Architectural defects in pronuclei of mouse nuclear transplant embryos

Abstract

Reprogramming somatic nuclear function by transplantation of nuclei into recipient oocytes is associated with a morphological remodeling of the somatic nucleus. Successful cloning of animals by nuclear transplantation (NT) demonstrates that reprogramming somatic cell function is possible. However, low pregnancy rates and high frequencies of lethal abnormalities in animals born suggest that reprogramming is rarely complete. To address this issue, we tested the hypothesis that nuclear transplantation leads to nuclear remodeling deficiencies. We report the identification of several markers of morphological remodeling, or lack thereof, of mouse cumulus cell nuclei after transplantation into oocytes. Notably, nuclear transplant mouse embryos exhibit nuclear assembly of the differentiated cell-specific A-type lamins at the one-cell stage, as a result of misregulation of lamin A gene expression. The transplanted nuclei also display enhanced concentration of the nuclear matrix-associated protein NuMA as a result of translation from maternal mRNA and de novo transcription. The A-kinase anchoring protein 95 (AKAP95), a marker of the nuclear envelope-chromatin interface, is of somatic origin. Furthermore, greater resistance of AKAP95 and DNA to in situ extractions of one-cell stage NT embryos with non-ionic detergent, DNase, RNase and NaCl reflects an enhanced proportion of heterochromatin in these embryos. Passage through first embryonic mitosis does not rescue the defects detected in one-cell stage embryos. We propose that somatic nuclear reprogramming deficiencies by NT might emanate from, at least in part, failure to remodel the somatic nucleus morphologically into a functional embryonic nucleus.

Identification of differentially expressed genes from the cross-subfamily cloned embryos derived from zebrafish nuclei and rare minnow enucleated eggs

“…Down-regulation of these three genes in the present study indicated that translation failure occurred in cloned embryos. In addition, some genes related to chromatin organization and cell cycle regulation, rcc1 and smc1a were down-regulated in the cloned embryos, in agreement with a previous study in somatic cell NT embryos [37][38][39]. Some other genes related to the regulation of cell cycle were also differentially expressed in the cloned embryos, e.g.…”

Comparison of the RNA Polymerase I-, II- and III-Dependent Transcript Levels Between Nuclear Transfer and In Vitro Fertilized Embryos at the Blastocyst Stage

“…Based on the reports that most NT embryos cease development at the pre-and peri-implantation stages [1,41], it is probable that the accumulation of different abnormalities observed in NT embryos at various developmental stages, such as failure to erase some of the features of donor cells [42][43][44], abnormal gene expression [4,8,10,25,[45][46][47], abnormal DNA methylation [4][5][6], abnormal formation of placentae [1,41,[48][49][50], and the abnormalities detected in this study, leads to gradual loss of NT embryos. Abnormalities that have not been detected in previous studies and/or small differences in gene expression may seriously affect embryonic development.…”

Fate of centrosomes following somatic cell nuclear transfer (SCNT) in bovine oocytes

“…After removal of cycloheximide 6 h following NT, extensive translation and expression of NuMA could occur. Consistently, NuMA reassembles as a result of translation from maternal mRNA and of de novo transcription from transplanted nuclear genomes in the mouse cloning (Moreira et al 2003). NuMA could be important for spindle pole organization in primates (Simerly et al 2003).…”

“…NuMA was also was detected in pronuclear stage embryos of mouse clones and in donor cumulus cells (Moreira et al 2003), as well as in normal bovine embryos (Degrouard et al 2004). In bovine clones produced by simultaneous fusion and activation, NuMA was weak or absent in donor nuclei, but strongly expressed in pronuclei following NT.…”