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Architectural defects in pronuclei of mouse nuclear transplant embryos
Pedro N Moreira1, James M Robl, Philippe Collas
1Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA 01003, USA.
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.
Insights
Nuclear transplantation (NT) for cloning often fails due to incomplete reprogramming. This study reveals that cloned embryos exhibit nuclear remodeling defects, including abnormal protein expression and chromatin structure, hindering successful reprogramming.
Area of Science:
- Reproductive biology
- Developmental biology
- Cell biology
Background:
- Nuclear transplantation (NT) is a technique used for cloning animals.
- While NT demonstrates the possibility of reprogramming somatic cells, low success rates and developmental abnormalities suggest incomplete reprogramming.
- Deficiencies in nuclear remodeling are hypothesized to contribute to incomplete reprogramming after NT.
Purpose of the Study:
- To investigate nuclear remodeling deficiencies in mouse cumulus cell nuclei following transplantation into oocytes.
- To identify morphological markers of nuclear remodeling failure in NT embryos.
- To understand the molecular basis of these remodeling defects.
Main Methods:
- Nuclear transplantation of mouse cumulus cell nuclei into recipient oocytes.
- Analysis of nuclear remodeling markers, including A-type lamins, NuMA, and AKAP95.
- Assessment of chromatin structure through in situ extraction resistance assays.
- Evaluation of defects at the one-cell stage and after the first embryonic mitosis.
Main Results:
- NT embryos show aberrant nuclear assembly of A-type lamins due to misregulated gene expression.
- Increased concentration of NuMA is observed, resulting from both maternal mRNA translation and new transcription.
- AKAP95, a marker of the nuclear envelope-chromatin interface, retains its somatic origin.
- NT embryos exhibit increased heterochromatin, indicated by enhanced resistance to extraction procedures.
- These nuclear defects persist through the first embryonic mitosis, indicating they are not transient.
Conclusions:
- Somatic nuclear reprogramming by NT is associated with significant morphological nuclear remodeling deficiencies.
- Misregulation of gene expression and protein localization contribute to these defects.
- Failure to adequately remodel the somatic nucleus into a functional embryonic nucleus is a key factor in incomplete reprogramming after NT.