scholarly journals 270 COMBINED ELECTRICAL AND CHEMICAL ACTIVATION OF ZONA-FREE PORCINE OOCYTES

2005 ◽  
Vol 17 (2) ◽  
pp. 284
Author(s):  
P.M. Kragh ◽  
N.R. Mtango ◽  
T.J. Corydon ◽  
L. Bolund ◽  
H. Callesen ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Culture Medium ◽  
Cytochalasin B ◽  
Chemical Activation ◽  
Cumulus Cells ◽  
Control Model ◽  
Parthenogenetic Development ◽  
Cloning Technique ◽  
Cattle Serum ◽  
Handmade Cloning

Activation is a crucial step in mammalian somatic cell nuclear transfer (SCNT). Recently we described the application of the handmade cloning technique for porcine SCNT that uses oocytes without zonaa pellucidae (zona-free) in a micromanipulation-independent procedure (Kragh et al. 2004 Reprod. Fertil. Dev. 16, 315–18). The purpose of the present study was to investigate the effect of a combined electrical and chemical activation of zona-free porcine oocytes. Cumulus-oocyte complexes were aspirated from ovaries of sows and matured for 41 h. Subsequently, the cumulus cells were removed by the addition of 1 mg/mL hyaluronidase in HEPES-buffered TCM-199. For zonae pellucidae removal, oocytes were incubated in 8 mg/mL pronase in HEPES-buffered TCM-199 supplemented with 20% cattle serum for 10 s. Three independent experiments with four treatments were conducted, and oocytes were activated by a combined electrical and chemical activation. Oocytes were washed once in activation medium (0.3 M mannitol, 0.1 mM MgSO4, 0.1 mM CaCl2, and 0.01% polyvinyl alcohol) and transferred to a chamber with two wires (0.5-mm separation) covered with activation medium. After the electrical pulse, oocytes were incubated in culture medium (NCSU-37 containing 4 mg/mL BSA) supplemented with 5 μg/mL cytochalasin B and 10 μg/mL cycloheximide for 6 h. Activated oocytes were cultured in culture medium using the wells of wells system (Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–64) in the submarine incubation system (Vajta et al. 1997 Theriogenology 48, 1379–85). The rate of development into blastocysts was checked on Day 7 of culture. In treatment 1, zona pellucida-intact oocytes were first activated by a single DC pulse of 1.25 kV/cm for 80 μs, and subsequently cultured in cytochalasin B and cycloheximide for 6 h. In treatments 2 and 3, oocytes without zonae pellucidae were activated by a single DC pulse of 1.25 and 0.85 kV/cm for 80 μs, respectively, and subsequently cultured in cytochalasin B and cycloheximide for 6 h. In treatment 4, oocytes without zonae pellucidae were bisected by hand under a stereomicroscope using a microblade in 5 μg/mL cytochalasin B in TCM-199 supplemented with 15 mg/mL BSA, re-fused/activated by a single DC pulse of 1.25 kV/cm for 80 μs in activation medium, and cultured in cytochalasin B and cycloheximide for 6 h. The rates of blastocyst formation from activated oocytes (mean ± SEM) in treatments 1, 2, 3, and 4 were 55 ± 4%, 40 ± 2%, 49 ± 1%, and 41 ± 8%, respectively. In conclusion, the combined electrical and chemical activation efficiently induced parthenogenetic development of zona-free oocytes. Also, a more authentic control model for activation during SCNT was established by activating and producing blasctocysts from re-fused bisected oocytes.

scholarly journals 340PRODUCTION OF TRANSGENIC PORCINE BLASTOCYSTS BY HANDMADE CLONING

2004 ◽  
Vol 16 (2) ◽  
pp. 290 ◽  
Author(s):  
P.M. Kragh ◽  
G. Vajta ◽  
T.J. Corydon ◽  
L. Bolund ◽  
H. Callesen
Keyword(s):  
Nuclear Transfer ◽  
Culture Medium ◽  
Fluorescent Protein ◽  
Uv Light ◽  
Calcium Ionophore ◽  
Ionophore A23187 ◽  
Transgenic Pigs ◽  
Reconstructed Embryos ◽  
Cattle Serum ◽  
Handmade Cloning

