33DEVELOPMENT OF BOVINE EMBRYOS CLONED WITH FETAL FIBROBLASTS ARRESTED 
AT G0/G1 PHASE BY DIFFERENT TREATMENTS

Numerous factors have an effect on the development of cloned embryos, and one of the most important might be the synchronization between donor nuclei and recipient ooplasts. The objective of this study was to examine the effect of donor cell treatments for G0/G1 synchronization and the donor cell type on development and incidence of apoptosis in cloned cattle embryos. Primary cultures were established from a female fetus on Day 50 of gestation and adult ear skin biopsies. The cells were used for assessements of cell cycle and apoptosis, and for nuclear transfer. Cells were randomly allocated into 3 experimental treatment groups after 6–8 passages: Group 1 (confluent), cells were cultured in DMEM supplemented with 10% FBS until 90% confluent; Group 2 (serum-starvation), cells were cultured in DMEM supplemented with 0.5% FBS for 5 days; Group 3 (Roscovitine), cells were cultured in DMEM supplemented with 10% FBS and 30μM Roscovitine for 12h. Cell cycle and apoptosis were analyzed using flow cytometry after labelling with DAPI and YO-PRO-1, respectively. At 19h post-maturation (hpm), enucleated oocytes were reconstructed with donor cells and fused by a single DC pulse (1.6kV/cm, 60μs) delivered by a BTX 200. After activation with the combination of ionomycin (5μM, 5min) and cycloheximide (10μgmL−1, 5h), the eggs were cultured in CR1aa medium for 3 days and additionally cultured in CR1aa medium supplemented with 30mgmL−1 BSA for 5 days at 39°C in a humidified atmosphere of 5% CO2 in air. Differences between groups were analyzed using one-way ANOVA after arc-sine transformation of the proportional data. There were no significantly differences in the incidence of cells arrested at G0/G1 for fetal fibroblasts cultured in the three treatment groups (87%, 83% and 80%; confluent, serum starvation and Roscovitine, respectively). More cells were apoptotic in Group 2 compared to the cells in Groups 1 and 3 (12% v. 6 and 6%, respectively) (P<0.05). Blastocyst development of cloned embryos was significantly (P<0.05) higher when fetal fibroblasts from Group 1 were used, compared to Groups 2 and 3 (35.1%, v. 31 and 29.7%, respectively). Similar results were observed in the use of ear skin fibroblasts as nuclear transfer donor cells (32.7%, v. 24 and 24%, respectively). These results suggest that fetal fibroblasts can be effectively synchronized at G0/G1 by three different treatments, including growth to confluence, serum-starvation and Roscovitine treatment. However, based on blastocyst development and levels of apoptosis, the use of confluent fetal fibroblasts as donor cells is more effective than using cells synchronized by serum-starvation or Roscovitine treatment in the production of cloned bovine embryos. [Supported by High Technology Development Project for Agriculture and Forestry Korea, MAF-SGRP, 30012-05-3-SB010 and Cho-A Pharm. Co. LTD.]

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68EFFECT OF ROSCOVITINE ON FIBROBLASTS' ABILITY TO FORM BLASTOCYSTS AND ESTABLISH PREGNANCIES AFTER BOVINE NUCLEAR TRANSFER

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab68 ◽

2004 ◽

Vol 16 (2) ◽

pp. 155

Author(s):

A.M. Powell ◽

P. Graininger ◽

N. Talbot ◽

R.J. Wall

Keyword(s):

