59 SYNCHRONIZATION OF CELL CYCLE STAGE OF BUFFALO (BUBALUS BUBALIS) FETAL FIBROBLAST CELLS BY DIFFERENT TREATMENTS

2011 ◽  
Vol 23 (1) ◽  
pp. 135
Author(s):  
N. L. Selokar ◽  
A. George ◽  
A. P. Saha ◽  
R. Sharma ◽  
M. Muzaffar ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bubalus Bubalis ◽  
Serum Starvation ◽  
Fibroblast Cells ◽  
Cloning Efficiency ◽  
Fetal Fibroblast ◽  
Cell Cycle Stage ◽  
Cell Cycle Synchronization ◽  
Donor Cells ◽  
Major Factors

Cell cycle stage of donor cells significantly influences the cloning efficiency during SCNT. Donor cells in G1/G0 stage have better capability to undergo nuclear reprogramming following transfer to an unfertilized oocyte. The lack of availability of cells synchronized at G1/G0 stage is one of the major factors limiting cloning efficiency in buffalo. The aim of this study was to compare the efficacy of various methods for cell cycle synchronization of buffalo fetal fibroblast cells for SCNT. Cells isolated from fetus, 2 to 3 months old, were cultured in DMEM + 10% FBS. The primary culture was sub-cultured 8 to 10 times. For cell cycle synchronization, the cells were cultured to 1) 60 to 70% confluence (controls), 2) 60 to 70% confluence followed by serum starvation (DMEM + 0.5% FBS) for 24 h (serum starved), 3), full confluence followed by culture for additional 3 to 5 days (full confluent), 4) full confluence followed by serum starvation (DMEM + 0.5% FBS) for 24 h (full confluent+serum starved) and 5) 60 to 70% confluence followed by treatment with roscovitine (10, 20, or 30 μM) for 24 h. The synchronization efficiency was examined by propidium iodide staining followed by analysis of DNA content using flow cytometry and the data were analysed by 1-way ANOVA followed by Fisher’s l.s.d. test after arcsine transformation. The percentage of cells in G0/G1 phase of cell cycle was significantly higher (P < 0.05) in the full confluent+serum starved and roscovitine treated (20 or 30 μM) groups than that in the full confluent group and that treated with 10 μM roscovitine which, in turn, was higher (P < 0.05) than that in the serum starved and control groups. These results suggest that buffalo fetal fibroblast cells can be synchronized by roscovitine treatment or by serum starvation of fully confluent cell cultures to obtain a high proportion of cells in G0/G1 stage for SCNT. Table 1.Buffalo skin fibroblast cells at various stages following different treatments for cell cycle synchronization Supported by grant No. 1(5)/2007-NAIP from ICAR, India.

73 IMPROVEMENT OF CANINE CLONING EFFICIENCY BY OPTIMIZED DONOR CELL PREPARATION

2010 ◽  
Vol 22 (1) ◽  
pp. 195
Author(s):  
S. W. Park ◽  
Y. W. Jeong ◽  
J. J. Kim ◽  
K. H. Ko ◽  
S. H. Jeong ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Electrical Stimulation ◽  
Somatic Cell ◽  
Somatic Cells ◽  
Contact Inhibition ◽  
Abnormal Development ◽  
Serum Starvation ◽  
Cell Cycle Synchronization ◽  
Donor Cells ◽  
Cloned Dog