The present study demonstrates the application of the recently developed handmade cloning (HMC) technique in production of transgenic porcine blastocysts. The HMC technique was originally established for bovine nuclear transfer (Vajta et al., 2003, Biol. Reprod. 68, 571–578), and has the advantages of being less demanding and more productive than traditional nuclear transfer techniques. Cumulus-oocyte complexes were aspirated from slaughterhouse ovaries and matured for 41h. Subsequently, the cumulus cells were removed by pipetting in 1mgmL−1 hyaluronidase in HEPES-buffered TCM-199; zonae pellucidae were removed by incubation in 2mgmL−1 pronase in HEPES-buffered TCM-199 supplemented with 2% cattle serum (T2) for 1min. Bisection was performed by hand under a stereomicroscope using a microblade in 5μgmL−1 cytochalasin B in TCM-199 supplemented with 20% cattle serum (T20). Demi-oocytes were incubated in 5μgmL−1 Hoechst 33342 in T20 for 10min, followed by examination under UV light to select the halves containing no chromatin, i.e., the cytoplasts. Porcine fibroblasts harvested from an ear skin biopsy were transfected with pN1-EGFP (Clontech) using Lipofectamine (Gibco, Life Technologies). G418 selection (0.8mgmL−1) was applied 48h after transfection, and well separated G418-resistant cell colonies originating from a single transfected cell were isolated, expanded, and cryopreserved. Days before, nuclear transfer cells were grown to a confluent monolayer in DMEM supplemented with 10% FCS. Fusions were performed 43h after start of maturation. One cytoplast was attached to one fibroblast in 500μgmL−1 phytohemagglutinin dissolved in T2. In the fusion chamber, covered with fusion medium (0.3M mannitol, 0.1mM MgSO4, 0.05mM CaCl2, and 0.01% PVA), one cytoplast-fibroblast pair was fused with one cytoplast in a single step. The fusions were performed with a double DC pulse of 65V, each pulse for 20μs and 0.1s apart from each other. Successfully fused embryos were activated 1h after the end of fusion by incubation in 2μM calcium ionophore A23187 in T20 for 5min followed by 3-h incubation in microdrops of culture medium (NCSU-23 with 4mgmL BSA) containing 2mM 6-dimethylaminopurine. Activated embryos were cultured individually in microdrops of culture medium for 7 days. In four independent experiments, 93% of attempted reconstructed embryos fused and survived activation (31/31, 15/23, 28/28, and 37/37, respectively). On Day 7 after activation, the blastocyst rates (per successfully reconstructed embryos) were 6% (2/31), and 7% (1/15), 7% (2/28), and 3% (1/37), respectively. Green Fluorescent Protein was expressed in all cells of the developing blastocysts. The results show that transgenic porcine blastocysts can be produced using HMC, and the technique may also be applied for the production of transgenic pigs.

scholarly journals 27BIRTH OF AFRICA'S FIRST NUCLEAR-TRANSFERRED ANIMAL PRODUCED WITH HANDMADE CLONING

2004 ◽  
Vol 16 (2) ◽  
pp. 136 ◽  
Author(s):  
P. Bartels ◽  
J. Joubert ◽  
M. de la Rey ◽  
R. de la Rey ◽  
R. Treadwell ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Calf Serum ◽  
Calcium Ionophore ◽  
Physiological Saline ◽  
Cumulus Cells ◽  
Animal Biotechnology ◽  
Additional Tool ◽  
Reconstructed Embryos ◽  
Cattle Serum ◽  
Hoechst Staining