Nuclear Transfer ◽

Donor Cell ◽

Culture Conditions ◽

Contact Inhibition ◽

Serum Starvation ◽

Blastocyst Development ◽

Variable Effect ◽

Return Rates ◽

Fetal Fibroblasts

Cloning efficiency of fibroblast nuclear transfer is dependent on donor cell chromatin status. Chromatin status is commonly regulated by serum starvation or contact inhibition. We have tested 3 methods of synchronizing chromatin activity, roscovitine exposure (in MEM + 10% serum) for 24h, with serum starvation (0.5% serum) for 5 days or growth to confluence in 10% serum prior to nuclear transfer. Roscovitine, a specific cyclin-dependent kinase (CDK)2 inhibitor, provides a means of precisely synchronizing bovine fetal fibroblasts (BFF) at G0/G1 cell cycle stage. Fibroblasts were from 100-day-old Jersey fetuses. Cells, frozen at passage 2, from fetus 10 are known to produce calves. Fetus 13 cells, frozen at passages 1 and 2, were compared for their ability to serve as nuclear donor cells. Oocytes, either purchased from Bomed or harvested from ovaries obtained from a local slaughterhouse and matured in Ham’s F10, were enucleated between 18–21h post-maturation initiation. Couplets were produced and fused by standard techniques. Embryos were activated 2 to 4 hours after fusion by exposure to ionomycin for 4min and DMAP for 4h. Embryos were then held in CR1aa for 12h before being cultured in G1 media for 3 days and then G2 media for another 3 days (38.5°C and 5% O2 + 5% CO2 + 90% N). On Day 7, good quality blastocysts were transferred to synchronized recipient heifers. The remaining embryos were evaluated after another day in culture. Blastocyst development [(100) X (total blastocysts/fused couplets)] was not influenced by fetus (BFF10, 31±3%; BFF13, 26±2%, P=0.126). However, a higher proportion of blastocysts were produced when fibroblasts were cultured in 0.5% serum (38±3%) compared to culture in 10% serum (29±3%) or in roscovitine (23±2%, P=0.001). Time in culture, as measured by passage, had a variable effect on the fibroblast’s ability to product blastocysts from the three fibroblast culture conditions tested. Passage 1 and 2 fibroblasts responded similarly to the 0.5% and 10% serum treatments (P>0.80). When cultured in roscovitine, passage 1 fibroblasts performed better then passage 2 fibroblasts (29±4% v. 16±3% blastocysts, P=0.010). Embryos have been transferred to 51 recipients to date. Ten recipients have given birth or are still pregnant. The 60-day non-return rate for those animals was 29%, 50%, and 31% for serum starvation, 10% serum, and roscovitine treatments, respectively. BFF10 and BFF13 cells have generated the same non-return rates (33%). In this study, of the 3 methods of synchronizing fibroblast chromatin, serum-starvation had an in vitro advantage. Cells cultured for different lengths of time (passages) responded differently to synchronization treatments. This may reflect a heterogeneous population of cells at early passages. Current non-return rates seem to favor synchronization by contact inhibition. Any advantage roscovitine offers may not be revealed until calving.

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38 RABBIT NUCLEAR TRANSFER AND IN VIVO-FERTILIZED EMBRYOS FAIL TO EXPRESS A MOUSE Oct-4 PROMOTER-DRIVEN EGFP REPORTER GENE

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab38 ◽

2007 ◽

Vol 19 (1) ◽

pp. 137 ◽

Cited By ~ 3

Author(s):

R. Hao ◽

A. Wuensch ◽

R. Klose ◽

E. Wolf ◽

V. Zakhartchenko

Keyword(s):

Dna Methylation ◽

Fluorescence Microscopy ◽

Reporter Gene ◽

Nuclear Transfer ◽

Donor Cell ◽

Bovine Embryos ◽

Egfp Gene ◽

Gene Construct ◽

Fetal Fibroblasts

Reprogramming of a donor cell genome during somatic cell nuclear transfer (SCNT) is largely dependent on appropriate expression of 'pluripotency'? genes, such as Oct-4 (POU5F1). Recently, we transfected bovine fetal fibroblasts with GOF18-ΔPE-EGFP, a reporter gene construct for the Oct-4 promoter and assessed the expression of Oct-4 after SCNT (Wuensch et al. 2006 Reprod. Fertil. Dev. 18, 144). Our previous study on DNA methylation reprogramming revealed that rabbit in vivo-fertilized and cloned embryos differ from bovine embryos in respect to this epigenetic modification (Shi et al. 2004 Biol. Reprod. 71, 340–347), suggesting differences in the mechanism of epigenetic reprogramming between these two species. In this study, we tested whether GOF18-ΔPE-EGFP could be used to monitor Oct-4 expression in rabbit cloned embryos. The reporter gene construct included the EGFP gene flanked by a 9-kb fragment of the murine Oct-4 upstream region with a deletion in the proximal enhancer (PE) and a 9-kb fragment containing the nontranscribed murine structural Oct-4 gene. The 21.2-kb fragment GOF18-DPE-EGFP was released from the vector backbone by NotI digestion and purified with QIAquickGel Extraction Kit (Qiagen, Hilden, Germany) after gel electrophoresis. Four stable transfected colonies of rabbit fetal fibroblasts (RFF), none of which exhibited green fluorescence, were used for SCNT. The resulting embryos were examined on Days 2–5 by fluorescence microscopy. To detect endogenous Oct-4 expression, in vivo-fertilized embryos were stained with anti-mouse Oct-3 antibody and then incubated with secondary Alexa 488-conjugated goat anti-mouse antibody. The most prominent endogenous Oct-4 expression was detected in in vivo-fertilized embryos at the morula and blastocyst stages. Depending on the donor cell line used for nuclear transfer, cleavage and blastocyst rates ranged from 56 to 97% and from 33 to 49%, respectively. When a total of 230 cloned embryos at the 2-to 16-cell stages and 93 cloned morulae and blastocycts were examined by fluorescence microscopy, none of the examined embryos exhibited fluorescence signals indicating the lack of Oct-4 promoter activity. Taking into account the fact that both cloned and in vivo-fertilized rabbit embryos have specific patterns of DNA methylation reprogramming, which are different from that of bovine embryos, we injected GOF18-ΔPE-EGFP gene constructs into pronuclei of in vivo-fertilized zygotes. None of the 74 injected embryos, which were examined at the 2-cell to blastocyst stages, showed fluorescence signals. Our results demonstrate that rabbit nuclear transfer and in vivo-fertilized embryos are unable to activate a mouse Oct-4 promoter-reporter construct. Potential reasons include incompatibilities between mouse Oct-4 promoter sequences and rabbit transcription factors as well as specific mechanisms of epigenetic reprogramming in the rabbit. This work was supported by the Bayerische Forschungsstiftung.

