scholarly journals Control of nuclear remodelling and subsequent in vitro development and methylation status of porcine nuclear transfer embryos

Reproduction ◽  
2008 ◽  
Vol 135 (5) ◽  
pp. 649-656 ◽  
Author(s):  
D J Kwon ◽  
C K Park ◽  
B K Yang ◽  
H T Cheong
Keyword(s):  
Nuclear Transfer ◽  
Apoptotic Cell ◽  
Formation Rate ◽  
Methylation Status ◽  
Subsequent Development ◽  
Treated Group ◽  
High Rate ◽  
Cell Stage ◽  
Methylation Patterns

We attempted to control the nuclear remodelling of somatic cell nuclear transfer embryos (NTs) and examined their subsequent development and DNA methylation patterns in pigs. Porcine foetal fibroblasts were fused to enucleated oocytes treated with either 5 mM caffeine for 2.5 h or 0.5 mM vanadate for 0.5 h. After activation, NTs were cultured in vitro for 6 days to examine their development. The nuclear remodelling type of the reconstituted embryos was evaluated 1 h after fusion. Methylated DNA of in vitro-fertilised (IVF) embryos and NTs at various developmental stages and of donor cells was detected using a 5-methylcytosine (5-MeC) antibody. Caffeine-treated NTs induced premature chromosome condensation at a high rate (P<0.05), whereas most vanadate-treated NTs formed a pronucleus-like structure. Although cleavage rates to the two-cell stage did not differ among groups, delayed cleavage was observed in the vanadate-treated group. The blastocyst formation rate was significantly reduced by vanadate treatment compared with caffeine-treated and non-treated (control) NT groups (P<0.05). The apoptotic cell index of NT blastocysts was lower in the caffeine-treated group than in other groups (P<0.05). The methylation patterns were similar among NTs, but more hypermethylated DNA was observed at the four-cell stage of control and vanadate-treated NTs when compared with that in IVF embryos (P<0.05). Thus, the nuclear remodelling type controlled by caffeine or vanadate treatment can affect in vitro development and the methylation status of NTs in relation to nuclear reprogramming.

47 DIFFERENTIAL DEVELOPMENTAL ABILITY OF EMBRYOS CLONED FROM TISSUE-SPECIFIC STEM CELLS

2007 ◽  
Vol 19 (1) ◽  
pp. 142
Author(s):  
K. Inoue ◽  
N. Ogonuki ◽  
H. Miki ◽  
S. Noda ◽  
S. Inoue ◽  
...  
Keyword(s):  
Stem Cells ◽  
Nuclear Transfer ◽  
Embryonic Stem ◽  
Donor Cell ◽  
Fibroblast Cell ◽  
High Rate ◽  
Full Potential ◽  
Cell Nuclei ◽  
Cell Stage

Although cloning animals by somatic cell nuclear transfer is generally an inefficient process, use of appropriate donor cell types may improve the cloning outcome significantly. Among the donor cells tested so far, mouse embryonic stem cells have given the best efficiency in terms of the development of reconstructed embryos into offspring. In this study, we examined whether 2 in vitro-produced pluripotent stem cells—neural stem cells (NSCs) and mesenchymal stem cells (MSCs)—could be better nuclear donors than other differentiated cells. Embryos were reconstructed by transfer of nuclei from NSCs or MSCs with full potential for differentiation in vitro. Most (76%) of the 2-cell NCS embryos developed to the 4-cell stage; 43% implanted and 1.6% developed to term after transfer to pseudopregnant recipients. These rates were very similar to those of embryos cloned from fibroblast cell nuclei. Interestingly, in the patterns of zygotic gene expression, NSC embryos were more similar to in vitro-fertilized embryos than fibroblast cloned embryos. By contrast, embryos reconstructed using MSC nuclei showed lower developmental ability and no implantation was obtained after embryo transfer. Chromosomal analysis of the donor MSCs revealed very high frequencies of monosomy and trisomy, which might have caused the very poor post-implantation development of embryos following nuclear transfer. Thus, in vitro-produced pluripotent cells can serve as donors of nuclei for cloning mice, but may be prone to chromosomal aberrations leading to a high rate of cloned embryo death.