Cloning technology has the potential to stimulate the development of the animal biotechnology industry in southern Africa, as well as provide conservationists with an additional tool to possibly assist with conserving critically endangered wildlife species sometime in the future. The aim of this study was to determine whether cloning could produce blastocysts and possibly live progeny in a field-type laboratory without micromanipulators and CO2 incubator. Approx. 1×1-cm ear skin notches were surgically removed from a physically immobilized 9-year-old Holstein cow, a former South African milk production record holder. The tissues were placed into physiological saline and transported to the laboratory at 4°C within 2h, cleaned with chlorohexidine gluconate and sliced finely in Minimal Essential Medium supplemented with 10% fetal calf serum. The resultant tissue explants were treated as previously described (Bartels et al., 2003 Theriogenology 59, 387) and actively growing fibroblast cultures were made available for the nuclear transfer process. Bovine oocytes from slaughterhouse-derived ovaries were collected and matured for 21h in modified TCM-199 medium supplemented with 15% cattle serum, 10IUmL−1 eCG and 15IUmL−1 hCG. Nuclear transfer was performed using the HMC technique (Vajta et al., 2003 Biol. Reprod. 68, 571–578). At 21h after the start of maturation, cumulus cells and zonae pellucidae were removed and oocytes were randomly bisected by hand. Cytoplasts were selected using Hoechst staining and a fluorescent microscope. After a two-step fusion, reconstructed embryos were activated with calcium ionophore and dimethylaminopurine. Culture was performed in SOFaaci medium supplemented with 5% cattle serum using WOWs (Vajta et al., Mol. Reprod. Dev. 50, 185–191). All incubations including culture of donor cells were performed in the submarine incubator system (SIS; Vajta et al., 1997 Theriogenology 48, 1379–1385). In two consecutive experiments, 6 blastocysts were produced from 52 reconstructed embryos. On Day 7, 5 blastocysts were selected for transfer into 3 previously synchronized recipients. All three recipients became pregnant, but two of the recipients aborted at six and seven months, respectively. Post-mortem examination on the first aborted fetus did not reveal any identifiable etiology, but coincided with 6 abortions from natural pregnancies during a heat wave, while the organism Brucella abortis was isolated from the second aborted fetus. The third pregnancy went to term, and a healthy calf, weighing 27kg, was delivered by Caesarean section. The three-month-old calf is being raised by a surrogate Jersey cow under standard dairy conditions and is expected to join the dairy in eighteen months’ time. The birth of ‘Futhi’, meaning ‘replicate’ in Zulu, is Africa’s first cloned animal and signifies an important milestone in the development of animal biotechnology in Africa.

236 PARTHENOGENETIC ACTIVATION OF IN VITRO-MATURED OVINE OOCYTES WITH STRONTIUM

2008 ◽  
Vol 20 (1) ◽  
pp. 197
Author(s):  
J. Zhu ◽  
K. H. S. Campbell
Keyword(s):  
Nuclear Transfer ◽  
Cytochalasin B ◽  
Blastocyst Stage ◽  
Oocyte Activation ◽  
Parthenogenetic Development ◽  
Activation Rate ◽  
Electrical Pulses ◽  
Blastocyst Rate ◽  
Activation Rates

