scholarly journals Remodeling of mouse thymocyte nuclei depends on the time of their transfer into activated, homologous oocytes

1988 ◽  
Vol 91 (4) ◽  
pp. 603-613
Author(s):  
D. Szollosi ◽  
R. Czolowska ◽  
M.S. Szollosi ◽  
A.K. Tarkowski
Keyword(s):  
Nuclear Envelope ◽  
Somatic Cell ◽  
Morphological Changes ◽  
Factor Activity ◽  
Cell Nuclei ◽  
Nuclear Envelope Breakdown ◽  
Somatic Cell Nucleus ◽  
Short Time ◽  
Nuclear Remodeling ◽  
Maturation Promoting Factor

The potential of parthenogenetically activated mouse oocytes to remodel somatic cell nuclei was studied by ultrastructural means using oocyte-thymocyte hybrids. Complete nuclear remodeling, initiated by nuclear envelope breakdown and chromosome condensation (which is followed by formation of pronucleus-like nucleus) is possible only during a short time gap between metaphase II and telophase of meiotic division. Maturation-promoting factor activity is high during this period. The thymocyte nucleus can follow the sequence of morphological changes only in concert with the development of the native nucleus and only after exposure of the chromatin to the ooplasm. If hybridization is effected with pronucleate oocytes, the thymocyte nucleus retains its interphase character but shows particular modifications in nucleolar morphology (identical to changes observed during reactivation of the nucleolus in stimulated lymphocyte) and in the activity of the nuclear envelope (blebbing). Thus the nucleus not exposed to maturation-promoting factor activity may be influenced by a ‘programme’ specific for oocyte (blebbing) and by a programme inherent in the introduced somatic cell nucleus.

Nuclear envelope removal/maintenance determines the structural and functional remodelling of embryonic red blood cell nuclei in activated mouse oocytes

Zygote ◽  
1998 ◽  
Vol 6 (1) ◽  
pp. 65-73 ◽  
Author(s):  
Daniel Szöllösi ◽  
Renata Czołowska ◽  
Ewa Borsuk ◽  
Maria S. Szöllösi ◽  
Pascale Debey
Keyword(s):  
Nuclear Envelope ◽  
Transcriptional Activity ◽  
Chromatin Condensation ◽  
Oocyte Activation ◽  
Cell Nuclei ◽  
Nuclear Envelope Breakdown ◽  
Mouse Oocytes ◽  
Female Pronucleus ◽  
Mouse Fetuses

SummaryNuclei of embryonic red blood cells (e-RBC) from 12-day mouse fetuses are arrested in Go phase of the cell cycle and have low transcriptional activity. These nuclei were transferred with help of polyethylene glycol (PEG)-mediated fusion to parthenogenetically activated mouse oocytes and heterokaryons were analysed for nuclear structure and transcriptional activity. If fusion proceeded 25–45 min after oocyte activation, e-RBC nuclei were induced to nuclear envelope breakdown and partial chromatin condensation, followed by formation of nuclei structurally identical with pronuclei. These ‘pronuclei’, similar to egg (female) pronuclei, remained transcriptionally silent over several hours of in vitro culture. If fusion was performed 1 h or later (up to 7 h) after activation, the nuclear envelope of e-RBC nuclei remained intact and nuclear remodelling was less spectacular (slight chromatin decondensation, formation of nucleolus precursor bodies). These nuclei, however, reinforced polymerase-II-dependent transcription within a few hours of in vitro culture. Our present experiments, together with our previous work, demonstrate that nuclear envelope breakdown/maintenance are critical events for nuclear remodelling in activated mouse oocytes and that somatic dormant nuclei can be stimulated to renew transcription at a time when the female pronucleus remains transcriptionally silent.

scholarly journals Maturation-promoting factor induces nuclear envelope breakdown in cycloheximide-arrested embryos of Xenopus laevis.

1983 ◽  
Vol 97 (1) ◽  
pp. 81-91 ◽  
Author(s):  
R Miake-Lye ◽  
J Newport ◽  
M Kirschner
Keyword(s):  
Xenopus Laevis ◽  
Nuclear Envelope ◽  
Nuclear Lamina ◽  
Embryonic Cell ◽  
Immunofluorescent Staining ◽  
Interphase Nuclei ◽  
Nuclear Envelope Breakdown ◽  
G2 Phase ◽  
Embryonic Cell Cycle ◽  
Maturation Promoting Factor

We have studied the effect of maturation-promoting factor (MPF) on embryonic nuclei during the early cleavage stage of Xenopus laevis development. When protein synthesis is inhibited by cycloheximide during this stage, the embryonic cell cycle arrests in an artificially produced G2 phase-like state, after completion of one additional round of DNA synthesis. Approximately 100 nuclei can be arrested in a common cytoplasm if cytokinesis is first inhibited by cytochalasin B. Within 5 min after injection of MPF into such embryos, the nuclear envelope surrounding each nucleus disperses, as determined histologically or by immunofluorescent staining of the nuclear lamina with antilamin antiserum. The breakdown of the nuclear envelope occurs at levels of MPF comparable to or slightly lower than those required for oocyte maturation. Amplification of MPF activity, however, does not occur in the arrested egg as it does in the oocyte. These results suggest that MPF can act to advance interphase nuclei into the first events of mitosis and show that the nuclear lamina responds rapidly to MPF.