The Tibetan Mastiff is the oldest dog breed in the world, and it is at the edge of extinction. Li et al. (2008) believe that protection of and research on the Tibetan Mastiff is extremely urgent, yet few studies have been carried out, particularly at the molecular level. Somatic cell nuclear transfer (SCNT) is an efficient technique for the conservation of endangered animals because it can increase the number of individuals within a population. Considering the virtually unlimited value of cloned canids in critical biotechnology applications, including gene conservation of endangered canids and disease models, the effect of cell-cycle synchronization methods, including the use of cycling canine adult skin fibroblasts (CASF), on the cell-cycle stage and viability of donor nuclei was analyzed. To improve the efficiency of cloned dog production, optimal conditions of donor cells were analyzed by culture duration (Days 1, 2, 3, and 4), passages (2, 4, 7, 10, and 11 passages) and mitotic regulator Plk-1/-4 gene expression. Simerly et al. (2003) reported that the depletion of microtubule motors and centrosomal proteins during enucleation of SCNT procedures caused abnormal development of SCNT embryos. We therefore analyzed Plk-1/-4-induced centriole biogenesis in CASF at different passages of donor cells. In this study, somatic cells were collected from a purebred 9-month-old male Mastiff and an 11-month-old female mastiff. In vivo-matured oocytes were retrieved from outbreed dogs by operation. Cycling cells cultured at Day 4 showed a similar effect to that of cells that were artificially synchronized (contact inhibition or serum starvation). It was also confirmed that fresh and short-term culture (<5 passages) resulted in fewer harmful effects and the same cell viability as control cells, using proliferation assays and expression levels of Plk-1/-4 genes. Therefore, 4 passage-cycling cells at Day 4 were used as donor cells of SCNT. A total of 289 oocytes were reconstructed with each male or female somatic cell and then simultaneously fused/activated with 2 DC pulses of 1.9 kV cm-1 for 30 s of electrical stimulation. Finally, 224 embryos were transferred to 16 naturally synchronized recipients. As a result, we were able to use somatic cells collected from both female and male Tibetan Mastiffs to produce 10 female and 6 male mastiffs. Moreover, one surrogate delivered a quartet of identical cloned female Tibetan Mastiffs puppies; each of 3 surrogates also delivered triplets. Microsatellite analysis demonstrated the genotypic identity of the cloned puppies. In conclusion, the present study shows that (1) cell-cycle synchronization of donor cells by serum starvation/contact inhibition is not required, (2) Plk-1/-4 mRNA can be used to select the donor cells, (3) electrical stimulation alone is sufficient for the activation of SCNT embryos for the production of SCNT cloned dogs, and (4) the cloned dog delivery efficiency (7.1%) was threefold higher than in previous reports. SWP and YWJ contributed equally to this work. WSH was corresponding author and SHH was co-corresponding author.

88 CELL CYCLE SYNCHRONIZATION OF FIBROBLASTS DERIVED FROM TRANSGENIC CLONED CATTLE EAR SKIN: EFFECTS OF SERUM STARVATION, ROSCOVITINE, AND CONTACT INHIBITION

2007 ◽  
Vol 19 (1) ◽  
pp. 161
Author(s):  
X.-Z. Sun ◽  
S.-H. Wang ◽  
Y.-H. Zhang ◽  
Y.-P. Dai ◽  
N. Li
Keyword(s):  
Cell Cycle ◽  
Fluorescent Protein ◽  
Critical Role ◽  
Contact Inhibition ◽  
Developmental Competence ◽  
G1 Phase ◽  
Serum Starvation ◽  
Becton Dickinson ◽  
Cell Cycle Stage ◽  
Cell Cycle Synchronization

