168 ESTABLISHMENT OF TROPHOBLAST STEM CELLS FROM MOUSE ANDROGENETIC AND PARTHENOGENETIC EMBRYOS

Abstract

Mouse androgenetic and parthenogenetic embryos, which have two sets of paternal and maternal genomes, respectively, are lethal until Day 9.5 of pregnancy, because the growth of the extraembryonic tissues including placenta is inadequate. These results suggest that both parental genomes are involved in placental development. However, the reason for placental deficiency in androgenetic and parthenogenetic embryos has been unclear. Trophoblast stem (TS) cells, which have the ability to differentiate into trophoblast lineage, have been used to elucidate the mechanism of differentiation and gene function in the development of the placenta. In this study, to characterize trophoblast lineage in androgenetic and parthenogenetic embryos, we examined TS cells from these embryos, and assessed their ability to differentiate into trophoblast lineage. The ovulated MII oocytes from B6D2F1 (C57BL/6 × DBA2) female mice were used for the uniparental embryo production. In order to produce parthenogenetic embryos, the oocytes were activated in SrCl₂ and cytochalasin B. The androgenetic embryos were produced by in vitro fertilization using enucleated oocytes. These uniparental embryos were cultured for 3.5–4.5 days to develop into blastocysts. TS-like cell lines were established from these blastocysts, as described previously (Tanaka et al. 1998 Science 282, 2072–2075). TS-like cells were cultured without feeder cells in conditioned medium containing FGF4 and heparin to maintain the stem cell conditions. The expression of 5 TS cell maker genes (Eomes, CdX2, Fgfr2, Ap2a, and Errb) in TS-like cells was analyzed by RT-PCR and northern blotting. In addition, the localization of CDX2 was detected using immunostaining. To cause their differentiation into giant cells, TS-like cells were cultured for 6 days in TS medium without FGF4 and heparin. The giant cells were detected by the expression of 2 giant cell marker genes, Hand1 and Pl-1. TS cells from fertilized embryos between B6D2F1 male and female mice were also established as controls. Five TS cell marker genes were expressed both in androgenetic TS-like cells (ATS) and in parthenogenetic ones (PTS). The CDX2 gene was localized in the nucleus in both ATS and PTS; however, the number of CDX2-positive cells decreased in PTS. After 6 days of differentiation, Hand1 and Pl-1 were expressed and giant cells were detected in differentiated derivatives in ATS. On the other hand, giant cells were not detected in differentiated derivatives in PTS. These results suggest that parental genomes may regulate the gene expression independently to differentiate into trophoblast lineage. In conclusion, TS-like cells from uniparental embryos are useful tools to aid the understanding of the function of the parental genomes during placental development.