58 EFFECTS OF CAFFEINE AND VANADATE ON NUCLEAR REMODELING AND MICROTUBULE DISTRIBUTION OF PORCINE NUCLEAR TRANSFER EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 146
Author(s):  
D. J. Kwon ◽  
C. K. Park ◽  
B. K. Yang ◽  
C. I. Kim ◽  
H. T. Cheong
Keyword(s):  
Nuclear Transfer ◽  
Laser Scanning ◽  
Morphological Changes ◽  
Laser Scanning Confocal Microscopy ◽  
Blastocyst Stage ◽  
Scanning Confocal Microscopy ◽  
Nuclear Envelope Breakdown ◽  
Nuclear Remodeling ◽  
Parthenogenetic Embryos

The present study was conducted to control nuclear remodeling types by treatment with caffeine or vanadate, and to examine the microtubule distribution of nuclear transfer embryos (NTs) after nuclear remodeling control and the mitotic spindle assembly and its morphological changes during the first mitosis of NTs in the pig. Enucleated oocytes were treated with 5 mM caffeine or 0.5 mM sodium orthovanadate (vanadate) for 2.5 or 0.5 h to increase or decrease MPF activity before injection of fetal fibroblast cells. Reconstituted eggs were fused by an electric stimulation (ES, 1.5 kV cm-1), activated by a combination of 2 pulses of ES (1.0 kV cm-1), and cultured for 3 h with 2 mM 6-dimethylaminopurine (6-DMAP) at 1 h after fusion treatment. Some matured oocytes were also treated by the same chemicals before parthenogenetic activation under the same conditions as NTs, and cultured in vitro to evaluate the effects of these chemicals on embryo development. NTs and parthenogenetic embryos were cultured in PZM-3 for 20 h or 6 days at 39�C, 5% CO2 in air, respectively. Nuclear remodeling types of NTs were examined at 1 h after fusion (before activation) by the whole-mount method. At least 3 replicates for each experiment were performed. Microtubules and DNA of NTs that were fixed at 1 h or 20 h after fusion were detected by indirect immunocytochemical technique. Images were captured using laser scanning confocal microscopy. Caffeine and vanadate did not affect the development to the blastocyst stage of porcine parthenogenetic embryos. When a nucleus was exposed to oocyte cytoplasm treated with caffeine, premature chromosome condensation (PCC) occurred at a higher rate (82/98, 83.7%) compared to control (42/73, 57.5%) and vanadate-treated (11/91, 12.1%) groups (P < 0.05). The proportion of embryos that did not undergo nuclear envelope breakdown (NEBD) was higher in the vanadate treatment group (87.9%) compared to the caffeine and control groups (16.3 and 42.5%, respectively; P < 0.05). The frequency of embryos showing a γ-tubulin only and both γ- and β-tubulins were 3.9–9.4% and 21.9–34.6%, respectively, in NTs (total 87 embryos) at 1 h after fusion regardless of caffeine and vanadate treatments. In the majority of NTs (61.5–68.6%), microtubules were not observed. At 20 h after fusion, the frequency of the embryos undergoing normal mitosis was similar in the control (17/45, 37.8%) and caffeine (19/43, 44.2%) groups, but it was significantly lower in the vanadate group (7/37, 18.9%; P < 0.05). The present study demonstrates that the nuclear remodeling type of NTs can be controlled by treatment with MPF regulators, caffeine and vanadate, and such treatment is not related to the microtubule distribution in porcine NTs. The finding, however, that the vanadate can delay the mitotic progression of porcine NTs at the first cell cycle may be due to the lack of NEBD and PCC. This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD;KRF-2005-042-F00030).

53 EVALUATION OF NUCLEAR REPROGRAMMING IN CLONED MINIATURE PIG EMBRYOS USING GENES OF Oct-4 FAMILY

2006 ◽  
Vol 18 (2) ◽  
pp. 135
Author(s):  
E. Lee ◽  
S. H. Lee ◽  
S. Kim ◽  
Y. W. Jeong ◽  
J. H. Kim ◽  
...  
Keyword(s):  
Germ Cell ◽  
Somatic Cell ◽  
Expression Patterns ◽  
Fibroblast Cell ◽  
Marker Genes ◽  
Miniature Pig ◽  
Cell Nuclei ◽  
Cell Clones ◽  
Somatic Cell Nucleus ◽  
Reliable Marker

