225 ANTI-APOPTOTIC EFFECT OF AGGREGATION ON PREIMPLANTATION DEVELOPMENT OF BOVINE IN VITRO-FERTILIZED EMBRYOS

Abstract

Apoptosis occurs during embryonic development, and is related to early embryonic loss. It is important to produce high-quality blastocysts in vitro for research on the establishment of embryonic stem (ES) cells and transgenic animal production. Therefore, our objectives were to compare the anti-apoptotic effect of bovine aggregate v. nonaggregate IVF embryos and to determine whether aggregation could improve the quality of bovine embryos. The cumulus–oocyte complexes were matured for 20–22 h, and the oocytes were fertilized with cryo-preserved bovine sperm using the swim-up method. After removal of the zona pellucida (ZP), three 4-cell-stage embryos (3X) were aggregated by co-culture in an aggregation hole that was made by an aggregation needle on the culture dish. Embryos were cultured either singularly (1X, ZP removed) or in aggregates of three (3X), and IVF intact embryos served as a control. Five days after aggregation, the developmental rate was observed. The numbers of total cells and apoptotic cells were determined by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) assay using blastocyst-stage embryos. Moreover, the mRNA expression pattern related to apoptosis and embryo quality was verified by real-time PCR of the aggregated (3X) and nonaggregated (1X) embryos (at least 3 embryos). The percentage of blastocysts was higher in the 3X aggregated embryos (41.3%) compared with that of the 1X ZP-free embryos (24.3%), whereas there was no significant difference in the 1X embryos and the intact controls (24.3 and 25.8%, respectively; P < 0.05). The total cell number of blastocysts also increased approximately threefold (P < 0.05) in 3X aggregated embryos compared with that of 1X controls. In contrast, the percentage of TUNEL-positive cells, an indication of apoptotic cells, was decreased by approximately threefold in 3X aggregated embryos when compared with that of 1X embryos (7.7 and 2.6%, respectively). The mRNA levels for the Oct-4, NANOG, and bcl-2 genes were higher (P < 0.05) and for the Bax gene were lower in the 3X aggregated embryos than for those of the 1X controls. Therefore, our results indicated that aggregation of bovine IVF embryos at a 4-cell stage could promote the quality and suppress the apoptosis of bovine pre-implantation-stage embryos produced in vitro. Further studies are required to investigate the quality of the aggregated embryos in terms of increasing the establishment rate of ES cell lines by seeding on the feeder layer and raising the efficiency of embryo transfer. This work was supported by the BioGreen 21 Program (#20070401034031, #20080401034031), Rural Development Administration, Republic of Korea (HK).