PLURIPOTENT BOVINE EMBRYONIC-CELL LINES DIRECT EMBRYONIC-DEVELOPMENT FOLLOWING NUCLEAR TRANSFER

Nuclear transfer (NT) procedures were used to determine the in vivo de velopmental capacity of bovine embryonic cell lines derived from both morula- and blastocyst-stage embryos. These cell lines differed in mor phology from trophoblast and endoderm-like cells. Regardless of initia l donor embryo stage, cells in the resulting bovine embryonic cell lin es had a small cytoplasmic/nuclear volume ratio and contained cytoplas mic vesicles. Developmental rates to blastocyst stage for NT embryos w ere improved when smaller cells (15 mu m) rather than larger cells (18 mu m or 21 mu m) were used in the NT procedure and the recipient oocy te was activated after the cell fusion step, NT embryos produced from these embryonic cell lines, both morula- and blastocyst-derived, initi ated pregnancies following transfer into recipient females. However, a ll of these pregnancies were lost prior to 60 days of gestation, These NT embryos were able to direct development through organogenesis, wit h one NT fetus reaching 55 days before death. When viable NT embryos w ere recovered during early gestation (38 days), an absence of cotyledo ns and a hemorrhagic response in the caruncles were observed. A chimer a produced by aggregating an NT embryo with two 8-cell-stage blastomer es from in vitro-produced embryos developed through the 85th day of ge station. However, this conceptus was also deficient of cotyledons. DNA markers indicated that 50% of the chimera conceptus tissues were deri ved from the embryonic cell line. Blastocyst- and morula-derived embry onic cell line nuclei are pluripotent in that they can direct developm ent through organogenesis, with subsequent pregnancy loss due, al leas t in part, to a deficiency in placentome development.