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156INFLUENCE OF SERUM AND BSA ON BLASTOCYST DEVELOPMENT AND HATCHING IN IVF AND NUCLEAR TRANSFER BOVINE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab156 ◽

2004 ◽

Vol 16 (2) ◽

pp. 200

Author(s):

G.F. Mastromonaco ◽

E. Semple ◽

C. Robert ◽

J. Rho ◽

D. Betts ◽

...

Keyword(s):

Fatty Acid ◽

Embryo Development ◽

Nuclear Transfer ◽

Culture Media ◽

Adverse Outcomes ◽

Bovine Embryos ◽

Blastocyst Development ◽

Treatment Groups ◽

Short Term Exposure ◽

Hatching Rates

Important differences exist between in vivo- and in vitro-produced bovine embryos. Studies have shown that various components in culture media affect embryo development, with serum producing some of the more detrimental effects. Efforts to develop a serum-free culture system have included looking at the effects of BSA, polyvinyl pyrrolidone and polyvinyl alcohol on embryo development. In this study, we compare serum and BSA during oocyte maturation and embryo culture of IVF and nuclear transfer (NT) embryos. Experiment A: Oocytes were aspirated from follicles and matured in either collection medium (Hams F-10+2% steer serum (SS); F-10) or in follicular fluid alone (FF). They were subjected to IVM-IVF-IVC as follows: 20–22h maturation in synthetic oviductal fluid +8mgmL−1 fatty acid-free BSA (SOF+BSA-FAF) supplemented with hormones, 18h co-incubation with sperm in IVF-TALP, and culture for 9 days in SOF+BSA-FAF. Experiment B: Oocytes were randomly distributed for IVM-IVF-IVC into the following treatment groups: (i) IVM and IVC in SOF+2% SS (SER), (ii) IVM in SOF+2% SS and IVC in SOF+BSA-FAF (SER-FAF), (iii) IVM and IVC in SOF+BSA-FAF (FAF), and (iv) IVM and IVC in SOF+BSA-FrV (FrV). Experiment C: Oocytes were matured for 18h in either SOF+2% SS (SER) or SOF+BSA-FAF (FAF). Couplets were constructed with adult skin fibroblasts, exposed to a single pulse of 1.5kVcm−1 for 40s and activated using ionomycin and cycloheximide. Embryos were cultured in SOF+BSA-FAF. Three replicates with 100–120 oocytes per treatment group were carried out. Results: Cleavage rates were similar among all treatments in experiments A and B. No differences were observed between oocytes collected in F-10 or FF indicating that short-term exposure to serum does not have long-term effects on embryo development. Although a higher number of blastocysts was observed in the SER group on Day 6 (3.2% v. <0.5%; P<0.05), no differences were seen in blastocyst development among the IVF treatment groups from Day 8 onwards (SER: 29.7%, SER-FAF: 21.1%, FAF: 20.4%, FrV: 19.9%). However, hatching rates on Days 8 and 9 were significantly greater (P<0.05) in groups with serum, with the exception of FAF on Day 9 (SER: 31.1%, 57.2%; SER-FAF: 29.4%, 50.6%; FAF: 23.1%, 46.4%; FrV: 18.5%, 34.2%). In the NT treatment groups, the presence of serum during IVM resulted in a higher proportion of MII oocytes at 18h, better oocyte quality for manipulation, and increased blastocyst development and hatching rates (SER: 31.4%, 18.2%; FAF: 21.7%, 4.8%). These results indicate that both serum and fatty acid-free BSA provide comparable embryo development during IVF. However, development in serum occurs at an accelerated rate as indicated by the shorter nuclear maturation times and blastocyst development on Day 6, which has been associated with adverse outcomes. Despite this, serum may provide the oocyte with factors that are important for membrane flexibility and repair, enabling greater survival after manipulation. Funding from NSERC and OMAFRA. Sperm provided by Gencor.

