scholarly journals 71EFFECT OF CALCIUM IONOPHORE AND CYTOCHALASIN D ON ACTIVATION AND IN VITRO DEVELOPMENT OF NUCLEAR TRANSFER BOVINE EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 157 ◽  
Author(s):  
S.J. Rzucidlo ◽  
S. Arat ◽  
S.L. Stice
Keyword(s):  
Nuclear Transfer ◽  
Calcium Ionophore ◽  
Cytochalasin D ◽  
Small Cells ◽  
In Vitro Development ◽  
Group Iii ◽  
Group I ◽  
Fetal Bovine ◽  
Vitro Development

The activation of oocytes is one of the most important steps for a successful cloning, and chemicals used for activation can affect the viability of cloned offspring. Therefore, some of them may be omitted for activation to eliminate their possible detrimental effect on nuclear transfer (NT) embryos. The objective of this study was to examine the effect of calcium ionophore (CaI, A23187, Sigma, St. Louis, MO, USA) and cytochalasin D (CD) on activation and in vitro development of nuclear transfer units derived from bovine granulosa cells (GCs) treated with the cell cycle inhibitor, roscovitine. Bovine oocytes isolated from slaughterhouse ovaries were matured in TCM199 supplemented with fetal bovine serum (FBS), sodium pyruvate, penicillin/streptomycin, rIGF-1, bFSH, and bLH. GCs were isolated from ovarian follicles and cultured in DMEM-F12 supplemented with 10% FBS at 37°C in 5% CO2 in air. Prior to NT, donor cells were exposed to 15mM roscovitine for 24 hours and small cells were used for NT. A single cell was inserted into the perivitelline space of the enucleated oocyte. Oocyte-cell couples were fused using a 20μs DC pulse of 40V/150μm. Two hours after fusion, NT units were assigned into four groups and activated by CaI (5μM for 10min.), and then incubated with cycloheximide (CHX, 10μgmL−1)+CD (2.5μgmL−1) for 1h, followed by CHX alone (without CD) for 5h (Group I) or CaI (for 10min.), followed by CHX alone for 6h (Group II). In the Group III, NT units were activated by CHX (10μgmL−1)+CD (2.5μgmL−1) for 1h, followed by CHX for 5h, and Group IV, CHX alone for 6h. The base activation medium was TCM199 with 1% FBS for CaI and 10% FBS for CHX and CD. After activation, NT units were cultured for 7 days in BARC medium. Differences in activation, cleavage and blastocyst formation rates among treatments were analyzed by one-way ANOVA after arcsin square transformation. The results are summarized in Table 1. Our data showed that CaI and CD did not affect the activation and in vitro development of NT embryos derived from roscovitine-treated GCs. It suggests that both chemicals may be redundant during cloning procedure. This study was supported by a grant from ProLinia, Inc and TUBITAK, Turkey (VHAG-1908-102V048). Table 1 In vitro development of NT embryos in different activation treatments

scholarly journals 25COLD STORAGE OF TISSUES AS SOURCE FOR DONOR CELLS DOES NOT REDUCE THE IN VITRO DEVELOPMENT OF BOVINE EMBRYOS FOLLOWING NUCLEAR TRANSFER

2004 ◽  
Vol 16 (2) ◽  
pp. 135 ◽  
Author(s):  
S. Arat ◽  
H. Bagis ◽  
F. Ergin ◽  
H. Sagirkaya ◽  
H.O. Mercan ◽  
...  
Keyword(s):  
Articular Cartilage ◽  
Cell Line ◽  
Nuclear Transfer ◽  
Calf Serum ◽  
Calcium Ionophore ◽  
Penicillin G ◽  
In Vitro Development ◽  
Viable Cells ◽  
Vitro Development