Cell cycle stage plays a critical role in somatic cell nuclear transfer (SCNT), and G0/G1 stage cells are preferred nuclear donors in attempts to produce cloned livestocks. Enhancement of survivability of cloned calves by roscovitine (the cyclin-dependent kinase-2 inhibitor) has been shown (Gibbons et al. 2002 Biol. Reprod. 66, 895–900). The purpose of this study was to evaluate the effects of serum starvation, roscovitine, and contact inhibition on cell cycle synchronization at the G0/G1 stage of transgenic cloned bovine ear skin-derived fibroblasts. The cell line was established from a cloned calf expressing green fluorescent protein (GFP). Data were analyzed by using SAS (8.0) with ANOVA (SAS Institute, Inc., Cary, NC, USA). At passage 2-6, cells were cultured in Dulbecco&apos;s modified Eagle's medium (DMEM) plus 10% fetal bovine serum (FBS) in T-25 culture flasks from immediately after subculture until monolayer cells reached 90% confluence at 39�C, under 5% CO2 in humidified air. Then cells grown in different flasks were randomly distributed to groups: Serum starvation (SS, culture medium changed to DMEM + 0.5% FBS), roscovitine (R, cultured in DMEM + 10% FBS + 15 �M roscovitine), and contact inhibition (CI, DMEM + 10% FBS). From Day 1 to Day 5 after treatment, 3 flasks of cells from each group were subjected to fixation and staining every day, followed by determination of cell cycle stage with Becton Dickinson FACScan (Kues et al. 2000 Biol. Reprod. 62, 412–419). At least 3 replicates were performed for each determination. The results showed that in the cycling fibroblasts (50–60% confluence), 59.29% of the cells were at the G0/G1 phase. For the SS group, the proportion of G0/G1 cells was significantly higher for treatment lasting 3 days, 4 days, and 5 days than for treatment lasting 1 day or 2 days (91.5%c, 91.7%c, and 93.5%c vs. 90.1%b and 88.8%a, respectively; P &lt; 0.05). No statistical differences were observed among 3-day, 4-day, and 5-day treatments. For the R group, one-day treatment was significantly lower in synchronizing cells to the G0/G1 stage than that from Day 2 to Day 5 (86.51%a vs. 91.1%a, 90.1%a, 89.4%a, and 91.3%b, respectively; P &lt; 0.05) during which similar rates of G0/G1 phase were observed. For the CI group, an increase of G0/G1 stage cells was found after 3 days of CI (from 89.4%a for Day 1 and 90.4%ab for Day 2, to 91.4%bc for Day 3; P &lt; 0.05), which was similar to 4 days (91.6%bc) and 5 days (92.1%c) for the CI group. We also found that the efficiency of obtaining G0/G1 phase cells was lower when roscovitine was employed to synchronize the cell cycle than when the SS and CI methods were used (89.7%b vs. 91.1%a and 91.0%a, respectively; P &lt; 0.05), after analysis of the most effective treatment duration of each group. Our data indicate that in attempts to harvest adequate G0/G1-stage cells for re-cloning of transgenic cattle from fibroblasts established from ear skin of a newborn transgenic cloned calf, 3 days of treatment is enough regardless of the method (SS, R, or CI). Further research is needed to evaluate the developmental competence of embryos cloned from cells prepared by the abovementioned treatments. [a-c mean statistical differences; P &lt; 0.05.]

scholarly journals 69 PRODUCTION OF CLONED PIGS BY NUCLEAR TRANSFER OF PREADIPOCYTES

2005 ◽  
Vol 17 (2) ◽  
pp. 184
Author(s):  
R. Tomii ◽  
M. Kurome ◽  
H. Ueda ◽  
S. Ueno ◽  
K. Hiruma ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Nuclear Transfer ◽  
Annexin V ◽  
The Other ◽  
Serum Starvation ◽  
Male Adult ◽  
Differentiation Induction ◽  
Cell Cycle Synchronization ◽  
Donor Cells ◽  
Fetal Fibroblasts

