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Nuclear transplantation in pigs: M-phase karyoplast to M-phase cytoplast fusion
1Development and Differentiation Laboratory, Babraham Institute, Cambridge, UK.
Abstract:
Porcine embryonic fibroblasts in M-phase were isolated from confluent cultures by selective detachment and fused to homologous metaphase II enucleated oocytes. The fusion products were analysed by light and electron microscopy (EM). The fusion treatment, per se, did not induce the disappearance of the maturation promoting factor (MPF) in the cloned embryos and the transferred chromatin remained condensed for up to 10 h. Spontaneous decondensation started after this time and, at about 20 h post-induction of fusion, a single large nucleus was present in the cytoplasm. The prolonged exposure of introduced chromatin to MPF resulted in a total morphological remodelling as assessed by the nucleolar morphology observed by EM in all the specimens.
Insights
Cloned porcine embryos were created by fusing M-phase fibroblasts with oocytes. Prolonged exposure to maturation promoting factor (MPF) in cloned embryos led to chromatin remodeling and nuclear formation.
Area of Science:
- Reproductive biology
- Cell biology
- Developmental biology
Background:
- Somatic cell nuclear transfer (SCNT) is a key technology in reproductive biology.
- Understanding early embryonic development post-SCNT is crucial for improving efficiency.
Purpose of the Study:
- To investigate the effects of maturation promoting factor (MPF) on chromatin behavior in cloned porcine embryos.
- To analyze the morphological changes of transferred chromatin after fusion with oocytes.
Main Methods:
- Porcine embryonic fibroblasts in M-phase were isolated and fused with metaphase II enucleated oocytes.
- Fusion products were analyzed using light and electron microscopy (EM).
- Maturation promoting factor (MPF) levels and chromatin decondensation were monitored over time.
Main Results:
- Fusion treatment did not immediately eliminate MPF in cloned embryos.
- Transferred chromatin remained condensed for up to 10 hours post-fusion.
- Spontaneous decondensation began around 10 hours, with nuclear formation by 20 hours.
- Prolonged MPF exposure induced significant morphological remodeling of the chromatin, including nucleolar changes observed via EM.
Conclusions:
- Maturation promoting factor (MPF) plays a critical role in regulating chromatin behavior and nuclear formation in early cloned porcine embryos.
- The observed morphological remodeling suggests MPF influences epigenetic reprogramming during early development.
- These findings provide insights into the developmental potential of SCNT-derived embryos.