The objective of the present experiments was to examine whether strontium could activate in vitro-matured ovine oocytes. Oocytes were collected and matured as previously described (Lee and Campbell 2006 Biol. Reprod. 74, 691–698). Briefly, selected cumulus–oocyte complexes were cultured in modified TCM-199 medium supplemented with 20% sheep serum and hormones for 22–23 h, at 39°C, 5% CO2 in air. Matured oocytes were randomly divided into four groups and treated as follows: (1) cultured in 10 mm strontium + 5 μg mL–1 cytochalasin B in Ca2+-free CZB medium for 4–5 h; (2) electrically activated in Ca2+-containing medium, then cultured in 10 mm strontium + 5 μg mL–1 cytochalasin B in Ca2+-free CZB medium for 4–5 h; (3) electrically activated in Ca2+-containing medium and then cultured in SOF medium containing 5 μg mL–1 cytochalasin B for 4–5 h; and (4) electrically activated in Ca2+-free medium and then transferred into SOF medium + 5 μg mL–1 cytochalasin B for 4–5 h. This experiment was repeated three times. Activation rates based on the number of pronuclear formations/the number of oocytes cultured were 96.7% (147/152), 95.9% (116/121), 75.9% (101/133), and 43.0% (56/107) in Groups 1–4, respectively. After 7 days of culture in SOF medium, 26.8%, 33.3%, 19.6%, and 0% of oocytes in Groups 1, 2, 3, and 4 developed to the blastocyst stage, respectively. Significant differences in blastocyst rate were observed across these groups except between groups 1 and 2 (P < 0.01). However, there were no significant differences in mean number of nuclei/blastocyst across Groups 1, 2, and 3 (P > 0.05). Our results demonstrated that in vitro-matured ovine oocytes can be effectively activated with strontium alone, resulting in an activation rate of 96.7% and a blastocyst rate of 26.8% (blastocysts/oocytes). Also, a combination of strontium and electrical pulses could benefit sheep oocyte activation and embryo development to the blastocyst stage (95.9% and 33.3%, respectively). We conclude that strontium is an effective activator for sheep oocyte activation and it could be used for sheep nuclear transfer. Table 1. Parthenogenetic development of oocytes activated by SrCl2+ and electrical pulses

scholarly journals 303PARTHENOGENETIC DEVELOPMENT OF PIG OOCYTES BY CHEMICAL ACTIVATION

2004 ◽  
Vol 16 (2) ◽  
pp. 271
Author(s):  
C.S. Park ◽  
D.I. Jin ◽  
M.Y. Kim ◽  
Y.J. Chang ◽  
Y.J. Yi
Keyword(s):  
Cytochalasin B ◽  
Polar Body ◽  
Chemical Activation ◽  
Formation Rate ◽  
Penicillin G ◽  
Oocyte Activation ◽  
Blastocyst Formation ◽  
Parthenogenetic Development ◽  
First Polar Body

Efficient activation is essential for the success of animal cloning by nuclear transfer. The aim of this study was to investigate the effects of chemical activation agents on parthenogenetic development of pig oocytes matured in vitro. The medium used for oocyte maturation was TCM-199 supplemented with 26.19mM sodium bicarbonate, 0.9mM sodium pyruvate, 10μgmL−1 insulin, 2μgmL−1 vitamin B12, 25mM HEPES, 10μgmL−1 bovine apotransferrin, 150μM cysteamine, 10IUmL−1 PMSG, 10IUmL−1 hCG, 10ngmL−1 EGF, 0.4% BSA, 75μgmL−1 sodium penicillin G, 50μgmL−1 streptomycin sulfate and 10% pFF. After about 22h of maturation, oocytes were cultured without cysteamine and hormones for 22h at 38.5°C, 5% CO2 in air. Cumulus-free oocytes showing first polar body were selected for activation. Oocytes were activated as follows. First, all oocytes were activated with 25mM HEPES buffered NCSU-23 medium containing 8% ethanol for 10min. After that, in treatment 1, oocytes were incubated in the NCSU-23 medium supplemented with 7.5μgmL−1 cytochalasin B for 3h. In treatment 2, oocytes were incubated in the NCSU-23 medium supplemented with 10μgmL−1 cycloheximide for 3h. In treatment 3, oocytes were incubated in the NCSU-23 medium supplemented with 7.5μgmL−1 cytochalasin B for 1.5h, and then were incubated in the NCSU-23 medium supplemented with 10μgmL−1 cycloheximide for 1.5h. In treatment 4, oocytes were incubated in the NCSU-23 medium supplemented with 7.5μgmL−1 cytochalasin B plus 10μgmL−1 cycloheximide for 3h. Following activation, oocytes were transferred into 500μL NCSU-23 culture medium containing 0.4% BSA for further culture for 20 and 144h. Activated oocytes were fixed and stained for evaluation of activation rate, cleaved oocytes, blastocyst formation rate and cell numbers per blastocyst. Data were analysed by ANOVA and Duncan’s multiple range test using the SAS program. The rate of oocyte activation was higher in treatment 4 (62.1%) than in treatment 1, 2 and 3 (52.0, 49.6 and 58.0%, respectively). The percentage of cleaved oocytes was lower in treatment 1 and 2 (56.9 and 55.2%) than in treatment 3 and 4 (68.8 and 68.5%). The rate of blastocyst formation from the cleaved oocytes was higher in treatment 3 and 4 (19.8 and 22.0%) than in treatment 1 and 2 (12.1 and 11.7%). Mean cells per blastocyst were lowest in treatment 2 (21.2±0.9) compared to treatment 1, 3 and 4 (27.3±2.2, 30.4±3.8 and 30.9±3.4, respectively). In conclusion, cytochalasin B combined with cycloheximide was more efficient for parthenogenetic development of pig oocytes matured in vitro.

