42 EFFECT OF S-ADENOSYLHOMOCYSTEINE, A NON-TOXIC EPIGENETIC MODIFYING REAGENT, ON PORCINE FEMALE DONOR CELLS AND CLONED EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 169 ◽  
Author(s):  
J. T. Kang ◽  
J. Y. Choi ◽  
S. J. Park ◽  
S. J. Kim ◽  
J. H. Moon ◽  
...  
Keyword(s):  
Dna Methylation ◽  
X Chromosome ◽  
Dna Demethylation ◽  
Control Group ◽  
Time Interval ◽  
Normal Donor ◽  
In Vitro Development ◽  
Donor Cells ◽  
Vitro Development

Despite great advances in the field of cloning techniques, the efficiency of production of cloning animals is very low. Maybe the poor outcome of somatic cell nuclear transfer (SCNT) is thought to be a consequence of incomplete reprogramming of the donor cell or cloned embryos. The objective of this study was to investigate the effects of treatment with S-adenosylhomocysteine (SAH), the reversible nontoxic inhibitor of DNA methyltransferases (DNMT), on porcine female fibroblast donor cells and in vitro development of cloned embryos. We hypothesized that SAH targeting DNA methylation could alter chromatin configuration and turn it more amenable to reprogramming. Thus, the female fibroblast donor cells were cultured in media containing respective concentrations of SAH [0 (control), 0.1, 0.5, and 1 mM) for 2 passages. One-way ANOVA was used to determine significant differences in the data and a Tukey test was done to determine statistical differences among groups. Compared with nontreated controls, the cells treated with SAH, especially 1 mM, revealed significantly (P < 0.05) reduced global DNA methylation, proved by commercial kit and immunocytochemistry analysis, and elevation of transcript levels for X chromosome-linked genes (XIST and HPRT), estimated by real-time PCR analysis compared with the control group. It was suggested that treatment with SAH in female cells could make cells into more valuable donor cells for cloning. In another trial, cloned embryos using normal donor cells were cultured in media containing 1 mM SAH for 0 (control), 12, and 24 h after activation on different time interval of DNMT inhibition, transferred to PZM5 media, and subsequently cultured for 7 days. Treatment with SAH for 12 h resulted in 13.0 ± 1.9% blastocyst production, which was significantly greater than cloned embryos treated with SAH for 24 h (11.2 ± 2.1%) and control cloned embryos (9.1 ± 1.2%). It was suggested that the appropriate DNMT inhibition might have an important role in in vitro development of porcine SCNT, and improving effects on developmental competency of cloned embryos. We concluded that SAH induced global DNA demethylation that partially reactivated the X chromosome and that a hypomethylated genome may facilitate the nuclear reprogramming process. This study was supported by IPET (no. 311011-05-1-SB010), MKE (no. 10033839-2012-21), Institute for Veterinary Science, the BK21 program, and TS Corporation.

75 EPIGENETIC EFFECTS OF VITRIFICATION ON PRONUCLEAR DOMESTIC CAT EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 131 ◽  
Author(s):  
J. H. Galiguis ◽  
C. E. Pope ◽  
M. N. Biancardi ◽  
C. Dumas ◽  
G. Wang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Genetic Material ◽  
Domestic Cat ◽  
Blastocyst Development ◽  
In Vitro Development ◽  
Epigenetic Effects ◽  
Donor Cells ◽  
Vitro Development