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32 CRYOPRESERVATION OF DONOR CELLS FOR NUCLEAR TRANSFER: EFFECT OF CELL FREEZING METHOD ON THE EFFICIENCY OF SOMATIC CELL NUCLEAR TRANSFER IN PIGS

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab32 ◽

2006 ◽

Vol 18 (2) ◽

pp. 125

Author(s):

J. Estrada ◽

E. Lee ◽

J. Piedrahita

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Donor Cell ◽

Fusion Rate ◽

Blastocyst Development ◽

Freezing Method ◽

Donor Cells ◽

First Polar Body ◽

Cell Freezing

Donor cell quality is one of the most important factors affecting somatic cell nuclear transfer (SCNT) in mammals. Many studies have been carried out to improve the donor cell characteristics in nuclear transfer, including studies on cell type, cell cycle stage, cell passage, and handling of donor cells before the SCNT. Even though most SCNT work is done with donor cells that have been previously frozen and thawed, no studies have been conducted to evaluate the effect of the cell freezing rate on the SCNT efficiency. The objective of this experiment was to evaluate the effect of the cell freezing method on development of pig SCNT embryos in vitro. Fibroblasts were collected from a 29-day-old female fetus, suspended in DMEM-F12 + 40% fetal bovine serum (FBS) + 10% dimethyl sulfoxide (DMSO), and placed in 1.6-mL cryovials for freezing. Vials were randomly assigned to two treatments: In treatment 1, cells were frozen at a controlled rate of 1�C/min in a programmable machine (P) until -40�C, and then plunged into liquid nitrogen (LN2; -196�C). In treatment 2, the traditional system (T), vials were placed in a styrofoam box and left overnight in a freezer at -80�C. The next day samples were plunged into LN2 (196�C). For each treatment, cells were thawed and cultured until confluence before being used for SCNT. Cells were used at passages 2 and 6. Cumulus-oocyte complexes (COCs) were aspirated from slaughterhouse ovaries and cultured for 39 h in TCM 199 supplemented with 10% porcine follicular fluid (pFF), 5 �g/mL insulin, 10 ng/mL epidermal growth factor (EGF), 0.6 mM cysteine, 0.2 mM pyruvate, 25 �g/mL gentamycin and 5 �g/mL each of equine and human chorionic gonadotropin (eCG and hCG). Oocytes were stained with bisbenzimide and enucleated in manipulation media with 7.5 �g/mL cytochalasin B by removing the first polar body and metaphase plate by means of a 16-�m beveled glass pipette. Cells from each treatment were injected into the perivitelline space of recipient enucleated oocytes and fused by two DC pulses of 140 V for 50 �s in fusion media. The fusion rate was evaluated 1 h later, and reconstructed oocytes were activated by two DC pulses of 120 V for 60 �s. After activation, oocytes were placed in bicarbonate-buffered NCSU-13 with 0.4% BSA and cultured at 38.5�C, 5% CO2 in a humidified atmosphere. Embryos were observed for cell cleavage at Day 2, and blastocyst development rate and cell number counting were done at Day 7 of culture. Every experiment was repeated three times. The temperature descending rate for P was slower and more linear (1�C/min vs. 2�C/min) than for the T method. Fusion rate was not significantly affected (P < 0.05) by the freezing method when they were evaluated either individually at each passage or accumulated regardless the passage (78.9 � 3.6% vs. 79.4 � 6.3%) for P and T, respectively. The same trends were observed for cleavage (61.2 � 5.2% vs. 64.3 � 5.2%), blastocyst development (4.2 � 1.8% vs. 5.0 � 2.8%), and number of cells at the blastocyst stage (19.4 � 3.1 vs. 19.8 � 6.2) for P and T, respectively. The present findings indicate that blastocyst development after SCNT does not differ when fetal fibroblasts donor cells are frozen by the two methods tested.