scholarly journals 61 CLONED MOUSE PRODUCED USING A ZONA FREE METHOD OF NUCLEAR TRANSFER

2005 ◽  
Vol 17 (2) ◽  
pp. 180
Author(s):  
R. Ribas ◽  
B. Oback ◽  
J. Taylor ◽  
A. Maurício ◽  
M. Sousa ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Cytochalasin B ◽  
Uv Light ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Strontium Chloride ◽  
Embryonic Cells ◽  
Reconstructed Embryos

Mice have been cloned from somatic and embryonic cells; however, only 0–3% of the reconstructed embryos develop into viable offspring. In addition, the piezo microinjection method widely used for mouse nuclear transfer (NT) is difficult to master. Our objective was to compare cumulus and ES cells as nuclear donors using a simplified method of zona-free NT. In cattle, zona-free NT is simpler, faster, easier to learn and more reproducible than zona-intact NT (Oback et al. 2003 Cloning Stem Cells 5, 3–12). Oocytes were recovered at metaphase II stage (13 h after hCG injection) from the oviducts of C57BL/6J × DBA/2 F1 females (8–10 weeks of age). Cumulus cells were removed with hyaluronidase (300 units/mL) and the zona pellucida digested with pronase (0.5%) at 37°C for 3 min. Oocytes were then enucleated under UV light in cytochalasin B (5 μg/mL) after a 5-min staining with Hoechst (5 μL/mL). The metaphase DNA was removed in an enucleation pipette (16–20 μm, perpendicular break) by separating karyoplast and cytoplast with a simple separation pipette (60–80 μm, perpendicular break, closed round tip). Embryonic stem (ES) cells were cultured for 3 days and serum-starved for 16 h before use. Cells from this line had yielded offspring by the piezo procedure. Cumulus cells were used freshly. Donor cells were attached to the cytoplasts with phytohemagglutinin (10 μg/mL) and couplets were electrically fused in 0.2 mM mannitol buffer. Reconstructed embryos were activated 1–2 h after fusion for 5–6 h in CZB medium containing 10 mM strontium chloride and 5 μg/mL of cytochalasin B. Embryos were cultured individually in 5-μL droplets in CZB. Morulae and blastocysts were transferred into the uteri (Day 2.5) of pseudopregnant surrogate mothers (C57BL/6J × CBA/2J). Recipient mothers were sacrificed at 19.5 days postcoitum and pups removed. Airways were cleaned to remove fluid and the pups were held in a warm box before being fostered by a lactating mother. During development of the technique, we assessed the frequency of fusion, cleavage of reconstructed embryos, and development to morula/blastocyst stage. Fusion (58.1 ± 6.7% vs. 24.2 ± 1.7%, P < 0.001) and cleavage (66.4 ± 4.2% vs. 50.5 ± 5.4%, P < 0.05), all respectively, were higher when cumulus cells were used as donors, as compared with ES cells. However, the percentage of embryos developing to morula/blastocyst stage was greater when ES cells were used (22.2 ± 4.2% vs. 5.3 ± 2.7%, P < 0.01). Using ES cells as donors, 19/94 (20.2%) reconstructed embryos reached compacted morula/blastocyst stage. After transfer to five recipients, one pup was born (5.2%). It was larger and heavier than uncloned pups of the same age. The pup is healthy and now 12 weeks old. Genotype was confirmed by microsatellite analysis. The birth of a healthy cloned mouse pup from zona-free NT provides “proof of principle” of a technology that promises to increase throughput, ease of operation, and reproducibility of mouse cloning.