115 APOPTOSIS EVENT OF PREIMPLANTATION DEVELOPMENT STAGES IN PORCINE IN VITRO-FERTILIZED EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 157
Author(s):  
S. M. Hong ◽  
S. H. Jeong ◽  
S. H. Hyun
Keyword(s):  
Cell Death ◽  
Actinomycin D ◽  
Apoptotic Cell ◽  
Formation Rate ◽  
Preimplantation Development ◽  
Preimplantation Embryos ◽  
Control Group ◽  
Cell Stage ◽  
Development Stages

Little is known about apoptosis events in porcine preimplantation embryos. In this study, we aimed to determine whether the evaluated markers of cell death could be found at particular developmental stages of normal porcine in vitro-fertilized (IVF) embryos. We investigated the characteristics of spontaneous and induced apoptosis during preimplantation development stages of porcine IVF embryos. In experiment 1, to induce apoptosis of porcine IVF embryos, porcine IVF embryos at 22 h postinsemination were treated at different concentrations of actinomycin D (0, 5, 50 and 500 ng mL–1 in NCSU medium). Four groups were incubated at 37°C in 5% CO2, 5%O2 for 8 h, and then washed to NCSU medium and incubated until blastocyst (BL) stage. We examined cleavage rate at 2 days and BL development rate at 7 days after in vitro culture (IVC). A significantly less rate of cleavage was found in the 500 ng mL–1 group compared with others (500 ng mL–1 v. 0, 5, 50 ng mL–1; 15.4% v. 48.6%, 40%, 32%). In the results of BL formation rate, there was a significantly less BL formation rate in 500 ng mL–1 compared with others (500 ng mL–1 v. 0, 5, 50 ng mL–1; 0% v. 10%, 8.8%, 9%). In experiment 2, to evaluate apoptotic cells at different stage (2-cell, 4-cell, 8-cell and BL stage) of all groups, we conducted TUNEL assay based on morphological assessment of nuclei and on detection of specific DNA degradation under fluorescence microscope. This result showed that apoptosis is a normal event during preimplantation development in control group (0 ng mL–1 actinomycin D). A high number of the BL derived control group contained at least one apoptotic cell. Actinomycin D treated BL responded to the presence of apoptotic inductor by a significant decrease in the average number of blastomeres and a significant increase in the incidence of apoptotic cell death. In the 500 ng mL–1 group, the incidence of apoptosis increased at the 4-cell stage and later. This result suggested that apoptosis is a process of normal embryonic development and actinomycin D is a useful tool for the apoptosis study of porcine preimplantation embryos. This work was supported by a grant (#20070301034040) from BioGreen 21 program, Rural Development Administration, Republic of Korea.

45 NUCLEAR LAMIN A/C EXPRESSION IN BOVINE PARTHENOTES AND NUCLEAR TRANSFER EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 131
Author(s):  
R. D. W. Kelly ◽  
R. Alberio ◽  
K. H. S. Campbell
Keyword(s):  
Nuclear Transfer ◽  
Expression Patterns ◽  
Subsequent Development ◽  
Lamin A ◽  
Santa Cruz ◽  
Cell Stage ◽  
Aberrant Expression ◽  
Fetal Fibroblasts ◽  
Bovine Oocytes