Xenotransplantation as a source of organs is a rapidly expanding field which can save thousands of human lives each year. Cloned miniature pigs have been considered as a model system for xenotransplantation. However, the efficiency of somatic cell nuclear transfer (SCNT) is extremely low, with most clones resulting in early lethality and several aberrancies. Possible explanation of the developmental failure of SCNT embryos is related to insufficient reprogramming of the somatic cell nucleus. In order to test this, we analyzed the reprogramming capacity of differentiated fibroblast cell nuclei and undifferentiated germ cell nuclei with Oct-4 and four Oct-4-related genes (Ndp5211, Dppa2, Dppa3, and Dppa5) as molecular markers using quantitative reverse transcription-polymerase chain reaction (RT-PCR). Oct-4 expression patterns were similar among IVF-derived embryos and cloned embryos derived from fibroblasts or germ cells during pre-implantation embryo development. However, the expression level was significantly lower (P < 0.05) in hatched blastocysts of fibroblast clones compared to other hatched blastocysts. Also, 9 of 13 cloned morulae and 12 of 40 cloned blastocysts failed to reactivate at least one of the five tested genes, whereas all of the germ cell clones and control embryos correctly expressed these genes. Analysis with miniature pig fetuses collected at Day 30 of gestation revealed that normal and cloned fetuses successfully expressed these genes. In conclusion, our results suggest that analysis of expression of Oct-4 and related genes could be a reliable marker for evaluating the reprogramming status of transplanted donor nuclei in cloned embryos. The reprogramming of fibroblast cloned embryos is highly error-prone. This may contribute to their embryonic lethality because cloned embryos that fail to reactivate the marker genes may fail to be successfully implanted. This study was supported by grants from the Ministry of Science and Technology (Top Scientist Fellowship), and the Biogreen 21-1000520030100000.

scholarly journals 52 NUCLEAR REMODELING AFTER SOMATIC CELL NUCLEAR TRANSFER (SCNT) IN THE RHESUS MONKEY

2005 ◽  
Vol 17 (2) ◽  
pp. 175
Author(s):  
S. Mitalipov ◽  
J. Byrne ◽  
M. Sparman ◽  
C. Ramsey ◽  
D. Wolf
Keyword(s):  
Rhesus Monkey ◽  
Nuclear Envelope ◽  
Somatic Cell ◽  
Nuclear Transfer ◽  
Chromosome Condensation ◽  
Advanced Technology ◽  
Lamin A ◽  
Spindle Formation ◽  
Fetal Fibroblasts ◽  
Nuclear Remodeling

Successful reprogramming of somatic cell nuclei after nuclear transfer requires active remodeling by factors present in the nonactivated cytoplast. High levels of maturation promoting factor (MPF) activity are associated with this remodeling process which includes nuclear envelope breakdown (NEBD), premature chromosome condensation (PCC), and spindle formation. MPF degradation, caused by fertilization or artificial activation, is in turn required for pronuclear formation and subsequent embryonic cleavage, and involves cyclin B catalyzed proteolysis by the proteasome system. In the rhesus monkey, SCNT results in the production of cleaving embryos, but development arrests at the morula stage presumably because of abnormal or incomplete reprogramming. We undertook this pilot study to examine the timing and extent of nuclear remodeling events (NEBD, PCC) in monkey SCNT embryos. The proteasome inhibitor MG-132 was employed to avoid or delay premature MPF degradation (Zhou et al. 2003 Science 302, 1179). Monkey fetal fibroblasts employed as nuclear donor cells were fused with nonactivated cytoplasts and incubated in the presence (n = 20) or absence (control; n = 35) of MG-132. Embryos were fixed and co-labeled with DAPI (DNA) and monoclonal antibody against lamin A/C (nuclear envelope). In monkey germinal vesicle-stage oocytes (n = 5) and zygotes (n = 6), a lamin A/C signal was detected at the nuclear periphery while matured MI (n = 6) and MII (n = 12) oocytes were negative for lamin A/C staining, consistent with the absence of a nuclear membrane. Donor fetal fibroblasts arrested at the G1 stage of the cell cycle exhibited a lamin A/C signal. Minimal or no changes were observed in donor nuclei within 1 h after fusion. The majority of control SCNT embryos sampled 4 h after fusion exhibited only slight chromatin condensation; however, they failed to form metaphase chromosomes. Positive lamin A/C staining indicated the presence of intact nuclear membranes. Following activation these SCNT embryos cleaved, but arrested at the 8–16 cell stage. In initial experimentation we determined the minimal efficient concentration of MG-132 to be 5 μM, that is capable of inhibiting first polar body extrusion during the MI–MII transition. Subsequent in vitro development to the blastocyst stage (53%) of fertilized oocytes treated with 5 μM MG-132 for up to 4 h was similar to that of nontreated controls. Incubation of SCNT embryos (n = 20) for 4 h with MG-132 resulted in robust chromosome condensation, spindle formation, and weak or partial lamin A/C signal. Our observations suggest that incomplete nuclear remodeling events in monkey SCNT embryos may be due to premature MPF inactivation perhaps caused by the fusion pulse. Future studies will address the developmental potential of monkey SCNT embryos exposed to MG-132. This work was supported by NIH grant NS04330, Core Grant RR00163 and a product donation from Ares Advanced Technology, Inc.

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