Study of implantation using molecular biological means using xeno-chimeric embryos.

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We investigated the mechanism of implantation using molecular biological techniques and transgenic animals. We have established "green rat" lines together with "red mouse" lines. The combination of these zeno-clilmeric embryos allows us to differentiate the contribution of cells from which species the cells are derived from.In order to have a sufficient source from rat embryos, we have established multipotent stem cells from "green rat" bone marrow cells (MAPC). We also succeeded to establish the stem cells of trophectoderm origin.In the middle of producing chimeric mice, We produced XX7XY chimeras by using embryos whose X chromosomes were tagged with EGFP (X^*), making the fluorescent green female (XX^*) germ cells easily distinguishable from their nonfluorescent male (XY) counterparts. Taking advantage of tagging with EGFP, the XX^* "prospermatogonia" were isolated from the testes, and the status of their genomic imprinting was examined. It was shown that these XX cells underwent a paternal imprinting, despite their chromosomal constitution. As previously indicated in sex-reversal XXsxr testes, we also found a few green XX^* germ cells developed as "eggs" within the seminiferous tubules of XX^*7XY chimeric testes. These cells were indistinguishable from XX^* prospermatogonia at birth but resumed oogenesis in a testicular environment. The biological nature of the "testicular eggs" was examined by recovering the eggs from chimeric testes.The testicular eggs not only formed an egg-specific structure, the zona pellucida, but also were able to fuse with sperm. The collected testicular eggs were indicated to undergo maternal imprinting, despite the testicular environment. The genomic imprinting did not always follow the environmental conditions of where the germ cells resided ; rather, it was defined by the sex that was chosen by the germ cells at early embryonic stage.