Vitrification remains a promising technique in the preservation of valuable genetic material; however, in the cat, success has varied. Live kittens have been produced from embryos vitrified at early cleavage stages, but phenotypic abnormalities in some kittens suggest possible epigenetic effects of the vitrification process. It has been reported that cryopreservation alters epigenetic events in somatic donor cells, which indirectly influences physical status of cloned offspring. However, extending post-warming in vitro culture of donor cells corrects these epigenetic modifications, resulting in normal embryos/clones. Accordingly, in the present study, vitrification was performed at the pronuclear stage to lengthen pretransfer culture time, and vitrified cat zygotes were assessed by analysing (1) histone acetylation/methylation, (2) global DNA methylation, (3) pluripotent gene expression, (4) in vitro development, and () in vivo viability. In vivo matured/IVF oocytes were vitrified in 15% dimethyl sulfoxide, 15% ethylene glycol, and 0.5 M sucrose at 16 h post-insemination (PI). After warming in 1.0 M sucrose at 38°C, embryos were fixed at 18 h or 40 h PI, and the nuclear intensity of either acetyl/dimethyl-H3K9 or 5-methylcytosine was determined by immunofluorescence. Results showed that at 18 h PI, mean H3K9ac intensity of vitrified embryos (11.8; n = 6) was higher than that of corresponding nonvitrified (fresh) controls (4.5; n = 6) and the fresh (3.2; n = 11) and vitrified (0.6; n = 7) 40-h groups (2-way ANOVA; P < 0.05). H3K9me2 in the fresh (36.9) and vitrified (32.5) 18-h embryos was similar but increased relative to both fresh (10.7) and vitrified (9.2) 40-h groups (P < 0.05). Mean DNA methylation (5MeC) in the fresh (31.6; n = 1) and vitrified (24.7; n = 3) 18-h groups was similar to that of the fresh 40-h group (19.8; n = 4) but higher than that of the vitrified 40-h group (15.0; n = 5; P < 0.05). To assess expression of POU5F1 and Nanog, qRT-PCR was performed on Day 8 blastocysts. Relative to controls (n = 9), mean POU5F1 and Nanog levels in vitrified blastocysts (n = 24) were 1.38- and 1.98-fold higher, respectively (one-way ANOVA; P > 0.05). In terms of in vitro development, Day 2 cleavage of vitrified zygotes (59%; n = 508) was similar to that of controls (66%; n = 340), but Day 8 blastocyst formation was reduced (9 v. 31%; t-test; P < 0.05). In vivo viability was assessed by oviducal transfer of 41 Day 1 embryos into 2 recipients. One pregnancy was established (50%), with 3 live kittens weighing 70, 79, and 131 g delivered without assistance on Day 65 of gestation. The 2 smaller kittens died within a few hours of birth, with the smallest exhibiting an umbilical hernia and organ exteriorization. The third kitten developed into a normal, healthy adult. In summary, mean H3K9me2, 5MeC, and POU5F1/Nanog expression of vitrified zygotes was similar to corresponding controls. H3K9ac increased at 18h PI as a result of vitrification, but was reduced after culture to 40 h PI. Although vitrified zygotes cleaved in vitro at rates similar to controls, blastocyst development was reduced. In vivo viability was demonstrated; however, postnatal survival of kittens produced was low.

26 EFFECT OF TREATMENT OF BOVINE DONOR CELLS WITH MOUSE EMBRYONIC STEM CELL EXTRACT ON THE DEVELOPMENT OF EMBRYOS AFTER NUCLEAR TRANSFER

2011 ◽  
Vol 23 (1) ◽  
pp. 119
Author(s):  
S. Akagi ◽  
E. Mizutani ◽  
Y. Inaba ◽  
M. Kaneda ◽  
T. Somfai ◽  
...  
Keyword(s):  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Control Group ◽  
In Vitro Development ◽  
Donor Cells ◽  
Effect Of Treatment ◽  
Vitro Development