Since the first success in producing cloned pigs, donor cells have been limited to fetal fibroblasts and a few other cell types. The aim of the present study was to determine if porcine preadipocytes can be efficient donor cells for somatic cell nuclear transfer (NT) in pigs. Preadipocytes established from subcutaneous adipose tissue of a male adult pig were used as nuclear donor cells. Cell cycle synchronization was carried out by serum starvation (5 days), confluency (5 days), roscovitine treatment (15 μM, 2 days), or differentiation induction by 0.5 mM 3-Isobutyl-1-methylxanthine, 0.25 μM dexamethasone, and 5 μg/mL insulin (5 days). Cell cycle synchronization and apoptosis of the donor cells were examined by flow cytometry and Annexin V staining and TUNEL. IVM oocytes were obtained from abattoir ovaries and matured in NCSU23. Donor cells were fused with the enucleated recipient oocytes by a single DC pulse of 200 V/mm for 10 μs in 0.28 M mannitol + 0.15 mM MgSO4. Reconstructed embryos were electrically activated at 1–1.5 h after the NT, followed by cytochalasin B treatment for 3 h. Development of the NT embryos was assessed by fixation/staining at 3 h after NT, culture for 7 days in NCSU23, and transfer to the oviducts of estrus-synchronized recipient gilts. The cells immediately entered the G0 phase by differentiation induction (92.5 ± 0.4%), with higher efficiency of synchronization than for the other methods (roscovitine: 80.3 ± 0.2%; confluency: 79.9 ± 0.3%, P < 0.05) except for serum starvation (89.8 ± 0.6%). The proportion of apoptotic cells in the differentiation group was significantly lower than the other groups (Annexin V: 7.7% vs. 15.7 to 19.3%, TUNEL: 8.3% vs. 12.8 to 14.0%, P < 0.05). Incidence of premature chromosome condensation following NT (88.0%) was as high as that observed after NT with fetal fibroblasts previously (data not shown). In vitro developmental rates of the NT embryos did not differ significantly among the cell cycle synchronization methods of the donor cells (7.2 to 10.8%). Cell number of the blastocysts was highest in the differentiation group (49.0 vs. 30.2 to 41.9, P < 0.05). Transfer of 1004 cloned embryos of the serum starvation group to 5 recipients resulted in the production of 4 live and 1 stillborn piglets from 1 recipient. Transfer of cloned embryos reconstructed of donor cells treated by differentiation induction is currently underway. These data demonstrate that preadipocytes collected from an adult pig are promising nuclear donor cells for pig cloning. This study was supported by PROBRAIN.

83 IN VITRO DEVELOPMENTAL ABILITIES OF PORCINE CLONED EMBRYOS RECONSTITUTED WITH CELL NUCLEI OF GENETICALLY TRANSFORMED FETAL FIBROBLASTS CYTOMETRICALLY DIAGNOSED ON CELL CYCLE AND APOPTOSIS

2007 ◽  
Vol 19 (1) ◽  
pp. 159
Author(s):  
M. Samiec ◽  
M. Skrzyszowska ◽  
M. Bochenek ◽  
D. Lipinski ◽  
R. Slomski
Keyword(s):  
Cell Cycle ◽  
High Efficiency ◽  
Fibroblast Cells ◽  
Apoptotic Cells ◽  
Facs Analysis ◽  
Developmental Potential ◽  
Fetal Fibroblast ◽  
Donor Cells ◽  
Fetal Fibroblasts