312 CHEMICAL ACTIVATION OF IN VITRO-MATURED PORCINE OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 263
Author(s):  
A. Bali Papp ◽  
E. Varga
Keyword(s):  
Cytochalasin B ◽  
Chemical Activation ◽  
Basic Medium ◽  
Ionophore A23187 ◽  
Strontium Chloride ◽  
Oocyte Activation ◽  
Parthenogenetic Development ◽  
Activation Rate ◽  
Significant Difference

Parthenogenetic oocyte activation is important for nuclear transfer and for the understanding of cell cycle regulation of oocytes. Several chemical agents, including ethanol, cycloheximide, strontium, cytochalasin B, 6 dimethylaminopurine, CaCl2 and ionophore A23187 can induce mammalian oocyte activation in vitro. The objectives of the present study were: (1) to assess the ability of strontium chloride (S), cytochalasin B (CB), cycloheximide (CX), and 6-dimethylaminopurine (D) to induce activation and parthenogenetic development in porcine oocytes; and (2) to verify whether the combinations of treatments (SB group = strontium combined with cytochalasin; SX group = strontium combined with cycloheximide, and SD group = strontium combined with 6-dimethylaminopurine) improves activation and parthenogenetic development rates. Oocytes from slaughterhouse ovaries were matured in vitro for 42 h at 39�C, in 5% CO2 in air. The basic medium used for oocyte maturation was TCM-199 supplemented with 10% pig follicular fluid, 1.25 mM L-glutamine, 0.9 mM Na-pyruvate, 100 �M cysteamine, 0.1 mg/mL streptomycin sulfate, 10 IU/mL pregnant mare serum gonadotropin (PMSG), and 10 IU/mL hCG (Werfft-Chemie GmbH, Vienna, Austria). Denuded MII oocytes were cultured in activation solution for 5 h. Thereafter the oocytes were cultured in NCSU37 for 6 days. At 48 h and 6 days after activation, oocytes, zygotes were fixed in acetic acid:alcohol (1/3 w/v), then stained with 0.1% (w/v) orcein in 45% (v/v) acetic acid, and evaluated under a phase contrast microscope. Each experiment was repeated four times. All data were analyzed by ANOVA, followed by Duncan's multiple range test (P < 0.05). A total of 2243 oocytes were activated in the different groups. In all groups, more than 45% of the oocytes were activated. No significant difference was observed in activation rate among SD (346/170, 49.13%), SX (302/164, 54.3%), and SB (318/182, 57.23%) groups. The activation rate for CB was significantly higher (P < 0.05) than for D or S (323/192, 59.44 � 6.84%; 366/176, 48.09 � 3.43%; and 319/183, 53.29 � 5.39%, respectively). The blastocyst rate for SX was significantly higher (P < 0.05) than that for D, SD, or SB (8.64 � 8.07%; and 0 � 0%; 0 � 0%; and 1.27 � 2.41%, respectively). In conclusion, this study suggests that chemical activation procedure is the most effective in strontium chloride combined with cycloheximide. The lowest oocyte fragmentation rates were in SX (28.40 � 1.26%) and CX (21.05 � 1.12%). This work was supported by the the Hungarian Scientific T 43131 Research Foundation and the Hungarian Science on Technology Foundation E 14/04.

