Blastocyst Formation and Chromosome Statuses of Reconstructed Embryos Derived from Interspecies Somatic Cell Nuclear Transfer (iSCNT)

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.

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Early development of reconstructed embryos after somatic cell nuclear transfer in a non-human primate

Theriogenology ◽

10.1016/j.theriogenology.2006.04.012 ◽

2006 ◽

Vol 66 (5) ◽

pp. 1300-1306 ◽

Cited By ~ 4

Author(s):

Naiqing Chen ◽

Swee-Lian Liow ◽

Wan-Yue Yip ◽

Lay-Geok Tan ◽

Guo-Qing Tong ◽

...

Keyword(s):

Somatic Cell ◽

Early Development ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Reconstructed Embryos ◽

Human Primate

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24 BUFFALO (BUBALUS BUBALIS) SOMATIC CELL NUCLEAR TRANSFER EMBRYOS PRODUCED FROM FROZEN–THAWED SEMEN-DERIVED SOMATIC CELLS: EFFECT OF TRICHOSTATIN A ON THE IN VITRO AND IN VIVO DEVELOPMENTAL POTENTIAL, QUALITY, AND EPIGENETIC STATUS

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab24 ◽

2015 ◽

Vol 27 (1) ◽

pp. 104

Author(s):

N. L. Selokar ◽

M. Saini ◽

H. Agrawal ◽

P. Palta ◽

M. S. Chauhan ◽

...

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Trichostatin A ◽

Somatic Cells ◽

Developmental Potential ◽

Reconstructed Embryos ◽

Frozen Semen ◽

Significant Difference

Cryopreservation of semen allows preservation of somatic cells, which can be used for the production of progeny through somatic cell nuclear transfer (SCNT). This approach could enable restoration of valuable high-genetic-merit progeny-tested bulls, which may be dead but the cryopreserved semen is available. We have successfully produced a live buffalo calf by SCNT using somatic cells isolated from >10 year old frozen semen (Selokar et al. 2014 PLoS One 9, e90755). However, the calf survived only for 12 h, which indicates faulty reprogramming of these cells. The present study was, therefore, carried out to study the effect of treatment with trichostatin A (TSA), an epigenetic modifier, on reprogramming of these cells. Production of cloned embryos and determination of quality and level of epigenetic markers in blastocysts were performed according to the methods described previously (Selokar et al. 2014 PLoS One 9, e90755). To examine the effects of TSA (0, 50, and 75 nM), 10 separate experiments were performed on 125, 175, and 207 reconstructed embryos, respectively. The percentage data were analysed using SYSTAT 12.0 (SPSS Inc., Chicago, IL, USA) after arcsine transformation. Differences between means were analysed by one-way ANOVA followed by Fisher's least significant difference test for significance at P < 0.05. When the reconstructed buffalo embryos produced by hand-made clones were treated with 0, 50, or 75 nM TSA post-electrofusion for 10 h, the cleavage percentage (100.0 ± 0, 94.5 ± 2.3, and 96.1 ± 1.2, respectively) and blastocyst percentage (50.6 ± 2.3, 48.4 ± 2.7, and 48.1 ± 2.6, respectively), total cell number (274.9 ± 17.4, 289.1 ± 30.1, and 317.0 ± 24.2, respectively), and apoptotic index (3.4 ± 0.9, 4.5 ± 1.4, and 5.6 ± 0.7, respectively) in Day 8 blastocysts were not significantly different among different groups. The TSA treatment increased (P < 0.05) the global level of H4K5ac but not that of H3K18a in embryos treated with 50 or 75 nM TSA compared with that in controls. In contrast, the level of H3K27me3 was significantly lower (P < 0.05) in cloned embryos treated with 75 nM TSA than in embryos treated with 50 nM TSA or controls. The ultimate test of the reprogramming potential of any donor cell type is its ability to produce live offspring. To examine the in vivo developmental potential of the 0, 50, or 75 nM TSA treated embryos, we transferred Day 8 blastocysts, 2 each to 5, 6, and 5 recipients, respectively, which resulted in 2 pregnancies from 75 nM TSA treated embryos. However, one pregnancy was aborted in the first trimester and the other in the third trimester. In conclusion, TSA treatment of reconstructed embryos produced from semen-derived somatic cells alters their epigenetic status but does not improve the live birth rate. We are currently optimizing an effective strategy to improve the cloning efficiency of semen-derived somatic cells.

