232 SEARCH FOR THE BOVINE HOMOLOG OF THE MURINE PED GENE

Abstract

The aim of the current study was to identify the bovine homolog of the murine Ped (preimplantation embryo development) gene, which regulates mouse preimplantation embryonic growth, including cleavage rate and embryo survivability, and to characterize the expression pattern of this gene during bovine preimplantation embryo development. Experiment (I): The National Center for Biotechnology Information (NCBI) GenBank/EBI EST databases were searched for bovine-expressed sequence tags (EST) that were homologous with the murine Ped gene (Accession number: NM_010394). The resulting ESTs were aligned and assembled in to one complete sequence (841 bp), which was shown to be homologous with the Murine Ped gene and the Bovine Major Histocompatibility Complex class I 4221.1 gene (Accession No.: AJ010865, length 1090 bp). The expression of the protein product of the Ped gene by bovine tissue was confirmed using Western Blot analysis. Experiment (II): The expression pattern of the Ped gene homolog during in vivo and in vitro bovine preimplantation embryo development was characterized using real time PCR. Embryos at the same stage for age were compared (Day 1: 2-cell; Day 2: 4-cell; Day 3: 8-cell; Day 4: 16-cell; Day 5: early morula;; Day 6, compact/late morula;; Day 7: blastocyst). The relative transcript abundance was consistently lower in the in vitro-cultured embryos at all stages of preimplantation development the differences were significant on Days 2, 4, 5, 6, and 7. The relative transcript abundance was significantly lower on Days 1, 2, and 3 of in vivo culture than on Days 4, 5, 6, and 7 and was significantly higher in Day 7 blastocysts than in Day 5 early morula. In in vitro-cultured embryos the relative transcript abundance was significantly higher in Day 7 blastocysts compared to all other stages of the preimplantation period. Experiment (III): A quantitative analysis of the Ped gene homolog was carried out on replicates of pools of ten 2-cell embryos collected at 25, 28, 32, and >36 hpi from three different fertilizations. Transcript relative abundance was highest in those embryos that had cleaved by 25 hpi. By 28 hpi abundance had decreased slightly;; as time to cleavage increased further to 32 and >36 hpi, the relative abundance decreased significantly. In conclusion, we have successfully identified a potential bovine homolog of the murine Ped gene. Furthermore, we have characterized the expression pattern of this gene during preimplantation embryo development in cattle and we have shown that a greater relative abundance of the gene transcript is associated with embryos of higher quality and greater developmental potential.