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Production of horse foals via direct injection of roscovitine-treated donor cells and activation by injection of sperm extract

Reproduction ◽

10.1530/rep.1.01095 ◽

2006 ◽

Vol 131 (6) ◽

pp. 1063-1072 ◽

Cited By ~ 44

Author(s):

K Hinrichs ◽

Y H Choi ◽

C C Love ◽

Y G Chung ◽

D D Varner

Keyword(s):

Nuclear Transfer ◽

Direct Injection ◽

Donor Cell ◽

Efficient Production ◽

Blastocyst Development ◽

Flow Cytometric ◽

Donor Cells ◽

Sperm Extract ◽

Sperm Factor ◽

Flow Cytometric Evaluation

We evaluated the effects of different donor cell treatments and activation methods on production of blastocysts after equine nuclear transfer. Nuclear transfer was performed by direct injection of donor cells, using a piezo drill, and standard activation was by injection of sperm factor followed by culture with 6-dimethylaminopurine. There was no difference in blastocyst development between embryos produced with roscovitine-treated or confluent donor cells (3.6% for either treatment). Addition of injection of roscovitine or culture with cycloheximide at the time of activation did not affect blastocyst development. Overall, transfer of eight blastocysts produced using roscovitine-treated donor cells and our standard activation protocol yielded three pregnancies, of which two (25% of transferred embryos) resulted in delivery of viable foals. Flow cytometric evaluation showed that roscovitine treatment significantly increased the proportion of cells classified as small, in comparison to growth to confluence or serum deprivation, but did not significantly affect the proportion of cells in G0/G1 (2N DNA content). Transfer of one blastocyst produced using roscovitine-treated donor cells, with addition of roscovitine injection at activation, yielded one pregnancy which was lost before 114 days’ gestation. Transfer to recipients of two blastocysts produced using confluent donor cells with addition of cycloheximide at activation gave no resulting pregnancies. We conclude that roscovitine treatment of donor cells yields equivalent blastocyst production after nuclear transfer to that for confluent donor cells, and that direct injection of roscovitine-treated donor cells, followed by activation using sperm extract, is compatible with efficient production of viable cloned foals.

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44 IMPROVED DEVELOPMENT OF BOVINE NUCLEAR TRANSFER EMBRYOS BY THE TREATMENT OF NUCLEAR DONOR CELLS WITH APOPTOSIS INHIBITORS DURING SERUM STARVATION

Reproduction Fertility and Development ◽

10.1071/rdv17n2ab44 ◽

2005 ◽

Vol 17 (2) ◽

pp. 171

Author(s):

C.-K. Lee ◽

Bon-sik Koo ◽

C.-H. Park ◽

S.-G. Lee ◽

D.-H. Choi ◽

...