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29 TREATMENT OF DONOR CELLS WITH XENOPUS OOCYTE EXTRACT ENHANCED SOMATIC CELL NUCLEAR TRANSFER EMBRYO DEVELOPMENT

Reproduction Fertility and Development ◽

10.1071/rdv24n1ab29 ◽

2012 ◽

Vol 24 (1) ◽

pp. 126

Author(s):

X. Yang ◽

J. Mao ◽

E. M. Walters ◽

M. T. Zhao ◽

K. Lee ◽

...

Keyword(s):

Somatic Cell ◽

Embryo Development ◽

Natural Science ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Control Group ◽

Blastocyst Formation ◽

Donor Cells ◽

Fetal Fibroblasts ◽

Frog Oocyte

Somatic cell nuclear transfer (SCNT) efficiency in pigs and other species is still very low. This low efficiency and the occurrence of developmental abnormalities in offspring has been attributed to incomplete or incorrect reprogramming. Cytoplasmic extracts from both mammalian and amphibian oocytes can alter the epigenetic state of mammalian somatic nuclei as well as gene expression to more resemble that of pluripotent cells. Rathbone et al. (2010) has showed that pretreating somatic donor cells with frog oocyte extract (FOE) increased live birth in ovine. Liu et al. (2011) also reported that treating donor cells with FOE enhanced handmade clone embryo development in pigs. The aim of this study was to evaluate the early development of cloned embryos produced with porcine GFP fibroblasts pre-treated with a permeabilizing agent, digitonin and matured frog oocyte extract. Frog egg cytoplasmic extract was prepared from one frog's oocytes after being matured in vitro to MII stage. The experiment included 2 groups. In the FOE-treated group, GFP-tagged fetal fibroblasts were permeabilized by digitonin (15 ng mL–1) and incubated in FOE containing an ATP-regenerating system (2.5 mM ATP, 125 μM GTP, 62.5 μg mL–1 of creatine kinase, 25 mM phosphocreatine and 1 mM NTP) at room temperature (24°C) for 2 h; cell membranes were re-sealed by culturing in 10% FBS in DMEM media for 2.5 h at 38.5°C before used as donor cells. In the control group, the same donor cells were treated with digitonin, but without frog oocyte extract incubation. The SCNT embryos were produced by using the 2 groups of donor cells as described above. In total, 305 control and 492 FOE oocytes were enucleated from 8 biological replicates. Two hundred fifty control and 370 FOE couplets were fused and cultured in porcine zygote medium 3. Percent cleavage was recorded on Day 2 and the percent blastocyst formation was determined on Day 7 (SCNT day = 0). In addition, the number of nuclei in the blastocysts was recorded on Day 7. Percent fusion, cleavage, blastocyst formation and number of nuclei in blastocysts were analysed by using SAS software (v9.2), with day and treatment class as main effects. There was no difference in percent fusion (FOE, 76.2 ± 2.5% vs control, 80.8 ± 2.8%) or in cleavage (FOE: 74.8 ± 2.5% vs control: 74.6 ± 2.9%). Only green blastocysts with 16 or more nuclei were considered to be a true SCNT blastocyst. The percent blastocyst was higher in the FOE group than that in the control (13.9 ± 0.8% vs 9.5 ± 0.9%, P < 0.05), whereas the number of nuclei in the blastocysts was not different between the 2 groups (39.7 ± 2.4, 35.9 ± 3.8 for FOE and control, respectively). In conclusion, our study demonstrated that pre-treatment of donor cells with digitonin and Xenopus MII oocyte extract increased porcine SCNT embryo development to blastocyst and cloning efficiency. Funded by the National Natural Science Foundation of China (NO. 31071311), Natural Science Foundation of Fujian Province of China (No. 2009J06017) and NIH U42 RR18877.

