Nucleolar ultrastructure in bovine nuclear transfer embryos

Nuclear transfer experiments in mammals have attempted to reprogram a donor nucleus to a state equivalent to the zygotic one, Reprogramming of the don or nucleus is, among other features, indicated by a synthesis of ribosomal RNA (rRNA). The initiation of rRNA synthesis is simultaneously reflected in nuclear morphology as a transformation of the nucleolus precursor body int o a functional rRNA synthesising nucleolus with a characteristic ultrastruc ture. We examined nucleolar ultrastructure in bovine in vitro produced (con trol) embryos and in nuclear transfer embryos reconstructed from a MII phas e (nonactivated) or S phase (activated) cytoplasts, Control embryos were fi xed at the two-, four-, early eight- and late eight-cell stages; nuclear tr ansfer embryos were fixed at 1 and 3 hr post fusion and at the two-, four-, and eight-cell stages. Control embryos possessed a nucleolar precursor bod y throughout all three cell cycles. In the eight-cell stage embryo, a prima ry vacuole appeared as an electron lucid area originating in the centre of the nucleolar precursor body. In nuclear transfer embryos reconstructed fro m nonactivated cytoplasts, the nuclear envelope was fragmented or completel y broken down at 1 hr after fusion and, by 3 hr after fusion, it was restor ed again. At this time, the reticulated fibrillo-granular nucleolus had an almost round shape. The nucleolar precursor body seen in the two-cell stage nuclear transfer embryos consisted of intermingled filamentous components and secondary vacuoles. A nucleolar precursor body typical for the two-cell stage control embryos was never observed. None of the reconstructed embryo s of this group reached the eight-cell stage. Nuclear transfer embryos reco nstructed from activated cytoplasts, in contrast, exhibited a complete nucl ear envelope at all time intervals after fusion. In the two-cell stage nucl ear transfer embryo, the originally reticulated nucleolus of the donor blas tomere had changed into a typical nucleolar precursor body consisting of a homogeneous fibrillar structure. A primary vacuole appeared in the four-cel l stage nuclear transfer embryos, which was one cell cycle earlier than in control embryos. Only nuclear transfer embryos reconstructed from activated cytoplasts underwent complete remodelling of the nucleolus. The reorganisa tion of the donor nucleolar architecture into a functionally active nucleol us was observed as early as in the four-cell stage nuclear transfer embryo. These ultrastructural observations were correlated with our autoradiograph ic data on the initiation of RNA synthesis in nuclear transfer embryos. Mel . Reprod. Dev. 52.253-263, 1999. (C) 1999 Wiley-Liss, Inc.