The efficiency of somatic cell cloning is very low, probably because of incomplete reprogramming of the somatic cell nucleus. In recent studies, it is suggested that transient exposure of donor somatic cells to mouse embryonic stem cell (ESC) extract enhances pluripotency of the cells in vitro (Bru et al. 2008 Exp. Cell Res. 314, 1634–1642; Xu et al. 2009 Anat. Rec. 292, 1229–1234). In the present study, we examined the effect of treatment of donor cells with mouse ESC extract on the in vitro development of bovine NT embryos. First, in order to examine effect of treatment of donor cells with streptolysin O (SLO), which reversibly permeabilizes the plasma membrane, we compared the in vitro development of NT embryos using donor cells treated with 5 μg mL–1 SLO (SLO group) and untreated donor cells (control group). As donor cells for NT, bovine fibroblast cells of passages 3 to 5 were used. Fibroblasts were treated with 5 μg mL–1 SLO for 45 min, and then incubated for resealing in DMEM including 2 mM CaCl2 for 60 min. NT was performed as previously described (Akagi et al. 2003 Mol. Reprod. Dev. 66, 264–272). After in vitro culture for 8 days, blastocyst formation and cell number of blastocysts were examined. There were no significant differences between SLO and control groups in the fusion rate (80% and 72%, respectively), cleavage rate (60% and 65%, respectively), developmental rate to the blastocyst stage of NT embryos (31% and 28%, respectively), and blastocyst cell number (127 ± 6 and 112 ± 14, respectively). These results suggest that SLO treatment of donor cells has no negative effect on the in vitro development of NT embryos. Next, we examined the in vitro developmental ability of NT embryos using donor cells treated with mouse ESC extract (ES extract group). After SLO treatment for 45 min, permeabilized fibroblast cells were treated with mouse ESC extract for 45 min, and then incubated in DMEM including 2 mM CaCl2 for 60 min, and used for producing NT embryos. There were no differences between ES extract and control groups in the fusion rate (68% and 69%, respectively), cleavage rate (86.7% and 80.6%, respectively), and developmental rate to the blastocyst stage of NT embryos (39.8% and 43.5%, respectively). The cell number of NT embryos at the blastocyst stage in ES extract group (201 ± 30) was significantly (t-test; P < 0.05) higher than that in control group (140 ± 14). In conclusion, treatment of bovine donor cell with mouse ESC extract did not affect the in vitro developmental ability of NT embryos, but improved the quality of blastocysts.

scholarly journals The HDAC Inhibitor LAQ824 Enhances Epigenetic Reprogramming and In Vitro Development of Porcine SCNT Embryos

2017 ◽  
Vol 41 (3) ◽  
pp. 1255-1266 ◽  
Author(s):  
Jun-Xue Jin ◽  
Sanghoon Lee ◽  
Anukul Taweechaipaisankul ◽  
Geon A. Kim ◽  
Byeong Chun Lee
Keyword(s):  
Dna Methylation ◽  
Formation Rate ◽  
Nuclear Reprogramming ◽  
In Vitro Development ◽  
Histone 3 ◽  
Expression Of Genes ◽  
Epigenetic Remodeling ◽  
Low Efficiency ◽  
Vitro Development

Background/Aims: Hypoacetylation caused by aberrant epigenetic nuclear reprogramming results in low efficiency of mammalian somatic cell nuclear transfer (SCNT). Many epigenetic remodeling drugs have been used in attempts to improve in vitro development of porcine SCNT embryos. In this study, we examined the effects of LAQ824, a structurally novel histone acetylase inhibitor, on the nuclear reprogramming and in vitro development of porcine SCNT embryos. Methods: LAQ824 treatment was supplemented during the culture of SCNT embryos. The reprogramming levels were measured by immunofluorescence and quantified by image J software. Relative expression levels of 18 genes were analyzed by quantitative real-time PCR. Results: 100 nM LAQ824 treatment of post-activation SCNT embryos for 24 h significantly improved the subsequent blastocyst formation rate. The LAQ824 treatment enhanced histone 3 lysine 9 (H3K9) levels, histone 4 lysine 12 (H4K12) levels, and reduced global DNA methylation levels as well as anti-5-methylcytosine (5-mC) at the pseudo-pronuclear and 2-cell stages. Furthermore, LAQ824 treatment positively regulated the mRNA expression of genes for histone acetylation (HAT1, HDAC1, 2, 3, and 6), DNA methylation (DNMT1, 3a and 3b), development (Pou5f1, Nanog, Sox2, and GLUT1) and apoptosis (Bax, Bcl2, Caspase 3 and Bak) in blastocysts. Conclusion: Optimum exposure (100 nM for 24 h) to LAQ824 post-activation improved the in vitro development of porcine SCNT embryos by enhancing levels of H3K9 and H4K12, reducing 5-mC, and regulating gene expression.