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15 Combination of RepSox with histone deacetylation inhibitors on invitro development competence of porcine somatic cell nuclear transfer embryos

Reproduction Fertility and Development ◽

10.1071/rdv32n2ab15 ◽

2020 ◽

Vol 32 (2) ◽

pp. 133

Author(s):

Z.-B. Luo ◽

M.-F. Xuan ◽

Z.-Y. Li ◽

X.-J. Yin ◽

J.-D. Kang

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Histone Deacetylase Inhibitors ◽

Formation Rate ◽

Epigenetic Reprogramming ◽

Histone Deacetylation ◽

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). In this study, we compared histone deacetylase inhibitors combined with the pluripotency inducer RepSox on invitro development of porcine embryos produced via SCNT. Porcine embryos were treated with valproic acid (VPA), mocetinostat, M344 and panobinostat (LBH589) after SCNT, respectively. The porcine embryo invitro-development competence, histone modification level, and pluripotency-related genes expression were analysed. The results showed that LBH589 significantly increased the blastocyst formation rate compared with mocetinostat, M344, and control. In addition, VPA treatment increased the blastocyst formation rate of SCNT porcine embryos; both VPA-treated and the untreated clones developed to term, but offspring from VPA-treated embryos had a lower survival to adulthood than those from control embryos (18.2 vs. 67.0%; P<0.05). Furthermore, cotreatment with 12.5mM RepSox and 50 nM LBH589 (RepSox+LBH589) for 24h significantly increased the blastocyst formation rate compared with that of untreated embryos (26.9 vs. 8.5%, respectively; P<0.05). Moreover, RepSox + LBH589 improved epigenetic reprogramming by histone acetylation and methylation. The expression of pluripotency-related genes NANOG and SOX2 was found to be 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. In summary, RepSox + LBH589 increases the expression of developmentally important genes, optimises epigenetic reprogramming, and improves the invitro development of porcine SCNT embryos.

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Glucose in a maturation medium with reduced NaCl improves oocyte maturation and embryonic development after somatic cell nuclear transfer and in vitro fertilization in pigs

Zygote ◽

10.1017/s0967199420000891 ◽

2021 ◽

pp. 1-8

Author(s):

Yongjin Lee ◽

Hanna Lee ◽

Joohyeong Lee ◽

Seung Tae Lee ◽

Geun-Shik Lee ◽

...

Keyword(s):

Embryonic Development ◽

Somatic Cell ◽

Oocyte Maturation ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Blastocyst Formation ◽

Nuclear Maturation ◽

Maturation Medium ◽

Glucose Supplementation

Summary This study was conducted to examine whether glucose in maturation medium containing reduced NaCl could improve oocyte maturation and embryonic development in pigs. The base medium was bovine serum albumin-free porcine zygote medium (PZM)-3 containing 10% (v/v) pig follicular fluid (FPZM) or 0.1% (w/v) polyvinyl alcohol (PPZM). Using each medium, the effects of NaCl concentrations (108 and 61.6 mM) and 5.56 mM glucose supplementation (designated as PZM108N, PZM108G, PZM61N, and PZM61G, respectively) were examined using a 2 × 2 factorial arrangement. When oocytes were matured in FPZM, glucose supplementation improved nuclear maturation compared with no supplementation, regardless of the NaCl concentrations. FPZM61G showed a higher blastocyst formation compared with FPZM108N and FPZM108G after parthenogenesis (PA). Blastocyst formations of somatic cell nuclear transfer (SCNT) embryos derived from FPZM61N and FPZM61G were higher compared with those of oocytes from FPZM108N. When oocytes were matured in PPZM, glucose added to PPZM108 and PPZM61 increased nuclear maturation compared with no supplementation. However, glucose added to PPZM108 did not alter embryonic development after PA. Additionally, oocytes matured in PPZM61G showed a higher blastocyst formation compared with those from PPZM61N. In SCNT, blastocyst formation was not influenced by glucose supplementation of PPZM108, but was increased by maturation in glucose-supplemented PPZM61. In embryonic development of in vitro fertilization (IVF), oocytes matured in medium with reduced NaCl and glucose showed significantly higher blastocyst formation compared with those matured in PPZM108G. Our results demonstrated that glucose in maturation medium containing 61.6 mM NaCl increased oocyte maturation and embryonic development after PA, SCNT, and IVF.