Despite the apparent successes of nuclear transfer (NT) technology, numerous recent reports have indicated de-regulation of key gene expression patterns in NT embryos as compared to their in vivo and IVF counterparts. Aberrant expression of lamin A/C has been reported in mouse (Moreira et al. 2003 J. Cell Sci. 116, 3713-3720) and bovine (Sullivan et al. 2004 Biol. Rep. 70, 146-153) NT embryos, leading to the hypothesis that the presence of lamin A/C might affect subsequent development. Lamin A/C expression is a potential marker for reprogramming due to the induced expression and remodeling during differentiation. Previously using immunofluorescence in bovine IVF embryos, we have demonstrated the persistence of lamin A/C until the 2-cell stage (Kelly et al. 2005 Reprod. Fertil. Dev. 17, 205-206). The present study was initiated to further characterize lamin A/C expression in bovine parthenogenetic and NT embryos using a monoclonal antibody specific to lamin A/C. Bovine oocytes were matured in vitro as previously described (Fouladi-Nashta et al. 1998 Biol. Rep. 59, 255-262). NT embryos were constructed using lamin A/C-positive primary bovine fetal fibroblasts and in vitro-matured, enucleated MII bovine oocytes. Oocyte cell couplets were fused at 24 h post onset of maturation 1 h prior to activation. Oocyte activation was achieved with 7% ethanol for 7 min followed by a 6 h incubation in mSOF containing 10 �g/mL cycloheximide and 7.5 �g/mL cytochalasin B for the production of both NT and parthenogenetic embryos. Embryos were cultured in mSOF supplemented with 10% FCS and collected at various stages for immunofluorescence staining. Prior to fixation, embryos were incubated in 2 mg/mL protease to remove the zona pellucida. Samples were fixed in 100% methanol at -20�C for 20 min and then blocked for 1 h (4% goat serum in PBS) at RT. Embryos were then incubated overnight at 4�C with mouse anti-lamin A/C antibody (IgM; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or with blocking solution as a control. Following the primary incubation, embryos were washed extensively in 1% BSA in PBS and then incubated with Cy3 goat anti-mouse IgM (1:400) (Chemicon International, Inc., Temecula, CA, USA) for 1 h at RT. Unbound secondary antibody was removed by washing with 1% BSA in PBS, and embryos were mounted in VectaShield mounting medium containing 42,6-diamidino-1-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA). Images were viewed using epifluorescence (Leica DMR; Leica Microsystems, Wetzlar, Germany) and confocal microscopy (Leica TCS). Inhibiting protein synthesis during the activation period with cycloheximide had no effect on lamin A/C assembly in 6 h post activation (hpa) parthenogenetic (35/35) and NT (7/7) embryos. The pronuclei of parthenogenetic (30/30) and NT (15/15) zygotes at 22 hpa were also positively labeled for lamin A/C. Nuclear labeling was observed in both parthenogenetic (25/25) and NT (12/12) 2-cell embryos. All parthenogenetic and NT embryos examined from the 4-cell stage through to blastocysts were stained negatively for lamin A/C. These results suggest that lamin A/C present in bovine NT zygotes is not due to aberrant reprogramming and that remodeling of the nuclear lamina occurs correctly in bovine NT embryos.

In vitro development of reconstructed bovine embryos and fate of donor mitochondria following nuclear injection of cumulus cells

Zygote ◽  
2001 ◽  
Vol 9 (3) ◽  
pp. 211-218 ◽  
Author(s):  
Jeong Tae Do ◽  
Kwon Ho Hong ◽  
Bo Yon Lee ◽  
Seung Bo Kim ◽  
Nam-Hyung Kim ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Formation Rate ◽  
Cumulus Cells ◽  
Control Group ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Cell Stage ◽  
Developmental Potential ◽  
Donor Cells

In this study we examined the developmental potential of reconstructed embryos and the fate of donor mitochondria during preimplantation development after nuclear transfer in cattle. Isolated cumulus cells were used as donor cells in nuclear transfer. Cumulus cells labelled with MitoTracker Green FM fluorochrome were injected into enucleated bovine MII oocytes and cultured in vitro. MitoTracker labelling on donor cells did not have a detrimental effect on blastocyst formation following nuclear transfer. Cleavage rate was about 69% (56/81) and blastocyst formation rate was 6.2% (5/81) at 7 days after nuclear transfer. The labelled mitochondria dispersed to the cytoplasm and became distributed between blastomeres and could be identified up to the 8- to 15-cell stage. Small patches of mitochondria were detected in some 8- to 15-cell stage embryos (5/20). However, donor mitochondria were not detected in embryos at the 16-cell stage and subsequent developmental stages. In the control group, mitochondria could be identified in arrested 1-cell embryos up to 7 days after nuclear transfer. These results suggest that disappearance of the labelled donor mitochondria in nuclear transfer bovine embryos is not due to fading of the fluorochrome marker, but is rather an as yet undefined cytoplasmic event.

scholarly journals 219 METHYLATION STATUS OF A DIFFERENTIALLY METHYLATED REGION (DMR) WITHIN THE BOVINE Igf2 GENE IN PREIMPLANTATION EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 260
Author(s):  
C. Gebert ◽  
C. Wrenzycki ◽  
D. Herrmann ◽  
R. Reinhardt ◽  
D. Gröger ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Vitro Fertilization ◽  
Nuclear Transfer ◽  
Methylation Status ◽  
Vitro Fertilization ◽  
Igf2 Gene ◽  
Donor Cells ◽  
Methylation Patterns