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Tunel Assay ◽

Serum Starvation ◽

Bovine Embryos ◽

Embryonic Fibroblasts ◽

Crown Rump Length ◽

Donor Cells

In somatic cell nuclear transfer, serum starvation is a widely used method to synchronize donor cells at the quiescent stage (Go) of the cell cycle. However, it has been shown that serum starvation during culture of mammalian cells could induce cell death via apoptosis by removing growth factors and increasing intracellular stress. Therefore, apoptosis caused by serum starvation in somatic cells could induce damages to nuclear DNA contributing to a lower efficiency of nuclear transfer. This study was performed to characterize apoptosis during serum starvation of bovine embryonic fibroblasts (BEFs) and to determine the effects of BEFs treated with apoptosis inhibitors on the development of bovine embryos after nuclear transfer. BEFs, collected from a fetus with a 3–4-cm crown-rump length, were cultured for 7 days in starvation medium consisting of Dulbecco's modified Eagle's medium containing 0.5% fetal bovine serum to induce quiescence. Cells were also placed in starvation medium containing the apoptosis inhibitors, β2-macroglobulin (broad-range protease inhibitor: MAC; 1.4 pM) and glutathione (GSH: reactive oxygen species scavenger; 2.0 mM). Apoptosis of serum starved BEFs with or without apoptosis inhibitors were analyzed morphologically with light and electron microscope, and biochemically using a TUNEL assay. Somatic cell nuclear transfer was performed by our standard procedure as follows. Bovine oocytes were matured in vitro and enucleated after 22 h. Nuclear donor cells were collected randomly before injection. The reconstructed embryos were placed into the fusion chamber in a solution containing 0.28 M mannitol and aligned manually. A double pulse of 1.8 kV/cm for 15 μs was used to fuse the cells and activate the embryos simultaneously. The fused embryos were cultured for 4 min in 5 μÂM ionomycin and 4 h in 2 mM 6-DMAP. Then, embryos were moved to culture media and cultured in 5% CO2 and 39°C in 100% humidity. Development of NT embryos was recorded at 120 h post NT (morulae) and 168 h (blastocysts) with experiments being repeated three times. Serum starved BEFs showed typical morphology of apoptotic cells such as chromatin condensation and membrane blebbing. Also, when stained for DNA fragmentation by TUNEL assay, 22.6% ofBEFs showed apoptosis, in contrast to 0.1% in actively growing cells. However, when BEFs were cultured with MAC and GSH, the proportions of apoptotic BEFs were greatly reduced, 6.0% and 2.1%, respectively. After nuclear transfer with BEFs, embryos reconstructed with BEF treated with apoptosis inhibitors showed significant improvement in in vitro development compared to the controls (Table 1). In conclusion, while there are a number of factors affecting the nuclear transfer procedure, damage to the donor nuclei by serum starvation is likely to reduce the efficiency of the procedure; the addition of apoptosis inhibitors could reduce this unnecessary damage to donor nuclei and result in improvement in the development of nuclear transferred embryos. Further experiments are needed to assess the effect of apoptosis inhibitors on improvement of overall nuclear transfer efficiency. Table 1. Development of bovine embryos nuclear transferred with embryonic fibroblasts treated with or without apoptosis inhibitors

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80 EFFECT OF DNA METHYLATION ON SOMATIC CELL NUCLEAR TRANSFER EMBRYO DEVELOPMENT IN RHESUS MONKEY

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab80 ◽

2006 ◽

Vol 18 (2) ◽

pp. 148

Author(s):

J. F. Yang ◽

S. H. Yang ◽

Y. Y. Niu ◽

Q. Zhou ◽

W. Z. Ji

Keyword(s):

Dna Methylation ◽

Cell Lines ◽

Nuclear Transfer ◽

Methylation Pattern ◽

Serum Starvation ◽

Global Dna Methylation ◽

Blastocyst Development ◽

Donor Cells ◽

Morula Stage ◽

Asymmetric Methylation

Up to now, no primate animals have been successfully cloned with somatic cell nuclear transfer (SCNT) and little is known about molecular events occurring in SCNT embryos. DNA methylation reprogramming is likely to have a crucial role in establishing nuclear totipotency in normal development and in cloned animals. Epigenetic characteristics of donor cell nuclei and their epigenetic reprogramming in oocyte cytoplasm have been supposed as major factors influencing the development of SCNT embryos. In Experiment 1, on donor cells used in a previous SCNT at our laboratory, global DNA methylation and histone 3 lysine 9 acetylation (H3K9ac) of three cell lines (S11, S1-04, and S1-03) derived from ear skin were examined after serum starvation by immunofluorescence with monoclonal antibody to 5-methyl cytosine (Oncogene, Science, Inc., Cambridge, MA, USA) and anti-acetyl-Histone H3 (Lys 9) (Upstate Jingmei Biotech, Ltd., Shenzhen, China). In the results, two cells lines, S11 and S1-04, supporting higher blastocyst development (about 20%) than that (7.8%) of S1-03, showed a higher level of H3K9ac than the S1-03 cell line. Global DNA methylation levels in the three cell lines were decreased after serum starvation, but no obvious correlation between the level and SCNT embryo developmental potential was found among the three cell lines. In Experiment 2, on SCNT and IVF embryos, global DNA methylation reprogramming during pre-implantation development was investigated with immunofluorescence and laser scanning microscopy techniques. In IVF embryos, active demethylation of paternal genome occurred soon after fertilization; subsequently, passive demethylation resulted in remarkably reduced global methylation level at the 8-cell stage and the morula stage. Thereafter, genomewide remethylation started at the late morula stage and an asymmetric methylation pattern was formed in blastocysts, with higher methylated trophectoderm than inner cell mass (ICM). Compared with IVF embryos, most SCNT 2-cell embryos and ICM in blastocysts showed higher methylation levels, and the asymmetric methylation pattern was not as evident as that in IVF blastocysts. Some SCNT 8-cell embryos showed higher methylation, but others were slightly stained, even lower than IVF embryos. In conclusion, the higher global H3K9 acetylation level of donor cells may benefit chromatin remolding and development of SCNT embryos. Abnormal methylation reprogramming in most SCNT embryos, especially in ICM of blastocysts, may be one main obstacle for primate cloning, although relatively high blastocyst development rate was obtained. DNA methylation reprogramming in rhesus monkey pre-implantation embryos, on the whole, was as conservative as that reported in other mammals.

