188 FATE OF CHIMERIC EMBRYONIC STEM CELLS RECONSTRUCTED WITH PARTHENOGENETIC MOUSE EMBRYOS

Abstract

Mouse parthenogenetic embryos have been known to be unable to develop to term and are absorbed from Day 11. However, Nagy et al. (1990 Development 110, 815–821) reported that the aggregated tetraploid with embryonic stem (ES) cell developed to term and survived after birth. Thus, our study investigated the developmental capacity of the aggregated ES cells with mouse parthenogenetic embryos. Oocytes obtained from superovulated female mice (BCF1) were treated with 7% ethanol and 5 μg mL⁻¹ cytochalasin B for the production of pathenotes and co-cultured with sperm (1 × 10⁶ mL⁻¹) for production of fertilized embryos. The reporter vector (pNeoEGFP) was introduced into ES cells (129S4/SvJae) by electroporation. At the 8-cell stage, pathenotes or fertilized embryos, from which the zona pellucida was removed, were co-cultured with ES cells for 4 h. The aggregated parthenotes or fertilized embryos with 5∼10 ES cells were cultured to the blastocyst stage, and transferred into the uteri of 2.5-day post-coitum pseudopregnant recipients. In experiment I, 144 parthenogenetic blastocysts were transferred into the uterine horns of 9 pseudopregnant recipients, and 5 recipients became pregnant. At Day 9, all fetuses were observed visible in uteri of pregnant fonder mice. At Days 10–11, many fetuses were observed in the progress of absorption in uteri of pregnant fonder mice, but a few fetuses were still alive. However, pathenogenetic fetuses were not detected alive beyond 11 days. In experiment II, the 171 aggregated fertilized embryos with ES cells were transferred (15–20 blastocysts/recipient) into 10 recipients and successfully produced 5 offspring from a recipient. We found that three newborn were chimeric mice derived from ES cells. In experiment III, the 209 aggregated parthenotes with ES cells failed to produce offspring, but inserted pNeoEGFP gene in ES cells was detected in the parthenogenetic 1 of 7 fetuses at 15-days of post-gestation by polymerase chain reactions. Therefore, this result suggests that the parthenotes show restricted development to fetus stage, but the aggregated parthenotes with ES cells might extend their developmental capacity. In the future, we will characterize the mechanism of this unusual phenomenon to understand the role of ES cells during development of chimeric parthenotes with ES cells.