Specific DNA regions within imprinted genes become differentially methylated on the maternal and paternal chromosomes during germ cell development. These DMRs play a crucial role in the regulation of imprinted gene expression. The murine insulin-like growth factor2 gene (Igf2) is imprinted and contains an intragenic DMR within the last exon. Recently it became known that the bovine Igf2 gene is also imprinted (Dindot et al. 2004 Biol. Reprod. 71, 470–478) where we have now identified an intragenic DMR in the last exon with the paternal allele being methylated. Aberrant methylation patterns within the bovine Igf2 gene could result in deregulated gene expression and could therefore be involved in the development of fetal abnormalities such as the large offspring syndrome (LOS) in cattle. We have studied the methylation status of 27 CG dinucleotides within this DMR in bovine pre-implantation embryos of different origin by bisulfite sequencing. DNA was isolated from expanded blastocysts collected in vivo and generated by in vitro fertilization (IVF), somatic nuclear transfer (NT), and parthenogenesis (PA). Additionally, DNA was obtained from fibroblasts derived from a female and a male adult animal and used as donor cells for NT and from zygotes and 4-cell embryos both produced by IVF. After PCR amplification of the bisulfite-treated DNA, PCR products were cloned and sequenced. Methylation percentages were calculated for each individual clone by division of the 27 CpGs with the number of methylated CpGs per sample. The methylation levels (%) from each sample were then used to obtain the global methylation levels of the analyzed region. Methylation decreased during the transition from the zygote (28.4% ± 3.8 SEM) to the 4-cell embryo (6.3% ± 2.2 SEM) indicating that the DMR is demethylated after fertilization. An increased methylation level was observed in expanded blastocysts (in vivo: 10.2% ± 1.2 SEM; IVF: 10.1% ± 0.7 SEM; female NT: 12.4% ± 1.4 SEM). Thus, remethylation starts before the blastocyst stage. The higher methylation level of male NT blastocysts (22% ± 1.9 SEM) in comparison to their in vivo and IVF counterparts could be due to an insufficient reprogramming of the donor cells after nuclear transfer. Female and male donor cells were both heavily methylated (77% ± 2.2 SEM; 72% ± 2.9 SEM, respectively). Parthenogenetic expanded blastocysts were less methylated (2.3% ± 1 SEM), probably due to their diploid maternal genome. Results show for the first time that the methylation status at this DMR is associated with the origin of the embryo. Analysis of methylation patterns in pre-implantation embryos could provide a diagnostic tool to unravel mechanisms involved in fetal malformations often observed after the use of in vitro fertilization and/or nuclear transfer.

Cotreatment with RepSox and LBH589 improves the in vitro developmental competence of porcine somatic cell nuclear transfer embryos

2018 ◽  
Vol 30 (10) ◽  
pp. 1342 ◽  
Author(s):  
Zhao-Bo Luo ◽  
Long Jin ◽  
Qing Guo ◽  
Jun-Xia Wang ◽  
Xiao-Xu Xing ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Formation Rate ◽  
Epigenetic Reprogramming ◽  
Developmental Competence ◽  
Abnormal Development ◽  
Control Group ◽  
Blastocyst Formation

Accumulating evidence suggests that aberrant epigenetic reprogramming and low pluripotency of donor nuclei lead to abnormal development of cloned embryos and underlie the inefficiency of mammalian somatic cell nuclear transfer (SCNT). The present study demonstrates that treatment with the small molecule RepSox alone upregulates the expression of pluripotency-related genes in porcine SCNT embryos. Treatment with the histone deacetylase inhibitor LBH589 significantly increased the blastocyst formation rate, whereas treatment with RepSox did not. Cotreatment with 12.5 μM RepSox and 50 nM LBH589 (RepSox + LBH589) for 24 h significantly increased the blastocyst formation rate compared with that of untreated embryos (26.9% vs 8.5% respectively; P < 0.05). Furthermore, the expression of pluripotency-related genes octamer-binding transcription factor 4 (NANOG) and SRY (sex determining region Y)-box 2 (SOX2) were found to significantly increased in the RepSox + LBH589 compared with control group at both the 4-cell and blastocyst stages. In particular, the expression of NANOG was 135-fold higher at the blastocyst stage in the RepSox + LBH589 group. Moreover, RepSox + LBH589 improved epigenetic reprogramming. In summary, RepSox + LBH589 increases the expression of developmentally important genes, optimises epigenetic reprogramming and improves the in vitro development of porcine SCNT embryos.

scholarly journals Dapagliflozin Alleviates Renal Fibrosis by Inhibiting RIP1-RIP3-MLKL-Mediated Necroinflammation in Unilateral Ureteral Obstruction