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32 EFFECTS OF FUSION/ACTIVATION METHODS ON DEVELOPMENT OF EMBRYOS PRODUCED BY NUCLEAR TRANSFER OF PORCINE FETAL FIBROBLASTS

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab32 ◽

2007 ◽

Vol 19 (1) ◽

pp. 134

Author(s):

P. Q. Cong ◽

E. S. Song ◽

E. S. Kim ◽

Z. H. Li ◽

Y. J. Yi ◽

...

Keyword(s):

Nuclear Transfer ◽

Polar Body ◽

Metaphase Plate ◽

Donor Cell ◽

Blastocyst Stage ◽

Pulse Number ◽

Donor Cells ◽

First Polar Body ◽

Fetal Fibroblasts

Pigs have become increasingly important in the field of biomedical research, and interest has grown in the use of transgenic cloned pigs as potential xenograft donors. The present study were carried out to investigate the effects of intensity of DC pulse, number of DC pulses, and equilibration before fusion/activation on developmental ability of porcine embryos derived from nuclear transfer. Porcine cumulus-oocyte complexes (COCs) were cultured in modified TCM-199 (mTCM-199) medium for 44 h at 38.5�C, 5% CO2 in air. After in vitro maturation (IVM), metaphase II oocytes were selected for enucleation. Porcine fetal fibroblasts were obtained from a porcine fetus on Day 35 of gestation as donor cells. Oocytes were enucleated by removing, with a micropipette, the first polar body along with adjacent cytoplasm containing the metaphase plate; then a donor cell was injected in contact with the cytoplasm of each oocyte. In experiment 1, several different fusion/activation intensities (two DC pulses of 0.4, 0.8, 1.2, 1.6, and 2.0 kV cm-1 for 30 �s) were carried out to investigate the effect on the development of nuclear transfer embryos. In experiment 2, the reconstructed oocytes were fused and activated with 1, 2, or 3 DC pulses of 1.2 kV cm-1 for 30 �s. In experiment 3, reconstructed oocytes were equilibrated in mTCM-199 medium at 38.5�C, 5% CO2 for 0, 1, 2, 3, 4, 5, and 6 h. After equilibration, the reconstructed oocytes were fused and activated with one DC pulse of 1.2 kV cm-1 for 30 �s in fusion medium. The reconstructed embryos were transferred into PZM-3 medium containing 0.3% BSA for further culture. The rates of embryo cleavage and development of blastocyst stage were evaluated at 48 h and 6-7 days, respectively. The cell numbers of blastocysts were counted by using Hoechst 33342 epifluorescence staining. Data were analyzed by ANOVA and Duncan

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Refrigeration of donor cells in preparation for bovine somatic nuclear transfer

Reproduction ◽

10.1530/rep.0.1220801 ◽

2001 ◽

pp. 801-808 ◽

Cited By ~ 5

Author(s):

JL Liu ◽

MK Wang ◽

QY Sun ◽

XR Zhang ◽

LK Jiang ◽

...

Keyword(s):