So far, most calves have been cloned from live adult cows or fresh fetal samples. There are few reports on using cells from a dead mammal for nuclear transfer (NT). This study was conducted to investigate whether different kind of viable cells could be obtained from tissues stored in cold for different duration and whether these cells could be used for NT. Bovine oocytes isolated from slaughterhouse ovaries were matured in TCM199 supplemented with 10% fetal calf serum (FCS), 50μgmL−1 sodium pyruvate, 1% v:v penicillin-streptomycin (10.000UmL−1 penicillin G, 10.000μgmL−1 streptomycin), 10ngmL−1 EGF, 0.5μgmL−1 FSH, and 5μgmL−1LH. First cell line (CC) was established from articular cartilage of the leg of a slaughtered cow stored at 0°C in a cold storage room for 48h. Second cell line (MC) was established from leg muscle of a cow carcass stored at 0°C for 24h. Tissues from articular cartilage and muscle were cut into small pieces. Tissue explants were cultured in DMEM-F12 supplemented with 10% FBS at 37°C in 5% CO2 in air. Bovine granulosa cells (GC) were isolated from ovarian follicles and used for NT as control cells. Prior to NT, all somatic cells were allowed to grow to confluency (G1/G0) in DMEM-F12 supplemented with 10% FBS. Cumulus cells were removed by vortexing with hyaluronidase at 18h after the start of maturation. Matured oocytes labeled with DNA fluorochrome Hoechst 33342 were enucleated under UV to ensure full removal of the chromatin. A single cell was inserted into the perivitelline space of the enucleated oocyte. Oocyte-cell couples were fused by a DC pulse of 133V/500μm for 25μs. After fusion, NT units were activated using a combination of calcium ionophore (5μM), cytochalasin D (2.5μgmL−1), and cycloheximide (10μgmL−1), and cultured for 7 days. Differences among groups were analyzed by one-way ANOVA after arcsin square transformation. The results are summarized in Table 1. The results suggest that viable cells can be obtained from articular cartilage and muscle of a cow carcass stored at cold temperature for 24 and 48h and these cells have ability to generate NT blastocysts at rates similar to that of the controls. This study was supported by a grant from TUBITAK, Turkey (VHAG-1908-102V048). F Ergin is a volunteer young researcher. Table 1 In vitro development of NT embroys from different cell lines

scholarly journals 64COMPARISON OF DEVELOPMENTAL POTENTIAL OF IN VIVO AND IN VITRO RECIPIENT OOCYTES AFTER NUCLEAR TRANSFER IN GOAT

2004 ◽  
Vol 16 (2) ◽  
pp. 154
Author(s):  
H.S. Park ◽  
M.Y. Lee ◽  
S.P. Hong ◽  
J.I. Jin ◽  
J.K. Park ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Electric Pulse ◽  
Serum Starvation ◽  
Cleavage Rate ◽  
Developmental Potential ◽  
In Vitro Development ◽  
Significant Difference ◽  
Vitro Development

Recent techniques in somatic cell nuclear transfer (SCNT) have been widely used for animal research. In addition, SCNT techniques may allow for the rescue of endangered species. Despite efforts for wildlife preservation, however, some threatened or endangered wild animal species will likely become extinct. As a preliminary experiment of a series in wildlife research, we tried to identify an improved method for the production of more transferable NT embryos in goats. Mature donor animals of Korean native goats (20–25kg) were synchronized with a CIDR (type G; InterAg, New Zealand) vaginal implant for 10 days followed by a total of 8 twice daily injections of 70mg of FSH (Folltropine, London, Ontario, Canada) and 400IU of hCG (Chorulon, Intervet, Moxmeer, The Netherlands). Oocytes were then collected surgically by retograde oviduct flush or direct aspiration from ovarian follicles in vivo at 29–34h after hCG. Oocytes collected from follicles were matured in TCM-199 containing 10% FBS and hormones. Prepared ear skin cells from the goat were cultured in TCM-199 containing 10% FBS at 39°C, 5% CO2 in air, and confluent monolayers were obtained. Oocytes were enucleated and donor cells from serum starvation (0.5%) culture were fused through a single electric pulse (DC 2.36kvcm−1, 17μs), and then activated by a single electric pulse (AC 5vmm−1, 5s+DC 1.56kvcm−1, 30μs) or chemical treatment (5μgmL−1 ionomycin 5min−1, 1.9mM 6-DMAP/4h). Reconstructed oocytes were cultured in M16 medium with 10% goat serum (GS) for 6–7 days. Data were analyzed by chi-square test. In in vitro development, significantly (P<0.05) more oocytes were cleaved (24/30, 80.0%) and developed (7/24, 29.2%) to morula or blastocyst stage, respectively, in NT oocytes activated by Iono + DMAP compared to electric stimulated oocytes (2/21, 40.0%; 0/2, 0%). There was a significant difference in in vitro development of NT embryos by the method of oocyte collection. Cleavage rate was higher (P<0.05) in NT embryos from in vivo oocytes (23/28, 82.1%) than in in vitro matured oocytes (19/35, 54.3%), and further development to morula or blastocyst was also significantly (P<0.05%) higher in NT embryos from in vivo oocytes (7/23, 30.4%) than in NT embryos from in vitro matured oocytes (0/19, 0%). When we compared NT embryos to parthenotes, developmental rate was not significantly different between NT embryos and parthenotes. These results strongly suggest that the in vivo oocytes will have superior developmental potential to oocytes matured in vitro. Table 1 Effect of different oocyte source on in vitro development following caprine SCNT

scholarly journals Streptolysin-O Treatment of Fetal Fibroblasts Improves Cell Fusion and In Vitro Development of Porcine Nuclear Transfer Embryos