2022 ◽  
Vol 12 ◽  
Author(s):  
Mei Ying Xuan ◽  
Shang Guo Piao ◽  
Jun Ding ◽  
Qi Yan Nan ◽  
Mei Hua Piao ◽  
...  
Keyword(s):  
Cell Death ◽  
Ureteral Obstruction ◽  
Renal Fibrosis ◽  
Apoptotic Cell ◽  
Subsequent Development ◽  
Unilateral Ureteral Obstruction ◽  
Apoptotic Cell Death ◽  
Inflammasome Activation ◽  
Renal Tubular

Dapagliflozin, a sodium-glucose cotransporter-2 inhibitor, offers renoprotection in diabetes. However, potential for use in nondiabetic kidney disease remains unknown. Herein, we assessed whether dapagliflozin alleviates renal fibrosis by interfering with necroinflammation in a rat model of unilateral ureteral obstruction (UUO) and in vitro. After induction of UUO, rats were administered dapagliflozin daily for seven consecutive days. UUO induced significant renal tubular necrosis and overexpression of RIP1-RIP3-MLKL axis proteins; these coincided with NLRP3 inflammasome activation, and subsequent development of renal fibrosis. Oxidative stress caused by UUO is tightly associated with endoplasmic reticulum stress and mitochondrial dysfunction, leading to apoptotic cell death through Wnt3α/β-catenin/GSK-3β signaling; all of which were abolished by both dapagliflozin and specific RIP inhibitors (necrostatin-1 and GSK872). In H2O2-treated HK-2 cells, dapagliflozin and RIP inhibitors suppressed overexpression of RIP1-RIP3-MLKL proteins and pyroptosis-related cytokines, decreased intracellular reactive oxygen species production and apoptotic cell death, whereas cell viability was improved. Moreover, activated Wnt3α/β-catenin/GSK-3β signaling was inhibited by dapagliflozin and Wnt/β-catenin inhibitor ICG-001. Our findings suggest that dapagliflozin ameliorates renal fibrosis by inhibiting RIP1-RIP3-MLKL-mediated necroinflammation via Wnt3α/β-catenin/GSK-3β signaling in UUO.

In vitro fertilisation of Chinese hamster oocytes by spermatozoa that have undergone ionophore A23187-induced acrosome reaction, and their subsequent development into blastocysts

Zygote ◽  
1996 ◽  
Vol 4 (2) ◽  
pp. 93-99 ◽  
Author(s):  
Hiroyuki Tateno ◽  
Yujiroh Kamiguchi
Keyword(s):  
Gas Phase ◽  
Acrosome Reaction ◽  
Chinese Hamster ◽  
Subsequent Development ◽  
In Vitro Fertilisation ◽  
Ionophore A23187 ◽  
Cell Stage ◽  
Developmental Arrest ◽  
Potential Use

SummaryTo enhance potential use of the Chinese hamster, Cricetulus griseus, in developmental and cytogenetic studies of mammalian gametes and embryos, techniques for in vitro fertilisation and embryo culture were developed in the species. Spermatozoa were recovered from the vasa deferentia of mature males, and incubated in modified TYH medium for 1 h at 37°C under 5% CO2 in air. They were then treated with ionophore A23187 (20¼M) for 10min to induce the acrosome reaction. Following ionophore treatment, superovulated oocytes were collected from hormonally stimulated females and incubated with the acrosome-reacted spermatozoa for 2 h at 37°C under 5% CO2 in air. In this study, 245 oocytes ova (98.0%) were determined to be monospermic. The monospermic ova were then cultured in TYH supplemented with 1mM hypotaurine under the same gas phase. Within 30h of fertilisation, 182 ova (93.8%) cleaved to the 2-cell stage, and subsequently 163 ova (84.0%) developed beyond the 2-cell stage. Thus, obstinate developmental arrest at the 2-cell stage(‘2-cell block’) was not observed in this species. Ultimately, 65.5% of monospermic ova reached morula to blastocyst stages.