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Porcine somatic cell nuclear transfer donor sources affect transplant success: A meta-analysis

Indian Journal of Animal Research ◽

10.18805/ijar.b-943 ◽

2018 ◽

Author(s):

Zhenhua Guo ◽

Lei Lv ◽

Di Liu ◽

Zhongqiu Li

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Meta Analysis ◽

Donor Cell ◽

Blastocyst Formation ◽

Embryo Viability ◽

Live Births ◽

Reconstructed Embryo

Herd boars, male domestic pigs used for stud, are economically important, and somatic cell nuclear transfer (SCNT) is a promising technology to expand herd boar yields. However, live births are dictated by donor cell source, and fetal donors may offer more advantages than adult donors. A meta-analysis was conducted to better understand how donor sources affect SCNT outcomes. Of the 1,431 records viewed, 10 were selected for review. Blastocyst formation rates, successful pregnancies, and live births were assessed to measure efficacy. SCNT blastocyst formation differed between adult and fetal donors among the studies. SCNT pigs had more malformed fetuses as well, which negatively affected the post-birth mortality. Organs of porcine fetuses are limited by deficiencies of maternal nutrient and growth hormones, which compromise post-birth adaptations. SCNT pregnancy success is neither determined by donor source nor by live births. Live births are also tied to donor age. Embryos from fetal donors are more frequently healthy likely due to less differentiation and less reprogramming of reconstructed embryos. Adult donors in contrast have more cell differentiation and as such accumulate more mutations and damage. This may reduce reconstructed embryo viability. Finally, SCNT efficiency may be improved with more in vitro passages, but more work is required to validate this concept.

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Effects of ovary storage temperature and embryo vitrification on somatic cell nuclear transfer outcomes in goats

Reproduction Fertility and Development ◽

10.1071/rd18529 ◽

2020 ◽

Vol 32 (4) ◽

pp. 419 ◽

Cited By ~ 1

Author(s):

Mehdi Hajian ◽

Farnoosh Jafarpour ◽

Sayed Morteza Aghamiri ◽

Shiva Rouhollahi Varnosfaderani ◽

Mohammad Hossein Nasr Esfahani

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Storage Temperature ◽

Storage Conditions ◽

Farm Animals ◽

Blastocyst Formation ◽

Term Pregnancy ◽

Developmental Potential ◽

Genetic Potential

Improving the genetic potential of farm animals is one of the primary aims in the field of assisted reproduction. In this regard, somatic cell nuclear transfer (SCNT) can be used to produce a large number of embryos from genetically elite animals. The aims of the present study were to assess the effects of: (1) ovary storage conditions on preimplantation development of recovered oocytes and the freezability of the derived blastocysts; and (2) vitrification of goat SCNT-derived blastocysts on postimplantation development. Goat oocytes were recovered from ovaries and stored under warm (25°C-27°C) or cold (11°C-12°C) conditions before being used to produce SCNT embryos. There were no differences in oocytes recovered from ovaries kept under cold versus warm storage conditions in terms of cleavage (mean (±s.d.) 95.68±1.67% vs 95.91±2.93% respectively) and blastocyst formation (10.69±1.17% vs 10.94±0.9% respectively) rates. The re-expansion rate of vitrified blastocysts was significantly lower for cold- than warm-stored ovaries (66.3±8.7% vs 90±11% respectively). To assess the effects of vitrification on postimplantation development, blastocysts from cold-stored ovaries only were transferred from fresh and vitrified–warmed groups. The pregnancy rate was comparable between the fresh and vitrified–warmed groups (41.65% and 45.45% respectively). In addition, established pregnancy in Day 28-38 and full-term pregnancy rates were similar between the two groups. In conclusion, this study shows similar invitro preimplantation developmental potential of warm- and cold-stored ovaries. This study introduces the vitrification technique as an appropriate approach to preserve embryos produced by SCNT for transfer to recipient goats at a suitable time.