2009 ◽  
Vol 55 (3) ◽  
pp. 236-239 ◽  
Author(s):  
Kenji NARUSE ◽  
Yan-Shi QUAN ◽  
Baek-Chul KIM ◽  
Su-Min CHOI ◽  
Chang-Sik PARK ◽  
...  
Keyword(s):  
Cell Fusion ◽  
Nuclear Transfer ◽  
In Vitro Development ◽  
Streptolysin O ◽  
Fetal Fibroblasts ◽  
Vitro Development

Effects of cycloheximide treatment and interval between fusion and activation on in vitro development of pig nuclear transfer embryos

2002 ◽  
Vol 14 (4) ◽  
pp. 191 ◽  
Author(s):  
M. A. Martinez-Diaz ◽  
K. Ikeda ◽  
Y. Takahashi
Keyword(s):  
Nuclear Transfer ◽  
Kinase Activity ◽  
Short Interval ◽  
Cumulus Cells ◽  
Cleavage Rate ◽  
In Vitro Development ◽  
M Phase ◽  
Cytostatic Factor ◽  
Vitro Development

The effects of cycloheximide (CHX) treatment and the interval between fusion and activation on the development of pig nuclear transfer (NT) embryos constructed with enucleated oocytes and serum-starved granulosa/cumulus cells were examined. One group of couplets was fused and activated simultaneously (FAS) by a single electrical pulse (activation pulse). Another three groups of couplets were fused electricaly 1.5, 2.5 or 4.5 h before being subjected to the activation pulse (FBA). Each group was divided into two subgroups and incubated with or without CHX. The NT embryos treated with CHX showed a high and stable cleavage rate, regardless of the interval between fusion and activation; however, development to blastocysts was improved only when the NT embryos were subjected to FAS with CHX. These results indicate that CHX-sensitive events occurring shortly after FAS may be responsible for the development to blastocysts. Fusion pulse rarely activated M II oocytes, but rapidly dropped the p34cdc2 kinase activity in NT embryos. A pronucleus-like structure was observed 2-2.5 h after the activation pulse with CHX in NT embryos of both the FAS and FBA groups. Therefore, successive inactivation of M-phase promoting factor and cytostatic factor at a certain short interval may also play an important role in the development of NT embryos.

54 IN VITRO DEVELOPMENT OF BOVINE CLONED EMBRYOS PRODUCED BY HANDMADE CLONING FROM DISTINCT CELL CULTURE CONFLUENCES AND AGGREGATION SCHEMES

2010 ◽  
Vol 22 (1) ◽  
pp. 185
Author(s):  
R. P. C. Gerger ◽  
E. S. Ribeiro ◽  
J. C. Mezzalira ◽  
L. U. Olwheiler ◽  
F. Forell ◽  
...  
Keyword(s):  
Cell Culture ◽  
Somatic Cell ◽  
Nuclear Transfer ◽  
Plateau Phase ◽  
In Vitro Development ◽  
Distinct Cell ◽  
Aggregation Scheme ◽  
Vitro Development ◽  
Handmade Cloning