Nuclear Transfer ◽

Cultured Cells ◽

Donor Cell ◽

Serum Starvation ◽

Fresh Tissue ◽

Reconstructed Embryos ◽

Donor Cells ◽

Somatic Nuclear Transfer

In mammals, preparation of donor cells for somatic nuclear transfer is very important because the character of the donor cell directly affects the efficiency and outcome of transfer. The protocols used most commonly for donor preparation are (i) disaggregating cells from fresh tissue 1-2 h before micromanipulation or (ii) trypsinizing cultured cells temporarily, after special treatments for 3-8 days (for example, serum starvation). In this study, a new simple protocol was designed, whereby the donor cells (cumulus cells) used in bovine somatic nuclear transfer were refrigerated. In brief, cultured cells at 80-100% confluency were detached using trypsin, washed by centrifugation, aliquoted into different vials and refrigerated at 4 degrees C. The density of viable cells was decreased after day 1 of refrigeration; however, the rate of decrease tended to slow down with increasing duration of refrigeration. Cells refrigerated for 15 days were seeded at a density of 5 x 10(4) ml(-1) and reached 70% confluency after day 2 of culture. Most cells had the normal number of chromosomes (2n = 60). Cells chilled at 4 degrees C for different durations were removed from refrigeration and immediately subjected to micromanipulation. The in vitro development of reconstructed embryos (fusion rates, cleavage rates, morula and blastocyst rates) indicated that there were no significant differences among treatment groups regardless of the duration of refrigeration (0-2 weeks) of the donor cells. Reconstructed embryos were transferred into the uteri of recipient cows. No significant differences were observed in established early pregnancies between embryos derived from the non-refrigerated donor cells and those derived from refrigerated donor cells. This study indicates that refrigeration of donor cells for 1-2 weeks is a feasible protocol for preparing donor cells for bovine somatic nuclear transfer, and does not compromise development in vitro and early development in vivo.

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29 BIRTH OF A FOAL CLONED BY ADULT SOMATIC CELL NUCLEAR TRANSFER WITH ROSCOVITINE-TREATED DONOR CELLS

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab29 ◽

2006 ◽

Vol 18 (2) ◽

pp. 123

Author(s):

Y. H. Choi ◽

Y. G. Chung ◽

D. D. Varner ◽

K. Hinrichs

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Direct Injection ◽

Donor Cell ◽

Mixed Gas ◽

Blastocyst Development ◽

Donor Cells ◽

Significant Difference

Only one horse foal produced from adult somatic cell nuclear transfer has been reported in the scientific literature (Galli et al. 2003 Nature 425, 680); a second foal from the same laboratory was reported in the popular press in 2005. In these reports, the blastocyst rates were 3 and 17%, and efficiency to birth of a live foal from total reconstructed oocytes was 0.1 and 0.5%, respectively. In cattle, roscovitine treatment of donor cells has been associated with a decrease in blastocyst development, but an increase in live births (Gibbons et al. 2002 Biol. Reprod. 66, 895-900). The present study was performed to determine the effect of roscovitine treatment of donor cells on blastocyst production after equine nuclear transfer and to evaluate the viability of pregnancies established via this treatment. In Experiment 1, fibroblasts were either grown to confluence or treated with 15 �g/mL roscovitine, for 24 h. Enucleated in vitro-matured oocytes were reconstructed by direct injection of fibroblasts using a piezo drill. Recombined oocytes were activated by injection of stallion sperm extract, followed by culture in the presence of 2 mM 6-DMAP for 4 h. They were then placed in culture in DMEM/F-12 with 10% fetal bovine serum (FBS) under mixed gas for 8 days and evaluated for blastocyst development. In Experiment 2, oocytes recombined with either confluent or roscovitine-treated donor cells were activated as above either alone or with the addition of 10 �g/mL cycloheximide at the time of 6-DMAP treatment. Resulting blastocysts from Experiment 2 were transferred transcervically to the uteri of recipient mares. One embryo was transferred per mare. In Experiment 1, there was no difference in rates of cleavage (73-19%) or blastocyst development between confluence and roscovitine treatments (2/55, 3.6% vs. 2/56, 3.6%, respectively). In Experiment 2, there was no significant difference in rates of cleavage (78-18%) or blastocyst development (0-1%; 4/105, 0/104, 0/106, 2/108) among donor cell or activation treatments. Six blastocysts were transferred to mares: two from confluent donor cells and four from roscovitine-treated donor cells. One mare, which received an embryo from the roscovitine donor/6-DMAP treatment, established pregnancy after transfer. The pregnancy continued normally and the mare delivered a colt with minimal assistance on Day 389. Typing for 13 equine microsatellites confirmed that the colt was of the same DNA type as the donor fibroblasts. The colt has grown and developed normally. Results of these studies show that roscovitine treatment of equine donor cells does not negatively affect the proportion of recombined oocytes that progress to the blastocyst stage. A viable colt resulted from an embryo produced with roscovitine-treated donor cells. More work is needed on methods to increase blastocyst rates after nuclear transfer in this species. This work was supported by the Link Equine Research Endowment Fund, Texas A&M University.

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