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28 TREATMENT WITH SUBEROYLANILIDE HYDOXAMIC ACID OR SODIUM BUTYRATE ON PORCINE SOMATIC CELL NUCLEAR TRANSFER EMBRYOS DERIVED FROM KIDNEY CELLS OF HUMAN HEME OXYGENASE-1 TRANSGENIC PIG

Reproduction Fertility and Development ◽

10.1071/rdv26n1ab28 ◽

2014 ◽

Vol 26 (1) ◽

pp. 128

Author(s):

K.-Y. Song ◽

J.-H. Moon ◽

E.-J. Park ◽

S.-J. Kim ◽

Y.-B. Choi ◽

...

Keyword(s):

Gene Expression ◽

Somatic Cell ◽

Sodium Butyrate ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Total Cell ◽

Heme Oxygenase 1 ◽

Kidney Cells ◽

Cloning Efficiency ◽

Blastocyst Formation

Because somatic cell nuclear transfer (SCNT) is influenced by many factors concerning a series of various steps, the cloning efficiency is low in so many species and it seems to be more serious in production of transgenic (TG) animals. Reprogramming of donor nucleus is one of the important factors that affects the developmental competence of SCNT embryos, and several epigenetic remodelling drugs have been used to improve the cloning efficiency. In this study, we examined the effect of suberoylanilide hydoxamic acid (SAHA) or sodium butyrate (NaBu) treatment on the development of porcine SCNT embryos derived from kidney cells of TG pig. Fully confluent porcine kidney cells expressing the human heme oxigenase-1 gene were used for nuclear donor. For SCNT, matured oocytes with 1st polar body were enucleated, electrically fused, and activated 1 h after fusion (Song et al. 2009 Mol. Reprod. Dev. 76, 611–619). Then, SCNT embryos were incubated in postactivation medium [PA; porcine zygote medium-5 (PZM-5) supplemented with 0.5% dimethyl sulfoxide] for 4 h (control), PA with 0.4 μg mL–1 demecolcine for 4 h (Dc), PA with 0.5 μM SAHA for 9 h (SAHA), or PA with 1 mM NaBu for 9 h (NaBu). After postactivation treatment, SCNT embryos were cultured in fresh PZM-5 for 7 days. The embryos were examined for cleavage and blastocyst formation on Days 2 and 7, respectively (the day of SCNT was designated Day 0). Total cell number of blastocysts was examined by counting the number of nuclei stained with Hoechst 33342 under ultraviolet light. Complementary DNA synthesised with total RNA extracted from blastocysts were used for qRT-PCR to determine HDAC2, HDAC6, and GAPDH gene expression. Data were analysed by one-way ANOVA followed by Tukey's multiple comparison test using GraphPad Prism version 5.01 (Graphpad Software, San Diego, CA, USA). The cleavage rates (77.0–82.9%) of treated embryos were not different from that of control embryos (79.0%). Blastocyst formation was slightly increased in Dc- (36/132, 27.3%), SAHA- (34/125, 28.6%), and NaBu- (36/133, 27.3%) treated embryos than in control embryos (32/128; 25.0%), but the difference was not significant. Total cell numbers (45.2–47.5) of treated embryos were not different from that of control embryos (51.8). Expression of HDAC2 was higher in SAHA-treated embryos than in control and Dc-treated embryos (P < 0.05), but it was not different from that of NaBu-treated embryos. The relative expression of HDAC6 transcript was increased in SAHA- and NaBu-treated embryos, but there was no significant difference among all groups. Although SAHA or NaBu did not improve the pre-implantational development of porcine SCNT embryos derived from kidney cells of TG pig as assessed in this study, additional studies are needed to determine the effect of SAHA or NaBu on gene expression of pig TG embryos and developmental competency following embryo transfer according to the origin of donor cells. This study was supported by IPET (#311011-05-2-SB010), MOTIE (#10033839-2012-21) and the TS Corporation.

