Development of Bovine Nuclear Transfer Embryos Made with Oogonia1

Abstract

The pluripotency of embryonic germ cells in the mouse suggests that mitotic bovine fetal germ cells might also be a source of pluripotent cells. To investigate the pluripotency of bovine oogonia, the development in vitro of bovine embryos reconstructed by fusing oogonia with enucleated oocytes was compared with that of embryos made similarly with either blastomeres or granulosa cells. The donor cells (fresh oogonia, cryopreserved oogonia, 16- to 32-cell-stage blastomeres, or granulosa cells) were fused to the enucleated oocytes electrically. The proportions of reconstructed embryos that had cleaved at 40 h after fusion using these types of donor cells were not significantly different (37%, 33%, 56%, and 31%, respectively; p > 0.05). However, the proportions of cleaved reconstructed embryos that developed to the blastocyst stage were 9%, 13%, 36%, and 3%, respectively, significantly higher (p < or = 0.05) with blastomeres than with the other three types of donor cells. After transfer of 3 morulae and 4 blastocysts made with oogonia into three recipient heifers, embryonic and extra-embryonic tissues developed in one animal. On recovery after 43 days gestation, this conceptus was shown to be genetically identical, at 11 microsatellite loci, to the fetus that had provided the oogonia. Cytological analysis of the embryos made with oogonia at 40-44 h after fusion and at the morula and blastocyst stages revealed that aberrant cytokinesis and nucleokinesis had given rise to multinucleated, anucleate, and polyploid cells in the reconstructed embryos. It is concluded that limited pluripotency of bovine oogonia has been demonstrated, warranting further study in this area.

Efficient Production of Cloned Bovine Embryos Using cdc2 kinase Inhibitor

“…The resulting reconstituted embryos developed to blastocyst stage with comparatively higher proportion, compared to those previously reported by others (1.8%, Moens et al. 1996; 4.3%Lavoir et al. 1997).…”

Cloning in cattle: from embryo splitting to somatic nuclear transfer

“…The pluripotency of bovine gonial cells has been tested through nuclear transfer by three separate laboratories, leading to the temporary conclusion that germ cells can direct in vitro development up to blastocysts in some cases (2-4 %), but no live offspring could be obtained after transfer to recipient heifers. Only two cases of initiated pregnancy were reported [29,35] [6] which claimed that cell quiescence is a prerequisite for somatic nuclear transfer, we and others [8,51] …”

Effect of Culture Media on In Vitro Development of Cloned Mouse Embryos

“…It is probable, therefore, that factors specific to the NT embryos such as the transferred donor nucleus are involved in causing this high rate of binucleation. A study in bovine NT embryos (Lavoir et al, 1997) revealed a twofold higher incidence of multinucleation in embryos reconstructed with differentiated oogonia versus embryos reconstructed with blastomere nuclei. Another possible explanation is that enucleation can coincide with a high amount of actin removal, which could interfere with proper cytokinesis (Peura et al, 1998).…”