39 Embryo Development of Kazakh Argali (Ovis ammon collium) by Handmade Cloning

2018 ◽  
Vol 30 (1) ◽  
pp. 159
Author(s):  
Y. Toishibekov ◽  
E. Asanova ◽  
M. Yermekova ◽  
A. Seisenbayeva ◽  
D. Toishybek ◽  
...  
Keyword(s):  
Embryo Development ◽  
Nuclear Transfer ◽  
Culture Medium ◽  
Calcium Ionophore ◽  
Blastocyst Stage ◽  
Critically Endangered ◽  
Domestic Sheep ◽  
Wildlife Species ◽  
Cattle Serum ◽  
Ovis Ammon

Wildlife conservation requires innovative preservation methods in order to preserve gene and species biodiversity. Nuclear transfer has the potential to preserve genes from critically endangered wildlife species where few or no oocytes are available from the endangered species, and where cryopreserved cell lines have been conserved in cryobanks. The purpose of this study was to investigate the developmental ability of embryos reconstructed with transfer of cryopreserved somatic cells from the Kazakh argali (Ovis ammon collium) to enucleated domestic sheep (Ovies aries) oocytes. Frozen-thawed fibroblasts were diluted with DMEM (1:5) and centrifuged at 300g for 7 to 10 min. Supernatants were removed, and cells were diluted with DMEM at a concentration of 2 × 106 cells mL−1. Fibroblasts were placed into culture Petri dishes containing DMEM supplemented with 20% (v/v) fetal bovine serum (FBS), and incubated at 5% CO2, 95% relative humidity, and 37°C. After 21 to 22 days of incubation, a fibroblast monolayer was observed, culture medium was removed, and cells were incubated for 7 to 10 min in presence of Dulbecco’s PBS + 0.25% trypsin. Dissociated fibroblasts were washed with DMEM by centrifugation at 300 × g for 10 min. Cumulus-oocyte complexes were aspirated from slaughterhouse ovaries. Subsequently, the cumulus cells were removed by pipetting in 1 mg mL−1 hyaluronidase in HEPES-buffered TCM-199; zonae pellucidae were removed by incubation in 2 mg mL−1 pronase in HEPES-buffered TCM-199 supplemented with 2% cattle serum (T2) for 1 min. Bisection was performed by hand under a stereomicroscope using a microblade in 5 μg mL−1 cytochalasin B in TCM-199 supplemented with 20% cattle serum (T20). Fusions were performed 24 to 28 h after the start of maturation. One cytoplast was attached to one fibroblast in 500 μg mL−1 phytohemagglutinin dissolved in T2. In the fusion chamber, covered with fusion medium (0.3 M mannitol, 0.1 mM MgSO4, 0.05 mM CaCl2, and 0.01% polyvinyl alcohol), one cytoplast-fibroblast pair was fused with one cytoplast in a single step. The fusions were performed with a single DC pulse of 100V, each pulse for 9 μs. Successfully fused embryos were activated 1 h after the end of fusion by incubation in 2 μM calcium ionophore (Sigma, St. Louis, MO, USA) in T20 for 5 min followed by 3-h incubation in microdrops of culture medium containing 2 mM 6-DMAP. After successful reconstruction, 79 nuclear transferred and activated embryos were cultured in well-of-the-wells in trigas (5% O2, 5% CO2, 90% N2) in Submarine incubation system for 7 days. All except 15 embryos cleaved; 35 (44.3%) developed to compacted morula, and 15 (18.9%) to the blastocyst stage. In conclusion, argali embryos developed from reconstruction using their frozen–thawed fibroblasts combined with domestic sheep cytoplasts; however, in vitro developmental ability to the blastocyst stage was limited. Additional research that establishes the early embryo development with optimising nuclear transfer techniques may have a potential role in the conservation of critically endangered wildlife species.