174 THE EFFECT OF DIFFERENT OVARY TRANSPORT TEMPERATURES ON IN VITRO DEVELOPMENT AND POST-THAW SURVIVAL OF BOVINE EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 203
Author(s):  
S. R. Cho ◽  
S. H. Choi ◽  
H. J. Kim ◽  
C. Y. Choe ◽  
H. J. Jin ◽  
...  
Keyword(s):  
Room Temperature ◽  
Development Rate ◽  
Control Group ◽  
Bovine Embryos ◽  
In Vitro Development ◽  
Embryo Freezing ◽  
Nuclear Maturation ◽  
Vitro Development ◽  
And Control

The present study was carried out to investigate the effect of different ovary transport temperatures on in vitro development and post-thaw survivability of bovine embryos. Bovine ovaries were collected at a local slaughterhouse and transported at 4 different temperature categories to the laboratory: 7–10�C (T1), 11–17�C (T2), 18–25�C (T3), and above 26�C (control group). The cumulus–oocyte complexes (COCs) were aspirated from 2–8 mm antral follicles using a syringe with an 18 gauge needle. Selected COCs were washed in HEPES-buffered tissue culture medium (TCM-199) supplemented with 5% FBS. Sets of 50 COCs were matured for 22 h in 4-well dishes of TCM-199 supplemented with 5% FBS, 10 �g mL-1 LH, and 10 �g mL-1 FSH, that had been previously covered with mineral oil and equilibrated in an atmosphere of 5% CO2 in air at 39�C. Mature COCs were fertilized with frozen–thawed semen treated with BO medium. To evaluate nuclear maturation to the metaphase II stage, the matured COCs were fixed in 1 : 3 acetic acid–ethanol for 30 s and stained with 3% basic Fuchsin. For embryo freezing, Day 7 and 8 blastocysts were equilibrated for 15 min in 1.8 M ethylene glycol as a cryoprotectant. Embryos were loaded into 0.25-mL straws at room temperature, plunged directly into a cooling chamber, kept at -7�C for 10 min, including time for seeding, and further cooled to -35�C at -0.3�C min-1; after 2 min at this temperature, they were plunged into liquid nitrogen. Thawing was performed by keeping straws at room temperature for 10 s, followed by immersion in a water bath at 37�C. The appearance of the embryos was evaluated immediately after warming and again at 24-h intervals for at least 3 days. The development rate was assessed by the re-expansion of the blastocoel and the hatching of blastocysts. Results were compared by ANOVA. The rates of maturation (to metaphase II), cleavage, and development to blastocysts were compared among treatment groups. Furthermore, frozen–thawed blastocysts were in vitro cultured to compare the survivability among groups. The maturation rates in the T1, T2, and T3 groups (24/40, 60.0%; 25/41, 61.0%; and 30/44, 68.2%, respectively) were significantly lower than that in the control group (36/44, 81.8%; P &lt; 0.05). The cleavage rates in the T1 and T2 groups (61/116, 52.6% and 66/121, 54.5%) were significantly lower than that in the control group (112/134, 83.6%; P &lt; 0.05). However, there was no difference in the development rate to blastocysts among all groups (27.9–33.0%; P &gt; 0.05). The survivability of frozen–thawed embryos was significantly lower in the T1 group (6/13, 46.2%) than in the T2 (11/16, 68.8), T3 (13/18, 72.2%), and control groups (19/26, 73.1%; P &lt; 0.05). In conclusion, the results suggest that ovary transport at 26�C may be optimal for better in vitro development and survival of frozen–thawed embryos produced in vitro. Furthermore, exposure of ovaries to temperatures below 10�C during transport may significantly decrease both in vitro development and survivability of frozen-thawed blastocysts.

