Pluripotent bovine embryonic cell lines direct embryonic development following nuclear transfer

Abstract

Nuclear transfer (NT) procedures were used to determine the in vivo developmental capacity of bovine embryonic cell lines derived from both morula- and blastocyst-stage embryos. These cell lines differed in morphology from trophoblast and endoderm-like cells. Regardless of initial donor embryo stage, cells in the resulting bovine embryonic cell lines had a small cytoplasmic/nuclear volume ratio and contained cytoplasmic vesicles. Developmental rates to blastocyst stage for NT embryos were improved when smaller cells (15 microns) rather than larger cells (18 microns or 21 microns) were used in the NT procedure and the recipient oocyte was activated after the cell fusion step. NT embryos produced from these embryonic cell lines, both morula- and blastocyst-derived, initiated pregnancies following transfer into recipient females. However, all of these pregnancies were lost prior to 60 days of gestation. These NT embryos were able to direct development through organogenesis, with one NT fetus reaching 55 days before death. When viable NT embryos were recovered during early gestation (38 days), an absence of cotyledons and a hemorrhagic response in the caruncles were observed. A chimera produced by aggregating an NT embryo with two 8-cell-stage blastomeres from in vitro-produced embryos developed through the 85th day of gestation. However, this conceptus was also deficient of cotyledons. DNA markers indicated that 50% of the chimera conceptus tissues were derived from the embryonic cell line. Blastocyst- and morula-derived embryonic cell line nuclei are pluripotent in that they can direct development through organogenesis, with subsequent pregnancy loss due, at least in part, to a deficiency in placentome development.