title: Preimplantational embryo development and incidence of blastomere apoptosis in bovine somatic cell nuclear transfer embryos reconstructed with long-term cultured donor cells.
abstract: This study was performed to investigate whether types and/or age of donor cells affect preimplantational embryo development and the incidence of apoptosis in bovine somatic cell nuclear transfer (SCNT) embryos. Bovine fetal or adult ear fibroblasts were isolated, cultured in vitro and categorized into fresh or long-term cultured cells in terms of population doublings (PD): in fetal fibroblasts, <16 being considered fresh and >50 being long-term cultured; in adult ear fibroblasts, <16 being considered fresh and >30 being long-term cultured. Bovine oocytes from slaughterhouse ovaries were matured in TCM-199, enucleated and reconstructed by SCNT. The reconstructed oocytes were fused, chemically activated, and cultured in modified synthetic oviduct fluid (mSOF) at 39 degrees C in a humidified atmosphere of 5% CO(2) air for 7 days. The early development of SCNT embryos was monitored under a microscope and the quality of blastocysts was assessed by differential counting of inner cell mass (ICM) and trophectoderm (TE) cells and by apoptosis detection in blastomeres using a terminal deoxynucleotidyl transferase-mediated d-UTP nick end-labeling (TUNEL) assay. As results, types and/or age of donor cells did not affect the rate of blastocyst formation and the number of ICM and TE cells. However, a significant increase in apoptotic blastomeres was observed in SCNT embryos reconstructed with long-term cultured fetal or adult ear fibroblasts compared to those in SCNT embryos derived from fresh fetal or adult ear fibroblasts. In conclusion, these results indicated that the long-term culture of donor cells caused increased the incidence of apoptosis in bovine SCNT embryos but did not affect the developmental competence and the cell number of blastocysts.
authors.affiliation: Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, South Korea.
authors.forename: Goo
authors.initials: G
authors.lastname: Jang
authors.forename: Eul-Soon
authors.initials: ES
authors.lastname: Park
authors.forename: Jong-Ki
authors.initials: JK
authors.lastname: Cho
authors.forename: M M U
authors.initials: MM
authors.lastname: Bhuiyan
authors.forename: Byeong-Chun
authors.initials: BC
authors.lastname: Lee
authors.forename: Sung-Keun
authors.initials: SK
authors.lastname: Kang
authors.forename: Woo-Suk
authors.initials: WS
authors.lastname: Hwang
mesh_terms: D000818:Animals; D017209:Apoptosis; D001755:Blastocyst / Q000502:physiology; D001757:Blastomeres / Q000166:cytology*; D002417:Cattle / Q000196:embryology*; D002478:Cells, Cultured; D047108:Embryonic Development; D005314:Embryonic and Fetal Development; D005260:Female; D005347:Fibroblasts / Q000648:ultrastructure; D020287:In Situ Nick-End Labeling; D053652:Nuclear Transfer Techniques; D011247:Pregnancy