 13/94, 13.8 ± 4.7%, respectively) compared to IVF and PT embryos (9/122, 7.4 ± 3.4% and 8/93, 8.6 ± 2.9%, respectively). TUNEL positive cells were detected in almost all blastocysts at Day 7 and were mainly observed in the ICM. The DNA fragmentation ratio of the ICM in the blastosysts at Day 7 (number of apoptotic nuclei in the ICM/total number of apoptotic nuclei in the blastocyst) was significantly (P < 0.05) higher in TG embryos (64.7 ± 21.4%) than in IVF (44.4 ± 28.0%), PT (50.0 ± 18.6%) or NT (53.0 ± 32.5%) embryos. These results indicate a higher occurrence of DNA fragmentation observed in NT and TG embryos when compared to IVF and PT embryos. In addition, ICM of TG blastocysts revealed a high DNA fragmentation ratio, which may be related to early embryonic loss after transfer of the resulting embryos. [Supported by High Technology Development Project for Agriculture and Forestry Korea, MAF-SGRP, 300012-05-3-SB010 and Cho-A Pharm. Co. LTD.]