297 PRODUCTION OF CHIMERIC PORCINE FETUSES BY AGGREGATION METHOD USING PARTHENOGENETIC EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 296
Author(s):  
K. Nakano ◽  
M. Watanabe ◽  
H. Matsunari ◽  
T. Matsuda ◽  
K. Honda ◽  
...  
Keyword(s):  
Cell Mass ◽  
Ips Cells ◽  
Large Animal ◽  
Aggregation Method ◽  
Cell Stage ◽  
In Vitro Development ◽  
Donor Cells ◽  
Vitro Development ◽  
Parthenogenetic Embryos

Porcine induced pluripotent stem (iPS) cells are considered to be an invaluable research tool in translational research with pigs as a large animal model. Pluripotency of the iPS cells needs to be verified by their competence to contribute to chimera formation. The aim of the present study is to establish feasible system to create chimeric pig fetuses using parthenogenetic embryos. In Experiment 1, inner cell mass (ICM) was isolated by immunosurgery from Day 6 blastocysts obtained by parthenogenetic activation of in vitro matured (IVM) oocytes. Isolated ICM were used as the donor cells after staining with fluorescent carbocyanine dye (DiI). Using parthenogenetic morulae or 4- to 8-cell embryos as the host embryos, chimeric embryos were prepared by injection or aggregation method. Injection of ICM was performed by micromanipulation: a single ICM was directly injected into the centre portion of the host morulae. In the aggregation method, a single ICM was aggregated with blastomeres isolated from 2 host embryos at the morula or 4- to 8-cell stage in a micro-well (400 µm diameter, 300 µm deep). The chimeric embryos were cultured in PZM-5 (Yoshioka et al. 2008) for 2 to 3 days to examine development to blastocysts and incorporation of donor ICM cells into the resultant blastocysts ICM (ICM chimerism). In Experiment 2, donor blastomeres isolated from a parthenogenetic morula or 4- to 8-cell embryo were stained by DiI and aggregated with a parthenogenetic host embryo at the morula or 4- to 8-cell stage, and the in vitro development to the blastocyst stage and the ICM chimerism were examined. In Experiment 3, ICM isolated from IVF blastocysts harboring humanized Kusabira-Orange (huKO) gene were used as donor cells. Donor ICM were aggregated with the host embryos at the morula or 4- to 8-cell stage, and the resultant blastocysts were transferred to 4 recipient gilts to collect fetuses on Day 18. Results of Experiments 1 and 2 are summarised in Table 1. Combination of the donor ICM and host morulae yielded high rates of blastocyst formation (~95%) and ICM chimerism (~85%), regardless of the method used (injection or aggregation). Transfer of 73 blastocysts developed from host morulae to 2 recipients (Experiment 3) gave rise to 25 (34.2%) fetuses, of which 6 (24.0%) were confirmed to be chimeric by their clear orange fluorescence and immunostaining by anti-huKO antibody. Of 22 (40.7%) fetuses obtained after transfer of 54 blastocysts derived from 4- to 8-cell host embryos to 2 recipients, 3 (13.6%) were chimeric. Contribution of the donor cells in the tissues of the chimeric fetuses measured by image analysis software (ImageJ, NIH, Bethesda, MD, USA) ranged between 16.1 and 65.2%. These results demonstrate that the aggregation method using parthenogenetic host embryos is an efficient means to produce chimeric pig fetuses, and thereby feasible for verification of pluripotent cells such as iPS cells. Table 1.In vitro development of injected or aggregated porcine embryos

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.

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

173 THE EFFECT OF AMINO ACIDS AND HYALURONAN ON BOVINE EMBRYO IN VITRO DEVELOPMENT AND GENE EXPRESSION PATTERN

2006 ◽  
Vol 18 (2) ◽  
pp. 194 ◽  
Author(s):  
A. T. Palasz ◽  
J. Beltrán Breña ◽  
P. De la Fuente ◽  
M. F. Martinez ◽  
A. Gutiérrez-Adán
Keyword(s):  
Gene Expression ◽  
Amino Acids ◽  
Embryo Development ◽  
Control Group ◽  
Bovine Embryo ◽  
In Vitro Development ◽  
Glut 1 ◽  
Vitro Development ◽  
And Control