The important factor that determines the development of mammalian cloned embryos is structuro-functional quality of nuclear donor cells. Analysis of nuclear DNA (nDNA) content of somatic cells undergoing apoptosis has become one of the most common methods for single-parameter flow cytometric measurement of this process. Apoptosis assessment is performed by quantification of hypodiploid cells. The aim of our study was to examine the in vitro developmental potential of porcine nuclear transfer (NT) embryos reconstituted with non-apoptotic fetal fibroblast cells expressing the eGFP transgene. The nuclear donor cells were derived from cell line populations whose representative random samples had been analyzed on both cell cycle and apoptosis through non-vital nDNA fluorescent dyeing and flow cytometry (FACS). Frozen-thawed fibroblast cells, which had been cultured up to a total confluency after 2–4 passages, were used for the diagnostics. The cells were fixed in ice-cold 70% ethanol. Then, the fetal fibroblasts were exposed to nDNA extraction buffer for 5 min at room temperature, and incubated in DNA staining solution (propidium iodide and RNAse) for 30 min. After fluorescent labeling, the cells were analyzed in the flow cytometer by reading nDNA fluorescence in the red band. In vitro-matured oocytes were the source of recipient cells. Fibroblast cell–ooplast couplets were simultaneously fused and activated. Reconstructed embryos were cultured in NCSU-23/BSA/FBS medium for 6–7 days. The rates of cleavage and development to morula/blastocyst stages were examined on Days 2 and 6/7, respectively. FACS analysis revealed that, out of all of the diagnosed fetal fibroblast cells, 54.7% were cycling, and up to 45.3% were late-apoptotic. In turn, from among the normal (i.e. non-apoptotic) cells, 82.2% were at G0/G1 stages of cell cycle, 17.0% at the S stage, and 0.8% at G2/M stages. A total of 150 enucleated oocytes were successfully fused with non-apoptotic transgenic nuclear donor cells. Out of 150 cultured NT embryos, 123 (82.0%) were cleaved. The frequencies of cloned embryos that reached the morula and blastocyst stages yielded 53/150 (35.3%) and 37/150 (24.7%), respectively. In conclusion, the FACS analysis for mitotic cycle of 100%-confluent transgenic fetal fibroblasts confirmed the high efficiency of the cell cycle synchronization at G0/G1 phases. However, a contact inhibition method induced the high frequency of late-apoptotic cells. Moreover, the relatively high percentage of NT blastocysts was developed from oocytes reconstructed with eGFP transgenic fetal fibroblast cells. This research was supported by the State Committee for Scientific Research as a Solicited Project number PBZ-MIN-005/P04/2002/6 from year 2003 to year 2006.

Effects of donor sex and different cell treatments on development of yak–bovine interspecies nuclear transfer embryos

2008 ◽  
Vol 5 (1) ◽  
pp. 55-60
Author(s):  
Liu Ying ◽  
Zhu Shi-En ◽  
Li Rong ◽  
Wang Li-Li ◽  
Wang Hai-Ping ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Nuclear Transfer ◽  
Developmental Competence ◽  
Cell Group ◽  
Serum Starvation ◽  
Cleavage Rate ◽  
Cell Cycle Synchronization ◽  
Donor Cells ◽  
Significant Difference ◽  
Interspecies Nuclear Transfer

AbstractThe purpose of this study was to evaluate the effects of donor sex, treatments of cell cycle synchronization and donor nuclei obtained from fresh or frozen–thawed conditions on developmental competence of yak–bovine interspecies nuclear transfer embryos. Bovine (Bos taurus) oocytes were used as recipients and yak (Bos grunniens) ear fibroblast cells were used as donors. Results indicated that the development rate of male blastocysts was higher than that of female (56.6% versus 39.5%, P<0.05), whereas cleavage and total cell number showed no difference between the two groups. No significant difference was observed in the development and quality of blastocysts with donor cells treated by serum starvation or contact inhibition, and there was no significant difference in embryo development with fresh or frozen–thawed donor cells, whereas the cleavage rate in the group of frozen–thawed cells was significantly lower than that of the fresh cell group (54.5% versus 78.2%, P<0.05). The results demonstrated that donor sex could impact the developmental competence of yak–bovine interspecies nuclear transfer embryos, whereas different treatments of cell cycle synchronization and freezing had little influence.

scholarly journals Creation of cloned pig embryos using contact-inhibited or serum-starved fibroblast cells analysed intravitam for apoptosis occurrence / Uzyskiwanie klonalnych zarodków świni z wykorzystaniem komórek fibroblastycznych poddanych inhibicji kontaktowej lub deprywacji troficznej oraz analizowanych przyżyciowo w kierunku apoptozy

2013 ◽  
Vol 13 (2) ◽  
pp. 275-293 ◽  
Author(s):  
Marcin Samiec ◽  
Maria Skrzyszowska ◽  
Jolanta Opiela
Keyword(s):  
Dermal Fibroblast ◽  
Somatic Cells ◽  
Fibroblast Cell ◽  
Serum Starvation ◽  
Fibroblast Cells ◽  
Blastocyst Formation ◽  
Cell Nuclei ◽  
Donor Cells ◽  
Cloned Pig