Relationships between timing of syngamy, female age and implantation potential in human invitro-fertilised oocytes

2007 ◽  
Vol 19 (3) ◽  
pp. 482 ◽  
Author(s):  
Celine Lawler ◽  
H. W. Gordon Baker ◽  
David H. Edgar
Keyword(s):  
Multiple Logistic Regression Analysis ◽  
Implantation Rate ◽  
Subsequent Development ◽  
Multiple Logistic Regression ◽  
Predictors Of Outcome ◽  
Cell Stage ◽  
Female Age ◽  
Early Entry ◽  
Early Developmental

Although early developmental markers are frequently used to select embryos for transfer in human assisted reproduction, their value as independent predictors of outcome is often unclear. In this study, the value of using early syngamy and first cleavage as predictors of implantation potential of Day 2 embryos was investigated by examining their interrelationships with subsequent development, female age and implantation. Implantation rates were higher when syngamy occurred before 23–24 h post insemination even when all embryos analysed were transferred 42 h post insemination at the 4-cell stage (25.8 v. 11.9% for the later syngamy group; P < 0.01). Although there was a significant (r = 0.682; P < 0.001) relationship between earlier entry into syngamy and female age, earlier syngamy was still associated with a significantly higher implantation rate in Day 2 embryos with four blastomeres in women under 36 years of age (31.4 v. 15.4% for the later syngamy group; P < 0.05). The ability of timing of syngamy to predict implantation independent of other variables was confirmed by multiple logistic regression analysis. Although related to both subsequent embryo development and female age, early entry into syngamy is a predictor of implantation potential independent of both correlates in human Day 2 in vitro-fertilised embryos.

30 NUCLEAR AND MICROTUBULE DYNAMICS IN SOMATIC CELL NUCLEAR TRANSFER SHEEP EMBRYOS ACTIVATED WITH T-DIMETHYLAMINOPURINE AND CYCLOHEXIMIDE

2006 ◽  
Vol 18 (2) ◽  
pp. 123
Author(s):  
G. Coppola ◽  
B.-G. Jeon ◽  
B. Alexander ◽  
E. St. John ◽  
D. H. Betts ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Laser Scanning ◽  
Cell Cycle Progression ◽  
Treated Group ◽  
Blastocyst Development ◽  
Fetal Fibroblasts

The early reprogramming events following somatic cell nuclear transfer (SCNT) determine the fate of the cloned embryo and its development to a healthy viable offspring. In the present study, we undertook a detailed immunocytochemical study of the patterns of both microtubules and chromatin during the first cell cycle of sheep nuclear transfer embryos after fusion and artificial activation using either 6-dimethylaminopurine (6-DMAP) or cycloheximede (CHX). Sheep oocytes were collected from abattoir ovaries and matured in vitro for 18-20 h and enucleated; fetal fibroblasts were transplanted using standard SCNT techniques. Reconstructed cell-cytoplast couplets were fused and activated with ionomycin, followed by culture in two separate groups containing 6-DMAP (2 mM) or CHX (10 �g/mL) for 3 h. Following activation, embryos were cultured in in vitro culture (IVC) medium for blastocyst development. Embryos (n = 15, 3 replicates) were randomly removed from culture at various time points and stained using standard immunocytochemical methods to observe microtubule and nuclear configurations. Images were captured using laser scanning confocal microscopy. Results reveled that at 1 h post-fusion, 63.3% of reconstructed embryos underwent nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC) was apparent as chromosomes were situated on a non-polar spindle. The remaining embryos showed abnormal spindle and DNA configurations including chromosome outliers, congression failure, and non-NEBD. At 1 h post-activation (hpa), the embryos treated with 6-DMAP had already formed a clearly visible pronucleus (diameter 6-8 �m), whereas in the CHX-treated group, none of the embryos were at pronuclear stage; instead most of the latter embryos showed two masses of chromatin. At 1 hpa, 6-DMAP- and CHX-treated embryos showed one swelled pronucleus with a mean diameter of 8.4 � 1.3 �m and 25.8 � 0.8 �m, respectively (P < 0.05). At 16 hpa, embryos from both treatment groups still showed one swelled pronucleus. In the 6-DMAP-treated embryos, most of the embryos showed a metaphase spindle with aligned chromosomes of the first mitotic division as early as 18-10 hpa, whereas in the CHX-treated group embryos were still at the pronuclear stage. Typical 2-cell division was seen in most of the 6-DMAP-treated embryos between 24 and 30 hpa, but it was slightly delayed in CHX-treated embryos (32-35 hpa). Blastocyst development rates in the 6-DMAP- and CHX-treated groups were 21.4 � 5.6% and 14.0 � 6.3%, respectively (P < 0.05). In summary, artificial activating agents 6-DMAP and CHX exhibited different effects on chromatin remodeling, cell cycle progression, and the degree of pronuclear swelling which may explain the poor developmental rates and abnormal chromosome complements observed for cloned embryos. This work was funded by NSERC, OMAF, and International Council for Canadian Studies.

Sign in / Sign up

Export Citation Format

Share Document