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49 EARLY ASPECTS OF NUCLEAR REPROGRAMMING FOLLOWING BOVINE SOMATIC CELL NUCLEAR TRANSFER

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab49 ◽

2008 ◽

Vol 20 (1) ◽

pp. 105

Author(s):

I. Petrovicova ◽

O. Svarcova ◽

F. Strejcek ◽

A. Lucas-Hahn ◽

E. Lemme ◽

...

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Transcriptional Activity ◽

Cell Complex ◽

Chromatin Dynamics ◽

Reconstructed Embryos ◽

Transmission Electron ◽

Single Block ◽

Fibrillar Component

This study was designed in order to evaluate the global transcription and morphology of reprogramming during somatic cell nuclear transfer (SCNT). Following 20–22 h of IVM, couplets of MII cytoplasts and starved bovine fibroblasts were, after 2 h co-culture, electrically fused, chemically activated by 5 µm ionomycin for 5 min, followed by a 3–4 h incubation in 2 mm 6-DMAP, and fixed at 0.5, 1, 1.5, 2, 3, 4, 8, 12, and 16 h post-activation (hpa). The reconstructed embryos were processed for lacmoid staining, autoradiography following [3H]uridine incubation, transmission electron microscopy (TEM), and immunofluorescence (4 and 12 hpa) in order to evaluate chromatin dynamics, transcriptional activity, nuclear and nucleolar ultrastructure, and localization of nucleolar proteins (upstream binding factor (UBF) and fibrillarin), respectively, during the first cell cycle. Likewise, starved fibroblasts were fixed and processed for autoradiography and TEM. The fibroblasts displayed strong transcriptional activity and active fibrillo-granular nucleoli. None of the reconstructed embryos, however, displayed transcriptional activity. During first 3 hpa, the majority of the embryos displayed a single block of condensed chromatin surrounded by a more or less complete nuclear envelope (NE) and an abundant population of elongated somatic cell mitochondria. This somatic cell complex was located peripherally in the ooplasm. At the subsequent time points, the embryos displayed pronuclear-like structures, which from 8 hpa were located centrally in the ooplasm. From about 4 hpa, the somatic cell complex had dispersed and the elongated mitochondria could no longer be tracked. The first nucleolus-related structures were observed at 1.5 hpa and only in nuclei with a complete NE. At 1.5 to 4 hpa, the nucleolus-related structures appeared either as bodies presenting a large fibrillar center and presumptive dense fibrillar component, but no granular component, or as electron-dense nucleolus precursor bodies (NPBs). From 4 hpa and onward, only compact NPBs were observed. At 4 and 12 hpa, UBF was localized into small discrete clusters of foci enclosed in a shell-like structure labeled by fibrillarin in the nucleus. In conclusion, at SCNT, the somatic cell cytoplasm remains structurally organized in a somatic cell complex over the initial 3–4 hpa. During the same period, the somatic cell chromatin undergoes condensation and the NE is partially dissolved. Subsequently, pronucleus-like euchromatic nuclei with typical NPBs are formed, although somatic cell nucleolar components may be temporarily seen. Throughout the process, transcription is repressed. This work was supported by Marie Curie Intra-European Fellowships (MEIF-CT-2006-021629), and by grant VEGA 1/3255/06 and DFG.

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