Abstract Somatic cell cloning efficiency is determined by many factors. One of the most important factors is the structure-functional quality of nuclear donor cells. Morphologic criteria that have been used to date for qualitative evaluation of somatic cells may be insufficient for practical application in the cloning. Biochemical and biophysical changes that are one of the earliest symptoms in the transduction of apoptotic signal may be not reflected in the morphologic changes of somatic cells. For this reason, adult cutaneous or foetal fibroblast cells that, in our experiments, provided the source of genomic DNA for the cloning procedure had been previously analysed for biochemical and biophysical proapoptotic alterations with the use of live-DNA (YO-PRO-1) and plasma membrane (Annexin V-eGFP) fluorescent markers. In Groups IA and IB, the generation of nucleartransferred (NT) embryos using non-apoptotic/non-necrotic contact-inhibited or serum-starved adult cutaneous fibroblast cells yielded the morula and blastocyst formation rates of 125/231 (54.1%) and 68/231 (29.4%) or 99/237 (41.8%) and 43/237 (18.1%), respectively. In Groups IIA and IIB, the frequencies of embryos reconstituted with non-apoptotic/non-necrotic contact-inhibited or serum-starved foetal fibroblast cell nuclei that reached the morula and blastocyst stages were 171/245 (69.8%) and 97/245 (39.6%) or 132/227 (58.1%) and 63/227 (27.8%), respectively. In conclusion, contact inhibition of migration and proliferative activity among the subpopulations of adult dermal fibroblast cells and foetal fibroblast cells resulted in considerably higher morula and blastocyst formation rates of in vitro cultured cloned pig embryos compared to serum starvation of either type of fibroblast cell line. Moreover, irrespective of the methods applied to artificially synchronize the mitotic cycle of nuclear donor cells at the G0/G1 phases, developmental abilities to reach the morula/blastocyst stages were significantly higher for porcine NT embryos that had been reconstructed with non-apoptotic/non-necrotic foetal fibroblast cells than those for NT embryos that had been reconstructed with non-apoptotic/non-necrotic adult dermal fibroblast cells. To our knowledge, the generation of cloned pig embryos using abattoir-derived oocytes receiving cell nuclei descended from contact-inhibited or serum-deprived somatic cells undergoing comprehensive vital diagnostics for the absence of biochemical and biophysical proapoptotic alterations within their plasmalemmas has not been reported so far.

70 FETAL FIBROBLAST CELLS PERMEABILIZED WITH STREPTOLYSIN O IMPROVE THE RATES OF FUSION AND IN VITRO DEVELOPMENT OF PORCINE RECONSTRUCTED EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 152
Author(s):  
K. Naruse ◽  
Y. M. Shin ◽  
Y. S. Quan ◽  
C. S. Park ◽  
D. I. Jin
Keyword(s):  
Cell Number ◽  
Fibroblast Cells ◽  
Fetal Fibroblast ◽  
In Vitro Development ◽  
Reconstructed Embryos ◽  
Streptolysin O ◽  
Donor Cells ◽  
Developmental Rates ◽  
Vitro Development