We have previously shown that bovine embryos cultured in SOFaa (BME + MEM amino acids) culture medium with hyaluronan (HA) + BSA are of better quality (Guti�rrez-Ad�n et al. 2005 Reprod. Fertil. Dev. 17, 219). Our objective was to examine the effect of essential (BME) or non-essential (MEM) amino acids with or without HA (MAP-5; Bioniche, Inc., Belleville, Ontario, Canada) on bovine embryo in vitro development and mRNA transcription of five developmentally important genes; apoptosis (Bax), growth factor (IGF-II), glucose (Glut-1) and fructose (Glut-5) transport and metabolism, and cell to cell adhesion (Cx-43). A total of 1474 presumptive zygotes (5 replicates) were initially cultured in 40 �L drops in the following groups: Group 1, control, SOFaa; Group 2, SOF-1 (MEM only); and Group 3, SOF-2 (BME only). On Day 4 (~96 h post-insemination (pi) the number of zygotes that had developed to d8 cells was recorded and 10 �L of SOF-1 and SOF-2, each with 2.5 mg/mL HA, was added to half of the embryos from Groups 2 and 3, respectively; the other half of Groups 2 and 3 and control group received 10 �L of corresponding medium without HA. Embryos were cultured under paraffin oil at 39�C and 5% CO2 in humidified air. Cleavage rates were recorded on Day 2 and the number of blastocysts on Days 7, 8, and 9. Five blastocysts from each replicate from each treatment were frozen for determination of gene expression patterns later. Cleavage rates and embryo development 96 h pi were compared among groups by chi-square analysis. The effects of HA and medium on blastocyst rates were analyzed by logistic regression and the data on mRNA expression by one-way repeated-measures ANOVA. Cleavage rates were 81.1% in SOFaa and 79.3% in SOF-1 (P = 0.48) and different from those in the SOF-2 group (72.4%; P < 0.02). The proportion of embryos that developed to d8 cells at Day 4 was higher in the control (46.7%) and SOF-1 (46.8%) groups than in the SOF-2 group (32.6%). The number of blastocysts that developed in SOFaa (37.0%), SOF-1 (37.7%), and SOF-1 + HA (37.8%) were higher (P < 0.001) than those in SOF-2 (19.6%) and SOF-2 + HA (21.8%). The level of expression of Glut-5 was not different among the groups. However, SOF-2 was the only group that had significantly lower expression of Glut-5, Igf II, and Cx43, and higher expression of BAX (P < 0.05) as compared to the control group and the SOF-1 groups with or without HA. Addition of HA to SOF-2 medium increased expression of Glut-1 and Igf II and decreased expression of BAX as compared to the SOF-1 only and control groups and the SOF-2 groups with or without HA (P < 0.05). The level of expression of Cx43 was higher in the control than in four remaining groups, and lower in the SOF-2 than in the SOF-1 group (P < 0.05). Addition of HA increased expression of Cx43 in both SOF-1 and SOF-2 groups but this level of expression was lower than in the control group; the level in the SOF-2 + HA group was lower (P < 0.05) than in the SOF-1 + HA group. We conclude that, within our protocol, MEM amino acids only stimulate embryo development to the blastocyst stage and the addition of HA to the SOF-MEM and SOF-BME media on Day 4 of culture improved embryo quality.

35 USE OF METAPHASE DONOR CELLS AND ACTIVATION WITH ROSCOVITINE FOR SOMATIC CELL NUCLEAR TRANSFER IN BOVINE

2017 ◽  
Vol 29 (1) ◽  
pp. 125
Author(s):  
G. V. Landschoot ◽  
V. Savy ◽  
N. Canel ◽  
S. Ferraris ◽  
D. Salamone
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cytochalasin B ◽  
M Cells ◽  
In Vitro Development ◽  
Donor Cells ◽  
G1 Cells ◽  
Vitro Development