The coordination of the cell cycle of the donor nucleus and the recipient cytoplasm is thought to be one of the major essential factors needed for successful development of cloned embryos and offspring from somatic cell populations. Cell cycle synchronization protocols used for somatic cell nuclear transfer (SCNT) vary in preference among groups, with the confluence inhibition by contact appearing to be one of the most widely used methods today. However, the relationship between the level of cell confluence in a culture dish at or near the plateau phase of growth and blastocyst yield following cloning by SCNT still needs to be better characterized. The aim of this study was to examine the effect of 3 distinct cell culture confluences before nuclear transfer and embryo aggregation on in vitro development of clone bovine embryos. In vitro-matured bovine COC were used for the production of clone embryos by handmade cloning, according to our established procedures (Ribeiro et al. 2009 Cloning Stem Cells 11). Oocytes were manually bisected following cumulus and zona removal. After selection of hemi-cytoplasts by DNA staining, 1 (50%) or 2 (100%) enucleated hemi-cytoplasts were paired with an adult Nelore skin somatic cell and then electrofused (15 V AC pre-pulse for 5 s, followed by a double 1.2 kV cm-1 DC pulse for 20 μs). Cells were selected from 1 out of 3 distinct culture confluences: (1) 70 to 80%; (2) 80 to 90%; and (3) >90%; assessed by morphological evaluation before the SCNT procedure. Reconstructed clone embryos and groups of zona-intact oocytes (parthenote controls) were activated in ionomycin and 6-DMAP. Clone embryos (100%) and hemi-embryos (50%) reconstructed from the 3 groups underwent IVC in the well of the well (WOW) system for 7 days, allocating 1 embryo (1 × 100%) or aggregating two hemi-embryos (2 × 50%) per WOW. After 11 replications, cleavage (Day 2) and blastocyst (Day 7) rates, on a per WOW basis, were compared using the chi-square test. Results are summarized in Table 1. Cleavage rates were similar for all groups. The aggregation scheme did not appear to have influenced, either positively or negatively, the developmental outcome to the blastocyst stage. However, blastocyst rates increased nonlinearly (7.0, 17.5, and 29.4%) with the increase in cell confluence. A highly confluent cell culture has already been shown to have a greater proportion of cells in G0/G1 than cycling cells at the log phase (>91% v. 59%; Sun et al. 2008 Zygote 16, 111-116). However, blastocyst development in this study was lower than anticipated for cells in the early plateau phase (70 to 80%), when predicting such development based on the expected G0/G1 proportion in that cell population. Table 1.In vitro development of bovine cloned embryos from distinct cell culture confluences and aggregation scheme This study was supported by FAPESP and CAPES/Brazil.

64 VALPROIC ACID ENHANCES IN VITRO DEVELOPMENT OF SOMATIC CELL NUCLEAR TRANSFER EMBRYOS IN PIGS

2010 ◽  
Vol 22 (1) ◽  
pp. 190
Author(s):  
Y. J. Kim ◽  
K. S. Ahn ◽  
M. J. Kim ◽  
H. Shim
Keyword(s):  
Stem Cells ◽  
Valproic Acid ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Developmental Competence ◽  
Control Group ◽  
In Vitro Development ◽  
Vitro Development ◽  
And Control

Epigenetic modification influences reprogramming and subsequent development of somatic cell nuclear transfer embryos. Such modification includes an increase of histone acetylation and a decrease of DNA methylation in transferred donor nuclei. Histone deacetylase inhibitors (HDACi) such as trichostatin A (TSA) and valproic acid (VPA) have been known to maintain high cellular levels of histone acetylation. Hence, the treatment of HDACi to NT embryos may increase efficiency of cloning. Indeed, TSA treatment has significantly enhanced the developmental competence of nuclear transfer embryos in several species including pigs (Zhang et al. 2007 Cloning Stem Cells 9, 357-363; Li et al. 2008 Theriogenology 70, 800-808). Valproic acid, another type of HDACi, has often been used to assist reprogramming of somatic cells into induced pluripotent stem cells in mice. In the present study, we tested the potency of VPA compared with TSA on the enhancement of in vitro development in porcine nuclear transfer embryos. Reconstructed embryos were produced by transferring nuclei of adult ear skin fibroblasts into enucleated oocytes. After electrical activation, these embryos were cultured in PZM-3 containing no HDACi (control), 5 mM VPA, or 50 nM TSA for 24 h, and another 5 days thereafter without HDACi. At least 3 replicates were conducted for the following experiments. The rates of cleavage were not different among the VPA, TSA, and control groups. However, the rate of blastocyst development was significantly higher (P < 0.05) in embryos treated with VPA than in those treated with TSA and without HDACi (125/306, 40.8% v. 94/313, 30.0% v. 80/329, 24.3%). Differential staining of inner cell mass (ICM) and trophectoderm (TE) also supported the beneficial effect of VPA treatment in NT embryos. Compared with the control group, the number of TE cells was significantly increased (P < 0.05) in the VPA and TSA treatment groups (79.3 ± 7.4 v. 74.6 ± 9.2 v. 40.0 ± 6.7). Moreover, VPA treatment significantly increased (P < 0.05) the number of ICM cells compared with the control (15.6 ± 1.7 v. 10.8 ± 2.6), whereas no differences were observed between the TSA treatment and control group (12.9 ± 3.0 v. 10.8 ± 2.6). The present study demonstrates that VPA enhances in vitro development of nuclear transfer embryos, in particular by an increase of blastocyst formation and the number of ICM cells, suggesting that VPA may be more potent than TSA in supporting developmental competence of cloned embryos. However, long-term effects of different HDACi in the development of nuclear transfer embryos, including any adverse outcome from destabilizing epigenetic condition, remain to be determined by further in vivo embryo transfer studies.

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