Streptolysin O (SLO) is known to bacterial proteins that form very large pores in the plasma membrane of mammalian cells. SLO has been used in the delivery of proteins into living cells following permeabilization. The objective of this study was to investigate the effect of permeabilization of donor cells using SLO on in vitro development of porcine reconstructed embryos. Porcine fetal fibroblast cells were treated with Ca2+-free DMEM medium containing 200 ng mL−1 of SLO for 50 min before or after trypsinization. Those SLO-treated donor cells were injected into enucleated oocytes, fused with 2 DC pulses (1.2 kV cm−1, 30 µs) and cultured in procine zygote medium-3 (PZM-3) for 6 days. In vitro development of the reconstructed embryos was examined. SLO treatment after trypsinzation significantly increased (P &lt; 0.05) the percentage of fusion rates and blastocyst developmental rates compared with that before trypsinization or in the nontreated group. Additionally there were no significant differences in fusion rates, cleavage rates, blastocyst developmental rates, and total cell number of blastocysts between the SLO-treated group before trypsinzation and the nontreated group. Next, after the trypsinzation treatment, fetal fibroblast cells were incubated in Ca2+-free DMEM containing 200 ng mL−1 of SLO for 0, 30, 50, and 70 min and SLO-treated donor cells were also tested for fusion rate and developmental capability following reconstruction. The 50-min group of SLO-treated cells significantly increased (P &lt; 0.05) the percentage of fusion rates (90.6 vs. 77.6, 85.4, and 78.5%) and blastocyst developmental rates (24.7 vs. 13.5, 11.2, and 13.5%) compared with the other groups (Table 1). However, there was no significant difference in the total cell number of blastocysts among SLO-treated groups. Although cleavage rates the in SLO-treated groups were not significantly different from those of the nontreated group, there the cleavage rates were slightly in SLO-treated groups. In conclusion, permeabilization of porcine fetal fibroblast cells with SLO improves the fusion rates and in vitro development of porcine reconstructed embryos. Table 1.Effects of SLO treatment of fetal fibroblasts by different exposure times on in vitro development of porcine reconstructed embryos

scholarly journals 65 PRODUCTION OF PORCINE NUCLEAR TRANSFER EMBRYOS USING FETAL FIBROBLAST CELLS ANALYZED ON APOPTOSIS

2005 ◽  
Vol 17 (2) ◽  
pp. 182 ◽  
Author(s):  
M. Skrzyszowska ◽  
M. Samiec
Keyword(s):  
Nuclear Transfer ◽  
Fibroblast Cell ◽  
State Committee ◽  
Fibroblast Cells ◽  
Developmental Potential ◽  
Fetal Fibroblast ◽  
Functional Quality ◽  
Donor Cells ◽  
Morphological Transformations

One of the most important factors that determine the developmental potential of mammalian cloned embryos is the structuro-functional quality of nuclear donor cells. Biochemical changes that are some of the earliest symptoms of apoptosis signal transduction are not reflected in the morphological features of somatic cells. Therefore, an appropriate system of cell selection would enable the sorting of donor nuclei with high morphological and biochemical susceptibility to somatic cloning. The aim of our study was to examine the in vitro developmental competencies of porcine nuclear transfer (NT) embryos reconstructed with fetal fibroblast cells that had been analyzed for apoptosis by live-fluorescent labelling. Frozen/thawed fetal fibroblast cells, which had been in vitro-cultured to a confluent state, were used for analysis. To detect the early apoptotic changes in the fibroblast cells, a single cell suspension of nuclear donor cells was subjected to dyeing with live-DNA green fluorochrome YO-PRO-1. The recipient cells were in vitro-matured oocytes. Maternal chromosomes were removed by a chemically assisted microsurgical technique. Then, single nuclear donor cells were inserted into the perivitelline space of enucleated oocytes. Fibroblast cell-ooplast couplets were simultaneously fused and activated with two consecutive DC pulses of 1.2 kV/cm for 60 μs. Reconstructed embryos were in vitro cultured in 50-μL drops of NCSU-23 medium supplemented with 0.4% BSA-V for 6 to 7 days at 38.5°C in a humidified atmosphere of 5% CO2 and 95% air. The rates of cleavage and development to morula/blastocyst stages were examined on Days 2 and 6/7, respectively. After fluorescent analysis of approximately 50 different random samples collected from the population of fetal fibroblast cells, that had been labelled with YO-PRO-1 dye, it was found that a relatively high proportion of donor cells revealed ultrastructural apoptotic changes. The percentage of late apoptotic cells with advanced morphological transformations was about 40% of the total pool of the fibroblast cells. A total of 262/270 (97.0%) enucleated oocytes were subjected to reconstruction and 141/262 (53.8%) were successfully fused with non-apoptotic nuclear donor cells. Following the simultaneous fusion/activation protocol, reconstituted oocytes were selected for in vitro culture. Out of 262, 133 (50.8%) cultured NT embryos cleaved. The frequencies of cloned embryos that reached the morula and blastocyst stages were 48/133 (36.0%) and 10/133 (7.5%), respectively. In conclusion, morphology is a sufficient selection factor for detection of apoptosis in the cultured (confluent) fetal fibroblast cells to be used for cloning. Moreover, it was found that YO-PRO-1 fluorochrome may be not able to detect the early phases of apoptosis, because only the morphologically abnormal cells emitted the YO-PRO-1-derived fluorescence. This research was supported by the State Committee for Scientific Research as a Solicited Project number PBZ-KBN-084/P06/2002/4.2 from years 2003 to 2005.