Cloning of domestic species by somatic cell nuclear transfer (SCNT) continues to be inefficient, probably due to an incomplete reprogramming of the reconstituted embryo. The ability of the embryonic cytoplasm to support reprogramming fluctuates within the cell cycle (Egli et al. 2007 Nature 447, 679–85). In this context, we compared the development capability and second polar body (2PB) extrusion of embryos produced by metaphase (M) cells, in comparison with G0/G1 cells, which are commonly used as nuclear donors. Because M cells have 2 sets of chromosomes (in contrast with G0/G1 cells, which have only 1 set), an activation protocol that impedes 2PB extrusion is required to produce reconstituted embryos with the correct ploidy. Therefore, we performed SCNT with M or G0/G1 cells, followed by different activation protocols, and evaluated in vitro development and 2PB extrusion of the reconstituted embryos. Cow oocytes were in vitro matured and enucleated as described by Gambini et al. (2014 PLoS One 14, 9). A group of cells at 70 to 80% confluence was synchronized in M stage using 0.05 μg mL−1 demecolcine for 3 to 4 h and used as nuclear donors for SCNT (M group). Another group of cells was induced into quiescence by serum starvation for 3 to 4 days before SCNT (G0/G1 group). For activation, reconstituted embryos were treated with 5 µM ionomycin (Io) for 4 min followed by 5-h incubation in 50 μM roscovitine for M group, or in 50 μM roscovitine and 5 μg mL−1 cytochalasin B for G0/G1 group. Parthenogenetic controls were activated with Io followed by 50 μM roscovitine alone (ROSCO) or with 5 μg mL−1 cytochalasin B (ROSCO/CB). Hoescht 33342 staining was performed 16 h post-Io to evaluate 2PB extrusion. Other activated oocytes were cultured in SOFaa medium and rates of cleavage, morulas, and blastocysts were evaluated at Days 2, 5 and 7 of in vitro development, respectively. Data were analysed by Fisher’s exact test (P < 0.05). Rates of 2PB extrusion were 72.72 (n = 33), 65.63 (n = 32), 80 (n = 15), and 42.86 (n = 14) for M, G0/G1, ROSCO, and ROSCO/CB, respectively. Results of in vitro development are shown in Table 1. In conclusion, somatic M cells can be used as donors to produce cloned embryos. The M and G0/G1 groups were able to induce cloned blastocysts, even though rates did not differed statistically from controls groups (ROSCO and ROSCO/CB). The M group was as effective as G0/G1. Although further analysis is required to establish the quality of the embryos, our results are encouraging for use in SCNT. Table 1.In vitro development of NT embryos produced with M and G0/G1 donor cells

In vitro development of green fluorescent protein (GFP) transgenic bovine embryos after nuclear transfer using different cell cycles and passages of fetal fibroblasts

2000 ◽  
Vol 12 (2) ◽  
pp. 1 ◽  
Author(s):  
Sangho Roh ◽  
Hosup Shim ◽  
Woo-suk Hwang ◽  
Jong-taek Yoon
Keyword(s):  
Green Fluorescent Protein ◽  
Nuclear Transfer ◽  
Fluorescent Protein ◽  
Blastocyst Formation ◽  
In Vitro Development ◽  
Donor Cells ◽  
Fetal Fibroblasts ◽  
Green Fluorescent ◽  
Vitro Development

Nuclear transfer using transfected donor cells provides an efficient new strategy for the production of transgenic farm animals. The present study assessed in vitro development of nuclear transfer embryos using green fluorescent protein (GFP) gene-transfected bovine fetal fibroblasts. In experiment 1, bovine fetal fibroblasts (BFF) were transfected with linearized pEGFP-N1 by electroporation, and the enucleated oocytes were reconstructed by nuclear transfer of transfected cells (BFF-GFP). The rates of blastocyst formation did not differ significantly between BFF and BFF-GFP (18.2% v. 15.6%). In experiment 2, before nuclear transfer, the donor cell stage was synchronized by serum deprivation or forming a confluent monolayer. The rates of cleavage (67.1% v. 71.8%) and blastocyst formation (15.8% v. 15.5%) did not differ between confluent and serum-starved cells after nuclear transfer. In experiment 3, the effects of different passages of donor fibroblast cells on the development of nuclear transfer embryos were investigated. Donor cells from ‘early’ (at passage 8–16) showed better blastocyst development (18.9%) than those from ‘late’ (at passage 17–32; 10.5%). In conclusion, this study suggests that transgenic somatic cell nuclei from early passages can be reprogrammed more effectively than those from late passages. In addition, GFP, a non-invasive selection marker, can be used to select transgenic nuclear transfer embryos.

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