Improved functional oocyte enucleation by actinomycin D for bovine somatic cell nuclear transfer

2019 ◽  
Vol 31 (8) ◽  
pp. 1321 ◽  
Author(s):  
Marcelo T. Moura ◽  
Jeferson Badaraco ◽  
Regivaldo V. Sousa ◽  
Carolina M. Lucci ◽  
Rodolfo Rumpf
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Caspase 3 ◽  
Actinomycin D ◽  
Cumulus Cells ◽  
Bovine Oocyte ◽  
Cleavage Stage ◽  
Animal Cloning ◽  
Parthenogenetic Development

Somatic cell nuclear transfer (SCNT) allows animal cloning but remains technically challenging. This study investigated limitations to functional oocyte enucleation by actinomycin D (AD) as a means of making SCNT easier to perform. Denuding oocytes or inhibiting transcription before AD treatment revealed that the toxicity of this compound during bovine oocyte maturation is mediated by cumulus cells. Exposure of denuded oocytes to higher concentrations of AD (5–20μgmL−1) and stepwise reductions of the incubation period (from 14.0 to 0.25h) led to complete inhibition of parthenogenetic development. Bovine SCNT using this improved AD enucleation protocol (NT(AD)) restored cleavage rates compared with rates in the parthenogenetic and SCNT controls (P(CTL) and NT(CTL) respectively). However, NT(AD) was associated with increased caspase-3 activity in cleavage stage embryos and did not recover blastocyst rates. The removal of AD-treated oocyte spindle before reconstruction (NT(AD+SR)) improved embryo development and reduced caspase-3 activity to levels similar to those in the P(CTL) and NT(CTL) groups. Furthermore, mid-term pregnancies were achieved using NT(AD+SR) blastocysts. In conclusion, improvements in AD functional enucleation for bovine SCNT circumvents most cellular roadblocks to early embryonic development and future investigations must focus on restoring blastocyst formation.

Mechanism of the adverse effect of hyaluronidase used for oocyte denudation on early development of bovine embryos

Zygote ◽  
2021 ◽  
pp. 1-5
Author(s):  
Shiori Ashibe ◽  
Kanade Irisawa ◽  
Ken Yokawa ◽  
Yoshikazu Nagao
Keyword(s):  
Treatment Group ◽  
Assisted Reproductive Technologies ◽  
Cumulus Cells ◽  
Reproductive Technologies ◽  
Control Group ◽  
Free Calcium ◽  
Parthenogenetic Development ◽  
Early Embryos ◽  
Bovine Oocytes

Summary Hyaluronidase is widely used in animal and human assisted reproductive technologies (ARTs) to remove cumulus cells around oocytes. However, adverse effects of hyaluronidase treatment, such as increased rates of degeneration and parthenogenesis, have been found after treatment of human and mouse oocytes. Currently, the mechanism(s) of the detrimental effects are unclear. The present study was initiated to identify the mechanism of adverse responses to hyaluronidase treatment in bovine oocytes and early embryos. Cumulus cells were removed from cumulus–oocyte complexes (COCs) with or without hyaluronidase and the oocytes were subjected to intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF). Significantly lower rates of blastocyst formation were obtained in the hyaluronidase treatment group after ICSI (22.4%) and IVF (21.2%) compared with the non-hyaluronidase control groups: 36.1% after ICSI and 30.4% after IVF. Next, we examined the effect of hyaluronidase on parthenogenetic development rates and on the cytoplasmic levels of free calcium ions (Ca2+), reactive oxygen species (ROS) and reduced glutathione (GSH). No differences in parthenogenesis rates were found between treated and untreated groups. Ca2+ levels in oocytes from the hyaluronidase treatment group indicated using mean fluorescence intensity were significantly higher (68.8 ± 5.3) compared with in the control group (45.0 ± 2.5). No differences were found in the levels of ROS or GSH between the treated and untreated groups. We conclude that hyaluronidase might trigger an increase in Ca2+ levels in oocytes, resulting in a decreased potential for normal embryonic development.

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