52 THE EFFECTS OF DONOR CELL CYCLE AND THE TIMING OF OOCYTE ACTIVATION ON DEVELOPMENT OF BOVINE NUCLEAR TRANSFERRED EMBRYOS IN VIVO

2011 ◽  
Vol 23 (1) ◽  
pp. 132 ◽  
Author(s):  
K. Matsukawa ◽  
S. Akagi ◽  
K. Fukunari ◽  
Y. Hosokawa ◽  
C. Yonezawa ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Embryo Transfer ◽  
Formation Rate ◽  
Donor Cell ◽  
Serum Starvation ◽  
Oocyte Activation ◽  
Blastocyst Formation ◽  
Donor Cells ◽  
G0 Phase ◽  
G1 Cells

The cell cycle of donor cells and recipient cytoplasts are important factors affecting development of nuclear transferred (NT) embryos. We previously showed that bovine NT embryos using pre-activated cytoplasts and early G1 cells had a high in vitro developmental rate (SSR, 2008, 41st Annual Meeting). The objective of the present study was to evaluate the effects of donor cell cycle (early G1 or G0 phase) and the timing of oocyte activation on fetal development of bovine NT embryos. Adult fibroblasts from ear skin tissue of Japanese black cattle were used as donor cells. The G0 phase cells were synchronized by serum-starvation, and the G1 phase cells were prepared from actively dividing M phase cells. NT embryo production was performed by 2 kinds of protocols as follows: 1) recipient oocytes were activated by Ca ionophore (CaI), followed with cycloheximide (CH) for 2 h, and fused with synchronized donor cells followed with cytochalasin D (CD) and CH for 1 h, then CH for 4 h (pre-activated), 2) unactivated oocytes were fused with synchronized donor cells and activation was performed by CaI 1 h after fusion, followed by with CD and CH 1 h, then CH for 4 h (post-activated). After activation treatments, NT embryos were cultured in IVD101 medium for 7 days. Then, blastocysts were transferred to recipient cows. Diagnosis of pregnancy was made by ultrasonography at days 30, 60, and 90 (Day 0 = the day of embryo transfer). As shown in Table 1, the blastocyst formation rate of the NT embryos derived from early G1 cells in the pre-activated group was higher than that from G0 cells in the post-activated group (36% v. 23%, P < 0.05). After embryo transfer, 29, 67, and 50% of recipient cows were pregnant at Day 30 in G0 post-, G1 post-, and G1 pre-activated groups, respectively. However, only 1 embryo (14%) of G0 post-activated group developed to term. In conclusion, bovine NT embryos using early G1 cells and pre-activated cytoplasts showed a high blastocyst formation rate, but the full-term development of bovine NT embryos could not be improved by using early G1 cells and pre-activated cytoplasts. Table 1.Effect of the timing of oocyte activation on developmental ability of bovine NT embryos derived from early G1 or G0 phase cells

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